Anti-canine CD20 antibodies

Abstract

Antibodies and antigen-binding portions thereof that bind to canine CD20 are provided. Also provided are compositions and methods for the treatment of conditions mediated by B cells in canine subjects.SOLUTION: Provided is an isolated canine antibody or antigen binding portion thereof that binds to canine CD20, wherein the antibody comprises or consists of: a) an HCCDR1 sequence comprising or consisting of a particular sequence; b) an HCCDR2 sequence comprising or consisting of a particular sequence; c) an HCCDR3 sequence comprising or consisting of a particular sequence; d) an LCCDR1 sequence comprising or consisting of a particular sequence; e) an LCCDR2 sequence comprising or consisting of a particular sequence; and f) an LCCDR3 sequence comprising or consisting of a particular sequence.SELECTED DRAWING: Figure 1

Description

[Background technology]

[0001] Canine lymphoma is one of the most common cancers diagnosed in dogs, representing approximately 7-14% of all cancers. As in humans, there are many different types of canine lymphoma, ranging from rapidly progressing cancers to chronic diseases.

[0002] CD20 is a cell surface protein thought to be involved in the regulation of B cell proliferation and differentiation. The antigen contains four transmembrane spanning regions and is present on the surface of almost all B cells, both normal and malignant.

[0003] Human antibodies that recognize human CD20, such as rituximab, are used to treat human diseases characterized by an excess number of B cells or overactive or dysfunctional B cells. These antibodies destroy B cells. Rituximab is considered a revolutionary advance in the treatment of B-cell lymphoma.

[0004] Since the development of antibodies such as rituximab for humans more than 20 years ago, many antibodies that recognize canine CD20 have been reported in the literature. However, none of them are currently in common clinical use. Therefore, there is still a need for different therapies. The present invention aims to address this need. Summary of the Invention

[0005] In a first aspect, the present invention relates to an isolated canine antibody, or antigen-binding portion thereof, that binds to canine CD20, the antibody comprising: a) an HC CDR1 sequence comprising or consisting of SEQ ID NO: 27, or an amino acid sequence having one or two amino acid differences compared to SEQ ID NO: 27; b) an HC CDR2 sequence comprising or consisting of SEQ ID NO: 28, or an amino acid sequence having 1, 2, 3, or 4 amino acid differences compared to SEQ ID NO: 28; c) an HC CDR3 sequence comprising or consisting of SEQ ID NO: 29, or an amino acid sequence having one or two amino acid differences compared to SEQ ID NO: 29; d) an LC CDR1 sequence comprising or consisting of SEQ ID NO: 30, or an amino acid sequence having one amino acid difference compared to SEQ ID NO: 30; e) an LC CDR2 sequence comprising or consisting of SEQ ID NO: 31, or an amino acid sequence having one amino acid difference compared to SEQ ID NO: 31; and f) comprises an LC CDR3 sequence comprising or consisting of SEQ ID NO: 32, or an amino acid sequence having one or two amino acid differences compared to SEQ ID NO: 32.

[0006] In an embodiment of another aspect or the first aspect of the invention, the antibody, or antigen-binding portion thereof, has a HC CDR1 sequence comprising or consisting of SEQ ID NO:7, a HC CDR2 sequence comprising or consisting of SEQ ID NO:8, a HC CDR3 sequence comprising or consisting of SEQ ID NO:9, a LC CDR1 sequence comprising or consisting of SEQ ID NO:10, a LC CDR1 sequence comprising or consisting of SEQ ID NO:11, and a LC CDR1 sequence comprising or consisting of SEQ ID NO:12.

[0007] In an embodiment of another aspect or the first aspect of the invention, the antibody has a HC CDR1 sequence comprising or consisting of SEQ ID NO: 17, a HC CDR2 sequence comprising or consisting of SEQ ID NO: 18, a HC CDR3 sequence comprising or consisting of SEQ ID NO: 19, a LC CDR1 sequence comprising SEQ ID NO: 20, a LC CDR2 sequence comprising or consisting of SEQ ID NO: 21, and a LC CDR3 sequence comprising or consisting of SEQ ID NO: 22.

[0008] In an embodiment of another aspect or the first aspect of the invention, the antibody has a HC CDR1 sequence comprising SEQ ID NO: 37, a HC CDR2 sequence comprising SEQ ID NO: 38, a HC CDR3 sequence comprising SEQ ID NO: 39, a LC CDR1 sequence comprising SEQ ID NO: 40, a LC CDR2 sequence comprising SEQ ID NO: 41, and a LC CDR3 sequence comprising SEQ ID NO: 42.

[0009] In an embodiment of another aspect or the first aspect of the invention, the antibody has a HC CDR1 sequence comprising or consisting of SEQ ID NO: 47, a HC CDR2 sequence comprising or consisting of SEQ ID NO: 48, a HC CDR3 sequence comprising or consisting of SEQ ID NO: 49, a LC CDR1 sequence comprising or consisting of SEQ ID NO: 50, a LC CDR2 sequence comprising or consisting of SEQ ID NO: 51, and a LC CDR3 sequence comprising or consisting of SEQ ID NO: 52.

[0010] In an embodiment of another aspect or the first aspect of the invention, the antibody has a HC CDR1 sequence comprising or consisting of SEQ ID NO: 82, a HC CDR2 sequence comprising or consisting of SEQ ID NO: 83, a HC CDR3 sequence comprising or consisting of SEQ ID NO: 84, a LC CDR1 sequence comprising or consisting of SEQ ID NO: 85, a LC CDR2 sequence comprising or consisting of SEQ ID NO: 86, and a LC CDR3 sequence comprising or consisting of SEQ ID NO: 87.

[0011] In an embodiment of another aspect or the first aspect of the invention, the antibody has a HC CDR1 sequence comprising or consisting of SEQ ID NO: 92, a HC CDR2 sequence comprising or consisting of SEQ ID NO: 93, a HC CDR3 sequence comprising or consisting of SEQ ID NO: 94, a LC CDR1 sequence comprising or consisting of SEQ ID NO: 95, a LC CDR2 sequence comprising or consisting of SEQ ID NO: 96, and a LC CDR3 sequence comprising or consisting of SEQ ID NO: 97.

[0012] In an embodiment of another aspect or the first aspect of the invention, the antibody has a HC CDR1 sequence comprising or consisting of SEQ ID NO: 102, a HC CDR2 sequence comprising or consisting of SEQ ID NO: 103, a HC CDR3 sequence comprising or consisting of SEQ ID NO: 104, a LC CDR1 sequence comprising or consisting of SEQ ID NO: 105, a LC CDR2 sequence comprising or consisting of SEQ ID NO: 106, and a LC CDR3 sequence comprising or consisting of SEQ ID NO: 107.

[0013] In an embodiment of another aspect or the first aspect of the invention, the antibody has a HC CDR1 sequence comprising or consisting of SEQ ID NO:112, a HC CDR2 sequence comprising or consisting of SEQ ID NO:113, a HC CDR3 sequence comprising or consisting of SEQ ID NO:114, a LC CDR1 sequence comprising or consisting of SEQ ID NO:115, a LC CDR2 sequence comprising or consisting of SEQ ID NO:116, and a LC CDR3 sequence comprising or consisting of SEQ ID NO:117.

[0014] In an embodiment of another aspect or the first aspect of the invention, the antibody has a HC CDR1 sequence comprising or consisting of SEQ ID NO: 122, a HC CDR2 sequence comprising or consisting of SEQ ID NO: 123, a HC CDR3 sequence comprising or consisting of SEQ ID NO: 124, a LC CDR1 sequence comprising or consisting of SEQ ID NO: 125, a LC CDR2 sequence comprising or consisting of SEQ ID NO: 126, and a LC CDR3 sequence comprising or consisting of SEQ ID NO: 127.

[0015] In an embodiment of another aspect or the first aspect of the invention, the antibody has a HC CDR1 sequence comprising or consisting of SEQ ID NO: 132, a HC CDR2 sequence comprising or consisting of SEQ ID NO: 133, a HC CDR3 sequence comprising or consisting of SEQ ID NO: 134, a LC CDR1 sequence comprising or consisting of SEQ ID NO: 135, a LC CDR2 sequence comprising or consisting of SEQ ID NO: 136, and a LC CDR3 sequence comprising or consisting of SEQ ID NO: 137.

[0016] In an embodiment of another aspect or the first aspect of the invention, the antibody has a HC CDR1 sequence comprising or consisting of SEQ ID NO: 142, a HC CDR2 sequence comprising or consisting of SEQ ID NO: 143, a HC CDR3 sequence comprising or consisting of SEQ ID NO: 144, a LC CDR1 sequence comprising or consisting of SEQ ID NO: 145, a LC CDR2 sequence comprising or consisting of SEQ ID NO: 146, and a LC CDR3 sequence comprising or consisting of SEQ ID NO: 147.

[0017] In an embodiment of another aspect or the first aspect of the invention, the antibody has a HC CDR1 sequence comprising or consisting of SEQ ID NO: 152, a HC CDR2 sequence comprising or consisting of SEQ ID NO: 153, a HC CDR3 sequence comprising or consisting of SEQ ID NO: 154, a LC CDR1 sequence comprising or consisting of SEQ ID NO: 155, a LC CDR2 sequence comprising or consisting of SEQ ID NO: 156, and a LC CDR3 sequence comprising or consisting of SEQ ID NO: 157.

[0018] In an embodiment of another aspect or the first aspect of the invention, the antibody has a HC CDR1 sequence comprising or consisting of SEQ ID NO: 162, a HC CDR2 sequence comprising or consisting of SEQ ID NO: 163, a HC CDR3 sequence comprising or consisting of SEQ ID NO: 164, a LC CDR1 sequence comprising or consisting of SEQ ID NO: 165, a LC CDR2 sequence comprising or consisting of SEQ ID NO: 166, and a LC CDR3 sequence comprising or consisting of SEQ ID NO: 167.

[0019] In an embodiment of another aspect or the first aspect of the invention, the antibody has a HC CDR1 sequence comprising or consisting of SEQ ID NO: 172, a HC CDR2 sequence comprising or consisting of SEQ ID NO: 173, a HC CDR3 sequence comprising or consisting of SEQ ID NO: 174, a LC CDR1 sequence comprising or consisting of SEQ ID NO: 175, a LC CDR2 sequence comprising or consisting of SEQ ID NO: 176, and a LC CDR3 sequence comprising or consisting of SEQ ID NO: 177.

[0020] In an embodiment of another aspect or the first aspect of the invention, the antibody has a HC CDR1 sequence comprising or consisting of SEQ ID NO: 182, a HC CDR2 sequence comprising or consisting of SEQ ID NO: 183, a HC CDR3 sequence comprising or consisting of SEQ ID NO: 184, a LC CDR1 sequence comprising or consisting of SEQ ID NO: 185, a LC CDR2 sequence comprising or consisting of SEQ ID NO: 186, and a LC CDR3 sequence comprising or consisting of SEQ ID NO: 187.

[0021] In an embodiment of another aspect or the first aspect of the invention, the antibody has a HC CDR1 sequence comprising or consisting of SEQ ID NO: 192, a HC CDR2 sequence comprising or consisting of SEQ ID NO: 193, a HC CDR3 sequence comprising or consisting of SEQ ID NO: 194, a LC CDR1 sequence comprising or consisting of SEQ ID NO: 195, a LC CDR2 sequence comprising or consisting of SEQ ID NO: 196, and a LC CDR3 sequence comprising or consisting of SEQ ID NO: 197.

[0022] In an embodiment of another aspect or the first aspect of the invention, the antibody has a HC CDR1 sequence comprising or consisting of SEQ ID NO:202, a HC CDR2 sequence comprising or consisting of SEQ ID NO:203, a HC CDR3 sequence comprising or consisting of SEQ ID NO:204, a LC CDR1 sequence comprising or consisting of SEQ ID NO:205, a LC CDR2 sequence comprising or consisting of SEQ ID NO:206, and a LC CDR3 sequence comprising or consisting of SEQ ID NO:207.

[0023] In an embodiment of another aspect or the first aspect of the invention, the antibody has a HC CDR1 sequence comprising or consisting of SEQ ID NO:212, a HC CDR2 sequence comprising or consisting of SEQ ID NO:213, a HC CDR3 sequence comprising or consisting of SEQ ID NO:214, a LC CDR1 sequence comprising or consisting of SEQ ID NO:215, a LC CDR2 sequence comprising or consisting of SEQ ID NO:216, and a LC CDR3 sequence comprising or consisting of SEQ ID NO:217.

[0024] In an embodiment of another aspect or the first aspect of the invention, the antibody has a HC CDR1 sequence comprising or consisting of SEQ ID NO:222, a HC CDR2 sequence comprising or consisting of SEQ ID NO:223, a HC CDR3 sequence comprising or consisting of SEQ ID NO:224, a LC CDR1 sequence comprising or consisting of SEQ ID NO:225, a LC CDR2 sequence comprising or consisting of SEQ ID NO:226, and a LC CDR3 sequence comprising or consisting of SEQ ID NO:227.

[0025] In an embodiment of another aspect or the first aspect of the invention, the antibody has a HC CDR1 sequence comprising or consisting of SEQ ID NO: 232, a HC CDR2 sequence comprising or consisting of SEQ ID NO: 233, a HC CDR3 sequence comprising or consisting of SEQ ID NO: 234, a LC CDR1 sequence comprising or consisting of SEQ ID NO: 235, a LC CDR2 sequence comprising or consisting of SEQ ID NO: 236, and a LC CDR3 sequence comprising or consisting of SEQ ID NO: 237.

[0026] In one embodiment, the antibody, or antigen-binding portion thereof, comprises an HC variable region sequence comprising SEQ ID NO: 24, or a sequence having at least 75%, 80%, 85%, or 90% sequence identity thereto, and an LC variable region sequence comprising SEQ ID NO: 26, or a sequence having at least 70%, 75%, 80%, 85%, 90%, or 95% sequence identity thereto.

[0027] For example, an antibody or antigen-binding portion thereof may a) an HC variable region sequence comprising SEQ ID NO: 4, and an LC variable region sequence comprising SEQ ID NO: 6; b) an HC variable region sequence comprising SEQ ID NO: 14, and an LC variable region sequence comprising SEQ ID NO: 16; c) an HC variable region sequence comprising SEQ ID NO: 34, and an LC variable region sequence comprising SEQ ID NO: 36; d) an HC variable region sequence comprising SEQ ID NO: 44, and an LC variable region sequence comprising SEQ ID NO: 46; e) an HC variable region sequence comprising SEQ ID NO: 79, and an LC variable region sequence comprising SEQ ID NO: 81; or an LC variable region sequence comprising SEQ ID NO: 238; f) an HC variable region sequence comprising SEQ ID NO: 89 and an LC variable region sequence comprising SEQ ID NO: 91, or an LC variable region sequence comprising SEQ ID NO: 239; g) an HC variable region sequence comprising SEQ ID NO: 99 and an LC variable region sequence comprising SEQ ID NO: 101, or an LC variable region sequence comprising SEQ ID NO: 240; h) an HC variable region sequence comprising SEQ ID NO: 109, and an LC variable region sequence comprising SEQ ID NO: 111, or an LC variable region sequence comprising SEQ ID NO: 241; i) an HC variable region sequence comprising SEQ ID NO: 119, and an LC variable region sequence comprising SEQ ID NO: 121; j) an HC variable region sequence comprising SEQ ID NO: 129, and an LC variable region sequence comprising SEQ ID NO: 131, or an LC variable region sequence comprising SEQ ID NO: 242; k) an HC variable region sequence comprising SEQ ID NO: 139, and an LC variable region sequence comprising SEQ ID NO: 141, or an LC variable region sequence comprising SEQ ID NO: 243; l) an HC variable region sequence comprising SEQ ID NO: 149, and an LC variable region sequence comprising SEQ ID NO: 151, or an LC variable region sequence comprising SEQ ID NO: 244; m) an HC variable region sequence comprising SEQ ID NO: 159, and an LC variable region sequence comprising SEQ ID NO: 161, or an LC variable region sequence comprising SEQ ID NO: 245; n) an HC variable region sequence comprising SEQ ID NO: 169, and an LC variable region sequence comprising SEQ ID NO: 171; o) an HC variable region sequence comprising SEQ ID NO: 179, and an LC variable region sequence comprising SEQ ID NO: 181, or an LC variable region sequence comprising SEQ ID NO: 246; p) an HC variable region sequence comprising SEQ ID NO: 189, and an LC variable region sequence comprising SEQ ID NO: 191, or an LC variable region sequence comprising SEQ ID NO: 247; q) an HC variable region sequence comprising SEQ ID NO: 199, and an LC variable region sequence comprising SEQ ID NO: 201; an LC variable region sequence comprising SEQ ID NO: 248; r) an HC variable region sequence comprising SEQ ID NO: 209, and an LC variable region sequence comprising SEQ ID NO: 211; an LC variable region sequence comprising SEQ ID NO: 249; s) an HC variable region sequence comprising SEQ ID NO: 219 and an LC variable region sequence comprising SEQ ID NO: 221; or t) has an HC variable region sequence comprising SEQ ID NO: 229, and an LC variable region sequence comprising SEQ ID NO: 231; and an LC variable region sequence comprising SEQ ID NO: 250.

[0028] For example, the antigen-binding portion is an scFv, Fv, heavy chain or single domain antibody.

[0029] The present invention also relates to isolated canine antibodies, or antigen-binding portions thereof, that bind to canine CD20 in competition with the above-described antibodies, or antigen-binding portions thereof.

[0030] For example, the antibody or antigen-binding portion thereof is conjugated to a therapeutic moiety.

[0031] For example, the therapeutic moiety is a second antibody or an antigen-binding portion thereof.

[0032] For example, the second antibody or antigen-binding portion thereof binds to a different target.

[0033] For example, the antibody or antigen-binding portion thereof is conjugated to a further moiety selected from a half-life extending moiety, a label, a cytotoxin, a liposome, a nanoparticle, or a radioisotope.

[0034] For example, the antibody, or antigen-binding portion thereof, is a-fucosylated.

[0035] The present invention also relates to pharmaceutical compositions comprising the above-described antibodies or antigen-binding portions thereof.

[0036] The present invention relates to a method of treating a B-cell mediated condition in a canine subject in need thereof, comprising administering an effective amount of the above-described antibody or antigen-binding portion thereof, or the above-described pharmaceutical composition.

[0037] For example, a condition mediated by B cells is a B cell lymphoma or leukemia.

[0038] For example, conditions mediated by B cells are immune-mediated diseases.

[0039] For example, the immune-mediated disease is an autoimmune disease.

[0040] For example, separate therapeutic agents may be administered separately to a subject.

[0041] For example, the therapeutic agent is a cytotoxic agent or a radiotoxic agent.

[0042] For example, the therapeutic agent is an immunosuppressant.

[0043] For example, the therapeutic agent is an immunomodulatory agent, such as a cytokine or chemokine.

[0044] The present invention also relates to an antibody or antigen-binding portion as described above, or a pharmaceutical composition as described above, for use in the treatment of a disease.

[0045] For example, the disease is a B cell mediated disease.

[0046] For example, a disease mediated by B cells is a B cell lymphoma or leukemia.

[0047] For example, diseases mediated by B cells are immune-mediated diseases.

[0048] For example, the immune-mediated disease is an autoimmune disease.

[0049] The present invention also relates to a method for inhibiting tumor growth or metastasis, comprising contacting tumor cells with an effective amount of the above-described antibody or antigen-binding portion thereof, or the above-described pharmaceutical composition.

[0050] The present invention also relates to a method of killing tumor cells that express CD20, comprising contacting the cells with an antibody as described above or a pharmaceutical composition as described above, such that killing of the cells that express CD20 occurs.

[0051] For example, the tumor cells are canine tumor cells.

[0052] The present invention also relates to nucleic acid sequences encoding the above-described antibodies or antibody antigen-binding portions thereof.

[0053] For example, the nucleic acid sequences may be any of SEQ ID NOs: 3, 5, 13, 15, 23, 25, 33, 35, 43, 45, 78, 80, 88, 90, 98, 100, 108, 110, 118, 120, 128, 130, 138, 140, 148, 150, 158, 160, 168, 170, 178, 180, 188, 190, 198, 200, 208, 210, 218, 220, 228, 230, 251, 252, 253, 254, 255, 256, 257, 258, 259, 260, 261, 262, 263, 264, 265, 266, 267, 268, 269, 270, 271, 272, 273, 274, 275, 276, 277, 278, 279, 280, 281, 282, 283, 284, 285, 286, 287, 288, 289, 290, 300, 301, 302, 303, 304, 305, 306, 307, 308, 309, 310, 311, 312, 313, 314, 315, 316, 317, 318, 319, 257, 258, 259, 260, 261, 262, 263, 264, 265, 266, 267, 268, 269, 270, 271, 272, 273, 274, 275, 276, 277, 278, 279, 280, 281, 282, 283, 284, 285, 286, 287, 288, 289, 290, 291, 292, 293, 294, 295, 296, 297, 298, 299, 300, or 301.

[0054] The present invention also relates to a vector comprising the above-described nucleic acid sequence.

[0055] The present invention also relates to a host cell comprising the above nucleic acid sequence or a vector thereof.

[0056] The present invention also relates to a kit comprising the above-mentioned antibody or antigen-binding portion thereof, or the above-mentioned pharmaceutical composition.

[0057] For example, it further includes reagents for detecting the antibody or antigen-binding portion thereof.

[0058] The present invention also relates to a method for producing a canine antibody that binds to CD20, comprising culturing the isolated host cells described above and recovering the antibody.

[0059] The present invention also provides a method for producing a canine antibody that binds to CD20, comprising the steps of: a) immunizing a transgenic mouse expressing a nucleic acid construct comprising a canine heavy chain V gene and a canine light chain V gene with a CD20 antigen; b) generating a library of antibodies from the mice; c) isolating antibodies from the library.

[0060] The present invention also relates to a method for detecting CD20 protein or the extracellular domain of CD20 protein in a biological sample from a canine subject, comprising contacting the biological sample with the above-described antibody, or antigen-binding portion thereof, wherein the antibody, or antigen-binding portion thereof, is linked to a detectable label.

[0061] For example, the biological sample is a biopsy, tissue, blood, serum, plasma, or lymph sample.

[0062] The present invention also relates to an antibody or antigen-binding portion thereof that binds to a dog at an epitope that includes one or more amino acid residues, for example, 1 to 10, for example, 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10, of the following amino acid residues ENLNLIKAPM (SEQ ID NO: 303; amino acid residues 150 to 159 in SEQ ID NO: 2), or consists of the following amino acid residues ENLNLIKAPM (SEQ ID NO: 303; amino acid residues 150 to 159 in SEQ ID NO: 2).

[0063] The invention is further described in the following non-limiting figures. [Brief explanation of the drawings]

[0064] [Figure 1] Antibody titers of canine CD20-immunized Ky9 mice. Post-immunization serum was serially diluted (as indicated) and added to canine CD20-expressing cells. Cells were then incubated with fluorophore-conjugated anti-mouse IgG1, IgG2a, or IgG2b secondary antibodies, and samples were evaluated by flow cytometry. Binding of pre-immunization serum to canine CD20-expressing cells is shown as the background line in the plots. [Figure 2-1](A) Amino acid sequence alignment of the variable regions of PMX001, PMX002, PMX003, PMX004, and PMX005 mAbs. Shading indicates the CDR1, CDR2, and CDR3 regions in both the heavy chain (HC) and light chain (LC) sequences of the mAbs. (B) Amino acid sequence alignment of the HC and LC variable regions of the mAbs. Boxes indicate the CDR1, CDR2, and CDR3 regions in both the heavy chain (HC) and light chain (LC) sequences of the mAbs. [Figure 2-2] Same as above. [Figure 2-3] Same as above. [Figure 3-1] Binding assessment of anti-canine CD20 mAb using flow cytometry. PMX001, PMX002, PMX003, PMX004, PMX005, 1E4-cIGGB, 4E1-7-cIGGB (see A), and PMX006, PMX007, PMX008, PMX009, PMX010, PMX011 (see B), and PMX003, PMX066, PMX067, PMX069, PMX070, PMX071, PMX072, PMX073, PMX074, PMX075, PMX076, PMX077, PMX078, PMX079, PMX080, PMX081, 1E4-cIGGB, 4E1-7-cIGGB (see C) were applied to cCD20-expressing MDCK II cells at 10 μg / ml. Rituximab-cIGGB (gray shading) was used as an isotype control and is presented within each plot. (D) Affinity determination of PMX003, PMX115, and PMX070 mAbs. Binding of 1:2 serially diluted antibodies to cCD20-expressing MDCK II cells was assessed in a binding assay, and the mean fluorophore signal was determined. Plots of mean fluorescence intensity (MFI) versus concentration were used to determine the EC50 of binding using nonlinear regression, which is the apparent Kd of the antibody for binding to cell surface-expressed canine CD20. [Figure 3-2] Same as above. [Figure 3-3] Same as above. [Figure 3-4] Same as above. [Figure 3-5] Same as above. [Figure 4]CDC activity of anti-canine CD20 antibodies. Both native and afucosylated formats of antibodies were analyzed in the CDC assay at a range of concentrations (as indicated). Rituximab-cIGGB chimeric antibody was used as an "isotype control." "F" refers to the afucosylated format of the antibody. [Figure 5-1] ADCC activity of anti-canine CD20 antibodies. Both native and afucosylated formats of antibodies were analyzed in the ADCC assay at a range of concentrations (as indicated). Rituximab-cIGGB chimeric antibody was used as an "isotype control." "F" refers to the afucosylated format of the antibody. EC50 values ​​are also presented for each antibody. [Figure 5-2] Same as above. [Figure 6] B cell depletion efficiency of anti-canine CD20 mAbs in whole canine blood. Freshly collected canine whole blood was diluted with culture medium and incubated for 24 hours with 10 μg / ml of PMX001, PMX003, 1E4-cIGGB, 4E1-7-cIGGB (see A), native or afucosylated formats of PMX003 and 4E1-7-cIGGB antibodies (see B), PMX003, PMX006, PMX007, PMX008, PMX009, PMX010, PMX011 (see C), or no antibody. Rituximab-cIGGB was used as an "isotype control" (Iso Ctrl). "F" refers to the afucosylated format of the antibody. [Figure 7-1]B cell depletion efficiency of anti-canine CD20 mAb in healthy beagles. (A-B) Three healthy beagles for each test group received the following antibodies intravenously: isotype control (rituximab-cIgGB, 2.5 mg / kg), low-dose PMX003 (0.5 mg / kg), high-dose PMX003 (2.5 mg / kg), and low-dose afucosylated PMX003 (0.5 mg / kg). "F" refers to the afucosylated format of the antibody. The percentages of CD21+ B cells (A) and CD8+ T cells (B) among lymphocytes were analyzed using flow cytometry on days 0, 1, 2, 5, 7, 15, 27, and 43. Data shown here are representative of four technical replicates. (C) Three healthy beagles for each test group received the following antibodies intravenously: isotype control (rituximab-cIgGB, 2 mg / kg), low-dose PMX115 (0.5 mg / kg), high-dose PMX115 (2 mg / kg), low-dose PMX070 (0.5 mg / kg), and high-dose PMX070 (2 mg / kg). The percentage of CD21+ B cells among lymphocytes was analyzed using flow cytometry on days 0, 1, 4, 7, 14, 21, and 28. Data shown here are representative of four technical replicates. [Figure 7-2] Same as above. [Figure 8] Epitope mapping strategy for anti-CD20 antibodies. (A) The canine CD20 extracellular domain sequence was mutated to the human CD20 sequence at the equivalent positions in vectors DH01-DH09. (B) The canine CD20 extracellular domain sequence was mutated to the mouse CD20 sequence at the equivalent positions in vectors DM01-DM09. (C) All residues in the small loop and all other residues in the large loop of the canine CD20 extracellular domain were mutated to alanine, generating vectors S01-S09 and L01-L25. Mutated residues are highlighted in bold. [Figure 9]The binding of PMX003, PMX115, and PMX070 mAbs to MDCK cells expressing canine-human chimeric CD20 or canine-mouse chimeric CD20 was assessed by flow cytometry. The black bars 01-09 in the figure represent binding to the DH01-DH09 sequences shown in Figure 8A, and the gray bars 01-09 in the figure represent binding to the DM01-DM09 sequences shown in Figure 8B. MDCK cells expressing wild-type canine CD20 were used as a positive control, and wild-type MDCK cells were used as a negative control. DETAILED DESCRIPTION OF THE INVENTION

[0065] Table 1. Examples of amino acid residues and conservative amino acid substitutions Table 2. Sequences Table 3. VH and VL gene usage Table 4. Summary of functional data for antibodies Table 5. Antibody Development Potential Profile

[0066] Detailed Description Embodiments of the present invention will now be further described. In the following sections, different embodiments are described. Each aspect so defined can be combined with any other aspect or aspects, unless expressly indicated to the contrary.

[0067] Generally, the nomenclatures used in connection with, and techniques of, cell and tissue culture, pathology, oncology, molecular biology, immunology, microbiology, genetics, and protein and nucleic acid chemistry and hybridization described herein are those well known and commonly used in the art. Unless otherwise indicated, the methods and techniques of the present disclosure are generally carried out according to conventional methods well known in the art and as described in various general and more specific references cited and discussed throughout the specification. See, for example, Green and Sambrook et al., Molecular Cloning: A Laboratory Manual, 4th ed., Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY (2012); Therapeutic Monoclonal Antibodies: From Bench to Clinic, Zhiqiang An (Editor), Wiley, (2009); and Antibody Engineering, 2nd Ed., Vols 1 and 2, Ontermann and Dubel, eds., Springer-Verlag, Heidelberg (2010).

[0068] Enzymatic reactions and purification techniques are performed according to manufacturer's specifications, as commonly accomplished in the art, or as described herein. The nomenclature used in connection with, and the laboratory procedures and techniques of, analytical chemistry, synthetic organic chemistry, and medicinal and pharmaceutical chemistry described herein are those well known and commonly used in the art. Standard techniques are used for chemical syntheses, chemical analyses, pharmaceutical preparation, formulation, and delivery, and treatment of patients.

[0069] The present inventors have developed fully canine antibodies that specifically bind to canine CD20. These antibodies were generated in transgenic rodents expressing canine V, D, and J genes. Therefore, the antibodies are less likely to be immunogenic upon administration to canine subjects than caninized or chimeric antibodies. Furthermore, because these antibodies can be used directly without further modification to their variable regions, there is no risk of reducing affinity or otherwise compromising the antibody. Other techniques risk introducing development or efficacy liabilities through the ex vivo combination of canine-derived antibody sequences with antibody sequences from another species, typically a rodent. Thus, the present invention relates to isolated canine antibodies, or antigen-binding portions thereof, that bind to canine CD20.

[0070] The properties of the antibodies and antigen-binding portions thereof of the present invention can be utilized in the therapeutic methods and uses and pharmaceutical formulations described herein.

[0071] The term CD20 refers to the B lymphocyte antigen CD20. The antibody and antigen-binding portion thereof specifically bind to wild-type canine CD20 as defined in SEQ ID NO: 1 (nucleotide sequence) and SEQ ID NO: 2 (amino acid sequence). Unless otherwise specified, the term CD20 as used herein refers to canine CD20. The B lymphocyte antigen CD20 or CD20 is expressed on the surface of all B cells, beginning at the pro-B stage (CD45R+, CD117+) and increasing in concentration progressively until maturity. In humans and dogs, CD20 is encoded by the MS4A1 gene.

[0072] The terms "CD20 binding molecule / protein / polypeptide / agent / moiety," "CD20 antigen-binding molecule molecule / protein / polypeptide / agent / moiety," "anti-CD20 antibody," and "anti-CD20 antibody or antigen-binding portion thereof" all refer to a molecule capable of specifically binding to the canine CD20 antigen. Binding can be demonstrated by standard methods, for example, by reference to a negative control test using an antibody of irrelevant specificity.

[0073] Antibodies or antigen-binding portions thereof of the invention, including multispecific, e.g., bispecific or trispecific, binding agents as described herein, that "bind" or "are capable of binding" to an antigen of interest, i.e., canine CD20, are antibodies or antigen-binding portions thereof that bind to the antigen with sufficient affinity such that the antibodies or antigen-binding portions thereof are useful as therapeutic agents in targeting cells or tissues that express the antigen CD20 described herein.

[0074] The antibodies, or antigen-binding portions thereof, according to the present invention specifically bind to canine CD20. In other words, binding to the CD20 antigen is measurably different from non-specific interactions. In particular, the antibodies described herein do not cross-react with mouse CD20.

[0075] As used herein, the terms "specific binding" or "specifically binds" or "specific for" a particular polypeptide or epitope on a particular polypeptide target means, for example, at least about 10 -6 M, alternatively at least about 10 -7 M, alternatively at least about 10 -8 M, alternatively at least about 10 -9 M, alternatively at least about 10 -10 M, alternatively at least about 10 -11 M, alternatively at least about 10 -12 In one embodiment, the KD is at least about 10 -8 M ~ about 10 -9 M, e.g., in one embodiment, KD is in the nanomolar range. In one embodiment, the term "specific binding" refers to binding of a molecule to a particular polypeptide or an epitope on a particular polypeptide without substantially binding to any other polypeptides or polypeptide epitopes. KD and K D The terms are used interchangeably herein.

[0076] As used herein, the term "antibody" refers broadly to any immunoglobulin (Ig) molecule, composed of four polypeptide chains, two heavy (H) chains and two light (L) chains, or an antigen-binding portion thereof, or any functional fragment, mutant, variant, or derivative thereof that retains the essential epitope-binding characteristics of an Ig molecule.

[0077] In a full-length antibody, each heavy chain is composed of a heavy chain variable region or domain (abbreviated herein as HCVR) and a heavy chain constant region. The heavy chain constant region is made up of three domains: C H 1. C H 2, and C H Each light chain is composed of a light chain variable region or domain (abbreviated herein as LCVR) and a light chain constant region. The light chain constant region consists of one domain, C L It consists of:

[0078] Heavy and light chain variable regions can be further subdivided into regions of hypervariability called complementarity-determining regions (CDRs), interspersed with more conserved regions called framework regions (FRs). Each heavy and light chain variable region is composed of three CDRs and four FRs, arranged from amino-terminus to carboxy-terminus in the following order: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4.

[0079] Immunoglobulin molecules generally can be of any isotype, class or subclass. H The three domains are C of canine IgG subtypes, e.g., IgG-A, IgG-B, IgG-C, and IgG-D. H There are three domains.

[0080] In dogs, there are four IgG heavy chains, designated A, B, C, and D. These heavy chains represent four different subclasses of canine IgG, designated IgG-A, IgG-B, IgG-C, and IgG-D. The DNA and amino acid sequences of these four heavy chains were first identified by Tang et al. (Vet. Immunol. Immunopathol. 80:259-270 (2001)). The amino acid and DNA sequences for these heavy chains are also available from the GenBank database (IgGA: Accession No. AAL35301.1, IgGB: Accession No. AAL35302.1, IgGC: Accession No. AAL35303.1, IgGD: Accession No. AAL35304.1). Canine antibodies also contain two types of light chains, kappa and lambda (GenBank Accession No. kappa light chain amino acid sequence ABY57289.1, GenBank Accession No. ABY55569.1). The antibodies herein may have lambda or kappa light chains. In one embodiment, the light chain is a lambda light chain.

[0081] The term "CDR" refers to a complementarity-determining region within an antibody variable sequence. In each of the heavy and light chain variable regions, there are three CDRs, designated CDR1, CDR2, and CDR3 for each variable region. The term "CDR set" refers to a group of three CDRs occurring in a single variable region that can bind to an antigen. The exact boundaries of these CDRs can be defined differently according to different systems known in the art.

[0082] The Kabat complementarity-determining regions (CDRs) are based on sequence variability and are most commonly used (Kabat et al., (1971) Ann. NY Acad. Sci. 190:382-391 and Kabat, et al., (1991) Sequences of Proteins of Immunological Interest, Fifth Edition, USDapartment of Health and Human Services, NIH Publication No. 91-3242). Chothia instead refers to the location of the structural loops (Chothia and Lesk J. Mol. Biol. 196:901-917 (1987)). The Kabat numbering system is generally used to refer to residues within the variable domain (approximately residues 1-107 of the light chain and residues 1-113 of the heavy chain). Another system is the ImMunoGeneTics (IMGT) numbering scheme. The IMGT numbering scheme is described in Lefranc et al., Dev. Comp. Immunol., 29, 185-203 (2005). Unless otherwise specified, the IMGT numbering scheme is used herein.

[0083] Chimeric antibodies are recombinant proteins that contain variable domains comprising the complementarity determining regions (CDRs) of an antibody derived from one species, preferably a rodent or human antibody, while the constant domains of the antibody molecule are derived from the constant domains of a canine antibody.

[0084] As used herein, the term "caninized antibody" refers to a form of recombinant antibody containing sequences from both canine and non-canine (e.g., murine) antibodies. Generally, caninized antibodies contain substantially all of at least one or more, typically two, variable domains, with all or substantially all of the hypervariable loops corresponding to those of a non-canine immunoglobulin, and all or substantially all of the framework (FR) regions (and typically all or substantially all of the remaining framework) from canine immunoglobulin sequences. A caninized antibody may contain both three heavy chain CDRs and three light chain CDRS from a murine or human antibody, together with a canine framework or a modified canine framework. The modified canine framework may contain one or more amino acid changes that can further optimize the effectiveness of the caninized antibody, for example, to increase binding to its target. For example, the non-canine sequence of the hypervariable loop may be further compared to the canine sequence, and as many residues as possible may be changed to resemble the authentic canine sequence.

[0085] In contrast, the complete canine antibodies of the present invention have canine variable regions and do not contain complete or partial CDRs or FRs from another species. Advantageously, the complete canine antibodies described herein are derived from transgenic mice containing canine immunoglobulin sequences. Antibodies produced in these immunized mice are developed through in vivo B cell signaling and development to allow natural affinity maturation, including in vivo V(D)J recombination, in vivo junctional diversification, in vivo pairing of heavy and light chains, and in vivo hypermutation. Complete canine antibodies produced in this manner generate antibodies with optimal properties for potential development and minimize lengthy lead optimization prior to large-scale production. Advantageously, such complete canine antibodies present the lowest possible risk of immunogenicity when introduced into patient animals, thereby facilitating repeated dosing regimes. Considering that ex vivo mAb engineering risks introducing developmental liability, immunogenicity, and reduced affinity (as outlined above), the complete canine antibodies of the present invention are therefore most likely to be effective therapeutics in clinical contexts. Thus, in embodiments, the term canine antibody refers to a whole canine antibody.

[0086] As used herein, the term "monoclonal antibody" refers to an antibody obtained from a population of substantially homogeneous antibodies, i.e., the individual antibodies comprising the population are identical except for possible naturally occurring mutations and / or post-translational modifications (e.g., isomerization, amidation, carbohydrate addition), which may be present in minor amounts. Monoclonal antibodies are highly specific, being directed against a single antigenic site. In contrast to polyclonal antibody preparations, which typically include different antibodies directed against different determinants (epitopes), each monoclonal antibody is directed against a single determinant on the antigen. In addition to their specificity, monoclonal antibodies are advantageous in that they are synthesized by hybridoma culture, uncontaminated by other immunoglobulins.

[0087] The term "antigen-binding site" refers to the portion of an antibody or antibody fragment that comprises the area that specifically binds to an antigen. An antigen-binding site may be provided by one or more antibody variable domains. An antigen-binding site is typically located within the relevant V domains of an antibody or antibody fragment. H and V L Included within.

[0088] The term "epitope" or "antigenic determinant" refers to a site on the surface of an antigen to which an immunoglobulin, antibody, or antibody fragment specifically binds. Generally, an antigen has several or many different epitopes and reacts with many different antibodies. This term specifically includes linear and conformational epitopes. Epitopes within a protein antigen can be formed both from contiguous amino acids (usually linear epitopes) or noncontiguous amino acids juxtaposed by tertiary folding of the protein (usually conformational epitopes). Epitopes formed from contiguous amino acids are typically, but not always, retained upon exposure to denaturing solvents, whereas epitopes formed by tertiary folding are typically lost upon treatment with denaturing solvents. An epitope typically includes at least 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15 amino acids in a unique spatial conformation. Methods for determining the epitope bound by a given antibody or antibody fragment (i.e., epitope mapping by alanine scanning mutagenesis or Pepscan) are well known in the art and include, for example, immunoblot and immunoprecipitation assays, in which overlapping or adjacent peptides are tested for reactivity with a given antibody or antibody fragment. An antibody binds to "essentially the same epitope" as a reference antibody when the two antibodies recognize the same or sterically overlapping epitopes. The most widely used and rapid method for determining whether two epitopes bind to the same or sterically overlapping epitopes is a competitive assay, which can be configured in different formats using either labeled antigen or labeled antibody.

[0089] In one embodiment, an antibody or antigen-binding portion thereof described herein comprises one or more amino acid residues, e.g., 1 to 19, e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, or 19, of the following amino acid residues: ITISHFFKMENLNLIKAPM (SEQ ID NO: 302; amino acid residues 141 to 159 in SEQ ID NO: 2), or binds to an epitope consisting of the following amino acid residues: ITISHFFKMENLNLIKAPM (SEQ ID NO: 302; amino acid residues 141 to 159 in SEQ ID NO: 2). In one embodiment, the epitope comprises one or more amino acid residues, for example, 1 to 10, for example, 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10, of the following amino acid residues ENLNLIKAPM (SEQ ID NO: 303, amino acid numbers 150 to 159 in SEQ ID NO: 2), or consists of the following amino acid residues ENLNLIKAPM (SEQ ID NO: 303, amino acid numbers 150 to 159 in SEQ ID NO: 2).

[0090] In one embodiment, the epitope is determined by site-directed mutagenesis as shown in the Examples, e.g., using alanine scanning. In one embodiment, the epitope is a linear epitope. In one embodiment, the epitope is a conformational epitope. In one embodiment, the epitope comprises a linear epitope within the major loop. In one embodiment, the epitope further comprises a conformational epitope within the minor loop. In one embodiment, the antibody is PMX003 or an antigen-binding portion thereof described herein, or an antibody having at least 80% sequence identity.

[0091] In another aspect, the present invention also relates to an antibody or antigen-binding portion thereof that binds to an epitope comprising one or more amino acid residues, for example, 1 to 19, for example, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, or 19, of the following amino acid residues: ITISHFFKMENLNLIKAPM (SEQ ID NO: 302; amino acid residues 141 to 159 in SEQ ID NO: 2), or consisting of the following amino acid residues: ITISHFFKMENLNLIKAPM (SEQ ID NO: 302; amino acid residues 141 to 159 in SEQ ID NO: 2). In one embodiment, the epitope comprises one or more amino acid residues, e.g., 1 to 10, e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10, of the following amino acid residues ENLNLIKAPM (SEQ ID NO: 303, amino acid numbers 150 to 159 in SEQ ID NO: 2), or consists of the following amino acid residues ENLNLIKAPM (SEQ ID NO: 303, amino acid numbers 150 to 159 in SEQ ID NO: 2). For example, the antibody, or antigen-binding portion thereof, can have a sequence described herein.

[0092] The present invention also relates to antibodies, or antigen-binding portions thereof, that compete with the antibodies, or antigen-binding portions thereof, described herein.

[0093] Proteolytic digestion of antibodies releases different fragments called Fv (fragment variable), Fab (fragment antigen binding), and Fc (fragment crystallization). The Fc fragment contains the carboxy-terminal portions of both H chains held together by disulfides. The constant domains of the Fc fragment are responsible for mediating the effector functions of the antibody.

[0094] The present invention extends to antigen-binding portions or antigen-binding fragments of antibodies. The terms "binding portion" and "fragment" are used interchangeably herein. Antibody fragments include any portion of an antibody, e.g., F(ab')2, Fab, Fv, scFv, heavy chain, light chain, variable heavy chain (V), H ), variable light chain (V L) domains. Functional fragments of a full-length antibody retain the target specificity of the full-length antibody. Thus, recombinant functional antibody fragments, such as Fab (fragment antibody), scFv (single-chain fragment variable), and single-domain antibodies (dAb), are being used to develop therapeutics as alternatives to mAb-based therapeutics.

[0095] The present invention also extends to antibody mimetics comprising the sequences described herein.

[0096] An "Fv" is the minimum antibody fragment that contains a complete antigen-recognition and antigen-binding site. This fragment consists of a dimer of one heavy-chain and one light-chain variable domain in tight, non-covalent association. The folding of these two domains generates six hypervariable loops (three loops each from the H and L chain) that contribute amino acid residues for antigen binding and confer antigen-binding specificity to the antibody. However, even a single variable domain (or half of an Fv containing only three HVRs specific for an antigen) has the ability to recognize and bind antigen, although at a lower affinity than the entire binding site.

[0097] A "single-chain Fv," also abbreviated as "sFv" or "scFv," is a polymer comprising two variable domains, V, connected to a single polypeptide chain. H and V L The antibody fragment consisting of scFv fragment (approximately 25 kDa) is naturally V H and V L The domains tend to associate and dissociate non-covalently through hydrophobic interactions, however, stable fragments can be engineered by linking the domains with flexible hydrophilic linkers to create single-chain Fvs (scFvs).

[0098] The smallest antigen-binding fragment is a single variable fragment, i.e., a variable heavy chain (V H ) or variable light chain (V L ) domain. V H and V LEach domain is capable of binding to an antigen. Binding to the respective light / heavy chain partner, or indeed the presence of other parts of the complete antibody, is not required for target binding. One single domain (V H or V L Antigen-binding entities of antibodies that have been reduced in size to a number of domains (corresponding to V domains) are commonly referred to as "single domain antibodies" or "immunoglobulin single variable domains." Thus, single domain antibodies (approximately 12-15 kDa) have V H or V L The term "dAb" for "domain antibody" generally refers to a single immunoglobulin variable domain (V) that specifically binds to an antigen. H , V HH , or V L ) polypeptide.

[0099] The term "isolated" refers to a moiety that is isolated from its natural environment. For example, the term "isolated" refers to an antibody or fragment thereof that is substantially free of other antibodies, antibodies, or antibody fragments. Furthermore, an isolated antibody may be substantially free of other cellular material and / or chemicals.

[0100] As used herein, the term "homology" or "identity" generally refers to the percentage of amino acid residues in a sequence that are identical to the residues of a reference polypeptide being compared, after aligning the sequences and, in some embodiments, introducing gaps as needed to achieve the maximum percentage homology, without considering any conservative substitutions as part of the sequence identity. Thus, the percentage homology between two amino acid sequences is equivalent to the percentage identity between the two sequences. Neither N-terminal nor C-terminal extensions, tags, or insertions should be interpreted as reducing identity or homology. Methods and computer programs for alignment are well known. The percent identity between two amino acid sequences can be determined using well-known mathematical algorithms.

[0101] As used herein, "amino acid" refers to one of the 20 naturally occurring amino acids or any non-natural analogue that may be present at a particular, defined position. Amino acid encompasses both naturally occurring and synthetic amino acids. However, in most cases, only naturally occurring amino acids are used when proteins are recombinantly produced.

[0102] As used herein, "substitution of an amino acid residue" with another amino acid residue in the amino acid sequence of a heterodimeric protein or polypeptide (e.g., an antibody) described herein is equivalent to "replacing an amino acid residue" with another amino acid residue and indicates that a particular amino acid residue at a particular position in the original (e.g., wild-type / germline) amino acid sequence has been replaced (or substituted) with a different amino acid residue. This can be done using standard techniques available to those of skill in the art, for example, recombinant DNA technology. Amino acids are altered compared to the native (wild-type / germline) sequence as found in nature in wild-type (wt), but can also be generated in IgG molecules that contain other changes compared to the native sequence. "Wild-type" or "WT" or "native" herein refers to an amino acid sequence or nucleotide sequence found in nature, including allelic variations. A WT protein, polypeptide, antibody, or immunoglobulin has an amino acid sequence or nucleotide sequence that has not been intentionally modified.

[0103] An antibody or antigen-binding portion thereof according to the invention has one or more of the following properties: a) specifically binds to canine CD20; b) binds to canine CD20 with a KD as measured in the Examples and as shown, for example, in the Figures; c) exhibit cell killing, such as CDC and / or ADCC, in CD20-expressing canine lymphoma cell lines, as determined in the Examples; d) promotes antibody-dependent cellular phagocytosis (ADCP); e) can effectively deplete CD20-positive B cells in dog tissues; f) capable of binding to cells expressing canine CD20; g) Preferably, cells expressing canine CD20 can be depleted by direct cell killing via apoptosis; h) provides good stability as shown in the examples; i) binding to an epitope comprising one or more amino acid residues, for example, 1 to 10, for example, 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10, of the following amino acid residues ENLNLIKAPM (SEQ ID NO: 303, amino acid numbers 150 to 159 in SEQ ID NO: 2), for example, ITISHFFKMENLNLIKAPM (SEQ ID NO: 302, amino acid numbers 141 to 159 in SEQ ID NO: 2); or consisting of the following amino acid residues ENLNLIKAPM (SEQ ID NO: 303, amino acid numbers 150 to 159 in SEQ ID NO: 2), for example, ITISHFFKMENLNLIKAPM (SEQ ID NO: 302, amino acid numbers 141 to 159 in SEQ ID NO: 2); j) For example, as shown in the Examples, it has a stronger binding ability to canine CD20 than the benchmark test antibodies 1E4 and 4E1-7 (5 to 10 times higher); k) have stronger ADCC and / or CDC activity than benchmark antibodies 1E4 and 4E1-7, e.g., as shown in the Examples and Table 4; and / or l) More efficient killing of B cells in whole dog blood compared to benchmark test antibodies 1E4 and 4E1-7 mAb, e.g., as shown.

[0104] In one embodiment, an antibody or antigen-binding portion thereof according to the invention has one or more of the above properties, and optionally one or more of the following properties: a) has CDC activity with an EC50 value of less than 20 nM; b) has ADCC activity with an EC50 value of less than 0.3 nM; c) having a transient expression yield of greater than 100ug / ml; d) has a Tm1 determined by Uncle of greater than 58°C; e) providing in vivo cell killing in dogs at a dose of about 0.5 mg / kg to 2.5 mg / kg; f) maintaining low levels of B cell depletion in vivo in dogs for at least 15 days; and / or g) Binds to an epitope comprising one or more amino acid residues, for example, 1 to 10, for example, 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10, of the following amino acid residues ENLNLIKAPM (SEQ ID NO: 303, amino acid numbers 150 to 159 in SEQ ID NO: 2), for example, ITISHFFKMENLNLIKAPM (SEQ ID NO: 302, amino acid numbers 141 to 159 in SEQ ID NO: 2), or consisting of the following amino acid residues ENLNLIKAPM (SEQ ID NO: 303, amino acid numbers 150 to 159 in SEQ ID NO: 2), for example, ITISHFFKMENLNLIKAPM (SEQ ID NO: 302, amino acid numbers 141 to 159 in SEQ ID NO: 2).

[0105] These properties can be measured by methods known in the art, such as those disclosed in the Examples, including in vivo studies in mouse models or dogs.

[0106] In particular, the inventors have found that the antibodies and antigen-binding portions thereof exhibit both CDC and ADCC activity, as demonstrated, for example, by cell killing in a CD20-expressing canine lymphoma cell line, as measured in the Examples.

[0107] In one aspect, the invention relates to an isolated canine antibody, or antigen-binding portion thereof, that binds to canine CD20, the antibody comprising: a) a heavy chain (HC) CDR1 sequence comprising or consisting of SEQ ID NO: 27, or an amino acid sequence having at least 60%, 70%, 80%, or 90% sequence identity thereto; b) an HC CDR2 sequence comprising or consisting of SEQ ID NO: 28, or an amino acid sequence having at least 60%, 70%, 80%, or 90% sequence identity thereto; c) an HC CDR3 sequence comprising or consisting of SEQ ID NO: 29, or an amino acid sequence having at least 60%, 70%, 80%, or 90% sequence identity thereto; d) a light chain (LC) CDR1 sequence comprising or consisting of SEQ ID NO: 30, having at least 60%, 70%, 80%, or 90% sequence identity thereto; e) an LC CDR2 sequence comprising or consisting of SEQ ID NO: 31, or an amino acid sequence having at least 60%, 70%, 80%, or 90% sequence identity thereto; and f) comprises an LC CDR3 sequence comprising or consisting of SEQ ID NO: 32, or an amino acid sequence having at least 60%, 70%, 80%, or 90% sequence identity thereto.

[0108] The present invention relates to an isolated canine antibody, or antigen-binding portion thereof, that binds to canine CD20, the antibody comprising: a) HC CDR1 sequence comprising or consisting of SEQ ID NO: 27 b) HC CDR2 sequence comprising or consisting of SEQ ID NO: 28 c) HC CDR3 sequence comprising or consisting of SEQ ID NO: 29 d) a LC CDR1 sequence comprising or consisting of SEQ ID NO: 30; e) a LC CDR2 sequence comprising or consisting of SEQ ID NO: 31, and f) LC CDR3 sequence comprising or consisting of SEQ ID NO: 32 or an isolated canine antibody or antigen-binding portion thereof having CDRs as described above, but having one or more CDRs that have one, two, three, or four amino acid substitutions compared to the CDR sequences described above.

[0109] Accordingly, the present invention relates to an isolated canine antibody, or antigen-binding portion thereof, that binds to canine CD20, the antibody comprising: a) a heavy chain (HC) CDR1 sequence comprising or consisting of SEQ ID NO: 27, or an amino acid sequence having one or two amino acid differences compared to SEQ ID NO: 27; b) an HC CDR2 sequence comprising or consisting of SEQ ID NO: 28, or an amino acid sequence having 1, 2, 3, or 4 amino acid differences compared to SEQ ID NO: 28; c) an HC CDR3 sequence comprising or consisting of SEQ ID NO: 29, or an amino acid sequence having one or two amino acid differences compared to SEQ ID NO: 29; d) a light chain (LC) CDR1 sequence comprising or consisting of SEQ ID NO: 30, or an amino acid sequence having one amino acid difference compared to SEQ ID NO: 30; e) an LC CDR2 sequence comprising or consisting of SEQ ID NO: 31, or an amino acid sequence having one amino acid difference compared to SEQ ID NO: 31; and f) comprises an LC CDR3 sequence comprising or consisting of SEQ ID NO: 32, or an amino acid sequence having one or two amino acid differences compared to SEQ ID NO: 32.

[0110] In one embodiment, the antibody, or antigen-binding portion thereof, has a HC CDR1 sequence comprising or consisting of SEQ ID NO:7, a HC CDR2 sequence comprising or consisting of SEQ ID NO:8, a HC CDR3 sequence comprising or consisting of SEQ ID NO:9, a LC CDR1 sequence comprising or consisting of SEQ ID NO:10, a LC CDR2 sequence comprising or consisting of SEQ ID NO:11, and a LC CDR3 sequence comprising or consisting of SEQ ID NO:12.

[0111] In one embodiment, the antibody, or antigen-binding portion thereof, has a HC CDR1 sequence comprising or consisting of SEQ ID NO: 17, a HC CDR2 sequence comprising or consisting of SEQ ID NO: 18, a HC CDR3 sequence comprising or consisting of SEQ ID NO: 19, a LC CDR1 sequence comprising SEQ ID NO: 20, a LC CDR2 sequence comprising or consisting of SEQ ID NO: 21, and a LC CDR3 sequence comprising or consisting of SEQ ID NO: 22.

[0112] In one embodiment, the antibody, or antigen-binding portion thereof, has a HC CDR1 sequence comprising SEQ ID NO: 37, a HC CDR2 sequence comprising SEQ ID NO: 38, a HC CDR3 sequence comprising SEQ ID NO: 39, a LC CDR1 sequence comprising SEQ ID NO: 40, a LC CDR2 sequence comprising SEQ ID NO: 41, and a LC CDR3 sequence comprising SEQ ID NO: 42.

[0113] In one embodiment, the antibody, or antigen-binding portion thereof, has a HC CDR1 sequence comprising or consisting of SEQ ID NO:47, a HC CDR2 sequence comprising or consisting of SEQ ID NO:48, a HC CDR3 sequence comprising or consisting of SEQ ID NO:49, a LC CDR1 sequence comprising or consisting of SEQ ID NO:50, a LC CDR2 sequence comprising or consisting of SEQ ID NO:51, and a LC CDR3 sequence comprising or consisting of SEQ ID NO:52.

[0114] In one embodiment, the antibody, or antigen-binding portion thereof, has a HC CDR1 sequence comprising or consisting of SEQ ID NO:82, a HC CDR2 sequence comprising or consisting of SEQ ID NO:83, a HC CDR3 sequence comprising or consisting of SEQ ID NO:84, a LC CDR1 sequence comprising or consisting of SEQ ID NO:85, a LC CDR2 sequence comprising or consisting of SEQ ID NO:86, and a LC CDR3 sequence comprising or consisting of SEQ ID NO:87.

[0115] In one embodiment, the antibody, or antigen-binding portion thereof, has a HC CDR1 sequence comprising or consisting of SEQ ID NO:92, a HC CDR2 sequence comprising or consisting of SEQ ID NO:93, a HC CDR3 sequence comprising or consisting of SEQ ID NO:94, a LC CDR1 sequence comprising or consisting of SEQ ID NO:95, a LC CDR2 sequence comprising or consisting of SEQ ID NO:96, and a LC CDR3 sequence comprising or consisting of SEQ ID NO:97.

[0116] In one embodiment, the antibody, or antigen-binding portion thereof, has a HC CDR1 sequence comprising or consisting of SEQ ID NO:102, a HC CDR2 sequence comprising or consisting of SEQ ID NO:103, a HC CDR3 sequence comprising or consisting of SEQ ID NO:104, a LC CDR1 sequence comprising or consisting of SEQ ID NO:105, a LC CDR2 sequence comprising or consisting of SEQ ID NO:106, and a LC CDR3 sequence comprising or consisting of SEQ ID NO:107.

[0117] In one embodiment, the antibody, or antigen-binding portion thereof, has a HC CDR1 sequence comprising or consisting of SEQ ID NO:112, a HC CDR2 sequence comprising or consisting of SEQ ID NO:113, a HC CDR3 sequence comprising or consisting of SEQ ID NO:114, a LC CDR1 sequence comprising or consisting of SEQ ID NO:115, a LC CDR2 sequence comprising or consisting of SEQ ID NO:116, and a LC CDR3 sequence comprising or consisting of SEQ ID NO:117.

[0118] In one embodiment, the antibody, or antigen-binding portion thereof, has a HC CDR1 sequence comprising or consisting of SEQ ID NO: 122, a HC CDR2 sequence comprising or consisting of SEQ ID NO: 123, a HC CDR3 sequence comprising or consisting of SEQ ID NO: 124, a LC CDR1 sequence comprising or consisting of SEQ ID NO: 125, a LC CDR2 sequence comprising or consisting of SEQ ID NO: 126, and a LC CDR3 sequence comprising or consisting of SEQ ID NO: 127.

[0119] In one embodiment, the antibody, or antigen-binding portion thereof, has a HC CDR1 sequence comprising or consisting of SEQ ID NO: 132, a HC CDR2 sequence comprising or consisting of SEQ ID NO: 133, a HC CDR3 sequence comprising or consisting of SEQ ID NO: 134, a LC CDR1 sequence comprising or consisting of SEQ ID NO: 135, a LC CDR2 sequence comprising or consisting of SEQ ID NO: 136, and a LC CDR3 sequence comprising or consisting of SEQ ID NO: 137.

[0120] In one embodiment, the antibody, or antigen-binding portion thereof, has a HC CDR1 sequence comprising or consisting of SEQ ID NO: 142, a HC CDR2 sequence comprising or consisting of SEQ ID NO: 143, a HC CDR3 sequence comprising or consisting of SEQ ID NO: 144, a LC CDR1 sequence comprising or consisting of SEQ ID NO: 145, a LC CDR2 sequence comprising or consisting of SEQ ID NO: 146, and a LC CDR3 sequence comprising or consisting of SEQ ID NO: 147.

[0121] In one embodiment, the antibody, or antigen-binding portion thereof, has a HC CDR1 sequence comprising or consisting of SEQ ID NO: 152, a HC CDR2 sequence comprising or consisting of SEQ ID NO: 153, a HC CDR3 sequence comprising or consisting of SEQ ID NO: 154, a LC CDR1 sequence comprising or consisting of SEQ ID NO: 155, a LC CDR2 sequence comprising or consisting of SEQ ID NO: 156, and a LC CDR3 sequence comprising or consisting of SEQ ID NO: 157.

[0122] In one embodiment, the antibody, or antigen-binding portion thereof, has a HC CDR1 sequence comprising or consisting of SEQ ID NO: 162, a HC CDR2 sequence comprising or consisting of SEQ ID NO: 163, a HC CDR3 sequence comprising or consisting of SEQ ID NO: 164, a LC CDR1 sequence comprising or consisting of SEQ ID NO: 165, a LC CDR2 sequence comprising or consisting of SEQ ID NO: 166, and a LC CDR3 sequence comprising or consisting of SEQ ID NO: 167.

[0123] In one embodiment, the antibody, or antigen-binding portion thereof, has a HC CDR1 sequence comprising or consisting of SEQ ID NO: 172, a HC CDR2 sequence comprising or consisting of SEQ ID NO: 173, a HC CDR3 sequence comprising or consisting of SEQ ID NO: 174, a LC CDR1 sequence comprising or consisting of SEQ ID NO: 175, a LC CDR2 sequence comprising or consisting of SEQ ID NO: 176, and a LC CDR3 sequence comprising or consisting of SEQ ID NO: 177.

[0124] In one embodiment, the antibody, or antigen-binding portion thereof, has a HC CDR1 sequence comprising or consisting of SEQ ID NO: 182, a HC CDR2 sequence comprising or consisting of SEQ ID NO: 183, a HC CDR3 sequence comprising or consisting of SEQ ID NO: 184, a LC CDR1 sequence comprising or consisting of SEQ ID NO: 185, a LC CDR2 sequence comprising or consisting of SEQ ID NO: 186, and a LC CDR3 sequence comprising or consisting of SEQ ID NO: 187.

[0125] In one embodiment, the antibody, or antigen-binding portion thereof, has a HC CDR1 sequence comprising or consisting of SEQ ID NO: 192, a HC CDR2 sequence comprising or consisting of SEQ ID NO: 193, a HC CDR3 sequence comprising or consisting of SEQ ID NO: 194, a LC CDR1 sequence comprising or consisting of SEQ ID NO: 195, a LC CDR2 sequence comprising or consisting of SEQ ID NO: 196, and a LC CDR3 sequence comprising or consisting of SEQ ID NO: 197.

[0126] In one embodiment, the antibody, or antigen-binding portion thereof, comprises a HC CDR1 sequence comprising or consisting of SEQ ID NO: 202, a HC CDR2 sequence comprising or consisting of SEQ ID NO: 203, a HC CDR3 sequence comprising or consisting of SEQ ID NO: 204, a LC CDR1 sequence comprising or consisting of SEQ ID NO: 205, a LC CDR2 sequence comprising or consisting of SEQ ID NO: 206, and a LC CDR3 sequence comprising or consisting of SEQ ID NO: 207.

[0127] In one embodiment, the antibody, or antigen-binding portion thereof, has a HC CDR1 sequence comprising or consisting of SEQ ID NO:212, a HC CDR2 sequence comprising or consisting of SEQ ID NO:213, a HC CDR3 sequence comprising or consisting of SEQ ID NO:214, a LC CDR1 sequence comprising or consisting of SEQ ID NO:215, a LC CDR2 sequence comprising or consisting of SEQ ID NO:216, and a LC CDR3 sequence comprising or consisting of SEQ ID NO:217.

[0128] In one embodiment, the antibody, or antigen-binding portion thereof, has a HC CDR1 sequence comprising or consisting of SEQ ID NO:222, a HC CDR2 sequence comprising or consisting of SEQ ID NO:223, a HC CDR3 sequence comprising or consisting of SEQ ID NO:224, a LC CDR1 sequence comprising or consisting of SEQ ID NO:225, a LC CDR2 sequence comprising or consisting of SEQ ID NO:226, and a LC CDR3 sequence comprising or consisting of SEQ ID NO:227.

[0129] In one embodiment, the antibody, or antigen-binding portion thereof, has a HC CDR1 sequence comprising or consisting of SEQ ID NO:232, a HC CDR2 sequence comprising or consisting of SEQ ID NO:233, a HC CDR3 sequence comprising or consisting of SEQ ID NO:234, a LC CDR1 sequence comprising or consisting of SEQ ID NO:235, a LC CDR2 sequence comprising or consisting of SEQ ID NO:236, and a LC CDR3 sequence comprising or consisting of SEQ ID NO:237.

[0130] In one embodiment, the antibody, or antigen-binding portion thereof, comprises a heavy chain (HC) variable region sequence comprising SEQ ID NO: 24, or a sequence having at least 75%, 80%, 85%, or 90% sequence identity thereto, and a light chain (LC) variable region sequence comprising SEQ ID NO: 26, or a sequence having at least 70%, 75%, 80%, 85%, 90%, or 95% sequence identity thereto. In one embodiment, the antibody, or antigen-binding portion thereof, comprises a heavy chain (HC) variable region sequence comprising SEQ ID NO: 24, and a light chain (LC) variable region sequence comprising SEQ ID NO: 26, or a LC variable region sequence comprising SEQ ID NO: 26, wherein the NVT sequence in FR1 of the LC variable region is modified, for example, an LC variable region having one, two, or three amino acid modifications in FR1, for example, an LC variable region sequence comprising FR1 having SEQ ID NO: 60, 61, 62, 63, 64, or 65, as set forth below, or an LC variable region sequence comprising SEQ ID NO: 53, 54, 55, 56, 57, or 58.

[0131] For example, an antibody or antigen-binding portion thereof may a) an HC variable region sequence comprising or consisting of SEQ ID NO: 4, and an LC variable region sequence comprising or consisting of SEQ ID NO: 6; b) an HC variable region sequence comprising or consisting of SEQ ID NO: 14, and an LC variable region sequence comprising or consisting of SEQ ID NO: 16; c) an HC variable region sequence comprising SEQ ID NO: 34, and an LC variable region sequence comprising or consisting of SEQ ID NO: 36; d) an HC variable region sequence comprising or consisting of SEQ ID NO: 44, and an LC variable region sequence comprising or consisting of SEQ ID NO: 46; e) an HC variable region sequence comprising SEQ ID NO: 79 and an LC variable region sequence comprising SEQ ID NO: 81, or an LC variable region sequence in which the NVT sequence described below has been modified, for example, an LC variable region sequence comprising SEQ ID NO: 238; f) an HC variable region sequence comprising SEQ ID NO: 89 and an LC variable region sequence comprising SEQ ID NO: 91, or an LC variable region sequence in which the NVT sequence described below has been modified, for example, an LC variable region sequence comprising SEQ ID NO: 239; g) an HC variable region sequence comprising SEQ ID NO: 99 and an LC variable region sequence comprising SEQ ID NO: 101, or an LC variable region sequence in which the NVT sequence described below has been modified, for example, an LC variable region sequence comprising SEQ ID NO: 240; h) an HC variable region sequence comprising SEQ ID NO: 109 and an LC variable region sequence comprising SEQ ID NO: 111, or an LC variable region sequence in which the NVT sequence described below has been modified, for example, an LC variable region sequence comprising SEQ ID NO: 241; i) an HC variable region sequence comprising SEQ ID NO: 119, and an LC variable region sequence comprising SEQ ID NO: 121; j) an HC variable region sequence comprising SEQ ID NO: 129 and an LC variable region sequence comprising SEQ ID NO: 131, or an LC variable region sequence in which the NVT sequence described below has been modified, for example, an LC variable region sequence comprising SEQ ID NO: 242; k) an HC variable region sequence comprising SEQ ID NO: 139 and an LC variable region sequence comprising SEQ ID NO: 141, or an LC variable region sequence in which the NVT sequence described below has been modified, for example, an LC variable region sequence comprising SEQ ID NO: 243; l) an HC variable region sequence comprising SEQ ID NO: 149 and an LC variable region sequence comprising SEQ ID NO: 151, or an LC variable region sequence in which the NVT sequence described below has been modified, for example, an LC variable region sequence comprising SEQ ID NO: 244; m) an HC variable region sequence comprising SEQ ID NO: 159, and an LC variable region sequence comprising SEQ ID NO: 161, or an LC variable region sequence in which the NVT sequence described below has been modified, for example, an LC variable region sequence comprising SEQ ID NO: 245; n) an HC variable region sequence comprising SEQ ID NO: 169, and an LC variable region sequence comprising SEQ ID NO: 171; o) an HC variable region sequence comprising SEQ ID NO: 179 and an LC variable region sequence comprising SEQ ID NO: 181, or an LC variable region sequence in which the NVT sequence described below has been modified, for example, an LC variable region sequence comprising SEQ ID NO: 246; p) an HC variable region sequence comprising SEQ ID NO: 189 and an LC variable region sequence comprising SEQ ID NO: 191, or an LC variable region sequence in which the NVT sequence described below has been modified, for example, an LC variable region sequence comprising SEQ ID NO: 247; q) an HC variable region sequence comprising SEQ ID NO: 199 and an LC variable region sequence comprising SEQ ID NO: 201, or an LC variable region sequence in which the NVT sequence described below has been modified, for example, an LC variable region sequence comprising SEQ ID NO: 248; r) an HC variable region sequence comprising SEQ ID NO: 209 and an LC variable region sequence comprising SEQ ID NO: 211, or an LC variable region sequence in which the NVT sequence described below has been modified, for example, an LC variable region sequence comprising SEQ ID NO: 249; s) an HC variable region sequence comprising SEQ ID NO: 219 and an LC variable region sequence comprising SEQ ID NO: 221; or t) has an HC variable region sequence comprising SEQ ID NO: 229, and an LC variable region sequence comprising SEQ ID NO: 231, or an LC variable region sequence in which the NVT sequence described below has been modified, for example, an LC variable region sequence comprising SEQ ID NO: 250.

[0132] In one embodiment, the antigen-binding portion is a F(ab')2, Fab, Fv, scFv, heavy chain, light chain, variable heavy (V H ) domain, or variable light (V L)

[0133] In another embodiment, the antibody or antigen-binding portion comprises an HC CDR1 sequence comprising or consisting of SEQ ID NO: 27, an HC CDR2 sequence comprising or consisting of SEQ ID NO: 28, an HC CDR3 sequence comprising or consisting of SEQ ID NO: 29, an LCDR1 sequence comprising or consisting of SEQ ID NO: 30, an LC CDR2 sequence comprising or consisting of SEQ ID NO: 31, and an LC CDR3 sequence comprising or consisting of SEQ ID NO: 32. The antibody or antigen-binding portion has framework regions with five amino acid changes in the heavy chain variable sequence and one amino acid change in the light chain variable sequence (SEQ ID NO: 76) compared to the germline sequence (SEQ ID NO: 74).

[0134] In one embodiment, the antibody, or antigen-binding portion thereof, has an HC CDR1 sequence comprising or consisting of SEQ ID NO:7, an HC CDR2 sequence comprising or consisting of SEQ ID NO:8, an HC CDR3 sequence comprising or consisting of SEQ ID NO:9, an LC CDR1 sequence comprising or consisting of SEQ ID NO:10, an LC CDR2 sequence comprising or consisting of SEQ ID NO:11, and an LC CDR3 sequence comprising or consisting of SEQ ID NO: 12. The antibody or antigen-binding portion has framework regions with two amino acid changes in the heavy chain variable sequence compared to the germline sequence (SEQ ID NO:74) and framework regions with three amino acid changes in the light chain variable sequence compared to the germline sequence (SEQ ID NO:76).

[0135] In one embodiment, the antibody, or antigen-binding portion thereof, has an HC CDR1 sequence comprising or consisting of SEQ ID NO: 17, an HC CDR2 sequence comprising or consisting of SEQ ID NO: 18, an HC CDR3 sequence comprising or consisting of SEQ ID NO: 19, an LC CDR1 sequence comprising or consisting of SEQ ID NO: 20, an LC CDR2 sequence comprising or consisting of SEQ ID NO: 21, and an LC CDR3 sequence comprising or consisting of SEQ ID NO: 22. The antibody or antigen-binding portion has framework regions with 10 amino acid changes in the heavy chain variable sequence compared to the germline sequence (SEQ ID NO: 74) and framework regions with no amino acid changes in the light chain variable sequence (SEQ ID NO: 76).

[0136] In one embodiment, the antibody, or antigen-binding portion thereof, has a HC CDR1 sequence comprising SEQ ID NO: 37, a HC CDR2 sequence comprising SEQ ID NO: 38, a HC CDR3 sequence comprising SEQ ID NO: 39, a LC CDR1 sequence comprising SEQ ID NO: 40, a LC CDR2 sequence comprising SEQ ID NO: 41, and a LC CDR3 sequence comprising SEQ ID NO: 42. The antibody or antigen-binding portion has framework regions with 12 amino acid changes in the heavy chain variable sequence compared to the germline sequence (SEQ ID NO: 74) and framework regions with no amino acid changes in the light chain variable sequence (SEQ ID NO: 76).

[0137] In one embodiment, the antibody, or antigen-binding portion thereof, has an HC CDR1 sequence comprising or consisting of SEQ ID NO: 47, an HC CDR2 sequence comprising or consisting of SEQ ID NO: 48, an HC CDR3 sequence comprising or consisting of SEQ ID NO: 49, an LC CDR1 sequence comprising or consisting of SEQ ID NO: 50, an LC CDR2 sequence comprising or consisting of SEQ ID NO: 51, and an LC CDR3 sequence comprising or consisting of SEQ ID NO: 52. The antibody or antigen-binding portion has framework regions with four amino acid changes in the heavy chain variable sequence compared to the germline sequence (SEQ ID NO: 74) and framework regions with no amino acid changes in the light chain variable sequence (SEQ ID NO: 76).

[0138] In embodiments, the antibody or antigen-binding portion has a framework region that has 1 to 10 amino acid changes in the heavy chain variable sequence compared to the germline sequence (SEQ ID NO: 74). For example, the antibody or antigen-binding portion has a framework region that can have 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acid changes in the heavy chain variable sequence compared to the germline sequence (SEQ ID NO: 74). In embodiments, the antibody or antigen-binding portion has a framework region that has 1 to 10 amino acid changes in the light chain variable sequence compared to the germline sequence (SEQ ID NO: 76). For example, the antibody or antigen-binding portion has a framework region that can have 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acid changes in the light chain variable sequence compared to the germline sequence (SEQ ID NO: 76).

[0139] The present invention also relates to an isolated canine antibody, or antigen-binding portion thereof, that binds to canine CD20, the antibody comprising: a) an HC CDR1 sequence comprising or consisting of SEQ ID NO: 112, or an amino acid sequence having 1, 2 or 3 amino acid differences compared to SEQ ID NO: 112; b) an HC CDR2 sequence comprising or consisting of SEQ ID NO: 113, or an amino acid sequence having 1, 2, 3, 4, or 5 amino acid differences compared to SEQ ID NO: 113; c) an HC CDR3 sequence comprising or consisting of SEQ ID NO: 114, or an amino acid sequence having 1, 2 or 3 amino acid differences compared to SEQ ID NO: 114; d) an LC CDR1 sequence comprising or consisting of SEQ ID NO: 115, or an amino acid sequence having one amino acid difference compared to SEQ ID NO: 124; e) an LC CDR2 sequence comprising or consisting of SEQ ID NO: 116, or an amino acid sequence having one or two amino acid differences compared to SEQ ID NO: 116; and f) comprises an LC CDR3 sequence comprising or consisting of SEQ ID NO: 117, or an amino acid sequence which has one or two amino acid differences compared to SEQ ID NO: 117.

[0140] The present invention also relates to an isolated canine antibody, or antigen-binding portion thereof, that binds to canine CD20, the antibody comprising: a) an HC CDR1 sequence comprising or consisting of SEQ ID NO: 122, or an amino acid sequence having 1, 2 or 3 amino acid differences compared to SEQ ID NO: 122; b) an HC CDR2 sequence comprising or consisting of SEQ ID NO: 123, or an amino acid sequence having 1, 2, 3, or 4 amino acid differences compared to SEQ ID NO: 123; c) an HC CDR3 sequence comprising or consisting of SEQ ID NO: 124, or an amino acid sequence having 1, 2, 3 or 4 amino acid differences compared to SEQ ID NO: 124; d) an LC CDR1 sequence comprising or consisting of SEQ ID NO: 125, or an amino acid sequence having one amino acid difference compared to SEQ ID NO: 125; e) an LC CDR2 sequence comprising or consisting of SEQ ID NO: 126, or an amino acid sequence having one amino acid difference compared to SEQ ID NO: 126; and f) comprises an LC CDR3 sequence comprising or consisting of SEQ ID NO: 127, or an amino acid sequence which has 1, 2 or 3 amino acid differences compared to SEQ ID NO: 127.

[0141] As used herein, an amino acid change is selected from an amino acid substitution, addition, or deletion. In one embodiment, the amino acid change is an amino acid substitution.

[0142] In one embodiment, the antibody is selected from the group consisting of PMX001, PMX002, PMX003, PMX004, PMX005, PMX006, PMX007, PMX008, PMX009, PMX010, PMX011, PMX066, PMX067, PMX068, PMX069, PMX070, PMX071, PMX072, PMX073, PMX074, PMX075, PMX076, PMX077, PMX078, PMX079, PMX080, PMX081, PMX082, PMX083, PMX084, PMX085, PMX086, PMX087, PMX088, PMX089, PMX090, PMX091, PMX092, PMX093, PMX094, PMX095, PMX096, PMX097, PMX098, PMX099, PMX100, PMX101, PMX102, PMX103, PMX104, PMX105, PMX106, PMX107, PMX1080, PMX1091, PMX1092, PMX1093, PMX1094, PMX1095, PMX1096, PMX1097, PMX1098, PMX1099, PMX110, PMX111, PMX112, PMX113, PMX114, PMX115, PMX116, PMX117, PMX118, PMX119, PMX120, PMX121, PMX122, PMX123, PMX124, PMX125, PMX126, PMX127, PMX MX073, PMX074, PMX075, PMX076, PMX077, PMX078, PMX079, PMX080, PMX081, PMX112, PMX113, PMX114, PMX115, PMX116, PMX117, PMX118, PMX119, PMX120, PMX121, PMX122, PMX123, or PMX124. In one embodiment, the antibody is selected from PMX001, PMX002, PMX003, PMX004, PMX005, PMX066, PMX067, PMX068, PMX069, PMX070, PMX071, PMX072, PMX073, PMX074, PMX075, PMX076, PMX077, PMX078, PMX079, PMX080, or PMX081. In one embodiment, the antibody is selected from PMX003, PMX066, PMX069, PMX070, PMX078, or PMX081. In one embodiment, the antibody is selected from PMX010, PMX112, PMX115, PMX122, or PMX124. In one embodiment, the antibody is selected from PMX069, PMX070, or PMX115.

[0143] Thus, in one embodiment, the antibody has a heavy chain variable region nucleotide sequence (SEQ ID NO:3) that encodes the corresponding amino acid sequence (SEQ ID NO:4), and a light chain variable region nucleotide sequence (SEQ ID NO:5) that encodes the corresponding amino acid sequence (SEQ ID NO:6). There are two amino acid changes in the heavy chain variable sequence compared to the germline sequence (SEQ ID NO:74), and three amino acid changes in the light chain variable sequence compared to the germline sequence (SEQ ID NO:76). The CDR sequences are as follows: CDR1 (SEQ ID NO:7), CDR2 (SEQ ID NO:8), and CDR3 (SEQ ID NO:9) for the heavy chain variable region, and CDR1 (SEQ ID NO:10), CDR2 (SEQ ID NO:11), and CDR3 (SEQ ID NO:12) for the light chain variable region.

[0144] In one embodiment, the antibody has a heavy chain variable region nucleotide sequence (SEQ ID NO:13) that encodes the corresponding amino acid sequence (SEQ ID NO:14) and a light chain variable region nucleotide sequence (SEQ ID NO:15) that encodes the corresponding amino acid sequence (SEQ ID NO:16). Compared to the germline sequence (SEQ ID NO:74), there are 10 amino acid changes in the heavy chain variable sequence and no amino acid changes in the light chain variable sequence (SEQ ID NO:76). The CDRs are: CDR1 (SEQ ID NO:17), CDR2 (SEQ ID NO:18), and CDR3 (SEQ ID NO:19) of the heavy chain variable region, and CDR1 (SEQ ID NO:20), CDR2 (SEQ ID NO:21), and CDR3 (SEQ ID NO:22) of the light chain variable region.

[0145] In one embodiment, the antibody has a heavy chain variable region nucleotide sequence (SEQ ID NO:23) that encodes the corresponding amino acid sequence (SEQ ID NO:24) and a light chain variable region nucleotide sequence (SEQ ID NO:25) that encodes the corresponding amino acid sequence (SEQ ID NO:26). Compared to the germline sequence (SEQ ID NO:74), there are five amino acid changes in the heavy chain variable sequence and one amino acid change in the light chain variable sequence (SEQ ID NO:76). The CDRs are: CDR1 (SEQ ID NO:27), CDR2 (SEQ ID NO:28), and CDR3 (SEQ ID NO:29) of the heavy chain variable region, and CDR1 (SEQ ID NO:30), CDR2 (SEQ ID NO:31), and CDR3 (SEQ ID NO:32) of the light chain variable region.

[0146] In one embodiment, the antibody has a heavy chain variable region nucleotide sequence (SEQ ID NO:33) that encodes the corresponding amino acid sequence (SEQ ID NO:34) and a light chain variable region nucleotide sequence (SEQ ID NO:35) that encodes the corresponding amino acid sequence (SEQ ID NO:36). Compared to the germline sequence (SEQ ID NO:74), there are 12 amino acid changes in the heavy chain variable sequence and no amino acid changes in the light chain variable sequence (SEQ ID NO:76). The CDRs are CDR1 (SEQ ID NO:37), CDR2 (SEQ ID NO:38), and CDR3 (SEQ ID NO:39) of the heavy chain variable region and CDR1 (SEQ ID NO:40), CDR2 (SEQ ID NO:41), and CDR3 (SEQ ID NO:42) of the light chain variable region.

[0147] In one embodiment, the antibody has a heavy chain variable region nucleotide sequence (SEQ ID NO:43) that encodes the corresponding amino acid sequence (SEQ ID NO:44) and a light chain variable region nucleotide sequence (SEQ ID NO:45) that encodes the corresponding amino acid sequence (SEQ ID NO:46). Compared to the germline sequence (SEQ ID NO:74), there are four amino acid changes in the heavy chain variable sequence and no amino acid changes in the light chain variable sequence (SEQ ID NO:76). The CDRs are CDR1 (SEQ ID NO:47), CDR2 (SEQ ID NO:48), and CDR3 (SEQ ID NO:49) of the heavy chain variable region and CDR1 (SEQ ID NO:50), CDR2 (SEQ ID NO:51), and CDR3 (SEQ ID NO:52) of the light chain variable region.

[0148] In one embodiment, the antibody has a heavy chain variable region nucleotide sequence (SEQ ID NO:78) that encodes the corresponding amino acid sequence (SEQ ID NO:79) and a light chain variable region nucleotide sequence (SEQ ID NO:80) that encodes the corresponding amino acid sequence (SEQ ID NO:81). The CDRs are CDR1 (SEQ ID NO:82), CDR2 (SEQ ID NO:83), and CDR3 (SEQ ID NO:84) of the heavy chain variable region, and CDR1 (SEQ ID NO:85), CDR2 (SEQ ID NO:86), and CDR3 (SEQ ID NO:87) of the light chain variable region.

[0149] In one embodiment, the antibody has a heavy chain variable region nucleotide sequence (SEQ ID NO:88) that encodes the corresponding amino acid sequence (SEQ ID NO:89) and a light chain variable region nucleotide sequence (SEQ ID NO:90) that encodes the corresponding amino acid sequence (SEQ ID NO:91). The CDRs are CDR1 (SEQ ID NO:92), CDR2 (SEQ ID NO:93), and CDR3 (SEQ ID NO:94) of the heavy chain variable region, and CDR1 (SEQ ID NO:95), CDR2 (SEQ ID NO:96), and CDR3 (SEQ ID NO:97) of the light chain variable region.

[0150] In one embodiment, the antibody has a heavy chain variable region nucleotide sequence (SEQ ID NO:98) that encodes the corresponding amino acid sequence (SEQ ID NO:99) and a light chain variable region nucleotide sequence (SEQ ID NO:100) that encodes the corresponding amino acid sequence (SEQ ID NO:101). The CDRs are CDR1 (SEQ ID NO:102), CDR2 (SEQ ID NO:103), and CDR3 (SEQ ID NO:104) of the heavy chain variable region, and CDR1 (SEQ ID NO:105), CDR2 (SEQ ID NO:106), and CDR3 (SEQ ID NO:107) of the light chain variable region.

[0151] In one embodiment, the antibody has a heavy chain variable region nucleotide sequence (SEQ ID NO: 108) that encodes the corresponding amino acid sequence (SEQ ID NO: 109) and a light chain variable region nucleotide sequence (SEQ ID NO: 110) that encodes the corresponding amino acid sequence (SEQ ID NO: 111). The CDRs are CDR1 (SEQ ID NO: 112), CDR2 (SEQ ID NO: 113), and CDR3 (SEQ ID NO: 114) of the heavy chain variable region, and CDR1 (SEQ ID NO: 115), CDR2 (SEQ ID NO: 116), and CDR3 (SEQ ID NO: 117) of the light chain variable region.

[0152] In one embodiment, the antibody has a heavy chain variable region nucleotide sequence (SEQ ID NO:118) that encodes the corresponding amino acid sequence (SEQ ID NO:119) and a light chain variable region nucleotide sequence (SEQ ID NO:120) that encodes the corresponding amino acid sequence (SEQ ID NO:121). The CDRs are CDR1 (SEQ ID NO:122), CDR2 (SEQ ID NO:123), and CDR3 (SEQ ID NO:124) of the heavy chain variable region, and CDR1 (SEQ ID NO:125), CDR2 (SEQ ID NO:126), and CDR3 (SEQ ID NO:127) of the light chain variable region.

[0153] In one embodiment, the antibody has a heavy chain variable region nucleotide sequence (SEQ ID NO: 128) that encodes the corresponding amino acid sequence (SEQ ID NO: 129) and a light chain variable region nucleotide sequence (SEQ ID NO: 130) that encodes the corresponding amino acid sequence (SEQ ID NO: 131). The CDRs are CDR1 (SEQ ID NO: 132), CDR2 (SEQ ID NO: 133), and CDR3 (SEQ ID NO: 134) of the heavy chain variable region, and CDR1 (SEQ ID NO: 135), CDR2 (SEQ ID NO: 136), and CDR3 (SEQ ID NO: 137) of the light chain variable region.

[0154] In one embodiment, the antibody has a heavy chain variable region nucleotide sequence (SEQ ID NO: 138) that encodes the corresponding amino acid sequence (SEQ ID NO: 139) and a light chain variable region nucleotide sequence (SEQ ID NO: 140) that encodes the corresponding amino acid sequence (SEQ ID NO: 141). The CDRs are CDR1 (SEQ ID NO: 142), CDR2 (SEQ ID NO: 143), and CDR3 (SEQ ID NO: 144) of the heavy chain variable region, and CDR1 (SEQ ID NO: 145), CDR2 (SEQ ID NO: 146), and CDR3 (SEQ ID NO: 147) of the light chain variable region.

[0155] In one embodiment, the antibody has a heavy chain variable region nucleotide sequence (SEQ ID NO: 148) that encodes the corresponding amino acid sequence (SEQ ID NO: 149) and a light chain variable region nucleotide sequence (SEQ ID NO: 150) that encodes the corresponding amino acid sequence (SEQ ID NO: 151). The CDRs are CDR1 (SEQ ID NO: 152), CDR2 (SEQ ID NO: 153), and CDR3 (SEQ ID NO: 154) of the heavy chain variable region, and CDR1 (SEQ ID NO: 155), CDR2 (SEQ ID NO: 156), and CDR3 (SEQ ID NO: 157) of the light chain variable region.

[0156] In one embodiment, the antibody has a heavy chain variable region nucleotide sequence (SEQ ID NO: 158) that encodes the corresponding amino acid sequence (SEQ ID NO: 159) and a light chain variable region nucleotide sequence (SEQ ID NO: 160) that encodes the corresponding amino acid sequence (SEQ ID NO: 161). The CDRs are CDR1 (SEQ ID NO: 162), CDR2 (SEQ ID NO: 163), and CDR3 (SEQ ID NO: 164) of the heavy chain variable region, and CDR1 (SEQ ID NO: 165), CDR2 (SEQ ID NO: 166), and CDR3 (SEQ ID NO: 167) of the light chain variable region.

[0157] In one embodiment, the antibody has a heavy chain variable region nucleotide sequence (SEQ ID NO: 168) that encodes the corresponding amino acid sequence (SEQ ID NO: 169) and a light chain variable region nucleotide sequence (SEQ ID NO: 170) that encodes the corresponding amino acid sequence (SEQ ID NO: 171). The CDRs are CDR1 (SEQ ID NO: 172), CDR2 (SEQ ID NO: 173), and CDR3 (SEQ ID NO: 174) of the heavy chain variable region, and CDR1 (SEQ ID NO: 175), CDR2 (SEQ ID NO: 176), and CDR3 (SEQ ID NO: 177) of the light chain variable region.

[0158] In one embodiment, the antibody has a heavy chain variable region nucleotide sequence (SEQ ID NO: 178) that encodes the corresponding amino acid sequence (SEQ ID NO: 179) and a light chain variable region nucleotide sequence (SEQ ID NO: 180) that encodes the corresponding amino acid sequence (SEQ ID NO: 181). The CDRs are CDR1 (SEQ ID NO: 182), CDR2 (SEQ ID NO: 183), and CDR3 (SEQ ID NO: 184) of the heavy chain variable region, and CDR1 (SEQ ID NO: 185), CDR2 (SEQ ID NO: 186), and CDR3 (SEQ ID NO: 187) of the light chain variable region.

[0159] In one embodiment, the antibody has a heavy chain variable region nucleotide sequence (SEQ ID NO: 188) that encodes the corresponding amino acid sequence (SEQ ID NO: 189) and a light chain variable region nucleotide sequence (SEQ ID NO: 190) that encodes the corresponding amino acid sequence (SEQ ID NO: 191). The CDRs are CDR1 (SEQ ID NO: 192), CDR2 (SEQ ID NO: 193), and CDR3 (SEQ ID NO: 194) of the heavy chain variable region, and CDR1 (SEQ ID NO: 195), CDR2 (SEQ ID NO: 196), and CDR3 (SEQ ID NO: 197) of the light chain variable region.

[0160] In one embodiment, the antibody has a heavy chain variable region nucleotide sequence (SEQ ID NO: 198) that encodes the corresponding amino acid sequence (SEQ ID NO: 199) and a light chain variable region nucleotide sequence (SEQ ID NO: 200) that encodes the corresponding amino acid sequence (SEQ ID NO: 201). The CDRs are CDR1 (SEQ ID NO: 202), CDR2 (SEQ ID NO: 203), and CDR3 (SEQ ID NO: 204) of the heavy chain variable region, and CDR1 (SEQ ID NO: 205), CDR2 (SEQ ID NO: 206), and CDR3 (SEQ ID NO: 207) of the light chain variable region.

[0161] In one embodiment, the antibody has a heavy chain variable region nucleotide sequence (SEQ ID NO:208) that encodes the corresponding amino acid sequence (SEQ ID NO:209) and a light chain variable region nucleotide sequence (SEQ ID NO:210) that encodes the corresponding amino acid sequence (SEQ ID NO:211). The CDRs are CDR1 (SEQ ID NO:212), CDR2 (SEQ ID NO:213), and CDR3 (SEQ ID NO:214) of the heavy chain variable region, and CDR1 (SEQ ID NO:215), CDR2 (SEQ ID NO:216), and CDR3 (SEQ ID NO:217) of the light chain variable region.

[0162] In one embodiment, the antibody has a heavy chain variable region nucleotide sequence (SEQ ID NO:218) that encodes the corresponding amino acid sequence (SEQ ID NO:219) and a light chain variable region nucleotide sequence (SEQ ID NO:220) that encodes the corresponding amino acid sequence (SEQ ID NO:221). The CDRs are CDR1 (SEQ ID NO:222), CDR2 (SEQ ID NO:223), and CDR3 (SEQ ID NO:224) of the heavy chain variable region, and CDR1 (SEQ ID NO:225), CDR2 (SEQ ID NO:226), and CDR3 (SEQ ID NO:227) of the light chain variable region.

[0163] In one embodiment, the antibody has a heavy chain variable region nucleotide sequence (SEQ ID NO:228) that encodes the corresponding amino acid sequence (SEQ ID NO:229) and a light chain variable region nucleotide sequence (SEQ ID NO:230) that encodes the corresponding amino acid sequence (SEQ ID NO:231). The CDRs are CDR1 (SEQ ID NO:232), CDR2 (SEQ ID NO:233), and CDR3 (SEQ ID NO:234) of the heavy chain variable region, and CDR1 (SEQ ID NO:235), CDR2 (SEQ ID NO:236), and CDR3 (SEQ ID NO:237) of the light chain variable region.

[0164] In one embodiment, the antibody, or antigen-binding portion thereof, comprises an Fc region, eg, a canine Fc region, eg, a canine IgGB Fc region.

[0165] The variable region sequences described herein, including but not limited to the amino acid and nucleotide sequences (and / or fragments thereof) set forth in Table 2, may be used in combination with one or more amino acid and / or nucleotide sequences encoding one or more constant chains (and / or fragments thereof) of an antibody molecule. For example, the variable region amino acid sequences set forth in Table 2 may be linked to the constant region of any antibody molecule of the same or a different species (e.g., human, goat, rat, sheep, chicken) from which the variable region amino acid sequence is derived. Preferably, the variable region amino acid sequences set forth in Table 2 are linked to the constant region of a canine antibody, which may be a constant region from any of canine IgG A, B, C, or D. In one embodiment, the constant region is a canine IgG B constant region. Canine IGGB (SEQ ID NO: 66), canine IGK (SEQ ID NO: 73), or canine IGLC5 (SEQ ID NO: 67) constant regions may also be used.

[0166] Variants of the above antibodies and antigen-binding portions are also within the scope of the present invention.

[0167] Variants of antibodies or antigen-binding portions thereof described herein have at least 80%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to the non-variant molecule. In one embodiment, the sequence identity is at least 95%. In one embodiment, the modifications are conservative sequence modifications.

[0168] As used herein, the term "conservative sequence modifications" is intended to refer to amino acid modifications that do not significantly affect or significantly alter the binding characteristics of the antibody containing the amino acid sequence. Such conservative modifications include amino acid substitutions, additions, and deletions. Modifications can be introduced into the antibodies of the present invention, or antigen-binding portions thereof, by standard techniques known in the art, such as site-directed mutagenesis and PCR-mediated mutagenesis. Conservative amino acid substitutions are those in which an amino acid residue is replaced with an amino acid residue having a similar side chain. Families of amino acid residues having similar side chains have been defined in the art. These families include amino acids with basic side chains (e.g., lysine, arginine, histidine), acidic side chains (e.g., aspartic acid, glutamic acid), uncharged polar side chains (e.g., glycine, asparagine, glutamine, serine, threonine, tyrosine, cysteine, tryptophan), nonpolar side chains (e.g., alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine), beta-branched side chains (e.g., threonine, valine, isoleucine), and aromatic side chains (e.g., tyrosine, phenylalanine, tryptophan, histidine). Thus, one or more amino acid residues within the CDR regions of an antibody of the invention can be replaced with another amino acid residue from the same side chain family, and the altered antibodies can be tested for retained function (i.e., CD20 binding) using the functional assays described herein.

[0169] Thus, these amino acid changes can typically be made without altering the biological activity, function, or other desired properties of the polypeptide, such as its affinity for an antigen or its specificity. Generally, single amino acid substitutions in non-essential regions of a polypeptide do not substantially alter biological activity. Furthermore, substitutions of amino acids that are similar in structure or function are unlikely to destroy the biological activity of the polypeptide. Abbreviations for the amino acid residues comprising the polypeptides and peptides described herein, as well as conservative substitutions for these amino acid residues, are set forth in Table 1 below. [Table 1]

[0170] In some embodiments, the invention provides antibodies or antigen-binding portions thereof that comprise one or more sequence modifications and have improved one or more properties, such as binding affinity, specificity, thermal stability, expression level, effector function, glycosylation, reduced immunogenicity, or solubility, compared to an unmodified antibody or fragment thereof, and are variants of the antibodies or antigen-binding portions thereof compared to a sequence described herein, e.g., selected from SEQ ID NOs: 3-65 or SEQ ID NOs: 78-237.

[0171] Suitable methods for measuring properties that may indicate that an antibody can be successfully developed on a large scale include first purification using chromatography such as affinity chromatography (Protein A: MabSelect Sure LX), anion exchange chromatography (Capto Q), cation exchange chromatography (Capto S), and buffer exchange (G-25 Fine), followed by assessment of whether the antibody remains intact (e.g., using SDS PAGE analysis to determine molecular weight, HPLC-SEC to calculate % monomer, and assess aggregation and thermal stability (Tm) studies).

[0172] For example, the consensus sequence for asparagine-linked glycosylation of proteins can be removed. Framework region 1 (FR1) of the light chain of PMX003 (SEQ ID NO: 26) contains the NVT sequon, which is the consensus sequence for asparagine-linked glycosylation of proteins. To remove the glycan attached to this site, the NVT sequon in FR1 of PMX003 mAb was mutated to QVT for PMX006 mAb, AVT for PMX007 mAb, EVT for PMX008 mAb, NVA for PMX009 mAb, SVT for PMX010 mAb, and TVT for PMX011 mAb. Such modifications are within the scope of the present invention.

[0173] The resulting light chain variable region amino acid sequences of PMX006 mAb (SEQ ID NO: 53), PMX007 mAb (SEQ ID NO: 54), PMX008 mAb (SEQ ID NO: 55), PMX009 mAb (SEQ ID NO: 56), PMX010 mAb (SEQ ID NO: 57), PMX011 mAb (SEQ ID NO: 58), and the FR1 sequences of PMX003 (SEQ ID NO: 59), PMX006 mAb (SEQ ID NO: 60), PMX007 mAb (SEQ ID NO: 61), PMX008 mAb (SEQ ID NO: 62), PMX009 mAb (SEQ ID NO: 63), PMX010 mAb (SEQ ID NO: 64), PMX011 mAb (SEQ ID NO: 65) are within the scope of the present invention and are listed in Sequence Table 2. These may also be used with the PMX003 heavy chain in place of SEQ ID NO: 26. The LC FR1 sequence for PMX003 is shown in SEQ ID NO:59, and the modified LC FR1 sequences are shown in SEQ ID NOs:60, 61, 62, 63, 64, and 65.

[0174] The NVT sequon is found at residue positions 11 to 13 in FR1 of the light chain of PMX001 to PMX005 (SEQ ID NO: 6, SEQ ID NO: 16, SEQ ID NO: 26, SEQ ID NO: 36, SEQ ID NO: 46) and PMX066 to PMX0081 (SEQ ID NO: 81, SEQ ID NO: 91, SEQ ID NO: 101, SEQ ID NO: 111, SEQ ID NO: 121, SEQ ID NO: 131, SEQ ID NO: 141, SEQ ID NO: 151, SEQ ID NO: 161, SEQ ID NO: 171, SEQ ID NO: 181, SEQ ID NO: 191, SEQ ID NO: 201, SEQ ID NO: 211, SEQ ID NO: 221, SEQ ID NO: 231). The NVT sequon found at residue positions 11 to 13 in FR1 of the light chain of PMX001 to PMX005 or PMX066 to PMX081 may be mutated to a sequence selected from QVT, AVT, EVT, NVA, SVT, and TVT. In embodiments, the NVT sequon found at residue positions 11-13 in FR1 of the light chain of PMX001-PMX005 or PMX066-PMX081 is mutated to SVT.

[0175] Framework region 1 (FR1) of the light chain of PMX066 (SEQ ID NO: 81), PMX067 (SEQ ID NO: 91), PMX068 (SEQ ID NO: 101), PMX069 (SEQ ID NO: 111), PMX071 (SEQ ID NO: 131), PMX072 (SEQ ID NO: 141), PMX073 (SEQ ID NO: 151), PMX074 (SEQ ID NO: 161), PMX076 (SEQ ID NO: 181), PMX077 (SEQ ID NO: 191), PMX078 (SEQ ID NO: 201), PMX079 (SEQ ID NO: 211), PMX081 (SEQ ID NO: 231) contains an NVT sequon, which is a consensus sequence for asparagine-linked glycosylation of proteins. To remove glycans attached to this site, PMX066, PMX067, PMX068, PMX069, PMX071, PMX072, PMX073, PMX074, PMX076, PMX077, PMX078, PMX079, and PMX081 The NVT sequon in FR1 of the mAbs was mutated to SVT to produce PMX112 (SEQ ID NO: 238), PMX113 (SEQ ID NO: 239), PMX114 (SEQ ID NO: 240), PMX115 (SEQ ID NO: 241), PMX116 (SEQ ID NO: 242), PMX117 (SEQ ID NO: 243), PMX118 (SEQ ID NO: 244), PMX119 (SEQ ID NO: 245), PMX120 (SEQ ID NO: 246), PMX121 (SEQ ID NO: 247), PMX122 (SEQ ID NO: 248), PMX123 (SEQ ID NO: 249), and PMX124 (SEQ ID NO: 250), respectively. Such modifications are within the scope of the present invention, and these light chains may be used with their respective heavy chain counterparts, for example, the HC of PMX066 (SEQ ID NO: 79) may be used with a modified LC (SEQ ID NO: 238), etc.

[0176] The resulting light chain variable region amino acid sequences of PMX112 mAb (SEQ ID NO: 238), PMX113 mAb (SEQ ID NO: 239), PMX114 mAb (SEQ ID NO: 240), PMX115 mAb (SEQ ID NO: 241), PMX116 mAb (SEQ ID NO: 242), PMX117 mAb (SEQ ID NO: 243), PMX118 mAb (SEQ ID NO: 244), PMX119 mAb (SEQ ID NO: 245), PMX120 mAb (SEQ ID NO: 246), PMX121 mAb (SEQ ID NO: 247), PMX122 mAb (SEQ ID NO: 248), PMX123 mAb (SEQ ID NO: 249), and PMX124 mAb (SEQ ID NO: 250) are within the scope of the present invention and are listed in the Sequence Listing.

[0177] Those skilled in the art will be aware that there are various methods for identifying, obtaining, and optimizing the antigen-binding molecules described herein, including in vitro and in vivo expression libraries. This is further described in the Examples. Optimization techniques known in the art, such as display (e.g., ribosome and / or phage display) and / or mutagenesis (e.g., error-prone mutagenesis), can be used. Thus, the present invention also encompasses sequence-optimized variants of the antibodies described herein.

[0178] In one embodiment, modifications can be made to reduce the immunogenicity of the antibody. For example, one approach is to restore one or more framework residues to the corresponding canine germline sequence. More specifically, an antibody that has undergone somatic mutation may contain framework residues that are different from the germline sequence from which the antibody is derived. Such residues can be identified by comparing the antibody framework sequence with the germline sequence from which the antibody is derived. In one embodiment, all framework sequences are germline sequences.

[0179] To return one or more amino acid residues in the framework region sequences to their germline configuration, somatic mutations can be "backmutated" to the germline sequence by, for example, site-directed mutagenesis or PCR-mediated mutagenesis.

[0180] Another type of framework modification involves mutating one or more residues within the framework regions, or within one or more CDR regions, to remove T-cell epitopes, thereby reducing the potential immunogenicity of the antibody.

[0181] In some embodiments, antigen-binding proteins, fragments and derivatives thereof, and fusion proteins of the present disclosure undergo post-translational modifications, for example, but not limited to, glutamine may be cyclized or converted to pyroglutamic acid; additionally or alternatively, amino acids may undergo deamidation, isomerization, glycation, and / or oxidation. Polypeptides of the present disclosure may undergo additional post-translational modifications, including glycosylation, e.g., N-linked or O-linked glycosylation, at sites known in the art. Changes can be made to the amino acid sequence of the polypeptide to prevent or minimize such modifications or to facilitate them in situations where such modifications are beneficial. Polypeptides of the present disclosure include, for example, polypeptides that have been modified to (1) reduce susceptibility to proteolysis, (2) reduce susceptibility to oxidation, (3) alter binding affinity for forming protein complexes, (4) alter binding affinity, and (5) impart or modify other physicochemical or functional properties.

[0182] Thus, in yet another embodiment, glycosylation is modified. For example, an aglycoslated antibody can be made (i.e., the antibody lacks glycosylation). In one embodiment, the light chain variable region amino acid sequence of the aglycosylated antibody comprises SEQ ID NO:53, SEQ ID NO:54, SEQ ID NO:55, SEQ ID NO:56, SEQ ID NO:57, SEQ ID NO:58, SEQ ID NO:238, SEQ ID NO:239, SEQ ID NO:240, SEQ ID NO:241, SEQ ID NO:242, SEQ ID NO:243, SEQ ID NO:244, SEQ ID NO:245, SEQ ID NO:246, SEQ ID NO:247, SEQ ID NO:248, SEQ ID NO:249, SEQ ID NO:250. The FR1 sequence is provided as SEQ ID NO:59, SEQ ID NO:60, SEQ ID NO:61, SEQ ID NO:62, SEQ ID NO:63, SEQ ID NO:64, or SEQ ID NO:65.

[0183] Glycosylation can also be modified, for example, to increase the affinity of the antibody for the antigen. Such carbohydrate modification can be achieved, for example, by modifying one or more glycosylation sites within the antibody sequence. For example, one or more amino acid substitutions can be made that result in the elimination of one or more variable region framework glycosylation sites, thereby eliminating glycosylation at that site. Such aglycosylation can increase the affinity of the antibody for the antigen.

[0184] In some applications, the binding agent can bind to canine CD20, but has a modified ability to bind to Fc receptors compared to standard binding agents. In one example, the binding agent is an antibody with a modified glycosylation pattern. For example, IgG molecules typically contain N-linked oligosaccharides, such as fucose.

[0185] In one embodiment, the antibody or antigen-binding portion thereof is afucosylated. In cancer immunotherapy, antibodies may rely on Fc-mediated immune effector function, antibody-dependent cellular cytotoxicity (ADCC), as their primary mode of action to deplete tumor cells. It is well known that this effector function is regulated by N-linked glycosylation in the Fc region of antibodies. In particular, the absence of core fucose on Fc N-glycans has been shown to increase IgG1 Fc binding affinity for FcγRIIIa present on immune effector cells such as natural killer cells, resulting in improved ADCC activity. Thus, afucosylated antibodies may be advantageous for improving therapeutic efficacy, and the absence / removal of fucose improves the ability of the antibody to interact with Fc receptors. This type of antibody may be referred to as "afucosylated." Such antibodies may be produced using techniques described herein and / or known in the art. In some embodiments, nucleic acid sequences encoding antibodies can be expressed in cell lines that have modified glycosylation capabilities (e.g., deleted, modified, or have reduced amounts of fucosyltransferase) and are unable to add typical fucose moieties.

[0186] In one embodiment, the antibody, or antigen-binding portion thereof, has CDC activity in the range of 0.9-4.5 ug / ml (6-30 nM), for example, with an EC50 value of less than 20 nM.

[0187] In one embodiment, the antibody, or antigen-binding portion thereof, has ADCC activity with an EC50 value of less than 0.3 nM, for example, 0.013 ug / ml (0.09 nM).

[0188] In one embodiment, an antibody or antigen-binding portion thereof according to the invention has one or more of the following properties: a) has CDC activity with an EC50 value of less than 20 nM; b) has ADCC activity with an EC50 value of less than 0.3 nM; c) having a transient expression yield of greater than 100ug / ml; d) has a Tm1 as determined by Uncle of greater than 58°C, and / or e) provides in vivo cell killing in dogs at doses of about 0.5 mg / kg to 2.5 mg / kg f) maintaining low levels of B cell depletion in vivo in dogs for at least 15 days; and / or g) Binds to an epitope comprising one or more amino acid residues, for example, 1 to 10, for example, 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10, of the following amino acid residues ENLNLIKAPM (SEQ ID NO: 303, amino acid numbers 150 to 159 in SEQ ID NO: 2), for example, ITISHFFKMENLNLIKAPM (SEQ ID NO: 302, amino acid numbers 141 to 159 in SEQ ID NO: 2), or consisting of the following amino acid residues ENLNLIKAPM (SEQ ID NO: 303, amino acid numbers 150 to 159 in SEQ ID NO: 2), for example, ITISHFFKMENLNLIKAPM (SEQ ID NO: 302, amino acid numbers 141 to 159 in SEQ ID NO: 2).

[0189] ADCC and CDC activity may be measured as described in the Examples.

[0190] In one embodiment, the antibodies and antigen-binding portions thereof exhibit both CDC and ADCC activity, e.g., as demonstrated by cell killing in a CD20-expressing canine lymphoma cell line as determined in the Examples.

[0191] In one embodiment, the antibody, or antigen-binding portion thereof, has one or more of the properties described above and has the HC and LC CDRs and / or the HC and LC CDRS of PMX069, PMX115, or PMX070. In one embodiment, the antibody, or antigen-binding portion thereof, is selected from PMX069, PMX115, or PMX070.

[0192] In one embodiment, the antibody is an antibody or antigen-binding portion thereof described in Table 4 and has an EC50 value for ADCC or CDC as shown in that table. EC50 values ​​may be measured as described in the Examples.

[0193] In one embodiment, the Fc portion of the antibody may be modified.

[0194] In one embodiment, one or more substitutions in the variant are in the CDR1, 2, and / or 3 regions. For example, there may be one, two, three, four, five, or more amino acid substitutions in the CDR1, 2, and / or 3 regions. In another example, there may be one or two amino acid deletions.

[0195] In one embodiment, the one or more substitutions are in a framework region, for example, there can be 1 to 20, e.g., 1 to 10, e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acid substitutions in the HC and / or LC variable region framework regions.

[0196] Antibodies of the present invention preferably have KD, IC50, and / or EC50 values, e.g., KDs further described herein in the Examples. Suitably, the KD value is sufficient for the antibody to have the desired biological effect. For example, the KD may be at least about 10 pM to 100 uM, about 100 pM to 10 nM or more. The EC50 value may be as shown in Table 4, e.g., 0.12 to 0.89 nM. The KD, IC50, and / or EC50 values ​​may be measured as described in the Examples.

[0197] The term "KD" refers to the "equilibrium dissociation constant," which is the value obtained in a titration measurement at equilibrium or by dividing the dissociation rate constant (Koff) by the association rate constant (Kon). "KA" refers to the affinity constant. The association rate constant, dissociation rate constant, and equilibrium dissociation constant are used to represent the binding affinity of an antibody to an antigen. Methods for determining the association and dissociation rate constants are well known in the art. The use of fluorescence-based techniques offers high sensitivity and the ability to examine samples in physiological buffer at equilibrium. Other experimental approaches and instruments, such as the BIAcore® SPR assay, can be used. The present invention also relates to isolated canine antibodies or antigen-binding portions thereof that bind to canine CD20 in competition with the above-described antibodies or antigen-binding portions thereof. Antibodies, antibody fragments, or antibody mimetics that bind to the same epitope or an overlapping epitope on canine CD20, or near any of the CD20 antibodies of the present invention, have the ability to cross-compete with any of the antibodies of the present invention for binding to CD20. Thus, the antibodies of the present invention can be used as reference antibodies to assess such cross-reactivity. Such cross-competing antibodies can be identified based on their ability to cross-compete with the antibodies described herein in standard CD20 binding assays. For example, BIAcore® analysis, ELISA assays, or flow cytometry can be used to demonstrate cross-competition with antibodies.

[0198] Nucleic acid sequences, vectors, and host cells The present invention also relates to nucleic acid sequences encoding the amino acid sequences of the antibodies or antigen-binding portions thereof described herein, e.g., the HC variable region or the LC variable region. Exemplary sequences are set forth in Table 2. In one embodiment, the nucleic acid is set forth in SEQ ID NO: 3, 5, 13, 15, 23, 25, 33, 35, 43, 45, 78, 80, 88, 90, 98, 100, 108, 110, 118, 120, 128, 130, 138, 140, 148, 150, 158, 160, 168, 170, 178, 180, 188, 190, 198, 200, 208, 210, 218, 220, 228, 230, 251, 252, 253, 254, 255, 256, 257, 258, 259, 260, 261 , 262, 263, 264, 265, 266, 267, 268, 269, 270, 271, 272, 273, 274, 275, 276, 277, 278, 279, 280, 281, 282, 283, 284, 285, 286, 287, 288, 289, 290, 291, 292, 293, 294, 295, 296, 297, 298, 299, 300, or 301, or a nucleic acid having at least 75%, 80%, or 90% sequence identity thereto. In one embodiment, the nucleic acid sequence is connected to a second nucleic acid sequence by a linker. In one embodiment, the second nucleic acid encodes an additional therapeutic moiety. In one embodiment, the linker is a nucleic acid linker. Exemplary nucleic acids are shown below. However, one of skill in the art will appreciate that due to the degeneracy of the genetic code, other sequences are contemplated.

[0199] Codon-optimized nucleotide sequences, including SEQ ID NOs: 68 and 69, are also within the scope of the present invention.

[0200] Nucleic acids according to the invention may comprise DNA or RNA and may be wholly or partially synthetic or recombinantly produced. Reference to a nucleotide sequence herein encompasses DNA molecules having the particular sequence, and RNA molecules having the particular sequence in which U is replaced by T, unless the context requires otherwise.

[0201] Furthermore, the present invention relates to a nucleic acid construct comprising at least one nucleic acid as defined above. The construct may be in the form of a plasmid, vector, transcription or expression cassette.

[0202] The present invention also relates to vectors containing nucleic acids encoding the CD20-binding molecules described herein. The term "vector" refers to a nucleic acid molecule, preferably a DNA molecule derived from, for example, a plasmid, bacteriophage, or vims, into which a nucleic acid sequence can be inserted or cloned. The vector preferably contains one or more unique restriction sites and may be capable of autonomous replication in a defined host cell, including a target cell or tissue or a progenitor cell or tissue thereof, or may be capable of integrating into the genome of a defined host so that the cloned sequence can be reproduced. Thus, the vector may be a self-replicating vector, i.e., a vector that exists as an extrachromosomal entity, the replication of which is independent of chromosomal replication, such as a linear or closed circular plasmid, an extrachromosomal element, a minichromosome, or an artificial chromosome. The vector may contain any means for ensuring self-replication. Alternatively, the vector may be one that, upon introduction into a host cell, integrates into the genome and replicates together with the chromosome into which it is integrated. The vector system may comprise a single vector or plasmid, or two or more vectors or plasmids, or a transposon, which together contain the total DNA to be introduced into the genome of the host cell. The selection of vector typically depends on the compatibility of the vector with the host cell that the vector is introduced into.Vector can also contain a selection marker, such as an antibiotic resistance gene, that can be used to select suitable transformants.Examples of such resistance genes are well known to those skilled in the art.In an embodiment, the vector is an adeno-associated virus (AAV) vector, such as that described in WO2021 / 176362.

[0203] In some embodiments, the nucleic acid may also include a leader sequence. In other embodiments, the nucleic acid does not include a leader sequence. Any suitable leader sequence may be used and may be selected to improve protein expression, including a native immunoglobulin germline leader sequence, such as SEQ ID NO: 71 for the heavy chain, SEQ ID NO: 72 for the light chain of PMX001-PMX005 and PMX066-PMX081 mAbs, or other sequences such as the Campath leader sequence (SEQ ID NO: 70) (see U.S. Pat. No. 8,362,208 B2).

[0204] In some embodiments, the nucleic acid may also contain a signal peptide, i.e., a short amino acid sequence (13-36 amino acids) at the N-terminus of a secretory protein (such as an immunoglobulin) that mediates translocation of the protein destined for secretion through the first membrane of the secretory pathway. This sequence is absent from the mature protein and is cleaved during co-translational events, but mediates secretion and correct expression of the protein. Suitable signal sequences may be used to optimize expression of the recombinant protein.

[0205] The present invention also relates to isolated recombinant host cells comprising one or more of the nucleic acid constructs described above. Host cells useful in the present invention can be prokaryotic, yeast, or higher eukaryotic cells, including, but not limited to, microorganisms such as bacteria (e.g., E. coli, B. subtilis) transformed with recombinant bacteriophage DNA, or plasmid or cosmid DNA expression vectors containing antibody coding sequences; yeast (e.g., Saccharomyces, Pichia) transformed with recombinant yeast expression vectors containing antibody coding sequences; insect cell lines infected with recombinant viral expression vectors (e.g., baculovirus) containing antibody coding sequences; plant cell lines infected with recombinant viral expression vectors (e.g., cauliflower mosaic virus, CaMV; tobacco mosaic virus, TMV) or transformed with recombinant plasmid expression vectors (e.g., Ti plasmid) containing antibody coding sequences; or mammalian cell lines (e.g., COS, CHO, BHK, 293, 3T3 cells) harboring recombinant expression constructs containing promoters derived from the genome of mammalian cells (e.g., metallothionein promoter) or from mammalian viruses (e.g., adenovirus late promoter; vaccinia virus 7.5K promoter).

[0206] Prokaryotes useful as host cells in the present invention include gram-negative or gram-positive organisms, such as E. coli, B. subtilis, Enterobacter, Erwinia, Klebsiella, Proteus, Salmonella, Serratia, and Shigella, as well as Bacilli, Pseudomonas, and Streptomyces. One cloning host is E. coli 294 (ATCC 31,446), although other strains such as E. coli B, E. coli X1776 (ATCC 31,537), and E. coli W3110 (ATCC 27,325) are suitable. In one embodiment, a method of producing an anti-CD20 antibody described herein is provided, comprising culturing a host cell under conditions suitable for expression of a polynucleotide encoding the antibody and isolating the antibody.

[0207] Nucleic acids encoding antibodies can be used to administer the antibodies to individuals to produce the encoded proteins in vivo and provide a therapeutic effect. Delivery of polynucleotides to a subject can be direct, such as by administering a polynucleotide or expression vector to an individual by directly introducing mRNA or DNA into cells, such as muscle cells. Indirect introduction is also contemplated, where a polynucleotide is transformed into cells in vitro prior to administration. Viral vectors, such as defective or attenuated viruses, can also be used.

[0208] In one embodiment, there is provided a method of making an anti-CD20 antibody described herein, the method comprising culturing a host cell under conditions suitable for expression of a polynucleotide encoding the antibody, and isolating the antibody.

[0209] The present invention also relates to heterologous assays or expression systems comprising canine CD20 and a cell line derived from a different species, for example a human cell line such as HEK.

[0210] The assay involves contacting canine CD20 with a cell line from a different species, e.g., a cell line from a different mammal, e.g., a rodent cell line or a human cell line such as HEK. For example, the cell line is transfected with canine CD20 so that it expresses canine CD20 in a stable or transient manner.

[0211] Immunoconjugates and other binding agents The present invention relates to immunoconjugates and other binding agents comprising an antibody or antigen-binding portion thereof according to the invention. For example, the antibody or antigen-binding portion thereof according to the invention may be conjugated to a therapeutic or non-therapeutic moiety.

[0212] In one embodiment, the therapeutic moiety is, for example, an antibody or antibody fragment (e.g., Fab, F(ab')2, Fv, single chain Fv fragment (scFv), or a single domain antibody, e.g., V H or V HH domain), or antibody mimetic proteins, that bind to a target antigen of interest.

[0213] In one embodiment, a protein or polypeptide comprising an antibody or antigen-binding portion thereof that binds to CD20 and a second moiety described herein is a fusion protein. In one embodiment, a protein or polypeptide comprising an antibody or antigen-binding portion thereof that binds to CD20 and a second moiety described herein is a drug conjugate.

[0214] As used herein, "conjugate" refers to a composition comprising an antibody that binds to CD20 as described herein that is linked / conjugated to a drug.

[0215] Such conjugates include "drug conjugates" which comprise an antibody that binds to CD20 to which a drug is covalently bound, and "non-covalent drug conjugates" which comprise an antibody that binds to CD20 min to which a drug is non-covalently bound.

[0216] As used herein, "drug conjugate" refers to a composition comprising an antibody to which a drug is covalently bound. The drug can be covalently bound to the antibody fragment directly or indirectly via a suitable linker moiety. The drug can be attached to the antibody at any suitable position, such as the amino terminus, the carboxyl terminus, or via a suitable amino acid side chain.

[0217] In one embodiment, the antibody is linked to the second moiety with a peptide linker or other suitable linker to connect the two moieties.

[0218] The term "peptide linker" refers to a peptide comprising one or more amino acids. Peptide linkers comprise 1 to 50, e.g., 1 to 20, amino acids. Peptide linkers are known in the art, and non-limiting examples are described herein. Suitable non-immunogenic linker peptides include, for example, linkers comprising G and / or S residues, (G4S)n, (SG4)n, or G4(SG4)n peptide linkers, where "n" is generally a number between 1 and 10, e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10.

[0219] The binding agent may be multispecific, eg, bispecific.

[0220] In one embodiment, the binding molecule is bispecific. Thus, in one aspect, the invention relates to a bispecific molecule comprising an antibody described herein linked to a second moiety having a different binding specificity than the antibody. Thus, the second antibody binds to a different target antigen, e.g., a target of interest.

[0221] In one embodiment, the binding molecule, eg, protein or construct, is multispecific and comprises further, ie, third, fourth, fifth, etc. moieties.

[0222] The therapeutic moiety may also be selected from a half-life extending moiety, a cytotoxin, or a radioisotope.

[0223] The non-therapeutic moiety may be selected from a label, a liposome, or a nanoparticle. The label is detectable or functional. The label may be any molecule that produces a signal or can be induced to produce a signal, including, but not limited to, a fluorophore, a fluorescent substance, a radioactive label, an enzyme, a chemiluminescent substance, a nuclear magnetic resonance active label, or a photosensitizer. Thus, binding may be detected and / or measured by detecting fluorescence or luminescence, radioactivity, enzyme activity, or light absorbance.

[0224] According to the present invention, antibodies and antigen-binding moieties that are linked to one moiety may be further linked to another moiety, for example, to a therapeutic moiety, and further linkage to a non-therapeutic moiety may be provided via either the antibody or the moiety.

[0225] In one embodiment, the binding agent or antibody, or antigen-binding portion thereof, according to the invention may comprise a half-life extending moiety, which may be selected from an antibody, or antigen-binding portion thereof, that binds to canine serum albumin. Alternatively, the extended half-life may be conferred through PEGylation.

[0226] The term "half-life" as used herein generally refers to the time required for the serum concentration of an amino acid sequence, compound, or polypeptide to decrease by 50% in vivo, for example, due to degradation of the sequence or compound and / or clearance or sequestration of the sequence or compound by natural mechanisms. The in vivo half-life of an amino acid sequence, compound, or polypeptide of the present invention can be determined in any manner known per se, such as by pharmacokinetic analysis. Suitable techniques will be apparent to those skilled in the art. Half-life can be expressed using parameters such as tl / 2-alpha, tl / 2-beta, and area under the curve (AUC). Half-life (talpha and tbeta) and AUC can be determined from the curve of serum concentration of the conjugate or fusion versus time. Thus, the term "half-life" as used herein particularly refers to tl / 2-beta or terminal half-life (tl / 2-alpha and / or AUC, or both, may be excluded from consideration).

[0227] For example, in the first phase (alpha phase), the drug composition (e.g., drug conjugate, noncovalent drug conjugate, drug fusion) is primarily distributed within the patient, with some elimination. The second phase (beta phase) is the terminal phase when the drug composition (e.g., drug conjugate, noncovalent drug conjugate, drug fusion) is distributed and the serum concentration decreases as the drug composition is cleared from the patient. The talpha half-life is the half-life of the first phase, and the tbeta half-life is the half-life of the second phase.

[0228] Pharmaceutical Composition In another aspect, a pharmaceutical composition is provided comprising an antibody or fragment described herein and, optionally, a pharmaceutically acceptable carrier. As used herein, the term "pharmaceutical composition" refers to a composition that is for veterinary use, that is used to treat companion animals, i.e., a veterinary composition. In a preferred embodiment, the animal being treated is a dog.

[0229] Pharmaceutical compositions may optionally include a pharmaceutically acceptable carrier. The antibody, protein, or construct or pharmaceutical composition may be administered by any convenient route, including, but not limited to, oral, topical, parenteral, sublingual, rectal, vaginal, ocular, intranasal, pulmonary, intradermal, intravitreal, intramuscular, intraperitoneal, intravenous, subcutaneous, intracerebral, transdermal, transmucosal, by inhalation, or topically, particularly to the ear, nose, eye, or skin, or by inhalation.

[0230] Parenteral administration includes, for example, intravenous, intramuscular, intraarterial, intraperitoneal, intranasal, rectal, intravesical, intradermal, topical, or subcutaneous administration. Preferably, the compositions are administered parenterally.

[0231] The pharmaceutically acceptable carrier or vehicle may be particulate, so that the composition is, for example, in tablet or powder form. The term "carrier" refers to a diluent, adjuvant, or excipient with which the drug-antibody conjugate of the present invention is administered. Such pharmaceutical carriers may be liquids, such as water and oils, including those of petroleum, animal, vegetable, or synthetic origin, such as peanut oil, soybean oil, mineral oil, sesame oil, and the like. Carriers may also be saline, acacia gum, gelatin, starch paste, talc, keratin, colloidal silica, urea, and the like. Additionally, auxiliary substances, stabilizers, thickeners, lubricants, and coloring agents may be used. In one embodiment, when administered to animals, the antibody or composition of the present invention and the pharmaceutically acceptable carrier are sterile. Water is a preferred carrier when the drug-antibody conjugate of the present invention is administered intravenously. Saline solutions and aqueous dextrose and glycerol solutions can also be used as liquid carriers, particularly for injectable solutions. Suitable pharmaceutical carriers also include excipients such as starch, glucose, lactose, sucrose, gelatin, malt, rice, flour, chalk, silica gel, sodium stearate, glycerol monostearate, talc, sodium chloride, dried skim milk, glycerol, propylene, glycol, water, ethanol, etc. The composition, if desired, can also contain minor amounts of wetting or emulsifying agents, or pH buffering agents.

[0232] The pharmaceutical composition of the present invention can be in the form of a liquid, for example, a solution, emulsion, or suspension.The liquid can be useful for injection, infusion (e.g., IV infusion), or subcutaneous delivery.When intended for oral administration, the composition is preferably in the form of a solid or liquid, and semi-solid, semi-liquid, suspension, and gel form are included in the form considered herein as either solid or liquid.

[0233] As a solid composition for oral administration, the composition can be formulated in the form of powder, granules, compressed tablets, pills, capsules, chewing gum, wafers, etc. Such solid compositions typically contain one or more inert diluents. In addition, one or more of the following may be present: binders such as carboxymethylcellulose, ethylcellulose, microcrystalline cellulose, or gelatin; excipients such as starch, lactose, or dextrin; disintegrating agents such as alginic acid, sodium alginate, or corn starch; lubricants such as magnesium stearate; glidants such as colloidal silicon dioxide; sweeteners such as sucrose or saccharin; flavors such as peppermint, methyl salicylate, or orange flavor; and colorants. When the composition is in the form of a capsule (e.g., a gelatin capsule), it can contain, in addition to the above-mentioned materials, a liquid carrier such as polyethylene glycol, cyclodextrin, or fatty oil.

[0234] The composition may be in the form of a liquid, such as an elixir, syrup, solution, emulsion, or suspension. This liquid may be useful for oral administration or delivery by injection. When intended for oral administration, the composition may contain one or more of a sweetener, a preservative, a dye / colorant, and a flavor enhancer. In a composition for administration by injection, one or more of a surfactant, a preservative, a wetting agent, a dispersing agent, a suspending agent, a buffer, a stabilizer, and an isotonic agent may also be included.

[0235] The compositions may be in the form of one or more dosage units. In certain embodiments, it may be desirable to administer the compositions locally to the area in need of treatment or by intravenous injection or infusion.

[0236] How to Treat a Disease The present invention further extends to methods for the treatment of disease, the administration of the pharmaceutical compositions or formulations described herein, or the antibodies or antigen-binding portions thereof of the invention. Also contemplated are pharmaceutical compositions or formulations described herein, or binding molecules or fusion proteins comprising the antibodies or antigen-binding portions thereof described herein, for use in the treatment of disease.

[0237] In particular, the present invention relates to a method of treating a B-cell mediated condition in a canine subject in need thereof, comprising administering an effective amount of an antibody, or antigen-binding portion thereof, described herein.

[0238] An aspect of the invention is also an antibody or antigen-binding portion thereof, or a pharmaceutical composition described herein for use in treating a B-cell mediated condition in a canine subject.

[0239] For example, the antibodies or antigen-binding portions thereof may be used to deplete dog blood and / or tissues of B-cell lymphoma cells. The B-cell mediated condition is selected from B-cell lymphoma (e.g., diffuse large B-cell lymphoma, Hodgkin's and non-Hodgkin's lymphoma, follicular lymphoma, mucosa-associated lymphoid tissue lymphoma (MALT), small cell lymphocytic lymphoma, chronic lymphocytic leukemia, Mantel cell lymphoma, Burkitt's lymphoma, mediastinal large B-cell lymphoma, Waldenstrom's macroglobulinemia, nodal marginal zone B-cell lymphoma (NMZL), splenic marginal zone lymphoma (SMZL), intravascular large B-cell lymphoma, primary effusion lymphoma, lymphomatoid granulomatosis), leukemia, or an immune-mediated disease. The immune-mediated disease may be an autoimmune disease. Examples may include, but are not limited to, autoimmune hemolytic anemia, immune-mediated thrombocytopenia, autoimmune bullous disease, immune-mediated arthritis and atopic dermatitis, rheumatoid arthritis, systemic lupus erythematosus (SLE), Sjogren's syndrome, vasculitis, multiple sclerosis, Graves' disease, idiopathic thrombocytopenia, dermatomyositis, immune-mediated thrombocytopenia, polymyocytosis, pemphigus, immune-mediated hemolytic anemia, and bullous pemphigus.

[0240] The amount of therapeutic agent that is effective / active in treating a particular disorder or condition depends on the nature of the disorder or condition and can be determined by standard clinical techniques. In addition, in vitro or in vivo assays may optionally be used to help identify optimal dosage ranges. The exact dose to be used in the composition also depends on the route of administration and the severity of the disorder or disease, and should be determined according to the judgment of the physician and each patient's circumstances. Factors such as age, body weight, sex, diet, administration time, excretion rate, host condition, drug combinations, reaction sensitivities, and disease severity should be taken into consideration.

[0241] Typically, this amount is at least about 0.01% of the antibody or fragment thereof of the present invention by weight of the composition. When intended for oral administration, this amount can vary from about 0.1% to about 80% by weight of the composition. Preferred oral compositions may contain from about 4% to about 50% of the antibody or fragment thereof of the present invention by weight of the composition.

[0242] Preferred compositions of the invention are prepared so that a parenteral dosage unit contains from about 0.01% to about 2% by weight of an antibody or fragment thereof of the invention.

[0243] For administration by injection, such as intravenous or subcutaneous injection, the composition may typically comprise about 0.01 mg / kg to about 250 mg / kg of the subject's body weight, e.g., 0.1 mg / kg to about 250 mg / kg, e.g., about 0.1 mg / kg to about 20 mg / kg of the animal's body weight, and more preferably about 1 mg / kg to about 10 mg / kg of the animal's body weight, although doses of less than 0.1 mg / kg are also contemplated. In one embodiment, the composition is administered at a dose of about 0.5 to 30 mg / kg, e.g., about 5 to 25 mg / kg, about 10 to 20 mg / kg, about 0.5 to 5 mg / kg, about 0.5 to 2.5 mg / kg, about 0.5 to 2.0 mg / kg, or about 2 or 3 mg / kg. In one embodiment, the composition is administered at a dose of 2 to 50 mg / ml. In one embodiment, the composition is administered at a dose of 0.5 mg / ml to 2.5 mg / ml, or 0.5 mg / ml to 5 mg / ml. The dosing schedule can vary, for example, from once weekly to once every 2, 3, or 4 weeks, or up to 8 weeks between doses. In one embodiment, the composition is administered at a dose of 0.5 mg / ml to 2.5 mg / ml every 3 to 4 weeks, for example, 0.5 mg / ml or 2.5 mg / ml every 3 to 4 weeks. Preferably, the dose is selected to provide long-term depletion of CD20-positive cells, allowing for a 3 to 4 week interval between doses. Multiple doses, preferably up to about 6 or more repeated doses, may be administered.

[0244] In one embodiment, after treatment, the subject is disease progression-free for at least 7 days, or at least 14 days, or at least 21 days, or at least 28 days, or at least 40 days, or at least 50 days, or at least 60 days. In one embodiment, after treatment, the subject is disease progression-free for at least 7 days, or at least 14 days, or at least 21 days, or at least 28 days, or at least 40 days, or at least 50 days, or at least 60 days.

[0245] In one embodiment, the number of days alive, disease free or disease progression free is at least 2 months, or at least 3 months, or at least 4 months, such as at least 5 months, for example at least 6 months.

[0246] In one embodiment, the number of days alive, disease-free, or disease progression-free is at least 9 months, 200 days, 300 days, or 3 years or more. In one embodiment, it is at least 1 year, 2 years, 3 years, or more. The invention provides a method of treating or preventing a CD20-mediated disease or disorder in a companion animal, e.g., a dog, comprising administering to the animal in need thereof an effective amount of an antibody or fragment of the invention.

[0247] As used herein, "treat," "treating," or "treatment" means inhibiting or alleviating a disease or disorder. For example, treatment can include postponing the onset of symptoms associated with a disease or disorder and / or reducing the severity of such symptoms that would or are expected to occur with the disease. These terms include amelioration of existing symptoms, prevention of additional symptoms, and amelioration or prevention of the underlying causes of such symptoms. Thus, these terms indicate that a beneficial result has been imparted to at least some of the mammalian, e.g., canine, patients being treated. Many medical treatments are effective in some, but not all, patients receiving the treatment. For example, in the treatment of B-cell lymphoma, improvement of symptoms can be assessed by measuring lymph nodes after treatment and observing a reduction in lymph node size as an indicator of successful treatment.

[0248] The term "subject" or "patient" refers to a dog who is the object of treatment, observation, or experiment. For the avoidance of doubt, the treatment of humans is excluded.

[0249] The molecules or pharmaceutical compositions of the present invention may be administered as the sole active ingredient or in combination with one or more other therapeutic agents, e.g., cancer therapy. A therapeutic agent is a compound or molecule useful in treating a disease. Examples of therapeutic agents include antibodies, antibody fragments, drugs, toxins, nucleases, hormones, immunomodulators, proapoptotic agents, antiangiogenic agents, boron compounds, photoactive agents or dyes, radioisotopes, immunosuppressants, or immunomodulators such as cytokines or chemokines. In one example, the molecules or pharmaceutical compositions of the present invention may be administered in combination with a multi-drug, CHOP-based chemotherapy protocol incorporating multiple injectable and oral drugs (lasparaginase, vincristine, cytoxin, prednisone, and doxorubicin), given more or less weekly over a period of several months. Administration may be simultaneous, before or after administration of the compounds of the present invention.

[0250] The present invention also relates to a method of inhibiting tumor growth or metastasis, comprising contacting tumor cells with an effective amount of an antibody or antigen-binding portion thereof, or pharmaceutical composition described herein. The method can be in vitro, in vivo, or ex vivo.

[0251] The present invention also relates to a method of killing tumor cells that express CD20, comprising contacting the cells with an antibody or pharmaceutical composition described herein, such that killing of the cells that express CD20 occurs. The tumor cells are canine tumor cells. The method can be in vitro, in vivo, or ex vivo.

[0252] Also provided are methods for eliminating cells expressing canine CD20 using the antibodies or pharmaceutical compositions described herein. The methods can be in vitro, in vivo, or ex vivo.

[0253] kit In another aspect, the invention provides kits for treating or preventing diseases or immune responses, e.g., as enumerated herein, and / or kits for detecting CD20 for the diagnosis, prognosis, or monitoring of disease, comprising an antibody of the invention and, optionally, instructions for use. Such kits may contain other components, packaging, instructions, or materials to aid in the detection of CD20 protein. The kits may include a labeled antibody that binds to CD20, or a binding molecule comprising an antibody that binds to CD20 and one or more compounds for detecting the label.

[0254] How to generate antibodies The antibodies described herein can be obtained from transgenic mammals, e.g., rodents, that express canine antibodies upon stimulation with the CD20 antigen. Such rodents are described in WO20018 / 189520 and WO2020 / 074874.

[0255] Thus, the antibodies or fragments described herein can be obtained from a mammal, e.g., a rodent, e.g., a transgenic animal, that expresses the antibody upon stimulation with a canine CD20 antigen. The transgenic rodent, e.g., a mouse, may have a reduced ability to express endogenous antibody genes. Thus, in one embodiment, the rodent has a reduced ability to express endogenous light and / or heavy chain antibody genes. Thus, the rodent, e.g., a mouse, may include modifications to disrupt expression of endogenous kappa and lambda light and / or heavy chain antibody genes, such that functional mouse light and / or heavy chains are not produced, for example, as further described below. Such transgenic rodents have been described in the art and are further described in the Examples below.

[0256] Also within the scope of the present invention is a method for producing a canine antibody capable of binding to CD20, said method comprising: a) immunizing a transgenic rodent, e.g., a mouse, with a CD20 antigen, wherein the rodent expresses a nucleic acid construct comprising unrearranged canine V, D, and J genes; b) isolating the canine antibody.

[0257] Also within the scope of the present invention is a method for producing an antibody capable of binding to canine CD20, said method comprising: a) immunizing a transgenic rodent, e.g., a mouse, with a CD20 antigen, wherein the rodent expresses a nucleic acid construct comprising unrearranged canine V, D, and J genes; b) generating a library of sequences comprising heavy and light chain sequences from said rodent, e.g., mouse; c) isolating antibodies comprising the heavy and light chain sequences from the library.

[0258] A further step may involve identifying antibodies that bind to CD20, for example, by using a functional assay as shown in the Examples.

[0259] Methods for preparing or producing the polypeptides, nucleic acids, host cells, products, and compositions described herein using in vitro expression libraries include: a) providing a set, collection, or library of nucleic acid sequences encoding amino acid sequences; b) screening said set, collection or library for amino acid sequences capable of binding to / having affinity for CD20; c) isolating amino acid sequences capable of binding to / having affinity for CD20.

[0260] In the above methods, the set, collection, or library of amino acid sequences can be displayed, for example, on a phage, phagemid, ribosome, or suitable microorganism (such as yeast) to facilitate screening. Suitable methods, techniques, and host organisms for displaying and screening (sets, collections, or libraries of) amino acid sequences will be apparent to those skilled in the art (see, for example, "Phage Display of Peptides and Proteins: A Laboratory Manual," Academic Press; 1st edition (October 28, 1996) Brian K. Kay, Jill Winter, John McCafferty). A library, such as a phage library, is generated by isolating cells or tissues expressing antigen-specific antibodies or fragments thereof, cloning sequences encoding the mRNA of the antibodies or fragments thereof from the isolated cells or tissues, and displaying the encoded proteins using the library. The sequences can be expressed in bacteria, yeast, or other expression systems.

[0261] Another aspect also relates to an isolated antibody obtained or obtainable by the above method.

[0262] Other Methods and Uses In another aspect, the antibodies or antigen-binding portions thereof described herein are used for non-therapeutic purposes, such as in diagnostic tests and assays. Accordingly, the present invention also relates to a method for detecting canine cells expressing canine CD20 or detecting canine CD20 protein in a biological sample from a canine subject, comprising contacting the biological sample with an antibody or antigen-binding portion thereof described herein, wherein the antibody or antigen-binding portion thereof is linked to a detectable label. The biological sample can be a biopsy, tissue, blood, serum, plasma, or lymph sample.

[0263] In certain embodiments, the methods may include comparing the amount of binding in the test biological sample with the amount of binding in a control biological sample, and increased binding to the test biological sample compared to the control biological sample may indicate the presence of one or more lymphoma cells in the test biological sample. In some embodiments, the biological sample is dog blood or needle aspirate. These methods are also provided in in vivo and / or in vitro formats.

[0264] The modification of antibodies for diagnostic purposes is well known in the art. For example, antibodies can be modified with a ligand group such as biotin, or a detectable marker group such as a fluorescent group, a radioisotope, or an enzyme. The compounds of the present invention can be used for diagnostic purposes, and can be labeled, for example, using conventional techniques. Suitable detectable labels include, but are not limited to, fluorophores, chromophores, radioactive atoms, electron-dense reagents, enzymes, and ligands with specific binding partners.

[0265] In another aspect, the antibodies or antigen-binding portions thereof described herein are used to isolate and / or identify cells that express canine CD20 or cells that contain cell surface proteins that react with these binding agents (e.g., B cells, B lymphoma cells, canine CD20).

[0266] The antibodies or antigen-binding portions thereof described herein can also be used in assays to determine the level of CD20 expression. The level of expression can then be correlated with baseline (e.g., control) levels to determine whether a particular disease is present in a patient, the patient's prognosis, or whether a particular treatment regimen is effective.

[0267] Unless otherwise defined herein, scientific and technical terms used in connection with the present disclosure shall have the meanings commonly understood by those skilled in the art. While the foregoing disclosure provides a general description of the subject matter encompassed within the scope of the present disclosure, including how to make and use the disclosure and its best mode, the following examples are provided to further enable those skilled in the art to practice the present disclosure. However, those skilled in the art will understand that the details of these examples should not be read as limiting the invention. The scope of the present invention should be understood from the claims appended hereto and their equivalents. Various further aspects and embodiments of the present disclosure will be apparent to those skilled in the art in light of the present disclosure.

[0268] All documents mentioned herein are incorporated by reference in their entirety, including references to gene accession numbers, scientific publications, and patent publications.

[0269] "And / or," as used herein, should be considered a specific disclosure of each of the two particular features or components with or without the other. For example, "A and / or B" should be considered a specific disclosure of (i) A, (ii) B, and (iii) each of A and B, as if each were individually set forth herein. Unless the context dictates otherwise, the above feature descriptions and definitions are not limited to any particular aspect or embodiment of the invention, but apply equally to all aspects and embodiments described.

[0270] The present invention is further illustrated in the following non-limiting examples. [Example]

[0271] Example 1: Cloning of canine CD20 A search of the CAMFAM_3.1 boxer reference genome was performed using the UCSC genome browser. The genomic sequence for CD20 (MS4A1) was downloaded along with the mRNA sequence AB210085.1. This sequence data was used to design primers to allow amplification of CD20 from cDNA with added sequence, allowing seamless cloning. This allowed for confirmation of the CD20 sequence in dog blood and seamless cloning into the piggyBac cloning vector.

[0272] Isolation of canine CD20 mRNA and generation of cDNA Beagle whole blood was provided by Envigo RMS (Alconbury, Huntingdon, UK), and PBMCs were isolated using a Ficoll gradient. Briefly, 10 ml of whole blood was diluted with 25 ml of phosphate-buffered saline (PBS) and layered over 15 ml of Ficoll-Paque Plus (Sigma-Aldrich), followed by centrifugation at 800 rcf for 10 minutes at room temperature with slow acceleration and no brake. The interphase disc was collected in PBS. Total RNA was isolated from PBMCs using standard procedures with the QIAGEN RNeasy Mini Kit (Qiagen, Hilden, DE) and on-column DNAse digestion. cDNA generation was performed using the SuperScript™ IV First-Strand Synthesis System and anchored oligo-dT primers (ThermoFisher, Massachusetts, US) according to standard procedures.

[0273] The nucleotide and amino acid sequences of full-length canine CD20 for cellular expression are shown below. nucleotide: ATGACAACACCCAGAAATTCAATGAGTGGAACTCTCCCGG TAGATCCTATGAAAAGCCCTACTGCCATGTATCCTGTTCA AAAAATAATTCCCAAAAGGATGCCTTCAGTGGTGGGCCCT ACACAAAACTTCTTCATGAGGGAATCTAAGACACTGGGGG CTGTCCAGATTATGAATGGGCTCTTCCACATTGCCCTAGG CAGCCTCCTGATGATTCACACGGATGTCTATGCGCCCATC TGTATAACTATGTGGTACCCTCTCTGGGGAGGCATTATGT TCATCATTTCTGGATCACTCCTGGCAGCAGCGGACAAAAA CCCCAGGAAGAGTTTGGTCAAAGGAAAAATGATAATGAAC TCATTGAGCCTCTTTGCTGCTATTTCTGGAATAATTTTTT TGATCATGGACATATTTAATATTACCATTTCCCATTTTTT TAAAATGGAGAATTTGAATCTTATTAAAGCTCCCATGCCA TATGTTGACATACACAACTGTGACCCAGCTAACCCCTCTG AGAAAAACTCTTTATCTATACAATATTGTGGCAGCATACG ATCTGTTTTCTTGGGCGTTTTTGCTGTGATGGTGATCTTT ACCTTTTTCCAGAAACTTGTGACAGCTGGCATTGTTGAGA ATGAATGGAAAAAACTGTGCTCTAAACCTAAATCTGATGT AGTTGTTCTGTTAGCTGCTGAAGAAAAAAAAGAACAGCCG ATTGAAACAACAGAAGAAATGGTTGAGCTGACTGAAATAG CTTCCCAACCAAAGAAAGAAGAAGACATTGAAATTATTCC AGTCCAAGAAGAAGAAGAGGAACTGGAAATAAACTTTGCA GAACCTCCCCAGGAGCAGGAATCTTCACCAATAGAAAACG ACAGCATCCCTTAA (SEQ ID NO: 1) protein: MTTPRNSMSGTLPVDPMKSPTAMYPVQKIIPKRMPSVVGP TQNFFMRESKTLGAVQIMNGLFHIALGSLLMIHTDVYAPI CITMWYPLWGGIMFIISGSLLAAADKNPRKSLVKGKMIMN SLSLFAAISGIIFLIMDIFNITISHFFKMENLNLIKAPMP YVDIHNCDPANPSEKNSLSIQYCGSIRSVFLGVFAVMVIF TFFQKLVTAGIVENEWKKLCSKPKSDVVVLLAAEEKKEQP IETTEEMVELTEIASQPKKEEDIEIIPVQEEEEELEINFA EPPQEQESSPIENDSIP (SEQ ID NO: 2)

[0274] Example 2: Canine CD20 expression Human embryonic kidney (HEK) 293 cells were grown on 90 mm round tissue culture plates as monolayers in DMEM / F12 (Life Technologies) supplemented with 10% fetal bovine serum (FBS, Sigma Aldrich) at 37°C with 5% CO2. HEK293 cells were co-transfected with CD20 cDNA and PiggyBac transposase using polyethyleneimine (PEI MAX: 40 kDa, Polysciences Inc., Eppelheim, Germany). 30 μl of PEI MAX (1 mg ml) was added. -1 ), 5 μg of cDNA, and 1 ml of DMEM / F12 were incubated at room temperature for 10 minutes and added dropwise to a 90 mm plate of 70-80% confluent HEK293 cells and incubated for 2 days before use. Stably transfected cells were selected after 48 hours using the appropriate antibiotic.

[0275] Mouse embryonic fibroblasts (MEFs) were grown as monolayers on 90 mm circular tissue culture plates in DMEM high glucose (Life Technologies) supplemented with 10% FBS, 1 mM sodium pyruvate (Sigma-Aldrich), 0.5 mM β-mercaptoethanol (Gibco), and 1% MEM non-essential amino acids (Sigma-Aldrich) at 37°C with 5% CO2. Cells were transfected with CD20 cDNA and PiggyBac transposase using Lipofectamine LTX with PLUS™ reagent (ThermoFisher Scientific) according to the manufacturer's recommended instructions. Stably transfected cells were selected after 48 hours using the appropriate antibiotic.

[0276] MDCK II (Madin-Darby canine kidney) cells were grown as monolayers on surface-treated tissue culture flasks (T25 / T75 / T175) in DMEM high glucose (Life Technologies) supplemented with 10% FBS and 1% MEM non-essential amino acids (Sigma-Aldrich) at 37°C with 5% CO. MDCK II cells were cotransfected with wild-type or mutant canine CD20 cDNA and piggyBac transposase using Lipofectamine™ LTX Reagent with PLUS™ Reagent (ThermoFisher Scientific) according to the recommended protocol.

[0277] Example 3: Immunization of mouse models using DNA and MEFs For example, Ky9™ mice were immunized substantially as described in WO2018 / 189520 and WO2020 / 074874. The transgenic mice have been modified by inserting a canine immunoglobulin variable gene repertoire into the corresponding loci of the mouse genome. This allows for the production of antibodies comprising variable antibody regions derived from the expression of canine DNA in mice in combination with mouse constant regions (in the case of heavy and kappa chains) or canine constant regions (in the case of lambda chains). Information about the variable regions of such chimeric antibody chains, or nucleic acids comprising the variable regions of such chimeric antibody chains, can be used to generate complete canine antibodies.

[0278] For DNA immunization, a prime and boost regimen was performed using hydrodynamic tail vein injection (HTVI), and tissues were harvested. For cell-based immunization, a prime and boost regimen was performed using MEF cells stably expressing CD20, and tissues were harvested. An additional immunization regime was performed using HTVI DNA immunization for the prime combined with a CD20-expressing MEF cell booster.

[0279] Serum titer determination: Mice were bled before immunization and 10 days after each subsequent boost. Serum was separated from clotted blood by centrifugation in a microvette 200 Z gel tube (Starstedt AG & Co. KG, Germany), and antibody titers against canine CD20 were assessed by flow cytometry. Serum was serially diluted 1:10 in FACS buffer (PBS + 3% FBS) and added to cells stably expressing canine CD20 or to canine CD20-negative control cells. Mouse antibodies against canine CD20 were detected with either a BB700 conjugate (BD Horizon Brilliant™ Blue700, BD Biosciences) or an FITC-conjugated secondary monoclonal antibody against isotypes IgG1, IgG2a, or IgG2b (BD OptiBuild™, Becton Dickinson). Data were acquired using a BD Accuri C6 flow cytometer (Becton Dickinson, NJ, USA) or a Beckman Coulter CytoFLEX. Pre-immunization serum was used to determine background. Antibody titers were determined as the highest dilution that gave a positive signal above background. Figure 1 shows the antibody titers of five immunized Ky9 mice.

[0280] Example 4: Isolation of antibody-producing cells, antibody sequencing, selection and sequence modification Tissue isolation: Spleens, lymph nodes, and bone marrow were harvested from mice. Splenocytes were prepared by cutting spleens into fragments and mashing them through a 40 μm cell strainer (Falcon) while rinsing with RPMI-1640 (Lonza, Basel, CH) + 10% FBS on ice. A similar process was used for lymphocytes from lymph nodes, with spleen and lymph node cells typically pooled. Bone marrow was harvested from femurs and tibias by flushing the bone marrow with RPMI-1640 using a 25-gauge needle through a 40 μm cell strainer pre-wetted with RPMI-1640. All cell types were pelleted at 300 g for 5 min and either used directly for flow sorting or resuspended in FBS + 10% dimethyl sulfoxide (DMSO) before freezing at -150°C.

[0281] Cell sorting: Generally, antigen-specific splenic B cells predominantly express transmembrane antibodies on their cell surface and can be captured by labeled antigen protein probes (e.g., extracellular domains) or antigen VLPs. On the other hand, antigen-specific plasmablasts or plasma cells are thought to be less susceptible to protein probes or VLPs due to their predominant expression of secreted antibodies. Therefore, plasmablasts and plasma cells isolated from splenic lymph node samples or bone marrow were subjected to the next step of antibody sequence recovery without the use of antigen probes to isolate antigen-specific subsets of these populations. Cell surface coexpression of CD138 and CD267 (TACI) was used to identify plasmablast and plasma cell populations. For bulk sorting of plasmablasts and plasma cells for 10X Genomics Chromium Single Cell Immune Profiling, a CD138 Plasma Cell Enrichment Kit (Miltenyi Biotech UK) was used to enrich these rare cells. Prior to antigen-specific cell sorting from spleen and lymph node cells, B cell enrichment was performed using a mouse pan B cell isolation kit (StemCell Technologies UK) according to the manufacturer's instructions or using an in-house biotinylated antibody cocktail and streptavidin rapidspheres (StemCell Technologies UK) according to the manufacturer's instructions.

[0282] Markers including CD19, IgM, IgA, IgD, CD138, and CD267 (TACI) are then used to identify isotype-switched B cells enriched within cells responding to immunization. Within this population, antigen-specific cells can be captured by staining with labeled protein probes or VLPs expressing the target antigen on their surface and flow sorting. VLPs are produced from HEK cells stably transfected with CD20, and the retroviral gag protein is fused to EGFP. Gag expression allows VLP budding from the cells, and EGFP labels the VLPs for fluorescent detection. Surface antigens on VLPs are expressed directly from recombinant cells without any purification or modification steps and are presented in their native form. Other mammalian cell lines, such as Chinese hamster ovary cells (CHO) or mouse embryonic fibroblasts (MEF), can also be used for VLP production. Markers to identify unwanted cell populations and dead cells (F4 / 80, Ly-6C / G, CD8a, CD4, CD11c, 7AAD, or Zombie-NIR / FVD EF780) are included in all staining panels to exclude these cells from the sorting procedure.

[0283] Next generation sequencing and candidate selection: Sorted cells were prepared for antibody profiling using the 10X Genomics Chromium Single Cell Immune Profiling System and V(D)J kit (10X Genomics) according to the manufacturer's instructions. For 10X, various Illumina platform instruments were used according to instructions from 10X Genomics. Sequences were analyzed using custom tools based on pRESTO / Change-O (Yale University) / IgBlast (NCBI, USA) / Enclone (10X Genomics) software to predict germline sequences and hypermutations.

[0284] Variable immunoglobulin regions include the VDJ regions of the immunoglobulin nucleotide sequences for heavy chain genes and the VJ regions of the immunoglobulin nucleotide sequences for Igκ and Igλ. Within a clonal family, there are subfamilies with shared mutations within their V(D)J segments that arise during immunoglobulin gene recombination and somatic hypermutation. Different clonal families that exhibit unique V(D)J segment usage typically exhibit distinct binding characteristics. During recombination and hypermutation, cells whose antibodies have higher affinity for antigen are selected. Affinity typically increases with further mutations; for example, clustered families are shown in Figure 6 of WO2015 / 040401.

[0285] A clonal family is generally defined by the related immunoglobulin heavy and light chain V(D)J sequences of two or more clonal cells. Related immunoglobulin V(D)J sequences can be identified by their shared use of V and J gene segments. An example of analysis of antibody sequences from sorted Ag-specific single B cells is shown in Figure 5 of WO2015 / 040401, which shows antibody sequences aligned by heavy chain V gene family usage and clustered to generate a displayed phylogenetic tree. Candidate clones are selected from phylogenetic trees such as these.

[0286] For example, anti-canine CD20 mAbs PMX001, PMX002, PMX003, PMX004, and PMX005 are all encoded by the same heavy chain V gene (cIGHV3-5), heavy chain J gene (cIGHJ4, with the germline sequence given in SEQ ID NO:75), light chain V gene (cIGLV3-3), and light chain J gene (cIGLJ3, with the germline sequence given in SEQ ID NO:77). The PMX001 variable region sequence was originally identified from canine CD20-immunized Ky9 mice. It has a heavy chain variable region nucleotide sequence (SEQ ID NO:3) that encodes the corresponding amino acid sequence (SEQ ID NO:4), and a light chain variable region nucleotide sequence (SEQ ID NO:5) that encodes the corresponding amino acid sequence (SEQ ID NO:6). There are two amino acid changes in the heavy chain variable sequence compared to the germline sequence (SEQ ID NO:74), and three amino acid changes in the light chain variable sequence compared to the germline sequence (SEQ ID NO:76). CDR1 (SEQ ID NO: 7), CDR2 (SEQ ID NO: 8), and CDR3 (SEQ ID NO: 9) of the heavy chain variable region and CDR1 (SEQ ID NO: 10), CDR2 (SEQ ID NO: 11), and CDR3 (SEQ ID NO: 12) of the light chain variable region were predicted using IMGT V-QUEST (http: / / www.imgt.org / IMGT_vquest / input). The CDR3 regions of the heavy and light chains of PMX mAb are 5 aa and 9 aa in length, respectively.

[0287] Hit expansion of the PMX001 mAb was subsequently performed in all canine CD20-immunized mice, and candidates sharing the same VJ gene usage and CDR3 length for both the heavy and light chains were selected for further screening. Compared to the PMX001 mAb, additional mutations may have occurred in the sequence. For example, PMX002, PMX003, PMX004, and PMX005 mAbs were selected from other immunization cohorts and therefore differ from the mice in which the PMX001 mAb was identified. This indicates convergent selection of successful gene rearrangements.

[0288] The PMX002 mAb has a heavy chain variable region nucleotide sequence (SEQ ID NO:13) that encodes the corresponding amino acid sequence (SEQ ID NO:14) and a light chain variable region nucleotide sequence (SEQ ID NO:15) that encodes the corresponding amino acid sequence (SEQ ID NO:16). Compared to the germline sequence (SEQ ID NO:74), there are 10 amino acid changes in the heavy chain variable sequence and no amino acid changes in the light chain variable sequence (SEQ ID NO:76). CDR1 (SEQ ID NO:17), CDR2 (SEQ ID NO:18), and CDR3 (SEQ ID NO:19) of the heavy chain variable region and CDR1 (SEQ ID NO:20), CDR2 (SEQ ID NO:21), and CDR3 (SEQ ID NO:22) of the light chain variable region are predicted using IMGT V-QUEST.

[0289] PMX003mAb has a heavy chain variable region nucleotide sequence (SEQ ID NO:23) that encodes the corresponding amino acid sequence (SEQ ID NO:24) and a light chain variable region nucleotide sequence (SEQ ID NO:25) that encodes the corresponding amino acid sequence (SEQ ID NO:26). Compared to the germline sequence (SEQ ID NO:74), there are five amino acid changes in the heavy chain variable sequence and one amino acid change in the light chain variable sequence (SEQ ID NO:76). CDR1 (SEQ ID NO:27), CDR2 (SEQ ID NO:28), and CDR3 (SEQ ID NO:29) of the heavy chain variable region and CDR1 (SEQ ID NO:30), CDR2 (SEQ ID NO:31), and CDR3 (SEQ ID NO:32) of the light chain variable region are predicted using IMGT V-QUEST.

[0290] PMX004 mAb has a heavy chain variable region nucleotide sequence (SEQ ID NO:33) that encodes the corresponding amino acid sequence (SEQ ID NO:34) and a light chain variable region nucleotide sequence (SEQ ID NO:35) that encodes the corresponding amino acid sequence (SEQ ID NO:36). Compared to the germline sequence (SEQ ID NO:74), there are 12 amino acid changes in the heavy chain variable sequence and no amino acid changes in the light chain variable sequence (SEQ ID NO:76). CDR1 (SEQ ID NO:37), CDR2 (SEQ ID NO:38), and CDR3 (SEQ ID NO:39) of the heavy chain variable region and CDR1 (SEQ ID NO:40), CDR2 (SEQ ID NO:41), and CDR3 (SEQ ID NO:42) of the light chain variable region are predicted using IMGT V-QUEST.

[0291] The PMX005 mAb has a heavy chain variable region nucleotide sequence (SEQ ID NO:43) that encodes the corresponding amino acid sequence (SEQ ID NO:44) and a light chain variable region nucleotide sequence (SEQ ID NO:45) that encodes the corresponding amino acid sequence (SEQ ID NO:46). Compared to the germline sequence (SEQ ID NO:74), there are four amino acid changes in the heavy chain variable sequence and no amino acid changes in the light chain variable sequence (SEQ ID NO:76). CDR1 (SEQ ID NO:47), CDR2 (SEQ ID NO:48), and CDR3 (SEQ ID NO:49) of the heavy chain variable region and CDR1 (SEQ ID NO:50), CDR2 (SEQ ID NO:51), and CDR3 (SEQ ID NO:52) of the light chain variable region are predicted using IMGT V-QUEST.

[0292] FIG. 2 shows an alignment of PMX antibody sequences.

[0293] Array Modification Framework region 1 (FR1) of the light chain of PMX003 (SEQ ID NO: 26) contains the NVT sequon, a consensus sequence for asparagine-linked glycosylation of proteins. To remove the glycan attached at this site, the NVT sequon in FR1 of PMX003 mAb was mutated to QVT for PMX006 mAb, AVT for PMX007 mAb, EVT for PMX008 mAb, NVA for PMX009 mAb, SVT for PMX010 mAb, and TVT for PMX011 mAb.

[0294] The resulting light chain variable region amino acid sequences of PMX006 mAb (SEQ ID NO: 53), PMX007 mAb (SEQ ID NO: 54), PMX008 mAb (SEQ ID NO: 55), PMX009 mAb (SEQ ID NO: 56), PMX010 mAb (SEQ ID NO: 57), PMX011 mAb (SEQ ID NO: 58), and the FR1 sequences of PMX003 (SEQ ID NO: 59), PMX006 mAb (SEQ ID NO: 60), PMX007 mAb (SEQ ID NO: 61), PMX008 mAb (SEQ ID NO: 62), PMX009 mAb (SEQ ID NO: 63), PMX010 mAb (SEQ ID NO: 64), PMX011 mAb (SEQ ID NO: 65) are listed in the Sequence Listing.

[0295] Framework region 1 (FR1) of the light chain of PMX066 (sequence number 81), PMX0067 (sequence number 91), PMX068 (sequence number 101), PMX0069 (sequence number 111), PMX071 (sequence number 131), PMX072 (sequence number 141), PMX073 (sequence number 151), PMX074 (sequence number 161), PMX076 (sequence number 181), PMX077 (sequence number 191), PMX078 (sequence number 201), PMX079 (sequence number 211), and PMX081 (sequence number 231) contains an NVT sequon. To remove glycans attached to this site, PMX066, PMX0067, PMX068, PMX0069, PMX071, PMX072, PMX073, PMX074, PMX076, PMX077, PMX078, PMX079, and PMX081 The NVT sequon in FR1 of the mAb was mutated to SVT to generate the resulting light chain variable region amino acid sequences PMX112 (SEQ ID NO: 238), PMX113 (SEQ ID NO: 239), PMX114 (SEQ ID NO: 240), PMX115 (SEQ ID NO: 241), PMX116 (SEQ ID NO: 242), PMX117 (SEQ ID NO: 243), PMX118 (SEQ ID NO: 244), PMX119 (SEQ ID NO: 245), PMX120 (SEQ ID NO: 246), PMX121 (SEQ ID NO: 247), PMX122 (SEQ ID NO: 248), PMX123 (SEQ ID NO: 249), and PMX124 (SEQ ID NO: 250), respectively. The sequences are listed in Table 2. Some antibodies contain naturally occurring sequence motifs that do not result in glycans being attached to the antibody. For example, PMX070 and PMX075 contain the sequence motif SVT in FR1 of the light chain, and PMX080 contains the sequence motif TVT in FR1 of the light chain.

[0296] Example 5: Generation of monoclonal antibodies from single cells The heavy and light chain V(D)J sequences of the selected candidate clones were synthesized and cloned into expression vectors containing the genomic sequences of the canine IgG constant region and the canine IGK or IGL constant region, respectively. For example, the PMX001 to PMX005 variable sequences were cloned into vectors encoding the canine IGGB (SEQ ID NO: 66) and canine IGLC5 constant regions (SEQ ID NO: 67). The expression vectors encoding the heavy and light chains were co-transfected into a suitable mammalian cell line, such as CHO cells, to obtain stable expression. Thus, a full-length canine antibody was generated.

[0297] For example, codon optimization can be performed on the nucleotide sequences of the heavy chain variable region (SEQ ID NO: 68) and light chain variable region (SEQ ID NO: 69) of PMX003 mAb to facilitate expression in CHO cells. The Campath leader (SEQ ID NO: 70) (see US Pat. No. 8,362,208 B2) can be introduced to replace the native leaders of PMX001-PMX005 mAbs (SEQ ID NO: 71 for the heavy chain and SEQ ID NO: 72 for the light chain).

[0298] Monoclonal antibodies 1E4 and 4E1-7 were also expressed. The 1E4 nucleotide sequence was obtained from WO2013 / 063186. The 4E1-7 amino acid sequence was obtained from Mizuno et al. Scientific Reports, 10, Article 11476 (2020) (https: / / doi.org / 10.1038 / s41598-020-68470-9). The variable regions of both antibodies were cloned into vectors encoding the canine IGGB (SEQ ID NO: 66) and canine IGK (SEQ ID NO: 73) constant regions. A rituximab-cIGGB control mAb was generated by synthesizing the variable region sequences (see US Pat. No. 5,736,137) and cloning them into vectors encoding the canine IGGB (SEQ ID NO: 66) and canine IGLC5 (SEQ ID NO: 67) constant regions.

[0299] Core fucose on N-linked glycans attached to the Fc portion of an antibody may affect the binding of Fc receptors to the Fc region of the antibody, thus reducing ADCC activity. To improve ADCC killing activity, Fut8 knockout (KO) CHO cells were generated in a pooled format by deleting exon 2 of the Fut8-encoding gene, as described in Yamane-Ohnuki et al., Biotechnol Bioeng. 2004 Sep 5;87(5):614-22. https: / / pubmed.ncbi.nlm.nih.gov / 15352059 / . KO cells were phenotypically selected using LCA staining.

[0300] For antibody production, 6 x 10 6 Selected CHO cells or Fut8 knockout CHO cells were seeded in 3 ml of culture medium and incubated at 32°C, 8% CO2 with shaking at 200 rpm. 4% HyClone Cell Boost 7a supplement + 0.4% HyClone Cell Boost 7b supplement + 1% glucose was added to the medium on days 1, 4, 7, and 10. Culture supernatants were collected on day 12, and IgG concentrations were determined using surface plasmon resonance (Biacore 8K, Cytiva Life Sciences).

[0301] Example 6: Binding assay Flow cytometry-based assays: HEK293 cells or MDCK II cells were stably transfected with a vector encoding the full-length canine CD20 cDNA. Antibody binding to these cells was assessed using flow cytometry. Briefly, 1-2 × 10 5Canine CD20-expressing cells were incubated with candidate mAbs at a fixed concentration of 1 μg / ml or 10 μg / ml for binding assays and at a range of concentrations (12-point 1:2 serial dilutions starting at 30 μg / ml, i.e., 200 nM) for affinity determinations for 1 hour at +4°C, followed by incubation with 5 μg / ml FITC-conjugated anti-canine IgG secondary antibody (Bethyl Laboratories) for 1 hour at +4°C. Cells incubated with anti-canine IgG FITC secondary antibody but without primary anti-canine CD20 antibody or with isotype control primary antibody served as negative controls. Data were acquired on either a Beckman Coulter CytoFLEX or BD Accuri C6 Plus flow cytometer and analyzed using FlowJo software. For affinity determination, stained cells were washed and fixed with 1% paraformaldehyde / 3% FBS / PBS for 15 minutes after incubation with candidate antibodies, and then fixed again 24 hours after incubation with secondary antibodies before data acquisition on a flow cytometer. Graphs using the mean fluorescence intensity (MFI) values ​​of the FITC channel versus antibody concentration were plotted in GraphPad Prism (Figure 3D). The apparent affinity (Kd) of candidate antibodies for cell-surface canine CD20 was determined as the concentration that stained 50% of CD20-expressing cells, i.e., the EC50 of binding, using the equation for log (agonist) versus response-variable slope (four parameters).

[0302] The results of the binding assay are shown in Figures 3A-C, which show that the PMX antibodies had stronger binding ability to canine CD20 than the test antibodies 1E4 and 4E1-7 at a single concentration point (10 μg / ml), except for PMX002, which was equivalent to the 4E1-7 antibody.

[0303] ELISA-based assays: The large loop of the extracellular domain (ECD) of the canine CD20 protein described below is expressed in CHO cells and secreted into the extracellular medium under the control of the CAG promoter before being purified using a protein A column.

[0304] CD20 ECD protein is coated onto assay plates. Antibodies are added at a range of concentrations and binding capacity is determined using ELISA.

[0305] SPR-based assays: Affinity (K d ) can be measured by SPR using recombinant mouse Fc-tagged CD20 extracellular domain.

[0306] The recombinant extracellular domain (ECD) (large loop) of CD20, designated dCD20LL-Fc (see WO2013 / 063186, page 37, SEQ ID NO: 62), is expressed in a secreted form with a mouse IgG2a Fc tag from stable CHO cells and purified from the clarified supernatant using protein A chromatography. The apparent K corresponds to the bivalent avidity of the interaction. d is derived by amine coupling 100-150 RU of dCD20LL-Fc onto flow cell 2 of a CM5 chip with NHS / EDC activation / deactivation added to flow cell 1. Three-fold dilutions of candidate IgG from 200 nM to 0.1 nM are made in HBS-EP+ buffer and injected over the chip surface at 30 μL / min for 180 seconds. Dissociation is monitored for 600 seconds at 30 μL / min, after which the surface is regenerated with a 60-second pulse of 10 mM glycine-HCl pH 2.0. A monovalent K corresponding to monovalent affinity is used. dis derived by repeating the above protocol using candidate antibody Fab fragments generated by papain (or SpeB) digestion of full-length IgG. Experiments are performed in HBS-EP+ running buffer at 25°C. All SPR experiments were performed on a Biacore 8K instrument, and the data were analyzed by fitting the data to a 1:1 binding model using Biacore Insight Evaluation software.

[0307] Example 7: Functional assays Complement-dependent cytotoxicity (CDC) activity The CLBL-1 canine lymphoma tumor cell line (University of Veterinary Medicine, Vienna), which naturally expresses canine CD20, was used as the target cell line for the CDC assay. 10,000 CLBL-1 cells per well of a 96-well plate (white with clear bottom) were incubated with anti-canine CD20 antibody and a titration of canine complement reserve serum (BioIVT) (11-point 1:3 dilutions, starting at 10 μg / ml) at a final dilution of 1:4 for 2 hours at 37°C and 5% CO2. The assay was set up using medium (RPMI + 1% L-glutamine + 20% fetal bovine serum) made with heat-inactivated serum, so that the canine complement reserve serum was the sole source of complement. Rituximab-cIGGB chimeric antibody was used as a negative isotype control.

[0308] Viable cells were then quantified using the CellTitre-Glo® Luminescent Cell Viability Assay (Promega) according to the assay protocol. This assay uses the ATP content of viable cells as an indicator of cell viability. Luminescence was measured on a CLARIOstar (BMG Labtech). Data was analyzed using MARS software (BMG Labtech), and the number of remaining viable cells was used to calculate the percentage of killing in the presence of antibody using Microsoft Excel, using wells without antibody as a baseline. Graphs were plotted in GraphPad Prism.

[0309] Figure 4 shows that CDC activity was observed for PMX003 in both its native and afucosylated forms. Under the same experimental setting using canine complement-preserved serum, no CDC activity was observed for the native and afucosylated forms of the 1E4 and 4E1-7 test antibodies. Table 4 summarizes the CDC activity for antibodies PMX066-PMX081.

[0310] Antibody-dependent cytotoxicity (ADCC) activity Canine cell lines, such as the MDCK II cell line (ATCC), were stably transfected with a construct encoding canine CD20 protein and a construct expressing a fluorescent protein (e.g., GFP). Either an MDCK II cell line expressing the fluorescent protein but not the antigen, or an isotype control antibody, was used as a negative control for the experiment.

[0311] Canine peripheral blood mononuclear cells (PBMCs, Envigo) were used as a source of effector cells. PBMCs were isolated from freshly collected whole blood using Ficoll-Paque plus (Cytiva, GE17-1440-02) density gradient centrifugation with heparin sodium anticoagulant according to the recommended protocol. PBMCs were resuspended in medium (RPMI + 10% heat-inactivated fetal bovine serum + 1% penicillin-streptomycin + 1% non-essential amino acids + 1% L-glutamine + 1% sodium pyruvate + 2% HEPES) supplemented with 50 ng / ml recombinant canine IL-2 (R&D systems) and incubated at 37°C for 24 hours before use in the ADCC assay.

[0312] To assess ADCC activity, 10,000 MDCK II cells were co-cultured with PBMCs in a 1:1 mixture of MDCK II medium (DMEM + 1% L-glutamine + 10% fetal bovine serum) and PBMC medium at a 35:1 effector:target ratio and antibody titration (11-point 1:3 dilutions, starting with 10 μg / ml antibody) for 24 h at 37°C. Rituximab-cIGGB was used as a negative (isotype) control antibody.

[0313] The GFP signal, which is proportional to the number of viable cells per well, was used as a measure of the number of viable cells remaining in the well at the end of the 24-hour incubation. The GFP signal was measured on a CLARIOstar (BMG Labtech). Data were analyzed using MARS software (BMG Labtech), and the percentage of killing in the presence of antibody was calculated using Microsoft Excel, using wells without antibody as a baseline. Graphs were plotted in GraphPad Prism.

[0314] Figure 5 shows the ADCC activity observed in the presence of PMX antibodies. The PMX003 antibody, in both its native and afucosylated forms, exhibits stronger ADCC activity than the tested antibodies 1E4 and 4E1-7. Table 4 summarizes the ADCC activity observed for antibodies PMX066-PMX081.

[0315] Antibody-dependent cellular phagocytosis (ADCP) activity A suitable method for measuring ADCP activity is described in Ito et al. Leuk Lymphoma. 2015 January;56(1):219-225.

[0316] Direct killing activity / apoptosis assay Direct killing can be measured as described in Mizuno et al. Scientific Reports, 10, Article 11476 (2020).

[0317] Example 8: Ex vivo whole blood assay To evaluate the efficiency of B cell depletion in an ex vivo canine model, 100 μl of freshly drawn canine whole blood was collected with heparin and diluted with 200 μl of RPMI 1640 + 10% heat-inactivated fetal bovine serum + 1% penicillin / streptomycin / L-glutamine + 1% non-essential amino acids + 1% sodium pyruvate + 2% HEPES buffer. The diluted blood was incubated with or without 10 μg / ml anti-canine CD20 antibody at 37°C for 2, 24, and 96 hours. After incubation, the samples were incubated with Versalyse solution (Beckman Coulter) at room temperature for 15 minutes to lyse red blood cells (RBCs). After washing, samples were stained with anti-canine CD21 and anti-canine CD8 antibodies (both from BioRad), and the numbers of CD21+ B cells and CD8+ T cells were analyzed on a CytoFLEX Flow Cytometer (Beckman Coulter). The percentage of B cell depletion was calculated based on the reduction in the ratio of CD21+ B cells to CD8+ T cells compared to the no-antibody control. The results in Figures 6A-B show that PMX003 mAb resulted in more efficient killing of B cells in whole dog blood compared to 1E4 and 4E1-7 mAbs. Table 4 summarizes the ex vivo specific B cell depletion activity observed for antibodies PMX066-PMX081.

[0318] Example 9: In vivo assay Tumor mouse model To evaluate the in vivo tumor-killing efficacy of anti-canine CD20 antibodies, a syngeneic tumor mouse model was generated. The genomic sequence of mouse 129Sv ES cells was modified by homologous recombineering to replace the mouse CD20 coding region with the canine CD20 coding region. The targeting vector contained the genomic DNA sequence of the canine CD20 coding region flanked on both sides by 4-4.5 kb of mouse genomic sequence. After microinjection and germline transmission, the mice thus generated were bred with Balb / c mice to produce mCD20 / dCD20 heterozygous offspring on a mixed genetic background.

[0319] The mouse lymphoma cell line A20 on a Balb / c genetic background was also modified to express canine CD20: the mouse CD20 coding region was replaced with the canine CD20 coding sequence by transfecting the A20 cell line with a linearized ES cell targeting vector.

[0320] To evaluate mouse tumor killing efficiency, 1 million canine CD20 knock-in A20 cells (in 100 μl of PBS) were subcutaneously implanted into mCD20 / dCD20 heterozygous mice (6-8 weeks old). Anti-canine CD20-mIGG2a chimeric antibody (150 μg in 500 μl of PBS) or PBS alone was injected intraperitoneally once on day 0. Tumor size was measured daily, and mice were euthanized when they reached a humane endpoint.

[0321] In vivo dog studies In dog study 1, 12 healthy male beagles (3 dogs per test group), aged 2-3 years and weighing 8-11 kg, were housed in Avogadro LS, France. Prior to the study, all of them were verified to be free of disease or other clinical abnormalities. The native format of the PMX003 antibody was produced in CHO cells and administered to two test groups: one group received 0.5 mg / kg antibody and the other group received 2.5 mg / kg antibody. An afucosylated version of the PMX003 antibody was produced by expression in Fut8 KO CHO cells and administered to one test group at 0.5 mg / kg. The control group received the isotype control, rituximab-cIGGB, at 2.5 mg / kg. All antibodies were administered on day 0 as a single intravenous infusion over 30 minutes. The percentage of CD21+ B cells was monitored and analyzed on the following days after antibody administration: Day 0 (pre-dose), Day 1, Day 2, Day 5, Day 7, Day 15, Day 27, and Day 43. 0.5 ml of freshly drawn blood using K3EDTA anticoagulant was fixed with 1 ml of 1x RBC lysis / fixation solution (BioLegend) for 30 minutes at room temperature in the dark and then diluted with 18 ml of 1x PBS. Fixed and diluted blood samples were kept at +4°C throughout storage and transport to the UK. Sample analysis was performed within 48 hours of blood collection. For each sample, cells were pelleted at 400 g for 5 minutes, the cell pellet was resuspended in 0.5 ml of buffer, and 50 μl of cells was used for staining each technical replicate. For each replicate, cells were stained with AF647-conjugated anti-canine CD21 antibody (BioRad, 1:10) and PE-conjugated anti-canine CD8 antibody (BioRad, 1:50). Data were acquired using a Beckman Coulter CytoFLEX by thresholding on DAPI to exclude debris and non-nucleated cells. FlowJo software was used to analyze the percentages of CD21+ B cells and CD8+ T cells in the lymphocyte population. B cell depletion was determined by comparing the percentage of CD21+ B cells on the day of evaluation after antibody administration with the percentage on day 0 (baseline) for each dog. The percentage of CD8+ T cells was also assessed and served as an internal control.Figures 7A-B show the results of B cell depletion in healthy beagles administered PMX and control antibodies. Depletion was observed when PMX003 was administered in its native and afucosylated forms. The afucosylated form was more effective than the native form. PMX003 demonstrated B cell killing at doses much lower than those observed in canine studies using 1E4 (Rue et al. Veterinary Immunology and Immunopathology 164 (2015), 148-159). In addition, B cell depletion was maintained at a low level for at least 15 days, longer than that observed in canine studies with 4E1-7 (Mizuno et al.).

[0322] In Canine Study 2, 15 healthy male beagles (3 dogs per test group), aged 2-3 years and weighing 8-11 kg, were housed in Avogadro LS, France. Prior to the study, all of them were verified to be free of disease or other clinical abnormalities. The native formats of the PMX070 and PMX115 antibodies were produced in CHO cells and administered to two test groups per antibody: one group received 0.5 mg / kg antibody and the other group received 2 mg / kg antibody. The control group received the isotype control, rituximab-cIGGB, at 2 mg / kg. All antibodies were administered on Day 0 as a single intravenous infusion over 30 minutes. The percentage of CD21+ B cells was monitored and analyzed on the following days after antibody administration: Day 0 (pre-dose), Day 1, Day 4, Day 7, Day 14, Day 21, and Day 28. One milliliter of freshly collected blood was fixed with K3EDTA anticoagulant in 2 ml of 1x RBC lysis / fixation solution (BioLegend) for 30 minutes at room temperature in the dark and then diluted with 12 ml of 1x PBS. Fixed and diluted blood samples were kept at +4°C throughout storage and transport to the UK. Sample analysis was performed 48-72 hours after blood collection. For each sample, cells were pelleted at 400 g for 5 minutes, the cell pellet was resuspended in 1 ml of buffer, and 50 μl of cells were used for staining each technical replicate. For each replicate, cells were stained with AF647-conjugated anti-canine CD21 antibody (BioRad, 1:10) and PE-conjugated anti-canine CD8 antibody (BioRad, 1:50). Data were acquired using a Beckman Coulter CytoFLEX by thresholding on DAPI to exclude debris and non-nucleated cells. FlowJo software was used to analyze the percentages of CD21+ B cells and CD8+ T cells in the lymphocyte population. B cell depletion was determined by comparing the percentage of CD21+ B cells on the day of evaluation after antibody administration with the percentage on day 0 (baseline) for each dog. The percentage of CD8+ T cells was also assessed and served as an internal control. Figure 7C shows the results of B cell depletion in healthy beagles administered PMX and control antibodies.Depletion was observed for PMX070 and PMX115 at doses of 0.5 mg / kg and 2 mg / kg, comparable to the depletion observed with the afucosylated form of PMX003.

[0323] For pharmacokinetic analysis, plasma concentrations of mAb drug were monitored and analyzed at the following times after antibody administration: pre-dose, 2 hours, 6 hours, 24 hours, 48 ​​hours, 72 hours, 120 hours, 168 hours, 360 hours, Day 27, and Day 43 in Dog Study 1, and pre-dose, 2 hours, 6 hours, 24 hours, Day 4, Day 7, Day 14, Day 21, and Day 28 in Dog Study 2. Immediately after blood collection, fresh blood with lithium heparin anticoagulant was placed on ice. Blood tubes were centrifuged at 2500 g at +5°C for 10 minutes. Plasma was collected into polypropylene tubes and kept on ice until stored at -20°C. Plasma was frozen until analysis by ELISA. The concentration of mAb is assessed by ELISA using an anti-idiotypic polyclonal antibody against PMX mAb as the capture antibody and anti-dog Fc-HRP (Sigma) or anti-dog Fc-biotin (Sigma) + streptavidin-HRP (BioLegend) as the detection antibody. Anti-idiotypic polyclonal or monoclonal antibodies against PMX mAb are generated in GenScript by immunizing rabbits with PMX Fab fragments, generating hybridoma cell lines, and selecting for anti-idiotypic antibodies that bind to PMX mAb, followed by removal of any cross-reactive antibodies that bind to an isotype control antibody.

[0324] For immunogenicity assessment, the concentration of anti-drug antibodies (ADAs) in frozen plasma is assessed by ELISA after acid dissociation of any preformed complexes between the candidate PMX antibody and the anti-drug antibody. Chimeric versions of the candidate antibody, such as PMX-mouse Fc or PMX-human Fc, are generated and fractions are labeled with biotin. The presence and concentration of anti-drug antibodies are determined using a sandwich ELISA assay format, using unlabeled chimeric antibodies as capture reagents and biotin-labeled chimeric antibodies with streptavidin-HRP for detection. The anti-idiotypic polyclonal antibodies generated for the pharmacokinetic assays described above are used as positive controls to generate standard curves.

[0325] Example 10: Epitope mapping Epitope mapping was performed to identify the site on the canine CD20 antigen to which the antibody specifically binds. Using site-directed mutagenesis, we divided the extracellular domain of canine CD20 into nine regions and generated a canine-human chimeric CD20 expression vector by replacing each region in the canine sequence with the equivalent position in the human sequence ( FIG. 8A ). Using the same strategy, we generated a canine-mouse chimeric CD20 ( FIG. 8B ). The mutated full-length cDNA was expressed in MDCK cells as described in Example 2. By comparing the binding of PMX003, PMX115, and PMX070 to these chimeric mutants with that of wild-type CD20, we identified the sequence ITISHFFKMENLNLIKAPM (SEQ ID NO: 303) as the epitope for PMX003, PMX115, and PMX070, because mutation at this position resulted in reduced binding in flow cytometry analysis ( FIG. 9 ). "ENLNLIKAPM" (SEQ ID NO: 303, amino acids 150-159 in SEQ ID NO: 2) was identified as a core epitope within the sequence. This epitope is distinct from "DIHNCD," which was described as the epitope of the benchmark 1E4 antibody. Another strategy for investigating epitopes is by alanine scanning, in which single alanine mutations are generated at every residue in the small loop and every other residue in the large loop (Figure 8C).

[0326] Example 11 Development feasibility criteria Antibody characterization encompasses several aspects, including: 1) transient expression levels during small-scale antibody production, which correlate with the final stable expression level, which impacts COGS (cost of goods sold). 2) thermal stability, which correlates with several properties, including the tendency to aggregate and compatibility with higher-concentration formulations. Thermal stability can be reflected by the Tm1 value, the temperature at which 50% of the protein is unfolded, and Tagg, the aggregation temperature. The Tm1 and Tagg values ​​of lead antibodies were measured using the UNcle protein stability screening platform (Unchained Labs). 3) size and purity. Fragmentation and aggregation can lead to issues with in vivo efficacy or immunogenicity. Antibody size was determined on non-reducing and reducing SDS-PAGE gels. HPLC-SEC showed that each candidate product was composed of more than 95% monomer. Particle diameter was detected using the UNcle machine, which can detect potential aggregation issues. The developability parameters for the PMX003, PMX066, PMX067, PMX069, PMX070, PMX071, PMX072, PMX073, PMX074, PMX075, PMX076, PMX077, PMX078, PMX079, PMX080, PMX081, PMX112, PMX115, PMX122, and PMX124 antibodies are shown in Table 5.

[0327] Example 12 Analysis of deglycosylated antibodies In addition to the above, in silico sequence analysis was used to predict sequence liability. An N-linked glycosylation motif (NVT) was identified in framework region 1 (FR1) of the light chain of PMX003 mAb and in 12 other mAbs from the lead panel (excluding PMX070, PMX075, and PMX080). To prevent glycosylation at this position and thereby reduce the liability, the NVT sequon in the light chain FR1 of PMX003 was mutated to QVT (PMX006), AVT (PMX007), EVT (PMX008), NVA (PMX009), SVT (PMX010), and TVT (PMX011). Deglycosylation was monitored by comparing the size of the light chain of the mutant antibodies with that of the original PMX003 mAb on a reducing SDS-PAGE gel. Following this, binding and functional evaluations were performed to assess the impact of these mutations. Flow cytometry analysis revealed that the binding ability to canine CD20-expressing cells remained unchanged except for the QVT and AVT mutants, which were slightly reduced (Figure 3B). Similarly, the killing potency in in vitro ADCC assays (Figures 5D and 5E) and ex vivo whole blood assays (Figure 6C) was not significantly affected by these mutations. Thus, it is possible to modify the NVT motif for deglycosylation without impairing binding and functional activity. Table 2: Sequences [Table 2-1] [Table 2-2] [Table 2-3] [Table 2-4] [Table 2-5] Table 2-6 Table 2-7 Table 2-8 Table 2-9 Table 2-10 Table 2-11 Table 2-12 Table 2-13 Table 2-14 Table 2-15 Table 2-16 Table 2-17 Table 2-18 Table 2-19 Table 2-20 Table 2-21 Table 2-22 Table 2-23 Table 2-24 Table 2-25 Table 2-26 Table 2-27 Table 2-28 Table 2-29 Table 2-30 Table 2-31 Table 2-32 Table 2-33 Table 2-34 Table 2-35 Table 3 Table 4 Table 5-1 Table 5-2 Table 5-3

Claims

1. An isolated canine antibody, or antigen-binding portion thereof, that binds to canine CD20, My body, a) a HC CDR1 sequence comprising or consisting of SEQ ID NO: 27, or SEQ ID NO: 2 an amino acid sequence having one or two amino acid differences compared to 7, b) a HC CDR2 sequence comprising or consisting of SEQ ID NO: 28, or SEQ ID NO: 2 an amino acid sequence having 1, 2, 3, or 4 amino acid differences compared to c) a HC CDR3 sequence comprising or consisting of SEQ ID NO: 29, or SEQ ID NO: 2 an amino acid sequence having one or two amino acid differences compared to d) an LC CDR1 sequence comprising or consisting of SEQ ID NO: 30, or SEQ ID NO: 3 an amino acid sequence having one amino acid difference compared to zero, e) an LC CDR2 sequence comprising or consisting of SEQ ID NO: 31, or SEQ ID NO: 3 an amino acid sequence having one amino acid difference compared to f) an LC CDR3 sequence comprising or consisting of SEQ ID NO: 32, or SEQ ID NO: 3 an antibody comprising an amino acid sequence having one or two amino acid differences compared to its antigen-binding portion.

2. the antibody, or antigen-binding portion thereof, i) a HC CDR1 sequence comprising or consisting of SEQ ID NO: 7, a HC CDR2 sequence comprising or consisting of SEQ ID NO: 8 HC CDR2 sequence consisting of or comprising SEQ ID NO:9; 3 sequences, a LC CDR1 sequence comprising or consisting of SEQ ID NO: 10, a LC CDR1 sequence comprising SEQ ID NO: 11 LC CDR2 sequence comprising or consisting of SEQ ID NO: 12 LC CDR3 sequence, ii) a HC CDR1 sequence comprising or consisting of SEQ ID NO: 17, HC comprising or consisting of CDR2 sequence, SEQ ID NO: 19 LC comprising CDR3 sequence, SEQ ID NO: 20; CDR1 sequence, SEQ ID NO: 21 or and an LC CDR2 sequence comprising or consisting of SEQ ID NO:

22. R3 sequence, iii) HC CDR1 sequence comprising SEQ ID NO: 37, HC CDR2 comprising SEQ ID NO: 38 HC CDR3 sequence comprising SEQ ID NO:39, LC CDR1 sequence comprising SEQ ID NO:40 , an LC CDR2 sequence comprising SEQ ID NO:41, and an LC CDR3 sequence comprising SEQ ID NO:

42. 、 iv) HC CDR1 sequence comprising or consisting of SEQ ID NO: 47, HC comprising or consisting of CDR2 sequence SEQ ID NO: 49 CDR3 sequence, SEQ ID NO: 50; LC CDR1 sequence, SEQ ID NO: 51; LC CDR2 sequences comprising or consisting of SEQ ID NO: 51, and LC CDR2 sequences comprising or consisting of SEQ ID NO:

52. a LC CDR3 sequence consisting of: v) HC CDR1 sequence comprising or consisting of SEQ ID NO: 82, including SEQ ID NO: 83 HC comprising or consisting of CDR2 sequence SEQ ID NO: 84 LC CDR1 sequence, SEQ ID NO: 8, comprising or consisting of CDR3 sequence, SEQ ID NO: 85 LC CDR2 sequence comprising or consisting of SEQ ID NO: 6, and SEQ ID NO:

87. a LC CDR3 sequence consisting of: vi) HC CDR1 sequence comprising or consisting of SEQ ID NO: 92, HC comprising or consisting of CDR2 sequence SEQ ID NO: 94 CDR3 sequence, SEQ ID NO: 95; LC CDR1 sequence, SEQ ID NO: 96; LC CDR2 sequences comprising or consisting of SEQ ID NO: 96, and LC CDR2 sequences comprising or consisting of SEQ ID NO:

97. a LC CDR3 sequence consisting of: vii) HC CDR1 sequence comprising or consisting of SEQ ID NO: 102, SEQ ID NO: 10 HC CDR2 sequence comprising or consisting of SEQ ID NO: 104; HC CDR3 sequence comprising or consisting of SEQ ID NO: 105; LC CDR1 sequence comprising or consisting of SEQ ID NO:

105. LC CDR2 sequence comprising or consisting of SEQ ID NO: 106, and SEQ ID NO: 107 a LC CDR3 sequence comprising or consisting of: viii) HC CDR1 sequence comprising or consisting of SEQ ID NO: 112, SEQ ID NO: 1 HC CDR2 sequence comprising or consisting of SEQ ID NO: 13, a HC CDR3 sequence comprising or consisting of SEQ ID NO: 115; a LC CDR1 sequence comprising or consisting of SEQ ID NO: 115; LC CDR2 sequences comprising or consisting of SEQ ID NO: 116, and SEQ ID NO: 117 a LC CDR3 sequence comprising or consisting of: ix) HC CDR1 sequence comprising or consisting of SEQ ID NO: 122, SEQ ID NO: 123 HC CDR2 sequence comprising or consisting of SEQ ID NO:

124. a HC CDR3 sequence comprising or consisting of SEQ ID NO: 125; a LC CDR1 sequence comprising or consisting of SEQ ID NO: 125; LC CDR2 sequences comprising or consisting of SEQ ID NO: 126, and a LC CDR3 sequence consisting of or consisting of a centipede; x) a HC CDR1 sequence comprising or consisting of SEQ ID NO: 132, SEQ ID NO: 133 HC CDR2 sequence comprising or consisting of SEQ ID NO: 134 HC CDR3 sequence, LC CDR1 sequence comprising or consisting of SEQ ID NO: 135, sequence LC CDR2 sequences comprising or consisting of sequence number 136, and SEQ ID NO: 137 a LC CDR3 sequence consisting of or comprising: xi) HC CDR1 sequence comprising or consisting of SEQ ID NO: 142, SEQ ID NO: 143 HC CDR2 sequence comprising or consisting of SEQ ID NO: 144 a HC CDR3 sequence comprising or consisting of SEQ ID NO: 145; a LC CDR1 sequence comprising or consisting of SEQ ID NO: 145; LC CDR2 sequences comprising or consisting of SEQ ID NO: 146, and a LC CDR3 sequence consisting of or consisting of a centipede; xii) HC CDR1 sequence comprising or consisting of SEQ ID NO: 152, SEQ ID NO: 15 HC CDR2 sequence comprising or consisting of SEQ ID NO: 154 HC CDR3 sequence comprising or consisting of SEQ ID NO: 155; LC CDR1 sequence comprising or consisting of SEQ ID NO: 155 LC CDR2 sequence comprising or consisting of SEQ ID NO: 156, and SEQ ID NO: 157 a LC CDR3 sequence comprising or consisting of: xii) HC CDR1 sequence comprising or consisting of SEQ ID NO: 162, SEQ ID NO: 16 HC CDR2 sequence comprising or consisting of SEQ ID NO: 164 HC CDR3 sequence comprising or consisting of SEQ ID NO: 165; LC CDR1 sequence comprising or consisting of SEQ ID NO:

165. LC CDR2 sequence comprising or consisting of SEQ ID NO: 166, and SEQ ID NO: 167 a LC CDR3 sequence comprising or consisting of: xiii) HC CDR1 sequence comprising or consisting of SEQ ID NO: 172, SEQ ID NO: 1 HC CDR2 sequence comprising or consisting of SEQ ID NO: 173, HC CDR3 sequence comprising or consisting of SEQ ID NO: 175; LC CDR1 sequence comprising or consisting of SEQ ID NO: 175; LC CDR2 sequences comprising or consisting of SEQ ID NO: 176, and SEQ ID NO: 177 a LC CDR3 sequence comprising or consisting of: xiv) HC CDR1 sequence comprising or consisting of SEQ ID NO: 182, SEQ ID NO: 18 HC CDR2 sequence comprising or consisting of SEQ ID NO: 184 HC CDR3 sequence comprising or consisting of SEQ ID NO: 185; LC CDR1 sequence comprising or consisting of SEQ ID NO:

185. LC CDR2 sequence comprising or consisting of SEQ ID NO: 186, and SEQ ID NO: 187 a LC CDR3 sequence comprising or consisting of: xv) HC CDR1 sequence comprising or consisting of SEQ ID NO: 192, SEQ ID NO: 193 HC CDR2 sequence comprising or consisting of SEQ ID NO:

194. a HC CDR3 sequence comprising or consisting of SEQ ID NO: 195; a LC CDR1 sequence comprising or consisting of SEQ ID NO: 195; LC CDR2 sequences comprising or consisting of SEQ ID NO: 196, and a LC CDR3 sequence consisting of or consisting of a centipede; xvi) HC CDR1 sequence comprising or consisting of SEQ ID NO: 202, SEQ ID NO: 20 HC CDR2 sequence comprising or consisting of SEQ ID NO: 3, comprising or consisting of SEQ ID NO: 204 HC CDR3 sequence comprising or consisting of SEQ ID NO: 205; LC CDR1 sequence comprising or consisting of SEQ ID NO:

205. LC CDR2 sequence comprising or consisting of SEQ ID NO:206, and SEQ ID NO:207 a LC CDR3 sequence comprising or consisting of: xvii) HC CDR1 sequence comprising or consisting of SEQ ID NO: 212, SEQ ID NO: 2 HC CDR2 sequence comprising or consisting of SEQ ID NO: 13, HC CDR3 sequence comprising or consisting of SEQ ID NO:215; LC CDR1 sequence comprising or consisting of SEQ ID NO:215; LC CDR2 sequences comprising or consisting of SEQ ID NO:216, and SEQ ID NO:217 a LC CDR3 sequence comprising or consisting of: xviii) HC CDR1 sequence comprising or consisting of SEQ ID NO: 222, SEQ ID NO: HC CDR2 sequence comprising or consisting of SEQ ID NO: 223, HC CDR3 sequence consisting of: LC CDR2 sequence comprising or consisting of the sequence SEQ ID NO:226, and SEQ ID NO:22 LC CDR3 sequence comprising or consisting of: xix) HC CDR1 sequence comprising or consisting of SEQ ID NO: 232, SEQ ID NO: 23 HC CDR2 sequence comprising or consisting of SEQ ID NO: 3, comprising or consisting of SEQ ID NO: 234 HC CDR3 sequence comprising or consisting of SEQ ID NO: 235; LC CDR1 sequence comprising or consisting of SEQ ID NO:

235. LC CDR2 sequence comprising or consisting of SEQ ID NO:236, and SEQ ID NO:237 2. The antibody of claim 1, or an antibody thereof, having a LC CDR3 sequence comprising or consisting of: Original connective part.

3. the antibody, or antigen-binding portion thereof, comprises a HC variable region sequence comprising SEQ ID NO:24, or a sequence having at least 75%, 80%, 85%, or 90% sequence identity to and an LC variable region sequence comprising SEQ ID NO: 26, or at least 70%, 75% thereof , 80%, 85%, 90%, or 95% sequence identity, e.g., SEQ ID NO: LC variable region sequences comprising sequences of SEQ ID NOs. 53, 54, 55, 56, 57, or 58. The antibody or antigen-binding portion thereof of claim 1 .

4. the antibody, or antigen-binding portion thereof, a) an HC variable region sequence comprising SEQ ID NO:4, and an LC variable region sequence comprising SEQ ID NO:6; b) an HC variable region sequence comprising SEQ ID NO: 14, and an LC variable region sequence comprising SEQ ID NO: 16 、 c) an HC variable region sequence comprising SEQ ID NO: 34, and an LC variable region sequence comprising SEQ ID NO:

36. 、 d) an HC variable region sequence comprising SEQ ID NO: 44, and an LC variable region sequence comprising SEQ ID NO: 46 、 e) a HC variable region sequence comprising SEQ ID NO: 79, and a LC variable region sequence comprising SEQ ID NO:

81. or an LC variable region sequence comprising SEQ ID NO: 238; f) a HC variable region sequence comprising SEQ ID NO: 89, and a LC variable region sequence comprising SEQ ID NO:

91. or an LC variable region sequence comprising SEQ ID NO: 239; g) an HC variable region sequence comprising SEQ ID NO: 99, and an LC variable region sequence comprising SEQ ID NO: 101; or an LC variable region sequence comprising SEQ ID NO: 240; h) a HC variable region sequence comprising SEQ ID NO: 109, and a LC variable region sequence comprising SEQ ID NO:

111. or an LC variable region sequence comprising SEQ ID NO: 241; i) a HC variable region sequence comprising SEQ ID NO: 119, and a LC variable region sequence comprising SEQ ID NO: 121 array, j) a HC variable region sequence comprising SEQ ID NO: 129, and a LC variable region sequence comprising SEQ ID NO:

131. or an LC variable region sequence comprising SEQ ID NO: 242; k) a HC variable region sequence comprising SEQ ID NO: 139, and a LC variable region sequence comprising SEQ ID NO:

141. or an LC variable region sequence comprising SEQ ID NO: 243; l) a HC variable region sequence comprising SEQ ID NO: 149, and a LC variable region sequence comprising SEQ ID NO:

151. or an LC variable region sequence comprising SEQ ID NO: 244; m) a HC variable region sequence comprising SEQ ID NO: 159, and a LC variable region sequence comprising SEQ ID NO:

161. or an LC variable region sequence comprising SEQ ID NO: 245; n) a HC variable region sequence comprising SEQ ID NO: 169, and a LC variable region sequence comprising SEQ ID NO:

171. array, o) a HC variable region sequence comprising SEQ ID NO: 179, and a LC variable region sequence comprising SEQ ID NO:

181. or an LC variable region sequence comprising SEQ ID NO: 246; p) a HC variable region sequence comprising SEQ ID NO: 189, and a LC variable region sequence comprising SEQ ID NO:

191. or an LC variable region sequence comprising SEQ ID NO: 247; q) a HC variable region sequence comprising SEQ ID NO: 199, and a LC variable region sequence comprising SEQ ID NO:

201. LC variable region sequence comprising sequence SEQ ID NO: 248; r) a HC variable region sequence comprising SEQ ID NO: 209, and a LC variable region sequence comprising SEQ ID NO:

211. LC variable region sequence comprising sequence SEQ ID NO:249; s) a HC variable region sequence comprising SEQ ID NO: 219, and a LC variable region sequence comprising SEQ ID NO: 221 an array, or t) a HC variable region sequence comprising SEQ ID NO: 229, and a LC variable region sequence comprising SEQ ID NO:

231. The antibody of claim 3 or its derivatives, having an LC variable region sequence comprising SEQ ID NO:

250. Antigen-binding portion.

5. The antigen-binding portion thereof is an scFv, Fv, heavy chain, or single domain antibody. The antibody or antigen-binding portion thereof of claim 1 .

6. A single antibody that binds to canine CD20 and competes with the antibody or antigen-binding portion thereof of the preceding claims. Isolated canine antibodies or antigen-binding portions thereof.

7. The antibody or antigen-binding portion thereof is conjugated to a therapeutic moiety. The antibody or antigen-binding portion thereof.

8. 8. The antibody or antibody of claim 7, wherein the therapeutic moiety is a second antibody or an antigen-binding portion thereof. its antigen-binding portion.

9. The antibody of claim 8 , wherein the second antibody or antigen-binding portion thereof binds to a different target. The body or antigen-binding portion thereof.

10. The antibody or antigen-binding portion thereof may be modified with a half-life extending moiety, a label, a cytotoxin, a liposome, a nanoparticle, a The compound of the preceding claim is conjugated to a further moiety selected from a nanoparticle or a radioisotope.

2. The antibody or antigen-binding portion thereof according to claim 1.

11. The antibody of the preceding claim, wherein the antibody or antigen-binding portion thereof is afucosylated. The body or antigen-binding portion thereof.

12. A pharmaceutical composition comprising an antibody or antigen-binding portion thereof according to the preceding claims.

13. An antibody or antibody according to any one of claims 1 to 11 for use in the treatment of a disease. or an antigen-binding portion thereof, or the pharmaceutical composition of claim 12.

14. Methods for treating B-cell mediated conditions in canine subjects in need thereof A method for treating a patient with an inflammatory bowel disease, comprising administering an effective amount of an antibody or its antigen-binding fragment according to any one of claims 1 to 11.

13. A method comprising administering a pharmaceutical composition according to claim 12.

15. The disease is a disease mediated by B cells, such as a B cell lymphoma, leukemia, or The antibody or antigen-binding portion thereof or medicament according to claim 13, which is for an immune-mediated disease. The composition or method of claim 14.

16. 16. The method of claim 13 or 15, further comprising separately administering to the subject another therapeutic agent. The antibody or antigen-binding portion thereof or pharmaceutical composition according to claim 14 or 15. The method described below.

17. The therapeutic agent may be a cytotoxic or radiotoxic agent, an immunosuppressant or immunomodulatory agent, e.g., a sarcopenic acid The antibody or antigen-binding portion thereof according to claim 16, which is a cytokine or a chemokine. or a pharmaceutical composition, or the method according to claim 16.

18. A gene encoding the antibody or antibody antigen-binding portion thereof according to any one of claims 1 to 11. Nucleic acid sequence.

19. SEQ ID NOs: 3, 5, 13, 15, 23, 25, 33, 35, 43, 45, 78, 80, 8 ８、９０、９８、１００、１０８、１１０、１１８、１２０、１２８、１３０、１３８、 １４０、１４８、１５０、１５８、１６０、１６８、１７０、１７８、１８０、１８８、 １９０、１９８、２００、２０８、２１０、２１８、２２０、２２８、２３０、２５１、 ２５２、２５３、２５４、２５５、２５６、２５７、２５８、２５９、２６０、２６１、 ２６２、２６３、２６４、２６５、２６６、２６７、２６８、２６９、２７０、２７１、 ２７２、２７３、２７４、２７５、２７６、２７７、２７８、２７９、２８０、２８１、 ２８２、２８３、２８４、２８５、２８６、２８７、２８８、２８９、２９０、２９１、 292, 293, 294, 295, 296, 297, 298, 299, 300, or 30 19. The nucleic acid sequence of claim 18, comprising a sequence selected from:

20. A vector comprising the nucleic acid sequence of claim 18 or 19.

21. 21. Comprising a nucleic acid sequence according to claim 18 or 19, or a vector according to claim 20. host cell.

22. The antibody or antigen-binding portion thereof according to any one of claims 1 to 11, or claim 1 A kit comprising the pharmaceutical composition according to claim 2.

23. 23. The method of claim 22, further comprising a reagent for detecting the antibody or antigen-binding portion thereof. tt.

24. 22. A method for producing a canine antibody that binds to CD20, comprising the steps of: culturing the host cells and recovering the antibody.

25. 1. A method for producing a canine antibody that binds to CD20, comprising: a) a transgene expressing a nucleic acid construct comprising a canine heavy chain V gene and a canine light chain V gene; immunizing the nicked mice with a CD20 antigen; b) generating a library of antibodies from said mice; c) isolating antibodies from said library.

26. CD20 protein or the extracellular domain of CD20 protein in biological samples from canine subjects A method for detecting an enzyme comprising: subjecting a biological sample to a method for detecting an enzyme according to any one of claims 1 to 11. wherein said antibody or antigen-binding portion thereof is , linked to a detectable label.

27. 3. The method of claim 2, wherein the biological sample is a biopsy, tissue, blood, serum, plasma, or lymph sample.

6. The method according to claim 6.

28. A method for inhibiting tumor growth or metastasis, comprising treating tumor cells with an effective amount of any one of claims 1 to 11. The antibody or antigen-binding portion thereof according to any one of claims 1 to 12, or the pharmaceutical composition according to claim 12. The method of claim 1, wherein the soluble component is a soluble component.

29. A method of killing tumor cells that express CD20, comprising: The cells are treated with an antibody or antibody according to any one of claims 1 to 11 so that killing occurs.

13. A method comprising contacting with the pharmaceutical composition of claim 12.

30. 29. The method of claim 28, wherein the tumor cells are canine tumor cells.

31. 1. An antibody, or antigen-binding portion thereof, wherein the epitope is selected from the following amino acid residues: ENLNLIK APM (SEQ ID NO: 303, amino acid numbers 150 to 159 in SEQ ID NO: 2) or more amino acid residues, for example, 1 to 10, for example, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or the following amino acid residues ENLNLIKAPM (SEQ ID NO: 3) 03, amino acid numbers 150 to 159 in SEQ ID NO: 2) or an antibody or antigen-binding Joint part.

32. The following amino acid residues ITISHFFKMENLNLIKAPM (SEQ ID NO: 302, SEQ ID NO: 2) (amino acid numbers 141 to 159), for example, 1 to 19, for example, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14 , 15, 16, 17, 18, or 19 amino acid residues, or the following: ITISH FFKMENLNLIKAPM (SEQ ID NO: 302, amino acid number 14 in SEQ ID NO: 2) 32. The antibody or antigen binding thereof according to claim 31, which binds to an epitope consisting of: portion.

Images