NZ623644B2 - Canine/feline cd20 binding epitope and compositions for binding thereto - Google Patents

Abstract

Disclosed is a method of preparing an antibody or an antigen binding fragment thereof that specifically binds to a cyclic polypeptide fragment of CD20 from a target species, the method comprising the steps of: - assessing the sequence of the framework regions of a donor antibody from a species other than the target species, wherein the donor antibody has binding specificity for CD20 from the target species; - comparing the sequence of the framework regions of the donor antibody with the sequence of the framework regions of an antibody or a pool of antibodies derived from the target species; and - modifying the framework regions of the donor antibody to substitute amino acid residues that are foreign at a corresponding position in an antibody or a pool of antibodies from the target species with amino acid residues present at the corresponding position in the target species; wherein the modified framework regions do not contain any amino acids in any position that would be foreign at that position in the target species. Also disclosed is a caninised/felinised antibody or an antigen binding fragment thereof which binds specifically to a cyclic polypeptide fragment of canine CD20, wherein the antibody comprises complementarity determining regions of a heavy and a light chain from a donor antibody from a species other than a canine, wherein the donor antibody has binding specificity for the cyclic polypeptide fragment of canine CD20, wherein the framework regions of the heavy and light chains from the donor antibody are modified to substitute amino acid residues that are foreign at a corresponding position in canine antibodies with amino acid residues present at the corresponding position in canine antibodies, and wherein the modified framework regions do not contain any amino acids in any position that would be foreign at that position in canine antibodies. her than the target species, wherein the donor antibody has binding specificity for CD20 from the target species; - comparing the sequence of the framework regions of the donor antibody with the sequence of the framework regions of an antibody or a pool of antibodies derived from the target species; and - modifying the framework regions of the donor antibody to substitute amino acid residues that are foreign at a corresponding position in an antibody or a pool of antibodies from the target species with amino acid residues present at the corresponding position in the target species; wherein the modified framework regions do not contain any amino acids in any position that would be foreign at that position in the target species. Also disclosed is a caninised/felinised antibody or an antigen binding fragment thereof which binds specifically to a cyclic polypeptide fragment of canine CD20, wherein the antibody comprises complementarity determining regions of a heavy and a light chain from a donor antibody from a species other than a canine, wherein the donor antibody has binding specificity for the cyclic polypeptide fragment of canine CD20, wherein the framework regions of the heavy and light chains from the donor antibody are modified to substitute amino acid residues that are foreign at a corresponding position in canine antibodies with amino acid residues present at the corresponding position in canine antibodies, and wherein the modified framework regions do not contain any amino acids in any position that would be foreign at that position in canine antibodies.

Description

WO 54127 CANINE/FELINE CD20 BINDING EPITOPE AND ITIONS FOR BINDING THERETO Field of the Invention The present invention relates to the identification of a binding epitope which is bound by antibodies which have binding specificity to CD20. The ion extends to the use ofthis epitope to generate antibodies and to the use of antibodies which bind thereto in the treatment and diagnosis of disease conditions, such as lymphoma and immune mediated diseases, in canines and felines. The invention 1O further extends to antibodies which bind to the identified epitope.

Background to the invention CD20 is an activated glycosylated phosphoprotein expressed on the surface of most B-cells, beginning at the pro-B phase and progressively increasing in concentration until maturity. The protein has no known natural ligand and its function is to enable an l B-cell immune response, specifically t T-cell independent ns.

It functions as a calcium channel in the cell membrane. It is involved in intracellular signal transduction and can also modulate cell growth and differentiation.

CD20 is an established marker or target for B-cell lymphomas due to the sion ofthis antigen at high levels on malignant B cells which are associated with B cell lymphoma. ing of CD20 (which is also known as Bp35) using monoclonal antibodies has been proven to be a successful therapeutic approach in the treatment of B-cell lymphomas in humans, and also in the treatment of immune-mediated conditions, such as rheumatoid arthritis. Human CD20 is the target of the monoclonal dies rituximab, Ibritumomab tiuxetan and tositumomab, which are all active agents in the ent of B-cell lymphomas.

Companion animals such as dogs and cats develop similar diseases to humans, including lymphoma, immune-mediated polyarthritidies, tic-lymphocytic synovitis, systemic lupus erythematosus, itis and a variety of autoimmune skin diseases. Canine lymphoma is the second most prevalent cancer in dogs whereas lymphoma is the most common malignancy diagnosed in cats. With current combination therapy, the expected al time for dogs with B-cell lymphoma is around 9 tolZ months. Side effects ofthe standard chemotherapeutic regimes are similar to those seen in humans and e ng, diarrhoea, lack of appetite, fever and sepsis.

Canine CD20 has been terised and predicted to contain two extracellular (EC) domains, four transmembrane (TM) domains, and three intracellular (1C) domains as in human CD20. While canine CD20 has ural homology with human CD20, 1O anti-human and anti-murine CD20 monoclonal antibodies are reportedly incapable of binding to canine CD20. The identification of antibodies which have g specificity to canine and/or feline CD20 could have ular utility in the treatment or sis of canines and felines with B-cell lymphoma.

Summary of the invention Following extensive experimentation, the present inventor has surprisingly identified a novel antigenic loop of the canine and feline CD20 polypeptide which is capable of being specifically bound by anti-human CD20 monoclonal antibodies when said polypeptide loop sequence is ained by a disulphide bond provided between first and second cysteine amino acid residues. As a result, the inventor has identified for the first time that certain monoclonal antibodies which have binding specificity to human CD20 can also have therapeutic and diagnostic applications in canines and felines, due to those antibodies having binding specificity for canine and/or feline CD20. Specifically, the identified novel epitope may have particular utility in the identification of novel anti-canine or anti-feline CD20 antibodies for use in the diagnosis, treatment and/or prophylaxis of CD20+ B-cell ma and immune mediated ions.

According to a first aspect ofthe invention, there is ed an antibody or an antigen binding fragment thereof that specifically binds to a cyclic polypeptide fragment of CD20 for use in the treatment or prevention of a condition mediated by B-cells in a canine or feline subject in need thereof, wherein the cyclic polypeptide fragment comprises, ts of or consists essentially of (i) a uous amino acid sequence sing, consisting of or consisting essentially of amino acid residues SEKNS (SEQ ID NO:67); (ii) a first cysteine residue which is present at a region N- terminal to the contiguous amino acid sequence and (iii) a second ne residue which is present at a region C-terminal to the contiguous amino acid sequence, wherein the cyclic polypeptide fragment is oxidised by the presence of a disulphide bond formed between the first and second cysteine residues.

According to a second aspect ofthe present invention there is provided a method for 1O treating or ting a condition mediated by B-cells in a canine or feline subject in need thereof, the method comprising the step of stering a eutically effective amount of an antibody or an antigen binding fragment thereof that specifically binds to a cyclic ptide fragment of CD20, wherein the cyclic polypeptide fragment comprises (i) a contiguous amino acid sequence consisting of amino acid residues SEKNS (SEQ ID NO:67); (ii) a first cysteine residue which is present at a region N-terminal to the uous amino acid sequence and (iii) a second cysteine residue which is present at a region C-terminal to the contiguous amino acid sequence, wherein the cyclic polypeptide fragment is oxidised by the presence of a disulphide bond formed between the first and second ne residues.

In certain embodiments the method of this aspect ofthe invention can further comprise the step of administering at least one immunosuppressive compound to the canine or feline subject. The immunosuppressive compound may be administered before, along with (simultaneously) or after (sequentially) the administration of the antibody or antigen g fragment. The immunosuppressive agent may be selected from the group ting of a growth factor inhibitor, an immunosuppressor such as an antibody, an anti-inflammatory, an enzymatic inhibitor, a steroid, a non-steroid anti-inflammatory drug, a metabolic inhibitor, a cytotoxic agent and a cytostatic agent.

In embodiments wherein the condition mediated by B cells is rheumatoid arthritis, the antibody can optionally be administered in conjunction with a second therapeutic agent, which is preferably methotrexate.

According to a third aspect of the present invention there is provided use of an antibody or an antigen binding fragment thereof that specifically binds to a cyclic polypeptide fragment of CD20 in the preparation of a medicament for the treatment or tion of a condition mediated by B-cells in a canine or feline subject in need thereof, wherein the cyclic polypeptide fragment comprises (i) a contiguous amino 1O acid sequence consisting of amino acid residues SEKNS (SEQ ID NO:67); (ii) a first cysteine residue which is present at a region N-terminal to the contiguous amino acid sequence and (iii) a second cysteine residue which is present at a region C- al to the contiguous amino acid sequence, wherein the cyclic ptide fragment is ed by the presence of a disulphide bond formed between the first and second cysteine residues.

The present inventor has identified that the cyclic polypeptide fragment of the above s ofthe invention forms an epitope. The epitope formed by the cyclic polypeptide fragment is common to humans, s, felines and mice. In certain embodiments the contiguous amino acid sequence comprises, consists of or consists essentially of amino acid residues SEKNSL (SEQ ID NO:68). The t inventor has identified that the epitope formed by the cyclic polypeptide fragment comprising SEQ ID NO:68 is common to canines and felines. In certain embodiments the contiguous amino acid sequence comprises, consists of or consists essentially of amino acid residues PSEKNS (SEQ ID NO:69). The present inventor has identified that the e formed by the cyclic ptide fragment comprising SEQ ID NO:69 is common to humans, canines and s. In certain embodiments the contiguous amino acid sequence comprises, consists of or ts ially of amino acid residues PSEKNSL (SEQ ID NO 1). The present inventor has identified that the epitope formed by the cyclic polypeptide fragment comprising SEQ ID NO:1 is common to s and felines.

In certain embodiments the cyclic polypeptide fragment comprises, consists of or consists essentially of SEQ ID NO:2 or an amino acid sequence having at least 85%, 90% or 95% sequence identity thereto. Typically the subject is a canine.

In certain embodiments the cyclic polypeptide fragment ses, consists of or consists essentially of SEQ ID NO:4 or an amino acid sequence having at least 85%, 90% or 95% sequence identity thereto. Typically the subject is a .

In certain embodiments the antibody is derived from a Type II anti-human or anti- 1O murine CD20 antibody, for example, Bl-H299, GA101 or Bly1. In certain embodiments the dy is a caninised or felinised antibody, for example, a caninised or felinised Type II anti-human or anti-murine CD20 antibody such as caninised or felinised 9, GA101 or Bly1 antibody.

In certain embodiments the antibody or antigen binding fragment thereof is derived from GA101. Typically where the subject is a , the antibody or antigen binding fragment may se a light chain variable region comprising at least one of an FR1 ork region consisting of or comprising the amino acid sequence of SEQ ID NO:7, an FRZ framework region consisting of or comprising the amino acid sequence of SEQ ID NO:8, an FR3 framework region consisting of or sing the amino acid sequence ofSEQ ID NO:9, and an FR4 ork region consisting of or comprising the amino acid sequence of SEQ ID NO:10, and/or a heavy chain variable region comprising at least one of an FR1 framework region consisting of or comprising the amino acid sequence of SEQ ID NO:11, an FRZ framework region consisting of or comprising the amino acid sequence of SEQ ID NO:12, an FR3 framework region consisting of or comprising the amino acid sequence of SEQ ID NO:13, and an FR4 framework region consisting of or comprising the amino acid sequence of SEQ ID NO:14. In certain embodiments the light chain variable region comprises all of the aforementioned light chain ork regions and/ or the heavy chain variable region comprises all of the aforementioned heavy chain framework regions. In n ments the antibody or antigen binding fragment thereof comprises a light chain le region comprising the amino acid sequence of SEQ ID NO:15 or an amino acid sequence which has an identity of at least 85%, 90%, 95% or 98% thereto and/or a heavy chain variable region comprising the amino acid sequence of SEQ ID NO:16 or an amino acid sequence which has an identity ofat least 85%, 90%, 95% or 98% thereto. In certain embodiments the antibody or antigen binding fragment thereof comprises a light chain comprising the amino acid sequence of SEQ ID NO:18 or an amino acid sequence which has an identity of at least 85%, 90%, 95% or 98% thereto and/or a heavy chain variable region comprising the amino acid ce of SEQ ID NO:17 or an amino acid sequence which has an identity of at least 85%, 90%, 95% or 98% 1O thereto.

In certain embodiments the antibody or antigen binding fragment fragment thereof comprises a heavy chain variable region comprising a complementarity determining region 1 (CDR1) region comprising the amino acid sequence ofSEQ ID NO:55, a CDRZ region comprising the amino acid ce of SEQ ID NO:56 and a CDR3 region sing the amino acid sequence of SEQ ID NO:57, a light chain variable region comprising a CDR1 region comprising the amino acid sequence of SEQ ID NO:58, a CDRZ region comprising the amino acid sequence of SEQ ID NO:59 and a CDR3 region sing the amino acid sequence of SEQ ID NO:60. An antibody having the above CDRs is termed RAZ.

In certain embodiments the antibody or antigen binding fragment fragment thereof is derived from RAZ, for example, the antibody or antigen binding fragment may be caninised or felinised RAZ.

In n embodiments wherein the subject is a canine and the RAZ is sed RAZ the heavy chain variable region comprises the amino acid sequence of SEQ ID N0:37 or an amino acid sequence which has an identity of at least 85%, 90%, 95% or 98% thereto and/or the light chain variable region comprises the amino acid sequence of SEQ ID N0:38 or an amino acid sequence which has an identity of at least 85%, 90%, 95% or 98% o. In certain embodiments wherein the subject is a canine the heavy chain comprises an amino acid sequence selected from the 2012/052532 group consisting of SEQ ID NO:39, SEQ ID NO:40, SEQ ID NO:41 and SEQ ID NO:42, or an amino acid sequence which has an identity of at least 85%, 90%, 95% or 98% to SEQ ID NO:39, SEQ ID NO:40, SEQ ID NO:41 or SEQ ID NO:42. In certain embodiments wherein the subject is a canine the light chain comprises the amino acid sequence of SEQ ID NO:43 or an amino acid sequence which has an identity of at least 85%, 90%, 95% or 98% thereto.

In certain embodiments wherein the subject is a feline and the RAZ is felinised the heavy chain variable region comprises the amino acid sequence of SEQ ID NO:51 or 1O an amino acid sequence which has an identity of at least 85%, 90%, 95% or 98% thereto and/or the light chain variable region comprises the amino acid sequence of SEQ ID NO:52 or an amino acid sequence which has an ty of at least 85%, 90%, 95% or 98% thereto.

In certain embodiments the antibody or antigen binding nt comprises a chimeric antibody, for example, comprising a constant domain of a canine or feline heavy and/or light chain. In certain embodiments wherein the subject is a canine the antibody or antigen binding fragment comprises chimeric RAZ, for e, the heavy chain comprises an amino acid ce selected from the group consisting ofSEQ ID NO:27, SEQ ID NO:28, SEQ ID NO:29 and SEQ ID NO:30, or an amino acid sequence which has an ty ofat least 85%, 90%, 95% or 98% to SEQ ID NO:27, SEQ ID NO:28, SEQ ID NO:29 or SEQ ID NO:30. In certain embodiments wherein the t is a canine the light chain comprises the amino acid sequence of SEQ ID NO:31 or an amino acid sequence which has an identity of at least 85%, 90%, 95% or 98% thereto.

In certain embodiments the antibody or antigen binding fragment fragment thereof comprises a heavy chain variable region comprising a complementarity determining region 1 (CDR1) region comprising the amino acid sequence ofSEQ ID NO:61, a CDRZ region comprising the amino acid sequence of SEQ ID NO:62 and a CDR3 region sing the amino acid sequence of SEQ ID NO:63, a light chain variable region comprising a CDR1 region comprising the amino acid sequence of SEQ ID NO:64, a CDRZ region comprising the amino acid sequence of SEQ ID NO:65 and a CDR3 region comprising the amino acid ce of SEQ ID NO:66. An antibody having the above CDRs is termed RFZ.

In n embodiments the antibody or antigen binding fragment thereof is derived from RFZ, for example, the antibody or antigen binding fragment may be caninised or felinised RFZ.

In certain embodiments wherein the subject is a canine and the antibody is 1O caninised RFZ the heavy chain variable region comprises the amino acid sequence of SEQ ID N0:44 or an amino acid sequence which has an identity of at least 85%, 90%, 95% or 98% thereto and/or the light chain le region comprises the amino acid sequence of SEQ ID NO:45 or an amino acid sequence which has an identity of at least 85%, 90%, 95% or 98% thereto. In certain embodiments wherein the subject is a canine the heavy chain comprises an amino acid sequence selected from the group consisting of SEQ ID NO:46, SEQ ID NO:47, SEQ ID NO:48 and SEQ ID NO:49, or an amino acid sequence which has an ty of at least 85%, 90%, 95% or 98% to SEQ ID NO:46, SEQ ID NO:47, SEQ ID NO:48 or SEQ ID NO:49. In certain embodiments wherein the subject is a canine the light chain comprises the amino acid sequence of SEQ ID NO:50 or an amino acid sequence which has an identity of at least 85%, 90%, 95% or 98% o.

In certain embodiments wherein the subject is a feline and the antibody is sed RFZ the heavy chain variable region comprises the amino acid sequence of SEQ ID NO:53 or an amino acid sequence which has an identity of at least 85%, 90%, 95% or 98% thereto and/or the light chain variable region comprises the amino acid sequence of SEQ ID NO:54 or an amino acid sequence which has an identity of at least 85%, 90%, 95% or 98% thereto.

In certain ments wherein the subject is a canine the antibody or n binding fragment comprises chimeric RFZ, for example, the heavy chain comprises an amino acid ce selected from the group consisting of SEQ ID NO:32, SEQ ID NO:33, SEQ ID NO:34 and SEQ ID NO:35, or an amino acid sequence which has an identity of at least 85%, 90%, 95% or 98% to SEQ ID NO:32, SEQ ID NO:33, SEQ ID NO:34 or SEQ ID NO:35. In certain embodiments n the subject is a canine the light chain comprises the amino acid sequence of SEQ ID NO:36 or an amino acid sequence which has an identity ofat least 85%, 90%, 95% or 98% thereto.

In n embodiments the B-cell mediated condition is a logic malignancy characterised by a high number of tumour cells in the blood or a B-cell lymphoma, in particular a CD20+ B-cell lymphoma. In certain embodiments the condition is a 1O hematologic ancy which is terised by lower B-cell CD20 expression levels, such as conditions selected from the group consisting of transformed non- Hodgkin’s lymphoma, precursor B-cell lymphoblastic leukemia/lymphoma and mature B-cell neoplasms, such as B-cell chronic lymphocytic leukemia small lymphocytic lymphoma (SLL), B-pro-lymphocytic leukaemia (B-PLL), lymphoplasmacytic lymphoma, mantle cell lymphoma (MCL), follicular ma (FL), including low-grade, intermediate-grade and high-grade FL, cutaneous follicle center lymphoma, marginal zone B-cell lymphoma (MALT type, nodal and splenic type), hairy cell leukemia, diffuse large B-cell lymphoma, Burkitt's lymphoma, plasmacytoma, plasma cell a, post-transplant lymphoproliferative disorder, Waldenstrom's macroglobulinemia and anaplastic large-cell lymphoma . In certain embodiments the condition is non-Hodgkin’s lymphoma, such as relapsed and previously treated low-grade non-Hodgkin’s lymphoma (NHL).

In certain embodiments the condition mediated by B-cells is an immune mediated disease. Typically the immune mediated disease is an mune disease, an immune disorder or an inflammatory disease and may be ed from the group consisting of rheumatoid arthritis, systemic lupus erythematosus (SLE), Sjogren’s syndrome, itis, multiple sis, Graves’ disease, idiopathic thrombocytopenia, dermatomyositis, immune mediated thrombocytopenia, polymyocytosis, pemphigus, immune ed haemolytic anaemia and bullous pemphigoid. In certain embodiments the immune mediated disease is selected from the group consisting ofjuvenile rheumatoid arthritis, Wegener's disease, inflammatory bowel disease, idiopathic thrombocytopenic purpura (ITP), thrombotic thrombocytopenic purpura (TTP), autoimmune ocytopenia, multiple sclerosis, psoriasis, IgA nephropathy, lgM polyneuropathies, myasthenia gravis, vasculitis, diabetes mellitus, Reynaud's syndrome, Crohn's disease, ulcerative colitis, gastritis, Hashimoto's thyroiditis, ankylosing spondylitis, hepatitis C- associated cryoglobulinemic vasculitis, chronic focal encephalitis, bullous pemphigoid, hemophilia A, noproliferative glomerulnephritis, adult and juvenile dermatomyositis, polymyositis, chronic uticaria, primary biliary cirrhosis, neuromyelitis optica, Graves' dysthyroid disease, membranoproliferative 1O glonerulonephritis, Churg- Strauss me, asthma, psoriatic arthritis, dermatitis, respiratory distress syndrome, meningitis, encephalitits, uveitis, eczema, atherosclerosis, yte adhesion deficiency, juvenile onset diabetes, Reiter's disease, Behcet's disease, hemolytic anemia, atopic dermatitis, pemphigus vulgaris, Wegener's granulomatosis, s syndrome, chronic renal failure, acute infectious mononucleosis, HIV and herpes-associated dieasese, systemic sclerosis and glomerulonephritis. In the foregoing conditions, it is understood that depleting B-cells may provide a therapeutic approach to treating such condition.

In certain embodiments a chemical agent or radioactive label may be conjugated to the antibody or antigen binding nt in order that the chemical agent or label is specifically delivered to stic B-cells. Said chemical agent or ctive label has the potential to destroy CD20 expressing cells. ing to a fourth aspect ofthe t invention there is provided a caninised or sed antibody or an antigen g fragment thereof which binds specifically to a cyclic polypeptide fragment of CD20, wherein the cyclic polypeptide fragment comprises, consists of or consists ially of (i) a contiguous amino acid sequence consisting of amino acid residues SEKNS (SEQ ID ; (ii) a first cysteine residue which is present at a region N-terminal to the contiguous amino acid sequence and (iii) a second ne residue which is present at a region C- terminal to the contiguous amino acid sequence, wherein the cyclic polypeptide fragment is oxidised by the presence of a disulphide bond formed n the first and second cysteine es.

In certain embodiments the antibody is a caninised antibody sing complementarity determining regions of a heavy and/or light chain from a donor antibody from a species other than a canine, wherein the donor dy has binding specificity for the cyclic polypeptide nt. In certain embodiments the antibody comprises framework regions of the heavy and/or light chain from the donor antibody. In n embodiments the framework regions of the heavy 1O and/or light chain from the donor antibody are modified to substitute amino acid residues that are foreign at a corresponding position in canine antibodies with amino acid es present at the corresponding position in canine antibodies.

In certain embodiments the antibody is a felinised antibody comprising complementarity determining regions of a heavy and/or light chain from a donor antibody from a s other than a feline, wherein the donor antibody has binding specificity for the cyclic polypeptide fragment. In certain embodiments the antibody comprises framework regions of the heavy and/or light chain from the donor antibody. In certain embodiments the framework regions of the heavy and/or light chain from the donor antibody are modified to substitute amino acid residues that are foreign at a corresponding position in feline antibodies with amino acid residues present at the corresponding position in feline antibodies.

Typically the amino acid residues that are foreign at the corresponding position in canine or feline antibodies are tuted with the amino acid residues present at the corresponding position which have the highest homology to the substituted amino acid residues.

Typically the antibody or antigen binding fragment ses constant domains of a heavy and/or light chain from a canine or feline antibody.

WO 54127 In certain embodiments the antibody is derived from (that is, a caninised or felinised version of) a Type II anti-human or anti-murine CD20 antibody, for example, the antibody may be selected from the group consisting of Bl-H299, GA101 and Bly1.

In certain ments the antibody is derived from (that is, a sed or felinised version of) RAZ or RFZ, for example, as described above.

According to a fifth aspect of the present invention there is provided a humanised 1O antibody or an antigen binding fragment thereof which binds specifically to a cyclic polypeptide fragment of CD20, wherein the cyclic polypeptide fragment comprises (i) a contiguous amino acid sequence ting of amino acid residues SEKNS (SEQ ID NO:67); (ii) a first cysteine residue which is present at a region N-terminal to the contiguous amino acid sequence and (iii) a second cysteine residue which is present at a region C-terminal to the contiguous amino acid sequence, wherein the cyclic ptide fragment is oxidised by the presence of a disulphide bond formed between the first and second cysteine residues, and wherein framework regions of the heavy and/or light chain are d from an dy obtained from a species other than human and the ork regions are modified to substitute amino acid residues that are n at a corresponding position in human antibodies with amino acid residues present at the corresponding position in human dies.

In certain embodiments the amino acid residues that are foreign at the corresponding position in human antibodies are substituted with the amino acid residues present at the corresponding position which have the highest homology to the one or more substituted amino acid residues.

Typically the antibody or antigen binding fragment comprises constant domains of a heavy and/or light chain from a human antibody.

According to a sixth aspect ofthe t invention there is provided a chimeric antibody or an antigen binding fragment thereof which binds specifically to a cyclic polypeptide fragment of CD20, wherein the cyclic polypeptide nt comprises (i) a contiguous amino acid sequence consisting of amino acid residues SEKNS (SEQ ID NO:67); (ii) a first cysteine residue which is present at a region N-terminal to the contiguous amino acid sequence and (iii) a second cysteine residue which is t at a region C-terminal to the contiguous amino acid sequence, wherein the cyclic polypeptide nt is oxidised by the presence of a disulphide bond formed between the first and second cysteine residues and wherein the antibody comprises a canine or feline constant domain. 1O According to a seventh aspect of the present invention there is provided an antibody or an antigen binding fragment thereof, which ically binds to CD20 wherein the antibody or fragment thereof comprises a heavy chain variable region sing a complementarity determining region 1 (CDR1) region comprising the amino acid sequence of SEQ ID NO:55, a CDRZ region comprising the amino acid sequence of SEQ ID and a CDR3 region comprising the amino acid sequence of SEQ ID NO:57, a light chain variable region comprising a CDR1 region comprising the amino acid sequence of SEQ ID NO:58, a CDRZ region comprising the amino acid sequence of SEQ ID NO:59 and a CDR3 region sing the amino acid sequence of SEQ ID NO:60.

In certain ments the heavy chain variable region ses the amino acid ce of SEQ ID NO:19 or an amino acid sequence which has an identity of at least 85%, 90%, 95% or 98% thereto and/or the light chain variable region comprises the amino acid sequence of SEQ ID NO:20 or an amino acid sequence which has an identity ofat least 85%, 90%, 95% or 98% thereto. In certain embodiments the heavy chain comprises the amino acid sequence of SEQ ID NO:23 or an amino acid sequence which has an identity of at least 85%, 90%, 95% or 98% thereto and/or the light chain comprises the amino acid sequence of SEQ ID NO:24 or an amino acid sequence which has an identity of at least 85%, 90%, 95% or 98% thereto.

In certain ments the antibody is a caninised antibody. Typically the heavy chain variable region ses the amino acid sequence of SEQ ID N0:37 or an amino acid sequence which has an identity ofat least 85%, 90%, 95% or 98% thereto and/or the light chain variable region comprises the amino acid ce of SEQ ID N0:38 or an amino acid sequence which has an identity of at least 85%, 90%, 95% or 98% thereto. In certain ments the heavy chain comprises an amino acid sequence selected from the group consisting of SEQ ID N0:39, SEQ ID NO:40, SEQ ID NO:41 and SEQ ID NO:42, or an amino acid sequence which has an identity of at least 85%, 90%, 95% or 98% to SEQ ID N0:39, SEQ ID N0:40, SEQ ID NO:41 or 1O SEQ ID NO:42. In certain embodiments the light chain ses the amino acid sequence of SEQ ID NO:43 or an amino acid sequence which has an identity of at least 85%, 90%, 95% or 98% thereto.

In certain embodiments the antibody is a felinised antibody. Typically the heavy chain variable region comprises the amino acid sequence of SEQ ID NO:51 or an amino acid sequence which has an identity ofat least 85%, 90%, 95% or 98% thereto and/or the light chain le region comprises the amino acid sequence of SEQ ID NO:52 or an amino acid sequence which has an identity of at least 85%, 90%, 95% or 98% thereto.

In certain embodiments the antibody is a humanised antibody.

In certain embodiments the antibody is a chimeric dy. Typically the heavy chain and/or light chain comprises a constant domain derived from a canine, feline or human antibody. In certain embodiments the heavy chain comprises an amino acid sequence selected from the group consisting of SEQ ID NO:27, SEQ ID NO:28, SEQ ID NO:29 and SEQ ID N0:30, or an amino acid sequence which has an identity of at least 85%, 90%, 95% or 98% to SEQ ID NO:27, SEQ ID NO:28, SEQ ID NO:29 or SEQ ID N0:30. In certain embodiments the light chain comprises the amino acid sequence of SEQ ID N0:31 or an amino acid sequence which has an identity of at least 85%, 90%, 95% or 98% thereto. lly the antibody or antigen binding fragment is reactive and specifically binds to human, murine, canine and feline CD20.

According to an eighth aspect ofthe present invention there is ed an antibody or an antigen binding fragment f, which specifically binds to CD20 wherein the antibody or fragment thereof comprises a heavy chain variable region comprising a complementarity determining region 1 (CDR1) region comprising the amino acid sequence of SEQ ID NO:61, a CDRZ region comprising the amino acid sequence of SEQ ID NO:62_and a CDR3 region comprising the amino acid sequence 1O of SEQ ID NO:63, a light chain variable region comprising a CDR1 region comprising the amino acid sequence of SEQ ID NO:64, a CDRZ region comprising the amino acid sequence of SEQ ID NO:65 and a CDR3 region comprising the amino acid ce of SEQ ID NO:66.

In certain embodiments the heavy chain variable region comprises the amino acid sequence of SEQ ID NO:21 or an amino acid sequence which has an identity of at least 85%, 90%, 95% or 98% thereto and/or the light chain variable region comprises the amino acid sequence of SEQ ID NO:22 or an amino acid ce which has an identity ofat least 85%, 90%, 95% or 98% o. In certain embodiments the heavy chain comprises the amino acid sequence of SEQ ID NO:25 or an amino acid sequence which has an identity of at least 85%, 90%, 95% or 98% thereto and/or the light chain comprises the amino acid sequence of SEQ ID NO:26 or an amino acid sequence which has an identity of at least 85%, 90%, 95% or 98% thereto.

In certain embodiments the dy is a caninised antibody. Typically the heavy chain variable region comprises the amino acid sequence of SEQ ID N0:44 or an amino acid sequence which has an identity ofat least 85%, 90%, 95% or 98% thereto and/or the light chain variable region comprises the amino acid sequence of SEQ ID NO:45 or an amino acid sequence which has an identity of at least 85%, 90%, 95% or 98% thereto. In certain embodiments the heavy chain comprises an amino acid sequence ed from the group consisting of SEQ ID NO:46, SEQ ID NO:47, SEQ ID NO:48 and SEQ ID NO:49, or an amino acid sequence which has an identity of at least 85%, 90%, 95% or 98% to SEQ ID NO:46, SEQ ID N0:47, SEQ ID NO:48 or SEQ ID NO:49. In certain embodiments the light chain comprises the amino acid sequence of SEQ ID NO:50 or an amino acid sequence which has an identity of at least 85%, 90%, 95% or 98% o.

In certain embodiments the antibody is a felinised antibody. Typically the heavy chain variable region comprises the amino acid sequence of SEQ ID NO:53 or an amino acid sequence which has an identity ofat least 85%, 90%, 95% or 98% 1O thereto and/or the light chain variable region comprises the amino acid sequence of SEQ ID NO:54 or an amino acid sequence which has an identity of at least 85%, 90%, 95% or 98% thereto.

In certain embodiments the antibody is a humanised antibody.

In certain embodiments the antibody is a chimeric antibody. Typically the heavy chain and/or light chain comprises a nt domain derived from a , feline or human antibody. In certain embodiments the heavy chain ses an amino acid ce selected from the group consisting of SEQ ID NO:32, SEQ ID NO:33, SEQ ID NO:34 and SEQ ID NO:35, or an amino acid sequence which has an ty of at least 85%, 90%, 95% or 98% to SEQ ID NO:32, SEQ ID NO:33, SEQ ID NO:34 or SEQ ID NO:35. In certain embodiments the light chain comprises the amino acid sequence of SEQ ID NO:36 or an amino acid sequence which has an identity of at least 85%, 90%, 95% or 98% thereto.

Typically the antibody or antigen binding fragment is cross-reactive and specifically binds to human, , canine and feline CD20.

According to a ninth aspect ofthe present invention there is provided an antibody or an antigen binding fragment thereof sing a heavy chain variable region comprising the amino acid sequence of SEQ ID NO:16 or an amino acid sequence which has an identity ofat least 85%, 90%, 95% or 98% thereto and/or a light chain variable region comprising the amino acid sequence of SEQ ID N0:15 or an amino acid sequence which has an identity of at least 85%, 90%, 95% or 98% thereto.

In certain embodiments the heavy chain comprises the amino acid ce of SEQ ID N0:17 or an amino acid ce which has an identity of at least 85%, 90%, 95% or 98% thereto and/or the light chain comprises the amino acid sequence of SEQ ID N0:18 or an amino acid sequence which has an ty of at least 85%, 90%, 95% or 98% thereto. 1O In certain ments of the fourth to ninth aspects of the invention the antibody or antigen binding fragment thereof specifically binds to canine and/or feline CD20 with a binding affinity having an equilibrium dissociation constant (KD) of 1x10'8 or less.

Typically the dy or antigen binding fragment is an ed antibody or antigen g nt thereof.

In certain embodiments the antibody or antigen binding fragment is selected from the group consisting of a single chain Fv (scFv) antibody fragment, a Fab antibody fragment, a Fab’ antibody fragment and a F(ab’)2 antibody fragment. In certain embodiments the antibody or antigen binding fragment is a pecific or multivalent antibody.

In certain embodiments a chemical agent may be conjugated to an antibody or antigen binding fragment according to any one of the fourth to ninth s in order that the chemical agent is specifically delivered to neoplastic B-cells. Said chemical agent or radioactive label has the potential to destroy CD20 expressing cells. Accordingly, the invention extends to immunoconjugates that consist of xic agents conjoined to an antibody or fragment thereofby means of a chemical linker, said immunoconjugates also being known as antibody-cytotoxic agent conjugates (ACC) or antibody drug conjugates (ADC). Such immunoconjugates allow the targeted delivery ofthe drug moiety to tumour cells.

Examples of drugs which are useful in this regard include methotrexate and vindesine, while toxins include, but are not d to, bacterial , plant toxins such as ricin and small molecule toxins such as geldanamycin.

In certain embodiments a radioactive label may be conjugated to an dy or antigen binding fragment according to any one of the fourth to ninth aspects in order that the radioactive label is specifically delivered to neoplastic s. In certain embodiments the label may be selected from the group comprising, but not limited to, a radiolabel, a hore, a chromophore, an imaging agent and a metal 1O ion. Typically the labelled antibody or fragment may have utility in diagnosis.

The invention therefore further provides a method for diagnosing a subject suspected of having a condition mediated by B-cells, said method sing administering to a subject an antibody or antigen binding fragment conjugated to a label and ing the distribution of the antibody or antigen binding fragment within the subject. In various embodiments the method of diagnosis includes diagnosing B-cell ed disorder, immune disorder, autoimmune disease or inflammatory disease selected from the list defined hereinbefore.

According to a tenth aspect ofthe present invention there is ed an isolated nucleic acid that encodes an antibody or antigen binding fragment according to any one of the fourth to ninth aspects of the invention.

Also provided is an expression vector comprising said nucleic acid and a host cell incorporating the expression vector. The ion further extends to a method for producing an antibody comprising the step of culturing said host cell to allow the cell to s the antibody.

The invention further extends to an antibody or antigen binding fragment ing to any one of the fourth to ninth aspects ofthe invention for use in the treatment or prevention of a condition mediated by B-cells.

Also provided is a method for treating or preventing a condition mediated by B-cells comprising the steps of administering a therapeutically effective amount of an antibody or antigen binding fragment ing to any one of the fourth to ninth s of the invention to a subject in need thereof.

The ion also extends to use of an antibody or antigen binding fragment according to any one ofthe fourth to ninth aspects ofthe invention in the preparation of a medicament for the treatment or prevention of a condition mediated by B-cells.

In certain embodiments the condition mediated by B-cells is any condition as described above in relation to the first to third aspects of the invention.

Also provided is an antibody or antigen g fragment according to any one of the fourth to ninth aspects of the invention for use in diagnosis.

The invention further provides a cyclic polypeptide fragment of CD20 comprising, consisting or consisting essentially of (i) a contiguous amino acid sequence consisting of amino acid residues SEKNS (SEQ ID NO:67); (ii) a first cysteine residue which is present at a region N-terminal to the contiguous amino acid ce and (iii) a second cysteine residue which is present at a region C-terminal to the contiguous amino acid sequence, wherein the cyclic polypeptide is oxidised by the presence of a disulphide bond formed between the first and second cysteine residues.

In certain ments the uous amino acid ce comprises, consists of or consists essentially of amino acid residues SEKNSL (SEQ ID NO:68). In certain embodiments the contiguous amino acid sequence comprises, consists of or ts essentially of amino acid residues PSEKNS (SEQ ID NO:69). In certain ments the contiguous amino acid sequence comprises, consists of or consists essentially of amino acid residues PSEKNSL (SEQ ID NO 1).

In certain embodiments the cyclic polypeptide fragment comprises less than 30, 28, , 24, 23, 22 or 21 amino acid residues.

In certain ments the cyclic polypeptide fragment ses, consists of or consists essentially of SEQ ID NO:2 or an amino acid sequence having at least 85%, 90% or 95% sequence identity thereto.

In n embodiments the cyclic polypeptide fragment comprises, consists of or consists essentially of SEQ ID NO:4 or an amino acid sequence having at least 85%, 1O 90% or 95% sequence identity thereto.

In certain embodiments the cyclic ptide fragment comprises, consists of or ts essentially of SEQ ID NO:3 or an amino acid sequence having at least 85%, 90% or 95% sequence identity thereto.

In certain embodiments the cyclic polypeptide fragment comprises, consists of or consists essentially of SEQ ID NO:6 or an amino acid sequence having at least 85%, 90% or 95% sequence identity thereto.

Typically binding of the cyclic polypeptide fragment by an nistic binding member antagonises CD20 biological activity.

The ion r extends to a pharmaceutical composition comprising the cyclic polypeptide fragment and a pharmaceutically acceptable carrier or excipient.

The invention further extends to a vaccine composition comprising the cyclic polypeptide fragment. In certain embodiments the cyclic polypeptide fragment is connected to or otherwise associated with one or more amino acid sequences comprising T-cell epitopes isable by the target species ofthe vaccine. Typical examples of such amino acid sequences include tetanus toxoid or fragments thereof, diphtheria toxoid or fragments thereof and keyhole limpet haemocyanin (KLH). In certain ments the vaccine will r comprise an adjuvant selected to increase the ude of the immune response ed to the vaccine. l examples of adjuvants include aluminium salts, oil-in-water and water-in-oil emulsions, saponins and Lipid A and its derivatives and homologues.

Also provided is use ofthe cyclic polypeptide fragment, such as a cyclic polypeptide fragment of SEQ ID NO:2, in a method for generating a binding member which ically binds to CD20.

In n ments the method is a method for ting a binding member 1O which specifically binds to canine CD20. In certain embodiments the method is a method for generating a binding member which specifically binds to feline CD20.

The ion further provides a method for generating a binding member which specifically binds to CD20, the method comprising the steps of: - administering to a subject a cyclic polypeptide fragment as described above, such as a cyclic polypeptide fragment of SEQ ID NO:2, and - isolating binding agents which bind specifically to said cyclic polypeptide fragment.

In certain embodiments the method is a method for generating a binding member which specifically binds to canine CD20. In certain embodiments the method is a method for generating a binding member which specifically binds to feline CD20.

The invention further provides a screening method for identifying a binding member which specifically binds to canine and/or feline CD20, the screening method comprising the steps of: - bringing a candidate compound into contact with a cyclic polypeptide fragment as described above, such as the cyclic polypeptide fragment comprising SEQ ID NO:2; and - assessing binding between the candidate compound and the cyclic polypeptide fragment; wherein binding between the candidate compound and the cyclic polypeptide fragment identifies the candidate compound as a binding member which specifically binds to canine and/or feline CD20.

In certain embodiments the candidate compound is selected from the group ting of proteins, such as antibodies, peptides, such as fusion protein, peptidomimetics, c acids, polynucleotides, ccharides, oligopeptides, carbohydrates, lipids, small le compounds and naturally occurring compounds.

The invention further provides a method for detecting the presence of canine or feline CD20 in a B-lymphocyte-containing sample comprising the steps of: - contacting one or more antibodies as described above with the sample under conditions that allow B-lymphocyte/antibody complexes to form; and - detecting B-lymphocyte/antibody complexes, wherein the ion of said complexes is an indication that canine or feline CD20 is present in the sample.

In certain embodiments, the method comprises the further step of determining whether the subject from which the sample was obtained has B-cell ma by means of detecting B-lymphocyte/antibody xes, wherein the presence of said complexes provides an indication that the subject from which the sample originated has a B-cell mediated condition, such as B-cell lymphoma.

A yet further aspect of the invention es a cell line, or a derivative or y cell thereof that produces an dy or an antigen binding fragment thereof according to any foregoing aspect ofthe invention.

A further still aspect provides a kit for the treatment or diagnosis of CD20+ B cell lymphoma in a canine comprising an anti-canine CD20 antibody according to any foregoing aspect of the invention. A further still aspect provides a kit for the WO 54127 treatment or diagnosis of CD20+ B cell lymphoma in a feline comprising an anti- feline CD20 antibody according to any foregoing aspect of the invention.

A yet further aspect provides a kit for the treatment of an immune mediated condition, comprising an anti-canine CD20 antibody according to any foregoing aspect ofthe invention. A yet further aspect provides a kit for the treatment of an immune mediated condition, comprising an anti-feline CD20 antibody according to any ing aspect of the invention. 1O Brief ption of the drawings Figure 1 shows the amino acid sequence of the canine CD20 derived cyclic polypeptide fragment having the amino acid ce of SEQ ID NO:2, with the disulphide bond between the two cysteine residues corresponding to ons 167 and 183 shown.

Figures 2A and 23 show the binding of H299 and BiogeneX Bl (BG) monoclonal antibodies to disulphide bonded human (H20), murine (M20) and canine (C20) CD20 polypeptide fragments.

Figure 3 shows the binding of H299 monoclonal antibody to both human and canine CD20 expressed on CHO cells. Figure 3A shows the binding of H299 monoclonal antibody (6ug/ml) to canine CD20 CHO cells. Figure SB shows the binding of H299 monoclonal antibody (6ug/ml) to human CD20 CHO cells. Figure 3C shows the binding of secondary anti-mouse PE dy (Sigma P9287) to canine CD20 CHO cells. Figure 3D shows the binding of secondary anti-mouse PE antibody (Sigma P9287) to human CD20 CHO cells.

Figure 4 shows a comparison of binding of two different Type II anti-human CD20 monoclonal antibodies (H299 and Bly1) to a hide bonded canine CD20 polypeptide fragment.

Figure 5 shows the binding by FACS of two different Type II uman CD20 monoclonal antibodies (H299 and Blyl) to human CD20- and canine CD20- expressing CHO cells.

Figure 6 shows that the g ofthe Type II monoclonal antibody H299 to a hide-bonded canine CD20 peptide is abolished by reduction of the disulphide bond with dithiothreitol (DTT), whereas the g to disulphide bonded human CD20 peptide is reduced, but not abolished, by DTT treatment. 1O Figure 7 shows the complete caninised GA101 VK kappa variable light chain amino sequence (Figure 7a - SEQ ID N0:15) and variable heavy chain (Figure 7b - SEQ ID N0:16) wherein the CDR region residues are shown in bold.

Figure 8 shows recombinant caninised GA101 (humanised Blyl) constructed from co-eXpressed SEQ ID N0:17 and 18 binds to canine CD20 peptide SEQ ID NO:2.

Figure 9 shows binding of novel murine MAbs RAZ and RFZ to cyclised canine, human, mouse and feline CD20 peptides - human CD20 cyclic peptide (SEQ ID N0:3) (RAZ weakly) and canine CD20 cyclised peptide (SEQ ID N0:2) as well as murine (SEQ ID N0:6) and feline (SEQ ID N0:4) CD20 peptides.

Figure 10 a to d show the derived variable domain heavy and light chain sequences of RAZ (a,b) and RFZ (c,d) monoclonal dies wherein CDRs are underlined.

Figure 10e shows an alignment of RAZ and RFZ variable s.

Figure 11 shows binding of recombinant mouse RAZ and RFZ MAbs to cyclised CD20 peptides.

Figure 12 shows an alignment of peptide loops from canine, feline, human and mouse CD20 and illustrates the shorter common peptide epitope SEKNS, a sub- epitope of L deduced from the common binding of Type II anti-human CD20 monoclonal antibodies to human and canine CD20.

Figure 13 shows the heavy (Figure 13a) and light chain (Figure 13b) variable domains of caninised RAZ.

Figure 14 shows the heavy (Figure 14a) and light (Figure 14b) chain variable s of caninised RFZ.

Figure 15 shows SDS—PAGE analysis ofCHO cell expressed and Protein A purified forms of murine, murine/canine chimeric and caninised RAZ and RFZ antibodies.

Figure 16 shows the g by ELISA of recombinant chimeric and caninised RAZ and RFZ monoclonal antibodies to canine, feline, mouse and human cyclised CD20 peptides.

Figure 17 shows binding of complement C1q to caCDZO e-immobilised chimeric and caninised RAZ and RFZ.

Figure 18 shows binding of soluble recombinant canine high affinity Fc receptor (scaCD64) to caCDZO peptide-immobilised chimeric and caninised RAZ and RFZ.

Detailed ption of the invention The present invention relates to the identification of a disulphide-bonded and structurally-constrained nic loop of canine CD20 that binds specifically to Type II anti-human CD20 monoclonal dies. The amino acid sequence of this novel epitope of CD20 is shown in Figure 1, where the hide bond between the two cysteine residues is shown. The defined epitope, when bound by a binding agent which binds specifically thereto, results in antagonism of the function of CD20.

Without wishing to be bound by , the inventor has singly identified that anti-human and/or anti-murine CD20 monoclonal antibodies can bind to a novel disulphide-bonded and structurally-constrained antigenic loop of canine CD20. In particular, the inventor has shown that the binding of monoclonal antibodies to canine CD20 is critically dependent on the conformation of the polypeptide, with the conformation being dependent on the presence of a disulphide bond ed between two cysteine residues.

In particular, the inventor has fied a polypeptide which is derived from canine CD20 which comprises the amino acid sequence of SEQ ID NO:2. In addition, ing the iation that the presence of cysteine es are necessary for the ce of a disulphide bond, the inventor has observed that cysteine residues which are used to form the disulphide bond are conserved across CD20 proteins of 1O l species. In particular, homologous disulphide bonded peptides from CD20 derived from different species may be useful in identifying diagnostic and therapeutic binding agents for use in the diagnosis and therapy of CD20+ B cell mediated conditions. Such species include human, feline, equine and murine having the polypeptide sequences set out in SEQ ID NO:3; SEQ ID NO:4; SEQ ID NO:5 and SEQ ID NO:6 respectively.

Anti-CD20 monoclonal antibodies can be classified as Type I and Type II anti-CD20 monoclonal antibodies. This classification is dependent on the mechanism of action used to kill malignant B-cells. Type I (rituXimab-like) monoclonal antibodies induce CD20 to redistribute into large lipid rafts or microdomains in the plasma membrane, whereas Type II (tositumomab-like) do not. Importantly, this ribution of CD20 impacts many of the binding properties and effector functions that control the therapeutic success of D20 monoclonal antibodies.

The inventor has surprisingly found that the Type II uman CD20 monoclonal antibodies H299 and GA101 (including its parent mouse monoclonal antibody Blyl) bind to not only human CD20, but also canine CD20 and that the polypeptide of SEQ ID NO:2 allows this determination to be made. The present or has also surprisingly shown that other Type II anti-human CD20 monoclonal antibodies bind to canine CD20. This is surprising as Type I anti-human CD20 monoclonal antibodies, such as Rituxan, do not bind canine CD20.

According to Polyak and Deans (Blood 99, 3256; 2002) the Type II anti-human CD20 monoclonal antibodies H299 and Bly1 bind to a similar epitope on human CD20.

Niederfellner et al. (2011) ined that H299 and Bly1 bind to human CD20 with dependence on each of contiguous residues 172-PSEKNSP-178. Type I anti-human CD20 monoclonal dies, such as mab and C2H7 have binding which is dependent on the more N-terminal contiguous residues 168-EPANPSEK-175. These residues are aligned with canine and murine CD20 es below (Type I underlined, Type II in italics): 1O Human IYNCEPANPSEKNSPSTQYC (SEQ ID NO:3) Canine IHNCDPANPSEKNSLSIQYC (SEQ ID NO:2) Murine IYDCEPSNSSEKNSPSTQYC (SEQ ID NO:6) Further investigation by the inventor has surprisingly identified an analogous sequence in felines: Feline IHTCQPESKPSEKNSLSIKYC (SEQ ID NO:4) The present invention therefore further relates to the identification of a disulphide- bonded and structurally-constrained antigenic loop of feline CD20.

Without wishing to be bound by theory, the inventor has identified that the lack of binding of RituXimab to canine CD20 is due to canine sequence amino acid residue D168 (E in the human sequence). Furthermore, the inventor has also identified that a al region for g Type II anti-human CD20 monoclonal antibodies such as H299 monoclonal antibody in human CD20 is the P178 residue, which is L in canine CD20. The inventor concludes that it is the P178L amino acid difference n human and canine CD20 polypeptides of SEQ ID NO:2 and SEQ ID NO:3 which explains why H299 binds more weakly to canine CD20 than it does to human CD20.

The inventor has taken the GA101 anti-human CD20 antibody which was not known to bind to canine CD20 and produced antibodies which bind specifically to canine WO 54127 CD20. Furthermore, the or has provided RAZ and RFZ anti-murine CD20 antibodies which bind specifically to human, canine, murine and feline CD20. The caninised and felinised and humanised antibodies of the invention, which are not produced using standard CDR grafting techniques, are shown to eXhibit high affinity binding to canine, human and/or feline CD20. The antibodies have been designed so that the framework and constant regions incorporate only residues present in canine, human or feline lgG molecules as appropriate so that when administered to a canine, human or feline, xenoantibodies are unlikely to be produced there against. 1O The process of ting the heavy and light chain variable domains for the antibodies of the invention which has been employed by the inventor results in the replacement of specific framework donor amino acid residues known to be n to the target species at that position with a residue which is found at that position in the target species (e.g. canine, feline or human) and which, based on the inventor’s is, will retain the conformation of the CDR regions and therefore maintain g specificity and avidity, while ng the presence of immunogenic es which may result in neutralising antibodies being generated against the antibody if it were to be administered to target species in an unaltered form.

Specifically, the method of preparing antibodies of the invention (known as PETisation) comprises assessing the sequence of the framework regions of a donor (e.g. human) antibody for suitability for administering to a target species by comparing the ce of the framework regions of the donor antibody with the ce of an antibody or a pool of antibodies derived from the target species.

Although the comparison may be between the donor sequence and a single member ofthe target sequence, it will be obvious that comparison with a pool oftarget sequences is preferred because this will eXpand the number of natural options at each Kabat on in the target species. Not only will this increase the chance of a “match" between the donor and the target, but it will also eXpand the options for replacement where a match does not eXist. As a result, a replacement with characteristics as close as possible to the donor will be able to be . Where the donor sequence and the target sequence differ at any Kabat number or corresponding position, the donor sequence is modified to substitute the amino acid 2012/052532 e in question with an amino acid residue which is known to be natural at that position in the target species.

Where substitution of an amino acid residue present in a donor immunoglobulin framework region is required, typically this is aken using the principle of conservative substitution wherein an amino acid residue is replaced with an amino acid residue which is natural at that Kabat on in a target species and is as closely d as possible in size, charge and hydrophobicity to the amino acid being substituted in the donor sequence. The intention is to choose a replacement 1O which would cause no, or at least only minimum, perturbation or disruption to the dimensional structure of the donor antibody. In certain situations, there will be no clear option and each choice will have benefits and downsides. A final decision may require three-dimensional modelling or even expression of various alternative sequences. However, generally, a clear preference will be available. As a result ofthis procedure, a change in the donor sequence is only made when that residue would be foreign in the target and the replacement amino acid is as y related as possible to that which it replaces. Thus, the creation of foreign es is avoided, but the overall three-dimensional structure is preserved and as a result, affinity and specificity are also preserved.

The term “epitope" as used herein relates to a portion or portions of a macromolecule which is capable ofbeing bound by a specific antibody, in this case, a portion of a ptide, in particular CD20. Epitopes generally consist of ally active surface groups and have specific three dimensional structural characteristics, as well as ic charge characteristics. Typically, the CD20 binding agent or binding compound antagonises the binding activity of CD20 and as such binds to an epitope known as an inhibiting e or an inhibitory epitope. An "inhibiting" or "inhibitory" epitope means an epitope present on CD20, that when bound by a binding compound such as a small molecule or an antibody, results in the loss ofbiological activity of CD20.

Epitopes may be defined from contiguous or non-contiguous sequences of amino acid residues comprised within a polypeptide sequence. The term “contiguous epitope" defines an epitope comprised of a linear series of amino acid residues within a polypeptide which define the epitope. A contiguous epitope may be conformational if the e is conformationally constrained e.g. by forming a loop.

A “non-contiguous epitope", which may also be referred to as a mational and discontinuous epitope, is an epitope which is comprised of a series of amino acid residues which are non-linear in alignment, that is that the residues are spaced or grouped in a non-continuous manner along the length of a polypeptide sequence. A 1O non-contiguous epitope can be a discontinuous e wherein the amino acid residues are grouped into 2 linear sequences, or alternatively the non-contiguous epitope can be a discontinuous scattered e wherein the residues which contribute to the e are provided in 3 or more groups oflinear amino acid ces arranged along the length of the polypeptide.

As herein d an "antibody" encompasses n-binding proteins which specifically bind to a target antigen ofinterest, in this case canine and/or feline CD20 including polypeptides that can be recombinantly prepared or which are genetically encodable by immunoglobulin genes, or fragments of immunoglobulin genes. The term "antibody" encompasses monoclonal and chimeric antibodies, in particular caninised and felinised antibodies, and further asses polyclonal antibodies or antibodies of any class or e. An "antibody" further extends to hybrid antibodies, bispecific dies, heteroantibodies and to functional fragments thereof which retain antigen binding.

The constant region of the antibody may be of any suitable immunoglobulin subtype, however it is preferred that the antibody e is lgG. Such an antibody may further belong to any subclass e.g. in the canine, lgG-A, lgG-B, lgG-C and IgG-D and in certain embodiments be either ofthe subclass lgG-B or lgG-C. However, in certain embodiments, the subtype ofthe antibody may be ofthe class lgA, lgM, IgD or lgE.

Fragments of a whole antibody can perform the function of antigen g.

Examples of such binding fragments are a Fab fragment sing the VL, VH, CL and CH1 antibody domains; an Fv fragment consisting of the VL and VH domains of a single antibody; a F(ab’)2 nt, a bivalent fragment comprising two linked Fab fragments; a single chain Fv le (scFv), wherein a VH domain and a VL domain are linked by a peptide linker which allows the two domains to associate to form an antigen binding site; or a bi-specific antibody, which may be multivalent or pecific fragments constructed by gene fusion. 1O A fragment of an antibody or of a polypeptide for use in the present invention, for e, a fragment of a polypeptide defining a canine CD20 binding epitope or an antibody which binds specifically to CD20 and in particular to CD20 at the epitope defined by SEQ ID NO:2, generally means a h of amino acid residues of at least to 7 contiguous amino acids, often at least about 7 to 9 contiguous amino acids, typically at least about 9 to 13 contiguous amino acids, more ably at least about 20 to 30 or more contiguous amino acids and most preferably at least about to 40 or more consecutive amino acids. Similarly, a fragment of a polypeptide defining a feline CD20 binding epitope or an antibody which binds specifically to CD20 and in particular to CD20 at the epitope defined by SEQ ID NO:4, generally means a stretch of amino acid es of at least 5 to 7 contiguous amino acids, often at least about 7 to 9 contiguous amino acids, typically at least about 9 to 13 contiguous amino acids, more preferably at least about 20 to 30 or more contiguous amino acids and most preferably at least about 30 to 40 or more consecutive amino acids.

A “derivative" of such an antibody or polypeptide, or of a fragment of a CD20 specific antibody means an antibody or polypeptide modified by varying the amino acid sequence of the protein, e.g. by manipulation of the nucleic acid encoding the n or by altering the n itself. Such derivatives of the natural amino acid sequence may involve insertion, addition, deletion and/or substitution of one or more amino acids, preferably while providing a peptide having CD20 binding activity. Preferably such derivatives involve the insertion, addition, deletion and/or 2012/052532 substitution of 25 or fewer amino acids, more preferably of 15 or fewer, even more preferably of 10 or fewer, more preferably still of 4 or fewer and most preferably of 1 or 2 amino acids only.

The term “derived from" as used herein to refer to an dy or antigen binding fragment being derived from a specific antibody is understood to mean that the antibody or antigen binding fragment may be a chimeric or modified (e.g. sed or sed) version of the specific antibody. Typically the antibody or antigen binding fragment which is derived from the specific antibody will retain the CDRs of 1O the specific antibody, but may comprise different nt and/or framework regions.

In certain embodiments the antibody is an "isolated antibody". This is understood to mean that the antibody is (1) free of at least some ns with which it would normally be found, (2) is essentially free of other proteins from the same source, e.g., from the same species, (3) is expressed by a cell from a different species, or (4) does not occur in nature.

The term “caninised" as used herein is understood to mean that the antibody has been modified for use in a canine, for e, by substituting one or more amino acids which would be foreign at a corresponding on in a canine with amino acids found at the corresponding position in a canine. The term "felinised" as used herein is understood to mean that the antibody has been modified for use in a feline, for example, by substituting one or more amino acids which would be n at a corresponding position in a feline with amino acids found at the corresponding position in a feline. The term "humanised" as used herein is understood to mean that the antibody has been modified for use in a human, for example, by substituting one or more amino acids which would be foreign at a corresponding position in a human with amino acids found at the corresponding position in a human.

Amino acids which are "foreign" in a target species may be identified by comparing an amino acid sequence of, for example, a framework region of a donor antibody with amino acid sequence(s) of, for example, a framework region of one or more antibodies from the target species to identify one or more amino acid residues in the donor framework region which are not present at the corresponding position in antibodies from the target species. The target species may be canine, feline or human.

The term "corresponding position" as used herein to refer to an amino acid residue that is present in a second sequence at a position corresponding to a ied amino acid residue in a first sequence is intended to refer to the on in the 1O second sequence which is the same position as the position in the first sequence when the two sequences are aligned to allow for maXimum sequence identity between the two sequences. Amino acid residues at corresponding ons have the same Kabat numbering.

The phrase "specifically binds to" refers to the binding of an dy to a specific protein or target which is present amongst a heterogeneous population of proteins.

Hence, when present in specific immunoassay conditions, the antibodies bind to a particular protein, in this case canine or feline CD20 and in ular to the e defined by SEQ ID NO:2 of 4, and do not bind in a significant amount to other ns present in the sample.

The term "subject" as used herein may refer to a mammal, e.g. a canine, feline or human. Typically the subject may be suffering from a B-cell mediated condition as described above.

The phrase "at a region inal to" as used herein is understood to mean that the first cysteine residue may be present directly adjacent the N-terminus of the contiguous amino acid sequence or, more typically, one or more amino acid residues may separate the first cysteine residue from the N-terminus of the contiguous amino acid sequence. Similarly, the phrase "at a region C-terminal to" as used herein is understood to mean that the second cysteine residue may be present directly nt the inus of the contiguous amino acid sequence or, more typically, one or more amino acid es may separate the second cysteine residue from the C-terminus of the contiguous amino acid ce.

The variable region other than the hypervariable region may also be derived from the variable region of a human antibody and/or may also be derived from a monoclonal antibody, such as a CD20 specific antibody. In such case, the entire variable region may be derived from a murine monoclonal antibody, such as a CD20 ic antibody and the antibody is said to be chimerised. Methods for making chimerised antibodies are known in the art.

It is possible to take monoclonal and other antibodies and use techniques of recombinant DNA technology to produce other antibodies or chimeric molecules which retain the specificity of the original antibody. Such techniques may involve introducing DNA encoding the immunoglobulin variable region, or the complementarity determining regions (CDRs), of an antibody to the constant regions, or constant regions plus framework regions, of a different immunoglobulin.

A oma or other cell producing an antibody may be t to genetic on or other changes, which may or may not alter the binding specificity of antibodies produced.

Production of Antibodies Certain methodologies for producing antibodies which have an affinity and binding specificity for the CD20 epitopes of the present invention are described hereinbefore.

The antibodies or antibody fragments of and for use in the present invention may also be ted wholly or partly by chemical synthesis. The antibodies can be readily ed according to well-established, standard liquid or, preferably, solid- phase peptide synthesis methods, general descriptions of which are y available and are well known by the person d in the art. Further, they may be prepared in solution, by the liquid phase method or by any combination of solidphase , liquid phase and solution chemistry.

Another convenient way of producing antibodies or antibody fragments suitable for use in the present invention is to express nucleic acid encoding them by use of nucleic acid in an expression system.

Nucleic acid for use in accordance with the present invention may comprise DNA or RNA and may be wholly or partially synthetic. Nucleic acid for use in the invention may code for antibodies or antibody fragments of the invention as defined above.

The skilled person will be able to determine tutions, deletions and/or 1O additions to such nucleic acids which will still provide an antibody or dy fragment of the t invention. c acid sequences encoding antibodies or antibody nts for use with the present invention can be readily prepared by the skilled person using the information and references contained herein and techniques known in the art given the nucleic acid sequences and clones available. These techniques include (i) the use ofthe polymerase chain reaction (PCR) to amplify samples of such c acid, e.g. from genomic sources, (ii) chemical synthesis, or (iii) preparing cDNA sequences. DNA encoding dy fragments may be ted and used in any suitable way known to those of skill in the art, including by taking encoding DNA, identifying suitable restriction enzyme ition sites either side of the n to be expressed, and cutting out said portion from the DNA. The portion may then be operably linked to a suitable promoter in a standard commercially available expression system. Another recombinant approach is to amplify the relevant portion of the DNA with suitable PCR primers. Modifications to the sequences can be made, e.g. using site directed mutagenesis, to lead to the expression of ed peptide or to take account of codon preferences in the host cells used to express the nucleic acid.

The nucleic acid may be comprised as constructs in the form ofa plasmid, , transcription or expression cassette which comprises at least one nucleic acid as described above. The construct may be comprised within a recombinant host cell which ses one or more ucts as above. Expression may conveniently be achieved by culturing under appropriate conditions recombinant host cells containing the nucleic acid. Following production by expression the antibody or antibody fragments may be isolated and/or purified using any suitable technique, then used as appropriate. s for cloning and expression of a polypeptide in a variety of different host cells are well known. Suitable host cells include bacteria, mammalian cells, yeast, insect and baculovirus systems. Mammalian cell lines available in the art for 1O expression of a heterologous ptide include Chinese hamster ovary (CHO) cells, HeLa cells, baby hamster kidney cells, NSO mouse myeloma cells. A common, preferred bacterial host is E. coli. The expression of antibodies and antibody fragments in prokaryotic cells such as E. coli is well established in the art.

Expression in eukaryotic cells in culture is also available to those skilled in the art as an option for production of a binding member.

Recombinant nucleic acids comprising an insert coding for a heavy chain variable domain and/or for a light chain variable domain of antibodies may be ed. By definition such nucleic acids comprise coding single stranded nucleic acids, double stranded nucleic acids consisting of said coding nucleic acids and of complementary nucleic acids thereto, or these mentary (single stranded) nucleic acids themselves. Furthermore, nucleic acids ng a heavy chain le domain and/or a light chain le domain of antibodies can be enzymatically or chemically synthesised nucleic acids having the authentic ce coding for a naturally-occurring heavy chain variable domain and/or for the light chain variable domain, or a mutant thereof.

Recombinant DNA technology may be used to improve the antibodies of the invention. Thus, chimeric antibodies may be constructed in order to se the immunogenicity thereof in stic or therapeutic applications. In order to reduce immunogenicity within a recipient, the invention may employ recombinant nucleic acids comprising an insert coding for a heavy chain variable domain of an antibody WO 54127 fused to a canine or feline constant domain. Likewise the invention concerns recombinant DNAs comprising an insert coding for a light chain variable domain of an antibody fused to a canine constant domain K or 7t.

Antibodies may moreover be generated by mutagenesis of antibody genes to produce artificial repertoires of antibodies. This technique allows the preparation of antibody libraries. Antibody libraries are also available commercially. Hence, the present invention advantageously employs artificial repertoires of immunoglobulins, preferably cial scFv repertoires, as an immunoglobulin 1O source in order to identify binding molecules which have specificity for the epitope of the present invention.

Antibody selection systems Immunoglobulins which are able to bind to the epitope ofthe present invention and which accordingly may be used in the methods of the ion can be identified using any technique known to the skilled person. Such immunoglobulins may be conveniently ed from libraries comprising cial repertoires of immunoglobulin polypeptides. A "repertoire" refers to a set of molecules generated by random, semi-random or directed variation of one or more template molecules, at the nucleic acid level, in order to provide a licity of binding specificities.

Methods for ting repertoires are well characterised in the art.

Any library selection system may be used in ction with the ion.

Selection protocols for isolating desired members of large libraries are known in the art, as typified by phage display techniques. Such systems, in which diverse peptide sequences are displayed on the surface of filamentous iophage, have proven useful for creating libraries of antibody fragments (and the nucleotide ces that encode them) for the in vitro selection and amplification of specific antibody fragments that bind a target antigen. The nucleotide sequences encoding the VH and VL regions are linked to gene fragments which encode leader signals that direct them to the periplasmic space ofE. coli and as a result the resultant dy fragments are displayed on the surface of the bacteriophage, typically as fusions to WO 54127 bacteriophage coat proteins (e. g.,plll or pVIII). Alternatively, dy fragments are displayed externally on lambda phage s (phage bodies). An advantage of phage-based display systems is that, because they are biological s, selected library members can be amplified simply by g the phage ning the selected library member in bacterial cells. Furthermore, since the nucleotide sequence that encodes the polypeptide library member is contained on a phage or phagemid vector, sequencing, expression and subsequent genetic manipulation is relatively straight forward. Methods for the construction of bacteriophage antibody display libraries and lambda phage expression libraries are well known in the art.

An alternative to the use of phage or other cloned libraries is to use nucleic acid, preferably RNA, derived from the B cells of an animal which has been immunised with the selected target, e.g. the CD20 epitope ofthe present invention. Isolation of V-region and on mRNA permits antibody fragments, such as Fab or Fv, to be expressed intracellularly. , RNA is isolated from the B cells of an immunised animal, for example from the spleen of an immunised mouse or the circulating B cells of a llama, and PCR primers used to amplify VH and VL cDNA selectively from the RNA pool. The VH and VL sequences thus ed are joined to make scFv dies. PCR primer sequences may be based on hed VH and VL ces.

Peptidomimetics Peptide analogues, such as peptidomimetics or peptide mimetics are non-peptide compounds with properties representative of a template peptide. Such peptide analogues are typically developed using computerised molecular modelling.

Peptidomimetics which are structurally similar to peptides which have affinity and binding specificity to the CD20 binding epitope of the t invention may be used to mediate similar diagnostic, lactic and therapeutic effects.

Peptidomimetics are typically structurally similar to a template peptide, but have one or more peptide linkages replaced by an alternative linkage, by methods which are well known in the art. A peptide may further be modified from the natural sequence to protect the peptides from protease attack. 2012/052532 Se uence homolo identi The hide-bonded epitope of the present invention comprises an amino acid sequence with a disulphide bond between two cysteine residues as defined in SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5 or SEQ ID NO:6.

The term “identity" or "sequence identity" as used herein, means that at any particular amino acid residue position in an aligned sequence, the amino acid residue is identical between the aligned sequences. The term “similarity" or 1O “sequence similarity" as used herein, indicates that, at any particular position in the aligned sequences, the amino acid residue is of a r type between the sequences. For example, leucine may be substituted for an isoleucine or valine residue. This may be referred to as vative substitution. Preferably when the amino acid sequences of the invention are modified by way of conservative substitution of any of the amino acid residues contained therein, these changes have no effect on the g specificity or onal activity ofthe resulting antibody when compared to the unmodified antibody.

Sequence identity with respect to a (native) polypeptide of the invention and its functional derivative relates to the percentage of amino acid residues in the ate ce which are identical with the residues of the corresponding native polypeptide, after ng the sequences and introducing gaps, if necessary, to e the m percentage homology, and not considering any conservative substitutions as part of the sequence identity. Neither N- or C- terminal extensions, nor insertions shall be construed as reducing sequence identity or homology. Methods and computer programs for ming an alignment of two or more amino acid sequences and determining their sequence identity or gy are well known to the person skilled in the art. For example, the percentage of identity or similarity of 2 amino acid sequences can be readily calculated using algorithms e.g. BLAST (Altschul et al. 1990), FASTA (Pearson & Lipman 1988), or the Smith-Waterman algorithm (Smith & Waterman 1981).

The term "consists essentially of" or "consisting essentially of" as used herein means that a polypeptide may have onal features or elements beyond those described provided that such additional features or elements do not materially affect the ability of an antibody or antibody fragment to bind to the epitope defined by the polypeptide. That is, the polypeptides may have additional features or elements that do not interfere with their ability to present an epitope which can be bound by g agents which are specific for canine and/or feline CD20. For example, a ptide consisting essentially of a specified sequence may contain one, two, three, four, five or more additional, deleted or tuted amino acids, at 1O either end or at both ends ofthe sequence provided that these amino acids do not interfere with, inhibit, block or interrupt the role ofthe antibody or fragment in binding to canine or feline CD20. Similarly, a polypeptide ofthe invention may be chemically modified with one or more onal groups provided that such functional groups do not interfere with the function of the polypeptide.

The terms "polypeptide", "peptide", or in" are used interchangeably herein to designate a linear series of amino acid es connected one to the other by peptide bonds n the alpha-amino and carboxy groups of adjacent residues.

The amino acid residues are usually in the natural "L" isomeric form. However, residues in the "D" isomeric form can be substituted for any L—amino acid residue, as long as the desired functional property is retained by the polypeptide.

The invention s to the use ofthe peptide which has been determined as contributing to the epitope in binding to CD20 ligands. As such, the invention extends to polypeptide fragments ofthe amino acid of SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5 or SEQ ID NO:6 ofvarying lengths, wherein the fragments define a binding epitope ing to the t invention, which when bound by a ligand with a specific binding affinity, results in an inhibition of CD20 ed signalling.

The epitope may be provided in an isolated form in order to assist in the production of antibodies and binding fragments which have affinity and binding specificity to the identified binding epitope. Accordingly, the present invention extends to naturally occurring fragments and variants as well as derived variants of a polypeptide having the amino acid sequence of SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5 or SEQ ID NO:6.

A "variant" of a polypeptide having the amino acid sequence of SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5 or SEQ ID NO:6 means a polypeptide ntially gous to a polypeptide having the amino acid sequence of SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5 or SEQ ID NO:6, but which has an amino acid 1O sequence ent from that of the polypeptide having the amino acid sequence of SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5 or SEQ ID NO:6 because of one or more deletions, insertions, or substitutions. The variant has an amino acid sequence that preferably is at least 80% identical to the ptide having the amino acid ce ofSEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5 or SEQ ID NO:6, most preferably at least 90% identical. The percentage identity may be determined, for example, by comparing sequence information using the GAP computer program, version 6.0 described by Devereux et al. (Nucl. Acids Res. 12:387, 1984) and available from the University of sin Genetics Computer Group (UWGCG).

The present invention extends to peptides which are variants, tes or homologues of a polypeptide having the amino acid sequence of SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5 or SEQ ID NO:6, such peptides may have a sequence which has at least about 30%, or 40%, or 50%, or 60%, or 70%, or 75%, or 80%, or 85%, or 90% or 95% gy to the sequence of a polypeptide having the amino acid sequence ofSEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5 or SEQ ID NO:6. Thus, a peptide fragment of any one ofthe peptides of the invention may include 1, 2, 3, 4, 5, or greater than 5 amino acid alterations.

Moreover, or in addition, the peptide may consist of a truncated version of a polypeptide having the amino acid sequence of SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5 or SEQ ID NO:6 which has been truncated by 1, 2, 3, 4 or 5 amino acids. A given amino acid may be replaced, for example, by a residue having similar physiochemical characteristics. Examples of such conservative tutions include substitution of one aliphatic residue for another, such as He, Val, Leu, or Ala for one r; substitutions of one polar residue for another, such as between Lys and Arg, Glu and Asp, or Gln and Asn; or substitutions of one aromatic e for another, such as Phe, Trp, or Tyr for one another. Other conservative substitutions, e.g., involving substitutions of entire regions having similar hydrophobicity characteristics, are well known. 1O Similarly, polynucleotides of the invention include variants that differ from a native polynucleotide ce because of one or more deletions, insertions or substitutions, but that encode a biologically active polypeptide. Expression, ion and purification of polypeptides defining the e of the invention and fragments thereof may be accomplished by any suitable technique.

A method for producing ptides comprises culturing host cells transformed with a recombinant expression vector encoding a polypeptide having the amino acid sequence ofSEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5 or SEQ ID NO:6 under conditions that e expression of the polypeptide in a disulphide bonded form, then recovering the sed polypeptides from the culture. The skilled person will recognise that the procedure for purifying the expressed polypeptides will vary according to such factors as the type of host cells employed, and whether the polypeptide is intracellular, membrane-bound or a soluble form that is secreted from the host cell.

Any suitable expression system may be employed. The vectors include a DNA encoding a polypeptide or fragment ofthe invention, operably linked to suitable transcriptional or translational tory nucleotide sequences, such as those derived from a mammalian, avian, microbial, viral, bacterial, or insect gene. tide sequences are operably linked when the regulatory sequence onally relates to the DNA sequence. Thus, a promoter nucleotide sequence is operably linked to a DNA sequence if the er nucleotide sequence controls the ription of the DNA sequence. An origin of replication that confers the ability to replicate in the desired (E.coli) host cells, and a selection gene by which transformants are identified, are generally incorporated into the expression vector.

In on, a sequence encoding an appropriate signal peptide (native or heterologous) can be incorporated into expression vectors. A DNA sequence for a signal peptide (secretory leader) may be fused in frame to the c acid ce of the invention so that the DNA is initially transcribed, and the mRNA translated, into a fusion protein comprising the signal peptide. A signal peptide that is 1O functional in the intended host cells promotes ellular secretion of the polypeptide. The signal peptide is cleaved from the polypeptide during translation, but allows secretion of polypeptide from the cell.

Suitable host cells for expression of polypeptides include higher eukaryotic cells and yeast. Prokaryotic systems are also suitable. Mammalian cells, and in particular CHO cells are particularly preferred for use as host cells.

Administration A monoclonal antibody or fusion protein of the present invention may be stered alone, but will preferably be administered as a pharmaceutical composition, which will generally comprise a suitable pharmaceutically acceptable excipient, diluent or carrier selected depending on the intended route of stration. Examples of suitable pharmaceutical carriers include; water, glycerol, ethanol and the like.

The antibody ofthe present invention may be administered to a canine or feline in need of treatment via any suitable route. As ed herein, it is preferred that the composition is administered parenterally by injection or on. Examples of preferred routes for parenteral administration include, but are not d to, intravenous, intracardial, intraarterial, intraperitoneal, intramuscular, avity, subcutaneous, transmucosal, tion or transdermal. Routes of administration WO 54127 may further include topical and enteral, for example, mucosal (including pulmonary), oral, nasal and rectal.

In preferred embodiments the composition is deliverable as an injectable composition. For intravenous, intradermal or subcutaneous application, the active ingredient will be in the form of a parenterally acceptable aqueous solution which is pyrogen-free and has suitable pH, isotonicity and stability. Those of relevant skill in the art are well able to e suitable solutions using, for example, isotonic vehicles such as sodium chloride injection, Ringer’s injection or Lactated Ringer’s 1O ion. Preservatives, stabilisers, buffers, antioxidants and/or other additives may be included as required. The composition may also be administered via microspheres, liposomes, other microparticulate delivery systems or sustained release formulations placed in certain tissues including blood.

The composition is preferably administered to an individual in a “therapeutically effective amount", this being sufficient to show benefit to the t to whom the composition is stered. The actual dose administered, and rate and time- course of stration, will depend on, and can be determined with due reference to, the nature and severity ofthe condition which is being treated, as well as factors such as the age, sex and weight of the canine to be treated and the route of administration. Further due consideration should be given to the properties of the ition, for example, its binding ty and in-vivo plasma life, the concentration of the fusion protein in the formulation, as well as the route, site and rate of ry.

Dosage regimens can include a single administration of the composition of the invention, or multiple administrative doses of the composition. The compositions can further be administered sequentially or separately with other eutics and ments which are used for the treatment of the condition for which the fusion protein of the present invention is being administered to treat.

Examples of dosage regimens which can be administered to a subject can be selected from the group comprising, but not limited to; lug/kg/day through to 20mg/kg/day, lug/kg/day h to 10mg/kg/day, 10ug/kg/day through to 1mg/kg/day.

The actual amount administered, and rate and time-course of administration, will depend on the nature and severity of what is being treated. Prescription of treatment, e.g. decisions on dosage etc, is ultimately within the responsibility and at the discretion of veterinary or medical s, and typically takes account of the 1O disorder to be treated, the condition ofthe canine, the site of delivery, the method of administration and other factors known to practitioners.

Definitions Unless otherwise defined, all technical and scientific terms used herein have the meaning commonly understood by a person who is skilled in the art in the field of the present invention.

Throughout the ication, unless the context s otherwise, the terms “comprise" or “include", or variations such as "comprises" or "comprising", "includes" or "including" will be understood to imply the inclusion of a stated integer or group of integers, but not the exclusion of any other integer or group of integers.

As used herein, terms such as "a", "an" and "the" include singular and plural nts unless the context y demands otherwise. Thus, for example, reference to "an active agent" or "a cologically active agent" includes a single active agent as well as two or more ent active agents in combination, while references to "a carrier" es mixtures of two or more carriers as well as a single carrier, and the like.

The nomenclature used to describe the cyclic polypeptide fragment of the present invention follows the conventional ce n the amino group (N) is presented to the left and the carboxy group to the right of each amino acid residue.

The expression “amino acid" as used herein is intended to include both natural and synthetic amino acids, and both D and L amino acids. A synthetic amino acid also encompasses chemically modified amino acids, including, but not limited to salts, and amino acid derivatives such as amides. Amino acids present within the polypeptides of the present invention can be modified by methylation, amidation, 1O acetylation or substitution with other al groups which can change the circulating half-life without adversely affecting their biological activity.

The terms "peptide", “polypeptide" and "protein" are used herein interchangeably to describe a series of at least two amino acids covalently linked by peptide bonds or modified peptide bonds such as isosteres. No limitation is placed on the maximum number of amino acids which may comprise a peptide or protein. Furthermore, the term polypeptide extends to fragments, analogues and tives of a e, wherein said fragment, analogue or derivative retains the same ical functional activity as the peptide from which the fragment, derivative or analogue is derived.

Furthermore the term “fusion protein" as used herein can also be taken to mean a fusion polypeptide, fusion peptide or the like, or may also be ed to as an immunoconjugate. The term “fusion protein" refers to a le in which two or more subunit molecules, typically polypeptides, are covalently or non-covalently .

As used herein, the term “therapeutically ive amount" means the amount of a binding agent of the invention which is required to reduce the severity of and/or ameliorate a B-cell mediated disease or at least one symptom thereof.

As used herein, the term “treatment" and associated terms such as “treat" and ing" means the reduction ofthe progression, severity and/or duration of a B- cell mediated ion of at least one symptom thereof, wherein said reduction or amelioration results from the administration of a binding compound which has specificity for the CD20 binding epitope of the present invention. The term “treatment" ore refers to any regimen that can t a subject. The treatment may be in respect of an eXisting condition or may be prophylactic (preventative treatment). Treatment may include curative, alleviative or lactic effects. References herein to "therapeutic" and "prophylactic" treatments are to be considered in their broadest context. The term "therapeutic" does not necessarily imply that a subject is treated until total recovery. Similarly, 1O "prophylactic" does not necessarily mean that the subject will not eventually contract a disease condition.

As defined herein, a e" may also be referred to as a dog. Canines can be categorised as belonging to the subspecies with the trinomial name Canis lupus familiaris (Canis familiaris icus) or Canis lupus dingo. Canines include any species of dog and includes both feral and pet varieties, the latter also being referred to as companion s.

As defined herein, a e" may also be referred to as a cat. Felines can be categorised as belonging to the subspecies with the trinomial name Felis silvestris catus. Felines include any species of cat and includes both feral and pet varieties, the latter also being referred to as companion animals.

The present invention will now be described with reference to the following examples which are provided for the purpose of illustration and are not intended to be construed as being limiting on the t invention. The methods and techniques of the present invention are generally performed according to conventional methods well known in the art and as bed in various general and more specific references that are cited and sed hout the present specification unless otherwise indicated.

EXAMPLES WO 54127 Example 1: Binding of murine anti-human CD20 antibodies to the cyclic polypeptide of SEQ ID NO:2 The peptide epitope was ed by chemical synthesis and oxidation with an N- terminal biotin moiety attached. This peptide and homologous biotinylated and oxidised human CD20 peptide (IYNCEPANPSEKNSPSTQYC (SEQ ID N0:3) and murine CD20 peptide (IYDCEPSNSSEKNSPSTQYC (SEQ ID NO:6), were coated onto streptaVidin coated ELISA plates, then washed and tested for binding of murine anti- human CD20 dies.

One antibody, Bl-H299 (Beckmann Coulter, hereafter referred to as H299 to distinguish it from the commercially available Biogenex Bl monoclonal antibody) was shown to bind ically to the canine (C20) and human (H20) CD20 es, but not to the murine (M20) CD20 peptide. The results ofthe binding of H299 to the epitope are shown in Figure 2A. The binding to the canine CD20 peptide e was approximately one sixth that ofthe binding to human CD20 peptide.

Although H299 was shown to bind to canine CD20, Biogenex Bl did not. Bl-H299 (H299) and Biogenex Bl (BG) monoclonal antibodies were incubated with canine CD20 peptide or to the homologous human CD20 peptide (IYNCEPANPSEKNSPSTQYC (SEQ ID NO:6)) and binding was detected using a secondary anti-mouse polyclonal HRP conjugate in an ELISA assay. The results are shown in Figure 2B. Binding to an isotype control antibody is shown (Iso). It is not known whether the d binding of the Biogenex Bl monoclonal antibody to peptide when compared with H299 is due to lack of affinity or r it simply reflects inaccuracy in the concentration of the Biogenex monoclonal antibody preparation supplied.

Example 2: H299 monoclonal antibody and intact ar CD20 binding study In order to confirm that the binding of the H299 monoclonal antibody to the canine CD20 epitope loop was ient for binding to intact cellular CD20, the canine and human CD20 were expressed on the surface of CHO cells and tested for binding of H299 by fluorescence activated cell sorter (FACS).

H299 binds both intact human and canine CD20 expressed on CHO cells. The results are shown in Figures 3A-D. Figure 3A shows the binding of H299 (6 ug/ml) to canine CD20 CHO cells. Figure SB shows the binding ofH299 (6 ug/ml) to human CD20 CHO cells. Figure 3C shows the binding of secondary anti-mouse PE antibody (Sigma P9287) alone to canine CD20 CHO cells. Figure 3D shows the binding of secondary anti-mouse PE antibody (Sigma P9287) alone to human CD20 CHO cells.

Example 3: ison of the binding of Blyl and H299 uman CD20 1O monoclonal antibodies to canine CD20 peptide SEQ ID NO:2 Binding of Bly1 and H299 monoclonal antibodies to the ed e loop of canine CD20 (SEQ ID NO:2) was compared by ELISA and on the e of transfected CHO cells by FACS. Microtitre plate wells were coated with oxidised peptide at 50ug/ml. H299 or Bly1 MAbs were added at increasing concentrations from 1.25-10 ug/ml and developed using polyclonal anti-mouse IgG horseradish dase (HRP).

The results are shown in Figure 4. The H299 monoclonal antibody bound with higher affinity than the Bly1 monoclonal antibody to the oxidised canine CD20 peptide under these conditions.

Example 4: H299 and Blyl onal antibodies and intact cellular CD20 binding study In order to confirm that the binding of the Type II anti-human CD20 monoclonal antibodies H299 and Bly1 to the canine CD20 epitope loop was sufficient for binding to intact ar CD20, the canine and human CD20 polypeptides (SEQ ID NO:2 and SEQ ID NO:3) were expressed on the surface of CHO cells and tested for binding of H299 or Bly1 as indicated by fluorescence intensity and the percentage of positive cells in the sample using a fluorescence activated cell sorter (FACS).

The results are shown in Figures 5A-F. The Type II anti-human CD20 monoclonal antibodies H299 and Bly 1 bind human and canine CD20 expressed on CHO cells to a similar extent. Figures 5A and 5B show the binding of the H299 monoclonal antibody ml) to canine and human CD20 CHO cells as indicated by mean fluorescence intensity (Fig 5A) and by the percentage of positive cells (Fig 5B).

Figures 5C and 5D show the binding of the Bly1 monoclonal antibody ml) to canine and human CD20 CHO cells as indicated by mean fluorescence intensity (Fig 5C) and by the percentage ofpositive cells (Fig 5D). Figures 5E and SF show FACS histograms of binding of H299 monoclonal antibody and Bly1 monoclonal antibody respectively to canine CD20-CHO cells; isotype control (Iso) and samples prepared without detection antibody (-D) are ted.

Example 5: Demonstration that the binding to canine CD20 by Type II monoclonal antibody H299 is critically dependent on hide g The biotinylated oxidised canine peptide SEQ ID NO:2 was bound to the surface of avidin-coated microtitre plate wells alongside wells coated with the homologous biotinylated human CD20 e (IYNCEPANPSEKNSPSTQYC (SEQ ID NO:3)). Half ofthe e coated wells were treated with dithiothreitol (DTT) to reduce the disulphide bonds and then washed. The binding of the H299 monoclonal dy was then assessed either in the ce or absence of continuing DTT.

An ELISA plate (Reacti-Bind NeutrAvidin Coated with blocker BSA, Thermo Scientific, catalogue number 15123) was incubated overnight at 4°C with 100ul or 50ug/ml of human or canine CD20 peptides in PBS. The plate was washed three times with PBS and the peptides reduced by incubating with 100ul of 15 mM DTT in PBS, at room temperature, for 30 minutes. The PBS/DTT solution was removed and the plate was incubated with 100 pl of B1 monoclonal antibody at 500ng/ml in either PBS or 5mM DTT/PBS, for 1 hour at room temperate. Mouse lgGZa K lsotype Control (ebiosciences, catalogue number 1681) was used as control antibody at 500ng/ml. The plate was washed three times with PBS and incubated with 100 pl detection dy (Sigma, Polyclonal Rabbit Anti-Mouse immunoglobulins/HRP catalogue number P 0161) diluted to 1 in 5000 in PBS. After washing three times with PBS, 100 pl of substrate was added and the reaction allowed to develop. Stop solution was added (100 pl, 2M H2S04) and the absorbance of the wells was read at 450 nm.

The results are shown in Figure 6 and these show that the binding of H299 to the canine CD20 epitope is ally ent on it being oxidised, since pre-treatment with DTT abolished all binding to the canine peptide. Binding to the homologous human CD20 peptide was reduced, but not eliminated by oxidation and this is tent with the ability of H299 to bind to linear peptides of the same region described previously (Teeling et al., 2006) in which the cysteine residues were each replaced by Alanine. The lack ofbinding of H299 to linear (reduced) canine peptide loop is consistent with the failure ofthe prior art to detect binding of uman CD20 monoclonal antibodies to a linear, larger peptide of the putative canine CD20 extracellular domain (Beall et al.). It is also consistent with the findings of Niederfellner at al. (2011) which showed binding of H299 to linear es (8mers) d from the human CD20 sequence.

Example 6: Example of sed form of the GA101 monoclonal antibody In this example, the inventor caninised the Type II anti-human CD20 onal antibody GA101 by way of substituting framework region amino acid sequences in order to reduce the immunogenicity ofthe antibody when it is administered to canines. No alteration was made to the amino acid sequence of the CDR domains.

Furthermore, the "caninised" heavy and light chain variable domains were conjoined to canine derived constant domains.

The amino acid sequence of the caninised GA101 light chain variable domain is shown in SEQ ID NO:15, with SEQ ID NO:7-10( FR1 - DIVMTQTPLSLSVTPEEPASISC (SEQ ID NO:7), FRZ — WYLQKPGQSPQLLIY (SEQ ID NO:8), FR3 — GVPDRFSGSGSGTDFTLKISRVEAEDVGVYYC (SEQ ID NO:9), FR4 — FGAGTKVEIK (SEQ ID NO:10), showing the ork regions (FR1 to FR4).

Further, the amino acid sequence ofthe caninised GA101 variable domain is shown in SEQ ID NO:16, with SEQ ID NO:11-14 (FR1- EVQLVQSGGEVVKPGGSLKVSCVASGF (SEQ ID NO:11), FRZ- WVRQAPGQGMEWVG (SEQ ID NO:12), FR3 — RVTITRDNSKSTAYLELSSLRSEDTAVYYCAR (SEQ ID NO:13), FR4- WGQGTLVTVSS (SEQ ID ) showing the ork regions (FR1 to FR4).

Figure 7 shows the complete caninised GA101 VK kappa variable light chain amino sequence (SEQ ID NO:15) n the CDR region residues are shown in bold and also the complete caninised GA101 VH variable heavy chain (SEQ ID NO:16) wherein the CDR region residues are shown in bold. Furthermore, Tables 1-8 show the amino acid residue substitutions used in the process of caninising the GA101 monoclonal antibody light and heavy chain variable domain framework regions.

Table 1 - Light chain variable domain FR1 residues Light chain Kabat light chain FR1 position numbering Caninised position GA101 WO 54127 -—-- “mn— -—-—— Table 2 - Light chain le domain FRZ residues Light Chain Kabal light chain Canine VK GA101 Caninised FR2 Position numbering system GA101 ——-l-l- Table 3 - Light chain variable domain FR3 residues Light chain Kabat light chain Canine GA101 Caninised FR3 position ing position VK WO 54127 Light chain Kabat light chain Canine GA101 Caninised FR3 position numbering position VK GA10 1 Table 4 - Light chain variable domain FR4 residues Light chain Kabat light chain Canine GA101 Caninised FR4 on numbering position Table 5 - Heavy chain variable domain FR1 residues WO 54127 Heavy chain Kabat heavy chain Canine GA101 Caninised FR1 position numbering position VH GA10 1 QHRAV EKLPS WO 54127 FLIDST Table 6 - Heavy chain variable domain FRZ residues Heavy chain Kabat heavy chain Canine VH FR2 position ing system GA101 Caninised GA101 ——-—— ——-n— ——--— Table 7 - Heavy chain variable domain FR3 residues Heavychahl Kabatheavychanl Cannua GA101 Cannused FR3 position ing position GA10 1 AGVSDP 75 KRENQ T K 11 76 NDSKH S S WO 54127 Heavy chain Kabat heavy chain Canine GA101 Caninised FR3 position numbering position VH GA101 ——-n— —-_lllll- VMILFT Table 8 - Heavy chain variable domain FR4 residues Heavy chain Kabat heavy chain Canine GA101 Caninised FR4 on numbering position VH GA101 DNA encoding full length caninised GA-101 heavy and light chains (SEQ ID NO:17 and SEQ ID NO:18) was transfected into CHO cells and the supernatant tested for binding by ELISA to canine CD20 cyclic peptide SEQ ID NO:2. The dose response indicates that like other Type II anti-human CD20 dies, and its parent mouse antibody Bly-1, the caninised GA101 also binds to canine CD20. Figure 8 shows that when expressed as a complete canine antibody the caninised variable domains of GA101 bind to the cyclic canine CD20 peptide of Figure 1 (SEQ ID NO:2). This confirms the data with Bly-1 above (and by extension its humanised variant GA101) that this is a further example of a Type II uman CD20 monoclonal antibody that also binds to a common epitope on canine CD20, thus validating the use of the structurally constrained shared epitope on canine CD20 as a novel target for monoclonal antibodies.

Example 7: Novel Type II monoclonal antibodies RAZ and RFZ Using a combination of cyclic human (SEQ ID N0:3) and canine (SEQ ID N0:2) CD20 peptides, monoclonal antibodies that would bind both human and canine peptides were screened for using tional oma screening of mice previously immunised with the cyclic canine CD20 peptide of Figure 1, coupled to diphtheria toxoid at the amino terminus as a source of T-cell help. Two antibody 1O hybridomas were selected, RAZ and RFZ and tested for binding to these as well as to murine (SEQ ID N0:6) and feline (SEQ ID N0:4) CD20 peptides. The results are shown in Figure 9. RAZ and RFZ bound strongly to canine CD20 and RFZ bound strongly to human CD20 peptide whereas RAZ binds more weakly. Unexpectedly RAZ and RFZ bound strongly to feline and also murine CD20 peptides. This novel binding n suggests an overlapping epitope with that of the Type II anti-human antibody H299 (also shown in Figure 9).

The mRNA encoding the variable heavy and light chains of RAZ and RFZ was extracted using a Qiagen kit and cDNA were prepared by RT-PCR using mouse immunoglobulin specific oligonucleotide primers (Novagen) and a cript III first strand synthesis system kit. The PCR products were sequenced using Novagen IgG kappa ic constant domain e primers. The derived ces are shown in Figure 10 (a,b muRAZ VH and VL; c,d muRFZ VH and VL. The sequences are related (Figure 10 e), but with several differences in the CDR regions.

The RAZ and RFZ variable heavy and light chain sequences (SEQ ID NO:23-26) were rebuilt as IgGZa/kappa dies by oligonucleotide-based gene synthesis, cloned into pcDNA3.3 vectors and co-expressed in appropriate pairs in CHO cells. The supernatant sed antibodies were purified by Protein A chromatography and tested for binding by CD20 peptide ELISA (Figure 11). Both purified recombinant forms of mouse RAZ and RFZ bound strongly to cyclic canine and feline CD20 peptides, and also to mouse and human CD20 peptides (the binding of RAZ to 2012/052532 human CD20 peptide was more pronounced than with the corresponding hybridoma supernatant in Figure 9).

Thus confirmation of binding of RAZ and RFZ antibodies to cyclic canine, human, mouse and feline CD20 epitopes suggests a more constrained epitope than that shared by the Type II anti-human CD20 antibodies exemplified by H299 and Bly1.

Figure 12 illustrates an alignment of the corresponding CD20 peptides from each species, from which the common epitope SEKNS can be derived (SEQ ID NO:67).

SEKNS (SEQ ID NO:67) is a sub-epitope of PSEKNSL (SEQ ID N0:1) ting an 1O unexpectedly small e common to each of these species was derived by immunisation and selection for RAZ and RFZ.

In order to make RAZ and RFZ useful in therapy of canines and felines, chimeric canine, caninised and sed antibodies were designed (as per caninised GA101 above) and expressed and purified from CHO cells using the techniques described above for the expression and purification of inant mouse RAZ and RFZ.

Example 8: Chimeric canine and caninised RAZ and RFZ antibodies Chimeric mouse-canine forms of RAZ heavy (isotypes A,B,C,D) and light chains are described by SEQ ID NO:27, 28,29, 30 and 31.

Chimeric mouse-canine forms of RFZ heavy pes A,B,C,D) and light chains are described by SEQ ID NO:32, 33, 34, 35 and 36.

Caninised RAZ variable heavy (VH) and light (VL) domains are described by SEQ ID NO:37 and 38. Full caninised RAZ antibody heavy (isotypes D) and light chains are described by SEQ ID NO:40, 41, 42 43 and 44.

Caninised RFZ variable heavy (VH) and light (VL) domains are described by SEQ ID NO:44 and 45. Full caninised RFZ antibody heavy (isotypes A,B,C,D) and light chains are described by SEQ ID NO:46, 47, 48, 49 and 50.

Figure 15 shows a GE gel experiment in which purified mouse RAZ and RFZ, chimeric RAZ (HCB Isotype SEQ ID NO:28 and 31) and RFZ (HCB isotype SEQ ID NO:33 and 36) and caninised RAZ (HCB isotype SEQ ID NO:40 and 43; HCC Isotype SEQ ID NO:41 and 43) and RFZ antibodies (HCB isotype SEQ ID N0:47 and 50; HCC isotype SEQ ID NO:48 and 50) were separated under reducing and non-reducing conditions. HCB and HCC isotypes were selected for their ability to recruit complement, a desirable feature in antibodies with potential use for example in the treatment and elimination of CD20-expressing lymphoma cells in vivo in dogs.

Tetrameric HZLZ antibodies are shown in the upper non-reducing gel at 1O approximately 150 kDa molecular weight. Bands corresponding to heavy and light chains can be seen in the lower reducing gel at approximately 50 kDa and 25 kDa respectively.

The purified antibodies shown in Figure 15 were tested for binding to cyclic CD20 es from different species by ELISA as above. Figure 16a shows binding of chimeric and sed RAZ and RFZ antibodies to cyclic canine CD20 e.

Figure 16b shows binding to peptides from ent species with binding specificities broadly similar to those ofthe parent mouse antibodies (c.f Figure 11).

Example 9: Chimeric and Caninised RAZ and RFZ anti-CD20 antibodies have active Fc domains Mechanisms that enable the elimination of canine CD20-expressing cells in therapy of disease (e.g. in dogs suffering from canine CD20+ lymphoma, or from B cell- mediated inflammatory diseases) e tment of the effector arms of the immune system following binding of anti-CD20 antibodies. Figures 17 and 18 demonstrate that the selected canine heavy chains HCB and HCC used in construction of the recombinant chimeric and caninised RAZ and RFZ antibodies are functional in ting complement (Figure 17, C1q ELISA) and in binding to canine CD64, the high affinity Fc receptor which es antibody dependent cellular xicity (Figure 18). Figure 17 shows binding of complement C1q to monoclonal antibodies constructed using four different heavy chain isotypes (HCA, HCB, HCC, HCD). Panel A shows, by way of example, that anti-canine NGF antibodies bearing canine HCA and HCD isotypes do not t complement, whereas those with HCB and HCC do recruit complement. Hence, we ucted the recombinant anti-canine CD20 antibodies (for which effector function is desirable) as HCB and HCC isotypes. As can be seen from Panels B and C, both the mouse lgGZa variant of RFZ (muRFZ) and each of the chimeric HCB (chiRAZ-B, chiRFZ-B), caninised HCB (caRAZ-B, caRFZ-B) and caninised HCC (caRAZ-C, caRFZ-C) isotype variants bind to complement as evidenced by the positive immunoreactivity to complement C1q by ELISA. Binding to C1q indicates that these antibodies will mediate complement- dependent cytotoxicity (CDC). All forms of chimeric and caninised antibodies based 1O on the HCB and HCC isotype were positive in this assay.

Figure 18 shows binding of soluble recombinant canine high affinity Fc receptor 64) to chimeric and caninised RAZ and RFZ. The binding to CD64 indicates that these antibodies will on via antibody-dependent ar cytotoxicity (ADCC). All forms of chimeric and caninised antibodies based on the HCB and HCC isotypes were positive in this assay.

Example 10: Felinised RAZ and RFZ antibodies Given the strong and unexpected binding of RAZ and RFZ to cyclic feline CD20 peptide and the success of design and construction of caninised versions therefrom, felinised and chimeric feline versions of RAZ and RFZ would be ble for the treatment of feline B-cell ed es. SEQ ID NO:51, 52, 53 and 54 respectively represent sed variable domains of RAZ and RFZ heavy and light chains respectively. These may be constructed into fully feline versions of RAZ and RFZ antibodies using feline constant domains using the methods illustrated using murine and canine versions above.

All documents referred to in this specification are herein incorporated by nce.

Various modifications and variations to the described embodiments of the inventions will be apparent to those skilled in the art without departing from the scope ofthe invention. Although the invention has been described in connection with specific preferred ments, it should be tood that the invention as claimed should not be unduly limited to such specific embodiments. Indeed, various cations of the described modes of carrying out the invention which are obvious to those skilled in the art are intended to be covered by the present invention.

Throughout this specification and the claims which follow, unless the context requires otherwise, the word "comprise", and variations such as ises" and "comprising", will be understood to imply the inclusion of a stated integer or step or group of integers or steps but not the exclusion of any other r or step or group of integers or steps.

The reference in this specification to any prior ation (or information derived from it), or to any matter which is known, is not, and should not be taken as an acknowledgment or admission or any form of suggestion that that prior publication (or information derived from it) or known matter forms part of the common general dge in the field of endeavour to which this specification relates.

Claims (3)

CLAIMS :

1. A method of preparing an antibody or an antigen binding fragment thereof that specifically binds to a cyclic polypeptide nt of CD20 from a target s, the method comprising the steps of: - assessing the sequence of the framework regions of a donor antibody from a species other than the target species, wherein the donor antibody has binding specificity for CD20 from the target species; - comparing the sequence of the framework regions of the donor antibody with the sequence of the framework regions of an antibody or a pool of antibodies derived from the target species; and - modifying the framework regions of the donor antibody to substitute amino acid residues that are foreign at a corresponding position in an antibody or a pool of antibodies from the target species with amino acid residues present at the corresponding position in the target species; n the modified framework regions do not contain any amino acids in any position that would be foreign at that position in the target species.

2. The method as claimed in claim 1, wherein the target species is selected from the group consisting of canine, feline and human.

3. A sed antibody or an antigen binding fragment thereof which binds specifically to a cyclic polypeptide fragment of canine CD20, wherein the antibody comprises complementarity determining regions of a heavy and a light chain from a donor antibody from a species other than a canine, wherein the donor antibody has binding specificity for the cyclic ptide fragment of canine CD20, wherein the ork regions of the heavy and light chains from the donor antibody are ed to substitute amino acid residues that are foreign at a corresponding on in canine antibodies with amino acid residues present at the corresponding position in canine dies, and wherein the ed framework regions do not contain any amino acids in any position that would be foreign at that position in canine antibodies. H:\amt\Interwoven\NRPortbl\DCC\AMT\7505592_1.docx-