CHIMERIC CANINE ANTI-CD20 ANTIBODY

Abstract

The disclosure relates to caninized chimeric anti-CD20 antibodies to the canine protein CD20 and methods of use to treat certain disorders such as non-Hodgkins B-cell lymphoma in dogs.

Description

SEQUENCE LISTING

[0001] The instant application contains a Sequence Listing which has been submitted electronically in ASCII format and is hereby incorporated by reference in its entirety. Said ASCII copy, created on Feb. 10, 2016, is named "P20914 sequence listing" and is 14 Kb in size.

[0002] The present invention relates to caninized chimeric anti-CD20 antibodies to the canine protein CD20 and methods of use to treat certain disorders such as non-Hodgkins B-cell lymphoma in dogs.

[0003] The present invention is in the field of treatment of cancer.

[0004] Canine lymphomas are a diverse group of cancers, and are among the most common cancers diagnosed in dogs. They collectively represent approximately 7-14% of all cancers diagnosed in dogs. There are over 30 described types of canine lymphoma, and these cancers vary tremendously in their behavior. Some progress rapidly and are acutely life-threatening without treatment, while others progress very slowly and are managed as chronic, indolent diseases. Lymphomas may affect any organ in the body, but most commonly originate in lymph nodes, before spreading to other organs such as the spleen, liver, and hone marrow.

[0005] CD20 is a cell-surface protein with four transmembrane spanning regions. The CD20 molecule is involved in regulation of B-cell proliferation and differentiation. The CD20 antigen is present exclusively on the surface of almost all B-cells, both normal and malignant.

[0006] Rituximab is a chimeric monoclonal antibody against the human protein CD20 which is primarily found on the surface of immune system B-cells. Rituximab destroys B-cells and is therefore used to treat diseases in humans which are characterized by excessive numbers of B-cells, overactive B-cells, or dysfunctional B-cells. This includes many lymphomas, leukemias, transplant rejection, and autoimmune disorders.

[0007] Rituximab destroys both normal and malignant human B-cells that have CD20 on their surfaces. The effect results in the elimination of B-cells (including the cancerous ones) from the body, allowing a new population of healthy B-cells to develop from phoid stem cells.

[0008] Unfortunately, a cure for canine lymphomas still remains elusive and there exists a need for more and different therapies that may prove to be effective in treating them.

[0009] K9L0-133 is a chimeric anti-CD20 antibody that targets mature B-lymphocytes in dogs. K9L0-133 binds to CD2( )on canine B-lymphocytes. K9L0-133 is potentially useful for treatment for non-Hodgkins B cell lymphoma and other types of lymphoma in dogs.

[0010] Accordingly, the present invention provides an antibody that specifically binds to canine CD20. The present invention also provides a method of treating lymphoma in a canine patient by administering to a lymphoma cancer canine patient in need of such treatment an effective amount of an antibody that specifically binds to canine CD20. The present invention provides for a pharmaceutical composition including an antibody that specifically binds to canine CD20, and one or more pharmaceutically acceptable carriers, diluents, or excipients. The present invention also provides an antibody that specifically binds to canine CD20 and also binds to protein A.

BRIEF DESCRIPTION OF DRAWINGS

[0011] FIG. 1 depicts the serum levels of B-lymphocytes CD2 I+ and CD22+ and T-lymphocytes CD4+ and CD8+ from -5 to 37 days in dogs treated with K9LO-133 antibody.

DETAILED DESCRIPTION

[0012] Unless indicated otherwise, the term "antibody" (Ab) refers to an immunoglobulin molecule comprising two heavy chains (HC) and two light chains (LC) interconnected by disulfide bonds. The amino terminal portion of each chain includes a variable region of about 100 to about 110 amino acids primarily responsible for antigen recognition via the complementarity determining regions (CDRst contained therein. The carboxy-terminal portion of each chain defines a constant region primarily responsible for effector function.

[0013] As used herein, the term "antigen-binding fragment" refers to any antibody fragment that retains the ability to bind to its antigen. Such "antigen-binding fragments" can be selected from the group consisting of Fv, scFv, Fab, F(ab').sub.2, Fab', scFv-Fc fragments and diabodies. An antigen-binding fragment of an antibody will typically comprise at least one variable region. Preferably, an antigen-binding fragment comprises a heavy chain variable region (HCVR) and a light chain variable region (LCVR). More preferably, an antigen-binding fragment as used herein comprises a HCVR and a LC\IR which confers antigen-binding specificity to canine CD20( )(i.e., a "canine CD20 binding fragment").

[0014] As used herein, the terms "complementarity determining region" and "CDR", refer to the non-contiguous antigen combining sites found within the variable region of LC and HC polypeptides of an antibody or an antigen-binding fragment thereof. These particular regions have been described by others including Kabat, et al., Ann. N.Y. Acad. Sci, 190:382-93(1971); Kabat et al, J. Biol. Chem, 252:6609-6616 (1977); Kabat, et al, Sequences of Proteins of Immunological Interest, Fifth Edition, U.S. Department of Health and Human Services, NIH Publication No, 91-3242 (1991); Chothia, et al, J. Mol. Biol. 196:901-917 (1987); MacCallum, et al., J. Mol. Biol., 262:732-745 (1996); and North, et al., J. Mol. Biol., 406, 228-256 (2011), where the definitions include overlapping or subsets of amino acid residues when compared against each other.

[0015] As used herein, the term "light chain variable region" refers to a portion of a LC of an antibody molecule that includes amino acid sequences of Complementarity Determining Regions (CDRs; i.e., LCDR1, LCDR2, and LCDR3), and Light Framework Regions (LERWs).

[0016] As used herein, the term "heavy chain variable region (HCNIR)" refers to a portion of a RC of an antibody molecule that includes amino acid sequences of Complementarity Determining Regions (CDRs; i.e., HCDR1, HCDR2, and HCDR3), and Heavy Framework Regions (HFRWs).

[0017] The CDRs are interspersed with regions that are more conserved, termed framework regions ("FRW"), Each LCVR and HCVR. is composed of three CDRs and four FRWs, arranged from amino-terminus to carboxy-terminus in the following order: FRW1, CDR1, FRW2, CDR2, FRW3, CDR3, FRW4. The three CDRs of the light chain are referred to as "LCDR I, :LCDR2, and LCDR3" and the three CDRs of the I1(are referred to as "HCDR1, HCDR2, and HCDR3." The CDRs contain most of the residues which form specific interactions with the antigen. The numbering and positioning of CDR amino acid residues within the LCVR and HCVR regions is in accordance with known conventions (e.g., Kabat (1991), Chothia (1987), and/or North (2011)). In different embodiments of the invention, the FRWs of the antibody may be identical to the germline sequences, or may be naturally or artificially modified.

[0018] In certain embodiments, the anti-CD2O Ab for the methods and/or uses of the present invention is altered to increase or decrease the extent to which the antibody is glycosylated. Addition or deletion of glycosylation sites to an antibody may be conveniently accomplished by altering the amino acid sequence such that one or more glycosylation sites is created or removed.

[0019] Unless indicated otherwise, when referring to an amino acid residue in an antibody by a number, the EU numbering system is used herein as it is conventionally used in the art (see, Kabat, et al., Sequences of Proteins of Immunological Interest, Fifth Edition, U.S. Department of Health and :Human Services, NIII Publication No, 91-3242 (1991), for example).

[0020] As used herein, the term "kit" refers to a package comprising at least two separate containers, wherein a first container contains a K9LO-133 Ab and a second container contains pharmaceutically acceptable carriers, diluents, or excipients. As used herein, the term "kit" also refers to a package comprising at least two separate containers, wherein a first container contains K9LO-133 Ab, and another antibody preferably for the treatment of cancers other than lymphomas. A "kit" may also include instructions to administer all or a portion of the contents of these first and second containers to a cancer patient. Optionally, these kits also include a third container containing a composition comprising a known chemotherapeutic agent.

[0021] As used herein, the terms "treating," "to treat," or "treatment" refers to restraining, slowing, stopping, reducing, or reversing the progression or severity of an existing symptom, disorder, condition, or disease.

[0022] As used herein, the term "effective amount" refers to the amount or dose of a. caninized anti-CD20 Ab which, upon single or multiple dose administration to the patient, provides an effective response in the patient under diagnosis or treatment.

[0023] As used herein, the terms "effective response" of a patient or a patient's "responsiveness" to treatment with a combination of agents, or "therapeutic effect" refers to the clinical or therapeutic benefit(s) imparted to a patient upon administration of a caninized anti-CD20 Ab. Such benefit(s) include any one or more of: extending survival (including overall survival and progression free survival); resulting in an objective response (including a complete response or a partial response); decreasing amount of B-cells, decreasing concentration of B-cells in a patient's blood or other tissues and fluids tumor regression, tumor weight or size shrinkage, longer time to disease progression, increased duration of survival, longer progression free survival, improved overall response rate, increased duration of response, and improved quality of life and/or improving signs or symptoms of cancer, etc.

[0024] An effective amount can be readily determined by the attending diagnostician, as one skilled in the art, by the use of known techniques and by observing results obtained under analogous circumstances. In determining the effective amount for a patient, a number of factors are considered by the attending diagnostician, including, but not limited to: the species or breed of patient; its size, age, and general health; the specific disease or disorder involved; the degree of or involvement or the severity of the disease or disorder; the response of the individual patient; the particular compound administered; the mode of administration;

[0025] the bioavailability characteristics of the preparation administered; the dose regimen selected; the use of concomitant medication; and other relevant circumstances.

K9LO-133

[0026] AME-133V is a second generation humanized IgG 1. monoclonal antibody. K9LO-133 is a partially caninized (human variable regions with canine constant regions) isotype C monoclonal antibody version of AME-133V that specifically binds to CD20 protein in dogs.

[0027] In an embodiment, K9LO-133 contains engineered variable regions of the heavy and light chains, CDRs and frameworks. In an embodiment, a residue variant in LCDR1 of K9LO-133 is introduced. In an embodiment, the residue variant in LCDR1of K9LO-133 is V165G. In an embodiment, a variant hinge region is introduced. In an embodiment, a variant region downstream of the hinge is added. In an embodiment, the variant region downstream of the hinge allows K9LO-133 to bind to protein A.

[0028] K9LO-133 has a heavy chain amino acid sequence corresponding to SEQ ID NO: 1 and a corresponding nucleotide sequence SEQ ID NO: 2 that encodes for the amino acid of SEQ ID NO: 1. K9LO-133 has a heavy chain variable region sequence of SEQ ID NO: 3. The heavy chain variable sequence region further consists of HFWK 1 (SEQ ID NO: 4), HCDR1 (SEQ ID NO: 5), HFWK 2 (SEQ ID NO: 6), HCDR2 (SEQ ID NO: 7), HFWK3 (SEQ ID NO: 8), and HCDR3 (SEQ ID NO: 9).

[0029] K9LO-133 has a light chain amino acid sequence corresponding to SEQ ID NO: 10 and a corresponding nucleotide sequence SEQ ID NO: 11 that encodes for the amino acid of SEQ ID NO: 10. K9LO-133 has a light chain variable region sequence of SEQ ID NO: 12. The light chain variable sequence region further consists of LFWK 1 (SEQ ID NO: 13), LCDR1 (SEQ ID NO: 14), LFWK 2 (SEQ ID NO: 15), LCDR2 (SEQ ID NO: 16), LFWK 3 (SEQ ID NO: 17), LCDR3 (SEQ.sub.. ID NO: 18), and a J region (SEQ ID NO: 19).

CD 2O Expression in Canine Lymphoma Tissue

[0030] CD 2O expression in canine lymphoma tissue can be evaluated using immunohistochemistry (IHC) with AME-133V and K9LO-133. In order to detect binding, AME-133V and K9LO-133 can be applied to cryosections of canine lymphoma samples at one concentration, for example10 .mu.g/mL. In addition, AME-133V and K9LO-133 can be substituted with an appropriate species and isotype matched negative control antibody which has a different antigenic specificity from that of detection antibodies, for example, either human IgG1, designated HuIgGl, (for AME-133V) and dog IgG, designated DgIgG (for

[0031] K9LO-133). Other controls can be produced by omission of the detection antibodies or negative control antibodies from the assay (assay control).

[0032] When tested essentially as described above, AME-133V and K9LO-133 produced 3+ staining of >75% of the positive control mononuclear leucocytes. For K9LO-133, 3+ staining was observed in lymphocytes in the white pulp in canine spleen. AME-133'V and K9LO-133 did not specifically react with the negative control smooth myocytes (specifically nuclei for K9LO-133) in canine spleen. The negative control antibodies, HuIg,G1 and DgIgG, did not specifically react with either the positive or negative control tissue elements in canine spleen. There also was no staining of the assay control slides. The specific reactions of AME-133V and K9LO-133 with the positive control tissue element and the lack of specific reactivity with the negative control tissue element, as well as the lack of reactivity of the negative control antibody, indicated that the assay was sensitive, specific, and reproducible.

[0033] CD20 was detected in >75% of the neoplastic cells in 8 of 10 canine lymphoma samples examined. In the other samples, 1-5% of neoplastic cells were positively stained for CD20.

K9LO -133 Treated Dogs

[0034] The human dose for Rituximab for the treatment of non-Hodgkin's lymphoma is 375 mg/m.sup.2. Generally, a canine chemotherapeutic dose is 40% of a human dose, irrespective of cancer type. Therefore, and without being bound by theory, an appropriate starting dose of an anti-CD20 antibody for canine non-Hodgkin's lymphoma is about 150 mg/m.sup.2. In order to adjust for body surface area of a 7 kg dog, the 150 mg/m.sup.2 number was corrected by multiplying by 0.37. The resulting dose of K9LO-133 administered to the dogs was about 57 mg/dog.

[0035] In an embodiment, the dose of K9LO-133 administered to a dog is from about 140 mg to about 160 mg per meter squared of body surface area. In an embodiment, the dose of K9LO-133 administered to a dog is from about 130 mg to about 170 mg per meter squared of body surface area. In an embodiment, the dose of K9LO-133 administered to a dog is from about 110 mg to about 190 mg per meter squared of body surface area. In an embodiment, the dose of K9LO-133 administered to a dog is from about 50 mg to about 250 mg per meter squared of body surface area.

[0036] In an embodiment, the dose of K9LO-133 administered to a dog is from about 50 mg/dog to about 60 mg/dog. In another embodiment, the dose of K.9LO-133 administered to a dog is from about 40 mg/dog to about 70 mg/dog. In an embodiment, the dose of K9LO-133 administered to a dog is from about 30 mg/dog to about 100 mg/dog. In an embodiment, the dose of K9LO-133 administered to a dog is from about 10 mg/dog to about 1000 mg/dog.

[0037] The efficacy of K.9LO-133 was determined in dogs by dosing them intravenously (IV) at about 150 mg/m.sup.2 on Days 0, 7, and 35. Three female Beagle dogs were acclimated to study conditions for seven days during which they were subjected to physical examinations; body weight measurements; and daily clinical observations. On dosing days.sub.; dogs were dosed with 57 mg of K9L0-133 via slow IV infusion at a rate of 0.5 to 1 mL/min.

[0038] Blood was collected for determination of lymphocyte proportions and serum retention on Days -5, 2, 9, 16, 23, 30, and 37.

[0039] As depicted in FIG. 1, there was a drop in the proportion of blood B-lymphocytes after treatment with K9LO-133. The proportion of B-lymphocytes (CD21+ and CD22+) dropped on Day 2, showed a recovery between Days 2 and 30, but had not yet returned to baseline levels by Day 37. CD8+ T-lymphocytes showed a decrease on Day 16 but had returned to baseline levels by Day 23. The proportion of CD4+ T-lymphocytes remained relatively constant.

[0040] Thus, IV administration of K9LO-133 reduced B-lymphocyte counts in circulation with a transient decrease in CD8+ T-lymphocytes in treated dogs.

[0041] In an aspect, disclosed herein is an antibody having a light chain iable region (LCVR) whose amino acid sequence is that given in SEQ ID NO: 12, and a heavy chain variable region (HCVR) whose amino acid sequence is that given in SEQ ID NO: 3. In an embodiment, the antibody specifically binds to canine CD20.

[0042] In another aspect, disclosed herein is an antibody having a light chain (LC) whose amino acid sequence is that given in SEQ ID NO: 10, and a heavy chain (HC) whose amino acid sequence is that given in SEQ ID NO: 1 is disclosed. In an embodiment, the antibody specifically binds to canine CD20.

[0043] In an aspect, disclosed herein is an antibody having two light chains (LC) each having an amino acid sequence that is given in SEQ ID NO: 10, and two heavy chains (HC) each having an amino acid sequence that is given in SEQ ID NO: 1, In an embodiment, the antibody specifically binds to canine CD20.

[0044] In another aspect, a method of treating lymphoma in a canine patient is disclosed that includes administering to a lymphoma cancer canine patient in need of such treatment an effective amount of an antibody having a light chain variable region (LCVR) whose amino acid sequence is that given in SEQ ID NO: 12, and a heavy chain variable region (HCVR) whose amino acid sequence is that given in SEQ ID NO: 3, and the antibody specifically binds to canine CD20. In an embodiment, the antibody has a light chain (LC) whose amino acid sequence is that given in SEQ ID NO: 10, and a heavy chain (HC) whose amino acid sequence is that given in SEQ ID NO: 1. In an embodiment, the antibody is K9LO-133.

[0045] In an aspect, a kit is disclosed that has a pharmaceutical composition containing K9LO -133 with one or more pharmaceutically acceptable carriers, diluents, or excipients.

[0046] In another aspect, an antibody is disclosed having a light chain (LC) whose amino acid sequence is that given in SEQ ID NO: 10, and a heavy chain (HC) whose amino acid sequence is that given in SEQ ID NO: 1, and the antibody specifically binds to canine CD20, and the antibody binds to protein A.

EXAMPLES

K9LO-133

[0047] K9LO-133 can be supplied in 10 mM citrate, 150 mM NaCL, pH 6.5 at 26.3 mg/mL. The vehicle can be physiological saline at 0.9% NaCI.

[0048] Dosing of K9LO-133 can be based on body surface area, which was estimated for the dogs in this study to be 0.37 m.sup.2. Each dog received 150 mg/m.sup.2 (57 mg) of K9LO-133 at each dose. Doses were prepared by drawing up 57 mg (2.2 mL) of K9LO-133 and diluting in 27.8 mL of physiologic saline to bring the total volume to 30 mL for infusion.

Flow Cytometry

[0049] T-lymphocyte (CD4+ and CD8+) and B-lymphocyte (CD21+ and CD22+) amounts can be determined by flow cytometry. Blood collection for flow cytometry was performed prior to feeding. Approximately 1 mL of EDTA anti-coagulated blood was provided from 3 dogs at baseline (day -5), and on Days 2, 9, 16, 23, 30 and 37. Red blood cells were lysed with ammonium chloride hypotonic buffer, and leukocytes were stained with antibodies reactive with canine CD4, CD8, C,D21. and CD22. Then, unbound antibodies were removed and the acquisition of 10,000 leukocyte events in a FACScan or a FACSAria flow cytometer were obtained.

Lymphoma Samples

[0050] Ten frozen canine lymphoma samples were trimmed and embedded in Tissue-Tek.RTM. OCT (Optimal Cutting Temperature) compound and stored in a freezer set to maintain -80 .degree. C. until sectioning. Sections were cut at approximately 5 .mu.m to generate an appropriate number of slides for subsequent IHC staining.

Immunohistochemistry Staining Sections from each lymphoma sample were stained using AME-133V, a human IgG1 monoclonal antibody directed against human CD20, and K9LO-133, a chimeric canine isotype C monoclonal antibody directed against canine CD20, using IHC as detailed below. AME-133V was in a 19.5 mg/mL solution in phosphate-buffered saline (PBS), pH 7.4. K9LO-133 was in a 2.5 mg/mL solution in PBS, pH 7.4.

[0051] IHC staining was performed including use of positive and negative control materials in each staining run and using additional slides for the negative control antibody and assay control to ensure stain specificity. The staining procedures, primary and secondary antibody dilutions, and controls for AME-133V were qualified in preliminary staining runs. The staining procedures, primary and secondary antibody dilutions, and controls for K9LO-133 were qualified in a methods development staining run.

AME-133V Staining

[0052] The indirect immunoperoxidase procedure can be used to stain the canine lymphoma tissue with AME-133V to detect CD20. Slides were fixed in acetone for 10 minutes at room temperature at the time of sectioning. Acetone-fixed ci osections were rinsed twice in phosphate-buffered saline, 0.15 M NaCl, pH 7.2 PBS. Endogenous peroxidase was then quenched by incubation of the slides with Biocare PeroxAbolish for 5 minutes at room temperature. Next, the slides were rinsed twice with PBS, incubated with the avidin solution for 15 minutes, rinsed once with PBS, incubated with the biotin solution for 15 minutes, and rinsed once with PBS. The slides were then treated with a protein block designed to reduce nonspecific binding for 2.0 minutes. The protein block was prepared as follows: PBS+1% bovine serum albumin (BSA); 0.5% casein; and 3% donkey serum.

[0053] Following the protein block, the primary antibodies AME-133V, Human IgG1, or none were applied to the slides at a concentration of 10 .mu.g/mL for 1 hour. Then, the slides were rinsed twice with PBS, and the biotinylated secondary antibody (donkey anti-human IgG) was applied to the slides for 30 minutes. Next, the slides were rinsed twice with PBS, reacted for 30 minutes with the ABC Elite reagent, and rinsed twice with PBS. Next, DAB was applied for 4 minutes as a substrate for the peroxidase reaction. All slides were rinsed with tap water, counterstained, dehydrated, and mounted. PBS+1% BSA served as the diluent for primary antibodies and ABC Elite reagent. (PBS+1% BSA)+canine IgG (1:25 dilution) served as the diluent for the secondary antibody.

K9-LO-133 Staining

[0054] The indirect immunoperoxidase procedure can be used to stain the canine lymphoma tissue with K9LO-133 to detect CD20. The requirement for labeling (e.g. biotin, peroxidase, or fluorescein) of K9LO-133 and preclusion of nonspecific reactivity between the secondary.sup., labeled anti-canine IgG and IgG endogenous to the tissues examined can be eliminated according to the following process. The labeled secondary antibody was allowed to attach specifically to the unlabeled primary antibody (K9LO-133, DgIgG, or none) by overnight incubation of the primary/secondary antibody mixtures prior to application to the tissue cryosections. The detection reagent or negative control antibody (at a concentration of 10 .mu.g/mL) was mixed with biotinylated rabbit anti-dog IgG, Fc fragment-specific antibody (Rb.alpha.DgIgG) at a concentration of 15 .mu.g/mL to achieve a primary:secondary antibody ratio of 1:1.5 on the day prior to staining.

[0055] Precomplexed antibodies were incubated overnight on a rocker mechanism in a refrigerator set to maintain 4.degree. C. Prior to use of the antibody on the subsequent day, dog gamma globulins were added to each vial to achieve a final concentration of 3 mg/mL, and then incubated for at least 2 hours on the rocker mechanism in a refrigerator set to maintain 4.degree. C. Slides were fixed in acetone for 10 minutes at room temperature at the time of sectioning, On the day of staining, the slides were rinsed twice with Tris-buffered saline, 0.15M NaCl, pH 7.6 TBS. Endogenous peroxidase was then quenched by incubation of the slides with an appropriate reagent such as Biocare PeroxAbolish reagent for 5 minutes at room temperature. Next, the slides were rinsed twice with TBS, incubated with the avidin solution for 15 minutes, rinsed once with TBS, incubated with the biotin solution for 15 minutes, and rinsed once with TBS. The slides were then treated with a protein block designed to reduce nonspecific binding for 20 minutes.

[0056] The protein block was prepared as follows: TBS+1% bovine serum albumin (BSA);

[0057] 0.5% casein; and 3% normal rabbit serum. Following the protein block, the precomplexed primary and secondary antibodies were applied to the slides for two hours. Next, the slides were rinsed twice with TBS, treated with the ABC Elite reagent for 30 minutes, rinsed twice with TBS, and then treated with DAB for 4 minutes as a substrate for the peroxidase reaction. All slides were rinsed with tap water, counterstained, dehydrated, and mounted. TBS -1- 1%

[0058] BSA served as the diluent for all antibodies and ABC reagent. Each staining run included a positive control (mononuclear leucocytes), and a negative control (smooth myocytes) of cryosections of canine spleen.

TABLE-US-00001 SEQUENCE LISTING PRT; Artificial Sequence SEQ ID NO: 1 EVQLVQSGAEVKKPGESLKISCKGSGRTFTSYNMHWVRQMPGKGLEWMGAI YPLTGDTSYNQKSKLQVTISADKSISTAYLQWSSLKASDTAMYYCARSTYV GGDWQFDVWGKGTTVTVSSASTTAPSVFPLAPSCGSQSGSTVALACLVSGY IPEPVTVSWNSGSLTSGVHTFPSVLQSSGLYSLSSMVTVPSSRWPSETFTC NVAHPATNTKVDKPVPKRENGRVPRPPDCPKCPAPELLGGPSVFIFPPKPK DTLLIARTPEVTCVVVDLDPENPEVQISWFVDSKQVQTANTQPREEQSNGT YRVVSVLPIGIGHQDWLSGKQFKCKVNNKALPSPIEKYRMTPPQLDEDGSY FLYSKLSVDKSRWQRGDTFICAVMHEALHNHYTQISLSHSPGK; DNA; Artificial Sequence SEQ ID NO: 2 GAGGTGCAGCTGGTGCAGTCTGGAGCAGAGGTGAAAAAGCCCGGGGAGTCT CTGAAGATCTCCTGTAAGGGTTCTGGCCGTACATTTACCAGTTACAATATG CACTGGGTGCGCCAGATGCCCGGGAAAGGCCTGGAGTGGATGGGGGCTATT TATCCCTTGACGGGTGATACTTCCTACAATCAGAAGTCGAAACTCCAGGTC ACCATCTCAGCCGACAAGTCCATCAGCACCGCCTACCTGCAGTGGAGCAGC CTGAAGGCCTCGGACACCGCCATGTATTACTGTGCGAGATCGACTTACGTG GGCGGTGACTGGCAGTTCGATGTCTGGGGCAAGGGGACCACGGTCACCGTC TCCTCAGCCTCCACCACGGCCCCCTCGGTTTTCCCGCTAGCGCCCAGCTGT GGGTCCCAATCCGGCTCCACGGTGGCCCTGGCCTGCCTGGTGTCAGGCTAC ATCCCCGAGCCTGTAACTGTGTCCTGGAATTCCGGCTCCTTGACCAGCGGT GTGCACACCTTCCCGTCCGTCCTGCAGTCCTCAGGGCTCTACTCCCTCAGC AGCATGGTGACAGTGCCCTCCAGCAGGTGGCCCAGCGAGACCTTCACCTGA ATGTGGCCCACCCGGCCACCAACACTAAAGTAGACAAGCCAGTGCCCAAAA GAGAAAATGGAAGAGTTCCTCGCCCACCTGATTGTCCCAAATGCCCAGCCC CTGAACTGCTGGGAGGGCCTTCGGTCTTCATCTTTCCCCCAAAACCCAAGG ACACCCTCTTGATTGCCCGAACACCTGAGGTCACATGTGTGGTGGTGGATC TGGACCCAGAAAACCCTGAGGTGCAGATCAGCTGGTTCGTGGATAGTAAGC AGGTGCAAACAGCCAACACGCAGCCTCGTGAGGAGCAGTCCAATGGCACCT ACCGTGTGGTCAGTGTCCTCCCCATTGGGCACCAGGACTGGCTTTCAGGGA AGCAGTTCAAGTGCAAAGTCAACAACAAAGCCCTCCCATCCCCCATTGAGG AGATCATCTCCAAGACCCCAGGGCAGGCCCATCAGCCTAATGTGTATGTCC TGCCGCCATCGCGGGATGAGATGAGCAAGAATACGGTCACCCTGACCTGTC TGGTCAAAGACTTCTTCCCACCTGAGATTGATGTGGAGTGGCAGAGCAATG GACAGCAGGAGCCTGAGAGCAAGTACCGCATGACCCCGCCCCAGCTGGATG AAGATGGGTCCTACTTCCTATACAGCAAGCTCTCCGTGGACAAGAGCCGCT GGCAGCGGGGAGACACCTTCATATGTGCGGTGATGCATGAAGCTCTACACA ACCACTACACACAGATATCCCTCTCCCATTCTCCGGGTAAATGATGATAG; PRT; Artificial Sequence SEQ ID NO: 3 EVQLVQSGAEVKKPGESLKISCKGSGRTFTSYNMHWVRQMPGKGLEWMGAI YPLTGDTSYNQKSKLQVTISADKSISTAYLQWSSLKASDTAMYYCARSTYV GGDWQTDVWGKGTTVTVSS; PRT; Artificial Sequence SEQ ID NO: 4 EVQLVQSGAEVKKPGESLKISC; PRT; Artificial Sequence SEQ ID NO: 5 KGSRTFTSYNMH; PRT; Artificial Sequence SEQ ID NO: 6 WVRQMPGKGLEWMG; PRT; Artificial Sequence SEQ ID NO: 7 AIYPLTGDTSYNQKSKL; PRT; Artificial Sequence SEQ ID NO; 8 QVTISADKSISTAYLQWSSLKASDTAMYYC; PRT; Artificial Sequence SEQ ID NO: 9 ARSTYVGGDWQFDV; PRT; Artificial Sequence SEQ ID NO: 10 EIVLTQSPGTLSLSPGERATLSCRASRSVPYIHWYQQKPGQAPRLLIYATS ALASGIPDRFSGSGSGTDFTLTISRLEPEDFAVYYCQQWLSNPPTFGQGTK LEIKRNDAQPAVYLFQPSPDQLHTGSASVVCLLNSFYPKDINVKWKVDGVI QDTGIQESVTEQDKDSTYSLSSTLTMSSTEYLSHELYSCEITHKSLPSTLI KSFQRSECQRVD; DNA; Artificial Sequence SEQ ID NO; 11 GAAATTGTGTTGACGCAGTCTCCAGGCACCCTGTCTTTGTCTCCAGGGGAA AGAGCCACCCTCTCCTGCAGGGCCAGCCGGAGTGTACCGTACATCCACTGG TACCAGCAGAAACCTGGCCAGGCTCCCAGGCTCCTCATCTATGCCACATCC GCTCTGGCTTCTGGCATCCCAGACAGGTTCAGTGGCAGTGGGTCTGGGACA GACTTCACTCTCACCATCAGCAGACTGGAGCCTGAAGATTTTGCAGTGTAT TACTGTCAGCAGTGGCTGAGTAACCCACCCACTTTTGGCCAGGGGACCAAG CTGGAGATCAAACGAAATGATGCCCAGCCAGCCGTCTATTTGTTCCAACCA TCTCCAGACCAGTTACACACAGGAAGTGCCTCTGTTGTGTGCTTGCTGAAT AGCTTCTACCCCAAAGACATCAATGTCAAGTGGAAAGTGGATGGTGTCATC CAAGACACAGGCATCCAGGAAAGTGTCACAGAGCAGGACAAGGACAGTACC TACAGCCTCAGCAGCACCCTGACGATGTCCAGTACTGAGTACCTAAGTCAT GAGTTGTACTCCTGTGAGATCACTCACAAGAGCCTGCCCTCCACCCTCATC AAGAGCTTCCAAAGGAGCGAGTGTCAGAGAGTGGAC; PRT; Artificial Sequence SEQ ID NO: 12 EIVLTQSPGTLSLSPGERATLSCRASRSVPYTHWYQQKPGQAPRLLIYATS ALASGIPDRFSGSGSGTDFTLTISRLEPEDFAVYYCQQWLSNPPTFGQGTK LEIK; PRT; Artificial Sequence SEQ ID NO: 13 EIVLTQSPGTLSLSPGERATLSC; PRT; Artificial Sequence SEQ ID NO: 14 RASRSVPYIH; PRT; Artificial Sequence SEQ ID NO; 15 WYQQKPGQAPRLLI; PRT; Artificial Sequence SEQ ID NO: 16 YATSALAS; PRT; Artificial Sequence SEQ ID NO: 17 GIPDRFSGSGSGTDFTLTISRLEPEDFAVYYC; PRT; Artificial Sequence SEQ ID NO; 18 QQWLSNPPT; PRT; Artificial Sequence SEQ ID NO: 19 FGQGTKLEIK;

Sequence CWU 1

1

191456PRTArtificial SequenceChimeric human/dog anti-CD20 antibody 1Glu Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Glu1 5 10 15Ser Leu Lys Ile Ser Cys Lys Gly Ser Gly Arg Thr Phe Thr Ser Tyr 20 25 30Asn Met His Trp Val Arg Gln Met Pro Gly Lys Gly Leu Glu Trp Met 35 40 45Gly Ala Ile Tyr Pro Leu Thr Gly Asp Thr Ser Tyr Asn Gln Lys Ser 50 55 60Lys Leu Gln Val Thr Ile Ser Ala Asp Lys Ser Ile Ser Thr Ala Tyr65 70 75 80Leu Gln Trp Ser Ser Leu Lys Ala Ser Asp Thr Ala Met Tyr Tyr Cys 85 90 95Ala Arg Ser Thr Tyr Val Gly Gly Asp Trp Gln Phe Asp Val Trp Gly 100 105 110Lys Gly Thr Thr Val Thr Val Ser Ser Ala Ser Thr Thr Ala Pro Ser 115 120 125Val Phe Pro Leu Ala Pro Ser Cys Gly Ser Gln Ser Gly Ser Thr Val 130 135 140Ala Leu Ala Cys Leu Val Ser Gly Tyr Ile Pro Glu Pro Val Thr Val145 150 155 160Ser Trp Asn Ser Gly Ser Leu Thr Ser Gly Val His Thr Phe Pro Ser 165 170 175Val Leu Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Met Val Thr Val 180 185 190Pro Ser Ser Arg Trp Pro Ser Glu Thr Phe Thr Cys Asn Val Ala His 195 200 205Pro Ala Thr Asn Thr Lys Val Asp Lys Pro Val Pro Lys Arg Glu Asn 210 215 220Gly Arg Val Pro Arg Pro Pro Asp Cys Pro Lys Cys Pro Ala Pro Glu225 230 235 240Leu Leu Gly Gly Pro Ser Val Phe Ile Phe Pro Pro Lys Pro Lys Asp 245 250 255Thr Leu Leu Ile Ala Arg Thr Pro Glu Val Thr Cys Val Val Val Asp 260 265 270Leu Asp Pro Glu Asn Pro Glu Val Gln Ile Ser Trp Phe Val Asp Ser 275 280 285Lys Gln Val Gln Thr Ala Asn Thr Gln Pro Arg Glu Glu Gln Ser Asn 290 295 300Gly Thr Tyr Arg Val Val Ser Val Leu Pro Ile Gly His Gln Asp Trp305 310 315 320Leu Ser Gly Lys Gln Phe Lys Cys Lys Val Asn Asn Lys Ala Leu Pro 325 330 335Ser Pro Ile Glu Glu Ile Ile Ser Lys Thr Pro Gly Gln Ala His Gln 340 345 350Pro Asn Val Tyr Val Leu Pro Pro Ser Arg Asp Glu Met Ser Lys Asn 355 360 365Thr Val Thr Leu Thr Cys Leu Val Lys Asp Phe Phe Pro Pro Glu Ile 370 375 380Asp Val Glu Trp Gln Ser Asn Gly Gln Gln Glu Pro Glu Ser Lys Tyr385 390 395 400Arg Met Thr Pro Pro Gln Leu Asp Glu Asp Gly Ser Tyr Phe Leu Tyr 405 410 415Ser Lys Leu Ser Val Asp Lys Ser Arg Trp Gln Arg Gly Asp Thr Phe 420 425 430Ile Cys Ala Val Met His Glu Ala Leu His Asn His Tyr Thr Gln Ile 435 440 445Ser Leu Ser His Ser Pro Gly Lys 450 45521377DNAArtificial SequenceChimeric human/dog anti--CD20 antibody 2gaggtgcagc tggtgcagtc tggagcagag gtgaaaaagc ccggggagtc tctgaagatc 60tcctgtaagg gttctggccg tacatttacc agttacaata tgcactgggt gcgccagatg 120cccgggaaag gcctggagtg gatgggggct atttatccct tgacgggtga tacttcctac 180aatcagaagt cgaaactcca ggtcaccatc tcagccgaca agtccatcag caccgcctac 240ctgcagtgga gcagcctgaa ggcctcggac accgccatgt attactgtgc gagatcgact 300tacgtgggcg gtgactggca gttcgatgtc tggggcaagg ggaccacggt caccgtctcc 360tcagcctcca ccacggcccc ctcggttttc ccgctagcgc ccagctgtgg gtcccaatcc 420ggctccacgg tggccctggc ctgcctggtg tcaggctaca tccccgagcc tgtaactgtg 480tcctggaatt ccggctcctt gaccagcggt gtgcacacct tcccgtccgt cctgcagtcc 540tcagggctct actccctcag cagcatggtg acagtgccct ccagcaggtg gcccagcgag 600accttcacct gcaatgtggc ccacccggcc accaacacta aagtagacaa gccagtgccc 660aaaagagaaa atggaagagt tcctcgccca cctgattgtc ccaaatgccc agcccctgaa 720ctgctgggag ggccttcggt cttcatcttt cccccaaaac ccaaggacac cctcttgatt 780gcccgaacac ctgaggtcac atgtgtggtg gtggatctgg acccagaaaa ccctgaggtg 840cagatcagct ggttcgtgga tagtaagcag gtgcaaacag ccaacacgca gcctcgtgag 900gagcagtcca atggcaccta ccgtgtggtc agtgtcctcc ccattgggca ccaggactgg 960ctttcaggga agcagttcaa gtgcaaagtc aacaacaaag ccctcccatc ccccattgag 1020gagatcatct ccaagacccc agggcaggcc catcagccta atgtgtatgt cctgccgcca 1080tcgcgggatg agatgagcaa gaatacggtc accctgacct gtctggtcaa agacttcttc 1140ccacctgaga ttgatgtgga gtggcagagc aatggacagc aggagcctga gagcaagtac 1200cgcatgaccc cgccccagct ggatgaagat gggtcctact tcctatacag caagctctcc 1260gtggacaaga gccgctggca gcggggagac accttcatat gtgcggtgat gcatgaagct 1320ctacacaacc actacacaca gatatccctc tcccattctc cgggtaaatg atgatag 13773121PRTArtificial SequenceChimeric human/dog anti-CD20 antibody 3Glu Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Glu1 5 10 15Ser Leu Lys Ile Ser Cys Lys Gly Ser Gly Arg Thr Phe Thr Ser Tyr 20 25 30Asn Met His Trp Val Arg Gln Met Pro Gly Lys Gly Leu Glu Trp Met 35 40 45Gly Ala Ile Tyr Pro Leu Thr Gly Asp Thr Ser Tyr Asn Gln Lys Ser 50 55 60Lys Leu Gln Val Thr Ile Ser Ala Asp Lys Ser Ile Ser Thr Ala Tyr65 70 75 80Leu Gln Trp Ser Ser Leu Lys Ala Ser Asp Thr Ala Met Tyr Tyr Cys 85 90 95Ala Arg Ser Thr Tyr Val Gly Gly Asp Trp Gln Phe Asp Val Trp Gly 100 105 110Lys Gly Thr Thr Val Thr Val Ser Ser 115 120422PRTArtificial SequenceChimeric human/dog anti-CD20 antibody 4Glu Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Glu1 5 10 15Ser Leu Lys Ile Ser Cys 20513PRTArtificial SequenceChimeric human/dog anti-CD20 antibody 5Lys Gly Ser Gly Arg Thr Phe Thr Ser Tyr Asn Met His1 5 10614PRTArtificial SequenceChimeric human/dog anti-CD20 antibody 6Trp Val Arg Gln Met Pro Gly Lys Gly Leu Glu Trp Met Gly1 5 10717PRTArtificial SequenceChimeric human/dog anti-CD20 antibody 7Ala Ile Tyr Pro Leu Thr Gly Asp Thr Ser Tyr Asn Gln Lys Ser Lys1 5 10 15Leu830PRTArtificial SequenceChimeric human/dog anti-CD20 antibody 8Gln Val Thr Ile Ser Ala Asp Lys Ser Ile Ser Thr Ala Tyr Leu Gln1 5 10 15Trp Ser Ser Leu Lys Ala Ser Asp Thr Ala Met Tyr Tyr Cys 20 25 30914PRTArtificial SequenceChimeric human/dog anti-CD20 antibody 9Ala Arg Ser Thr Tyr Val Gly Gly Asp Trp Gln Phe Asp Val1 5 1010216PRTArtificial SequenceChimeric human/dog anti-CD20 antibody 10Glu Ile Val Leu Thr Gln Ser Pro Gly Thr Leu Ser Leu Ser Pro Gly1 5 10 15Glu Arg Ala Thr Leu Ser Cys Arg Ala Ser Arg Ser Val Pro Tyr Ile 20 25 30His Trp Tyr Gln Gln Lys Pro Gly Gln Ala Pro Arg Leu Leu Ile Tyr 35 40 45Ala Thr Ser Ala Leu Ala Ser Gly Ile Pro Asp Arg Phe Ser Gly Ser 50 55 60Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Arg Leu Glu Pro Glu65 70 75 80Asp Phe Ala Val Tyr Tyr Cys Gln Gln Trp Leu Ser Asn Pro Pro Thr 85 90 95Phe Gly Gln Gly Thr Lys Leu Glu Ile Lys Arg Asn Asp Ala Gln Pro 100 105 110Ala Val Tyr Leu Phe Gln Pro Ser Pro Asp Gln Leu His Thr Gly Ser 115 120 125Ala Ser Val Val Cys Leu Leu Asn Ser Phe Tyr Pro Lys Asp Ile Asn 130 135 140Val Lys Trp Lys Val Asp Gly Val Ile Gln Asp Thr Gly Ile Gln Glu145 150 155 160Ser Val Thr Glu Gln Asp Lys Asp Ser Thr Tyr Ser Leu Ser Ser Thr 165 170 175Leu Thr Met Ser Ser Thr Glu Tyr Leu Ser His Glu Leu Tyr Ser Cys 180 185 190Glu Ile Thr His Lys Ser Leu Pro Ser Thr Leu Ile Lys Ser Phe Gln 195 200 205Arg Ser Glu Cys Gln Arg Val Asp 210 21511648DNAArtificial SequenceChimeric human/dog anti-CD20 antibody 11gaaattgtgt tgacgcagtc tccaggcacc ctgtctttgt ctccagggga aagagccacc 60ctctcctgca gggccagccg gagtgtaccg tacatccact ggtaccagca gaaacctggc 120caggctccca ggctcctcat ctatgccaca tccgctctgg cttctggcat cccagacagg 180ttcagtggca gtgggtctgg gacagacttc actctcacca tcagcagact ggagcctgaa 240gattttgcag tgtattactg tcagcagtgg ctgagtaacc cacccacttt tggccagggg 300accaagctgg agatcaaacg aaatgatgcc cagccagccg tctatttgtt ccaaccatct 360ccagaccagt tacacacagg aagtgcctct gttgtgtgct tgctgaatag cttctacccc 420aaagacatca atgtcaagtg gaaagtggat ggtgtcatcc aagacacagg catccaggaa 480agtgtcacag agcaggacaa ggacagtacc tacagcctca gcagcaccct gacgatgtcc 540agtactgagt acctaagtca tgagttgtac tcctgtgaga tcactcacaa gagcctgccc 600tccaccctca tcaagagctt ccaaaggagc gagtgtcaga gagtggac 64812106PRTArtificial SequenceChimeric human/dog anti-CD20 antibody 12Glu Ile Val Leu Thr Gln Ser Pro Gly Thr Leu Ser Leu Ser Pro Gly1 5 10 15Glu Arg Ala Thr Leu Ser Cys Arg Ala Ser Arg Ser Val Pro Tyr Ile 20 25 30His Trp Tyr Gln Gln Lys Pro Gly Gln Ala Pro Arg Leu Leu Ile Tyr 35 40 45Ala Thr Ser Ala Leu Ala Ser Gly Ile Pro Asp Arg Phe Ser Gly Ser 50 55 60Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Arg Leu Glu Pro Glu65 70 75 80Asp Phe Ala Val Tyr Tyr Cys Gln Gln Trp Leu Ser Asn Pro Pro Thr 85 90 95Phe Gly Gln Gly Thr Lys Leu Glu Ile Lys 100 1051323PRTArtificial SequenceChimeric human/dog anti-CD20 antibody 13Glu Ile Val Leu Thr Gln Ser Pro Gly Thr Leu Ser Leu Ser Pro Gly1 5 10 15Glu Arg Ala Thr Leu Ser Cys 201410PRTArtificial SequenceChimeric human/dog anti-CD20 antibody 14Arg Ala Ser Arg Ser Val Pro Tyr Ile His1 5 101514PRTArtificial SequenceChimeric human/dog anti-CD20 antibody 15Trp Tyr Gln Gln Lys Pro Gly Gln Ala Pro Arg Leu Leu Ile1 5 10168PRTArtificial SequenceChimeric human/dog anti-CD20 antibody 16Tyr Ala Thr Ser Ala Leu Ala Ser1 51732PRTArtificial SequenceChimeric human/dog anti-CD20 antibody 17Gly Ile Pro Asp Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr1 5 10 15Leu Thr Ile Ser Arg Leu Glu Pro Glu Asp Phe Ala Val Tyr Tyr Cys 20 25 30189PRTArtificial SequenceChimeric human/dog anti-CD20 antibody 18Gln Gln Trp Leu Ser Asn Pro Pro Thr1 51910PRTArtificial SequenceChimeric human/dog anti-CD20 antibody 19Phe Gly Gln Gly Thr Lys Leu Glu Ile Lys1 5 10

Claims

1. An antibody comprising a light chain variable region (LCVR) whose amino acid sequence is that given in SEQ :ID NO: 12, and a heavy chain variable region (HCVR) whose amino acid sequence is that given in SEQ ID NO: 3.

2. The antibody of claim 1, wherein the antibody specifically binds to canine CD20.

3. An antibody comprising a light chain (LC) whose amino acid sequence is that given in SEQ ID No: 10, and a heavy chain (HC) whose amino acid sequence is that given in SEQ ID NO: 1.

4. The antibody of claim 3, wherein the antibody specifically binds to canine CD20.

5. An antibody comprising two light chains (LC) each having an amino acid sequence that is given in SEQ ID NO: 10, and two heavy chains (HC) each having an amino acid sequence that is given in SEQ ID NO: 1.

6. The antibody of claim 5, wherein the antibody specifically binds to canine CD20.

7. A method of treating lymphoma in a canine patient, comprising administering to a lymphoma cancer canine patient in need of such treatment an effective amount of an antibody comprising a light chain variable region (LCVR) whose amino acid sequence is that given in SEQ ID NO: 12, and a heavy chain variable region (HCVR) whose amino acid sequence is that given in SEQ ID NO: 3, wherein the antibody specifically binds to canine CD20.

8. The method according to claim 7 wherein the antibody comprises a light chain (LC) whose amino acid sequence is that given in SEQ ID NO: 10, and a heavy chain (HC) whose amino acid sequence is that given in SEQ ID NO: 1.

9. The method according to claim 7, wherein the antibody is K9LO-133.

10. A kit, comprising a pharmaceutical composition, comprising K9LO-133, with one or more pharmaceutically acceptable carriers, diluents, or excipients.

11. An antibody comprising a light chain (LC) whose amino acid sequence is that given in SEQ ID NO: 10, and a heavy chain (HC) whose amino acid sequence is that given in SEQ :11) NO: 1 and wherein the antibody specifically binds to canine CD20, and wherein the antibody binds to protein A.

12. Use of an antibody comprising a light chain variable region (LCVR) whose amino acid sequence is that given in SEQ ID NO: 12, and a heavy chain variable region (HCVR) whose amino acid sequence is that given in SEQ ID NO: 3, wherein the antibody specifically binds to canine CD20, for treating lymphoma in a canine patient.

13. The use according to claim 12 wherein the antibody comprises a light chain (LC) whose amino acid sequence is that given in SEQ ID NC): 10, and a heavy chain (HC) whose amino acid sequence is that given in SEQ ID NO: 1.

14. The use according to claim 12, wherein the antibody is K9LO-133.

15. An antibody comprising a light chain variable region (LCVR) whose amino acid sequence is that given in SEQ ID NO: 12, and a heavy chain variable region (HCVR) whose amino acid sequence is that given in SEQ ID NO: 3, wherein the antibody specifically binds to canine CD20, for use in treating lymphoma in a canine patient.