U.S. patent application number 13/674489 was filed with the patent office on 2013-03-21 for monoclonal antibodies.
The applicant listed for this patent is Genevieve HANSEN. Invention is credited to Genevieve HANSEN.
Application Number | 20130071385 13/674489 |
Document ID | / |
Family ID | 41797723 |
Filed Date | 2013-03-21 |
United States Patent
Application |
20130071385 |
Kind Code |
A1 |
HANSEN; Genevieve |
March 21, 2013 |
MONOCLONAL ANTIBODIES
Abstract
The invention provides heterochimeric antibodies and/or
fragments thereof comprising (i) hypervariable region sequences
wholly or substantially corresponding to sequences found in
antibodies from a donor species; (ii) constant region sequences
wholly or substantially corresponding to sequences found in
antibodies from a target species which is different from the donor
species; and (iii) heavy and/or light chain variable framework
sequences which contain at least three non-CDR residues
corresponding to sequences found in antibodies from a target
species and at least three contiguous non-CDR residues
corresponding to sequences found in antibodies from a donor
species. The invention further provides antibody to canine or
feline or equine antigens, e.g., CD20 or CD52, and methods of
making and using antibodies as described.
Inventors: |
HANSEN; Genevieve; (Del Mar,
CA) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
HANSEN; Genevieve |
Del Mar |
CA |
US |
|
|
Family ID: |
41797723 |
Appl. No.: |
13/674489 |
Filed: |
November 12, 2012 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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12584390 |
Sep 4, 2009 |
8337842 |
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13674489 |
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61094333 |
Sep 4, 2008 |
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61163188 |
Mar 25, 2009 |
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Current U.S.
Class: |
424/133.1 |
Current CPC
Class: |
C07K 2317/24 20130101;
C07K 2317/565 20130101; A61P 35/00 20180101; C07K 2317/34 20130101;
C07K 16/2887 20130101; C07K 2317/73 20130101; A61K 39/39558
20130101; C07K 14/70596 20130101; A61K 45/06 20130101; C07K 16/2893
20130101; A61P 39/00 20180101; C07K 2317/567 20130101; A61K 31/56
20130101 |
Class at
Publication: |
424/133.1 |
International
Class: |
A61K 39/395 20060101
A61K039/395; C07K 16/30 20060101 C07K016/30 |
Claims
1.-22. (canceled)
23. A method of treating canine lymphoma comprising administering a
therapeutically effective amount of an antibody which binds to
canine lymphocytes.
24. A method of claim 23, wherein said antibody is a heterochimeric
antibody, or fragment thereof, comprising amino acid sequences of
antibodies from a donor species of mammal and from a target species
of a different mammal, comprising: a) a constant region sequence
from a heavy chain and light chain identical to, or a conservative
variant of, a sequence from the target species; and b) a variable
domain sequence from a heavy chain and light chain, wherein said
variable domain sequence comprises: i.) Hypervariable region
sequences comprising three complementarity determing regions (CDRs)
as definied by Kabat, which are identical to, or a conservative
variant of, CDRs from the donor species, and ii.) FR1, FR2, FR3,
and FR4 framework sequences, wherein the FR1 and/or the FR4
sequence is identical to, or a conservative variant of, a FR1
and/or FR4 sequence from the target species, and the FR2 and FR3
sequences are identical to, or a conservative variant of, FR2 and
FR3 sequences from the donor species; wherein said variable domain
sequence comprises at least three contiguous non-CDR residues
corresponding to residues from the target species and at least
three contiguous non-CDR residues corresponding to residues from
the donor species.
25. A method of claim 23, wherein said antibody binds to canine
CD20 or CD52.
26. A method of treating a patient suffering from a disease or
condition characterized by the presence of abnormal cells
expressing a target antigen comprising administering a
therapeutically effective amount of an antibody binding to such
target antigen wherein the antibody is a heterochimeric antibody,
or fragment thereof, comprising amino acid sequences of antibodies
from a donor species of mammal and from a target species of a
different mammal, comprising: a) a constant region sequence from a
heavy chain and light chain identical to, or a conservative variant
of, a sequence from the target species; and b) a variable domain
sequence from a heavy chain and light chain, wherein said variable
domain sequence comprises: i.) Hypervariable region sequences
comprising three complementarity determing regions (CDRs) as
definied by Kabat, which are identical to, or a conservative
variant of, CDRs from the donor species, and ii.) FR1, FR2, FR3,
and FR4 framework sequences, wherein the FR1 and/or the FR4
sequence is identical to, or a conservative variant of, a FR1
and/or FR4 sequence from the target species, and the FR2 and FR3
sequences are identical to, or a conservative variant of, FR2 and
FR3 sequences from the donor species; wherein said variable domain
sequence comprises at least three contiguous non-CDR residues
corresponding to residues from the target species and at least
three contiguous non-CDR residues corresponding to residues from
the donor species.
27. The method of claim 26, wherein said canine antibody binds to
canine CD20.
28. The method of claim 26, wherein said canine antibody binds to
canine CD52.
29. The method of claim 26, wherein the disease or condition
characterized by the presence of abnormal cells overexpressing
CD20.
30. The method of claim 26, wherein the disease or condition
characterized by the presence of abnormal cells expressing a target
antigen is selected from the group consisting of: acute
inflammation, rheumatoid arthritis, transplant rejection, asthma,
allergic inflammation, restenosis, arterial restenosis,
inflammatory bowel disease, uveitis multiple sclerosis, psoriasis,
wound healing, lupus erythematosus, allergic rhinitis, atopic
dermatitis, food allergies, diabetes mellitus, dermatitis,
thrombotic, thrombocytopenic purpura, encephalitis, leukocyte
adhesion deficiency, rheumatic fever, psoriatic arthritis,
osteoarthritis, ocular inflammatory disorders, progressive systemic
sclerosis, primary biliary cirrhosis, CNS inflammatory disorder,
antigen-antibody complex mediated diseases, autoimmune hemolytic
anemia, ischemic heart disease, atherosclerosis, post-dialysis
syndrome, leukemia, acquired immune deficiency syndrome, septic
shock, lipid histiocytosis, and cancer.
31. The method of claim 26, further comprising the administration
of chemotherapy.
32. The method of claim 31, wherein the chemotherapy comprises
administration of one or more agents selected from
cyclophosphamide, doxorubicin, vincristine, prednisone,
L-asparaginase, and adriamycin.
33. The method of claim 26, further comprising the administration
of an anti-inflammatory agent.
34. The method of claim 33, wherein the anti-inflammatory agent is
a corticosteroid.
35. The method of claim 26, further comprising the administration
of radiation.
36. The method of claim 26, comprising the co-administration of
antibody to CD20 and CD52.
37. The method of claim 26, wherein the antibody is administered to
treat or inhibit recurrence of cancer following treatment with
radiation or chemotherapy.
38. The method of claim 26, wherein the disease is canine lymphoma
and the heterochimeric antibody binds to an epitope on the
extracellular loop of canine CD52.
39. The method of claim 38, further comprising the administration
of a therapeutically effective amount of a heterochimeric antibody
to canine CD20.
40. The method of claim 26, wherein the heterochimeric antibody
comprises a light and heavy chain, wherein the light chain is
selected from the group of antibody variable domains consisting of:
FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4.sub.T-Lambda-C.sub.T-Lambda;
FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4.sub.T-Kappa-C.sub.T-Lambda;
FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4.sub.T-Lambda-C.sub.T-Kappa;
FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4.sub.T-kappa-C.sub.T-Kappa;
FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4.sub.T-kappa-C.sub.T-Kappa;
FR1.sub.T-Kappa-CDR1-FR2-CDR2-FR3-CDR3-FR4-C.sub.T-Lambda;
FR1.sub.T-Lambda-CDR1-FR2-CDR2-FR3-CDR3-FR4-C.sub.T-Kappa;
FR1.sub.T-kappa-CDR1-FR2-CDR2-FR3-CDR3-FR4-C.sub.T-Kappa;
FR1.sub.T-Lambda-CDR1-FR2-CDR2-FR3-CDR3-FR4.sub.T-Lambda-C.sub.T-Lambda;
and
FR1.sub.T-kappa-CDR1-FR2-CDR2-FR3-CDR3-FR4.sub.T-kappa-C.sub.T-Kappa;
wherein T=Target species; Lambda=lambda light chain; Kappa=kappa
light chain; C=Constant domain; FR=Framework region;
CDR=Complementarity Determining Region; and wherein a FR is a donor
species unless otherwise marked as a target species.
41. The method of claim 26, wherein the heterochimeric antibody
comprises a light and heavy chain, wherein the light chain is
selected from the group of antibody variable domains consisting of:
FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4.sub.T-C.sub.T;
FR1.sub.T-CDR1-FR2-CDR2-FR3-CDR3-FR4-C.sub.T; and
FR1.sub.T-CDR1-FR2-CDR2-FR3-CDR3-FR4.sub.T-C.sub.T; wherein
T=Target species; Lambda=lambda light chain; Kappa=kappa light
chain; C=Constant domain; FR=Framework region; CDR=Complementarity
Determining Region; and wherein a FR is a donor species unless
otherwise marked as a target species.
42. The method of claim 40, wherein said heterochimeric antibody is
an anti-CD20 or anti-CD52 monoclonal antibody.
43. The method of claim 41, wherein said heterochimeric antibody is
an anti-CD20 or anti-CD52 monoclonal antibody.
44. The method of claim 23, wherein said antibody binds to canine
CD20, and wherein the canine CD20 comprises SEQ ID NO: 67.
Description
RELATED APPLICATIONS
[0001] This application claims the benefit of U.S. Provisional
Application Ser. No. 61/094,333 filed Sep. 4, 2008 and U.S.
Provisional Application Ser. No. 61/163,188 filed Mar. 25, 2008,
the entire disclosures of which are incorporated herein by
reference.
FIELD OF THE INVENTION
[0002] This invention generally relates to monoclonal antibodies
for the treatment of diseases, e.g., in mammals and particularly in
companion animals, such as dogs, cats and horses. More
particularly, the invention provides "heterochimeric" antibody
constructs, and antibodies encoded by the constructs, which react
with targets useful for detection of targets, diagnosis of disease
and treatment of companion animals.
BACKGROUND OF THE INVENTION
[0003] The use of antibodies as therapeutic treatment for a variety
of diseases and disorders are rapidly increasing because they have
shown to be safe and efficacious therapeutic agents. Approved
therapeutic monoclonal antibodies for human use include Trastuzumab
(antigen: HER2/neu), Edrecolomab (antigen: Ep-CAM), Anti-human milk
fat globules (HMFG1) (antigen: HMW Mucin), Cetuximab (antigens: EGF
receptor), Alemtuzumab (antigen: CD52), and Rituximab (antigen:
CD20). Additional monoclonal antibodies are in various phases of
clinical development for humans for a variety of diseases with the
majority targeting various forms of cancer.
[0004] Antibodies target an antigen through its binding of a
specific epitope on an antigen by the interaction with the variable
region of the antibody molecule. Furthermore, antibodies have the
ability to mediate and/or initiate a variety of biological
activities. For example, antibodies can modulate receptor-ligand
interactions as agonists or antagonists. Antibody binding can
initiate intracellular signalling to stimulate cell growth,
cytokine production, or apoptosis. Antibodies can deliver agents
bound to the Fc region to specific sites. Antibodies also elicit
antibody-mediated cytotoxicity (ADCC), complement-mediated
cytotoxicity (CDC), and phagocytosis.
[0005] While the properties of antibodies make them very attractive
therapeutic agents, there are a number of limitations. There are
several methods being utilized to generate antibodies including
hybridoma technology, ribosome display, bacterial and yeast
display, and others known in the art. The vast majority of
monoclonal antibodies (mAbs) are of rodent origin. When such
antibodies are administered in a different species, patients can
mount their own antibody response to such xenogenic antibodies.
Such response may result in the eventual neutralization and
elimination of the antibody. One solution to this challenge
involves the process of engineering an antibody with sequences
compatible with the species subjected to the treatment. This
process can prevent or greatly delay the patient developing an
immune response against the administered therapeutic monoclonal
antibody and extends the half-life of that antibody in the
circulation of the treated subject. Such approaches, however,
require careful balancing so that the antibody retains specificity
and binding.
[0006] These limitations have prompted the development of
engineering technologies known as "humanization". Humanized
antibodies can be generated as chimeric antibodies or fragments
thereof which contain minimal sequence derived from non-human
immunoglobulin. For the most part, humanized antibodies are human
antibodies (i.e. "recipient antibody" or "target species antibody")
in which residues from a complementarity determining region (CDR)
of the recipient are replaced by residues from a CDR of a non-human
species (i.e. "donor antibody" or "originating species antibody")
such as mouse, having the desired properties such as specificity,
affinity, and potency. In some instances, framework region (FR)
residues of the human immunoglobulin are replaced by corresponding
non-human residues. This humanization strategy is referred to as
"CDR grafting" as reported for the making of humanized antibodies
(Winter, U.S. Pat. No. 5,225,539). Back mutation of selected target
framework residues to the corresponding donor residues might be
required to restore and/or improved affinity. Structure-based
methods may also be employed for humanization and affinity
maturation, for example as described for humanization in U.S.
patent application Ser. No. 10/153,159 and related applications.
Comparison of the essential framework residues required in
humanization of several antibodies, as well as computer modeling
based on antibody crystal structures revealed a set of framework
residues termed as "Vernier zone residues" (Foote, J. Mol. Biol.
224:487-499 (1992)). In addition, several residues in the VH-VL
interface zone have been suggested to be important in maintaining
affinity for the antigen (Santos, Prog Nucleic Acid Res Mol Biol.
60:169-94 (1998); Kettleborough, et al., Protein Engin., 4:773-783
(1991)). Similar strategies for "caninization" of antibodies for
use in dogs are described in WO 03/060080.
[0007] Alternatively, humanized antibodies may contain the CDRs
from a non-human sequence grafted into pools (e.g. libraries) of
individual human framework regions. This newly engineered antibody
is able to bind to the same antigen as the original antibody. The
antibody constant region is derived from a human antibody. The
methodology for performing this aspect is generally described as
framework shuffling (Dall'Acqua, Methods, 36:43-60 (2005)).
Furthermore, the humanized antibody may contain sequences from two
or more framework regions derived from at least two human antibody
germline sequences with high homology to the donor species.
Antibodies designed using this method are described as hybrid
antibodies (Rother et al., U.S. Pat. No. 7,393,648).
[0008] The approaches described above utilize essentially entire
framework regions from one or more antibody variable heavy chains
or variable light chains of the target species which are engineered
to receive CDRs from the donor species. In some cases, back
mutation of selected residues in the variable region is used to
enhance presentation of the CDRs. Designing antibodies that
minimize immunogenic reaction in a subject to non-native sequences
in the antibody, while at the same time preserving antigen binding
regions of the antibody sufficiently to maintain efficacy, has
proven challenging.
[0009] Another challenge for developing therapeutic antibodies
targeting proteins is that epitopes on the homologous protein in a
different species are frequently different, and the potential for
cross-reactivity with other proteins is also different. As a
consequence, antibodies have to be made, tested and developed for
the specific target in the particular species to be treated.
SUMMARY OF THE INVENTION
[0010] The invention provides heterochimeric antibodies and/or
fragments thereof comprising (i) Hypervariable region sequences
wholly or substantially corresponding to sequences found in
antibodies from a donor species; (ii) constant region sequences
wholly or substantially corresponding to sequences found in
antibodies from a target species which is different from the donor
species; and (iii) heavy and/or light chain variable framework
sequences which contain at least three, e.g. at least four, or at
least five, or at least six contiguous non-CDR residues
corresponding to sequences found in antibodies from a target
species and at least three, e.g. at least four, or at least five,
or at least six contiguous non-CDR residues corresponding to
sequences found in antibodies from a donor species. Heterochimeric
antibodies as described herein include heterochimeric hybrid
antibodies wherein the target antibody sequences are from different
antibodies from the target species. In one embodiment, the
heterochimeric antibody comprises FRI and/or FR4 variable region
sequences wholly or substantially corresponding to FRI and/or FR4
variable region sequences found in antibodies from a target
species, and CDR, FR2 and FR3 sequences wholly or substantially
corresponding to sequences found in the donor species antibody.
Methods of making and using these antibodies and fragments are also
provided.
[0011] In another embodiment the invention provides therapeutic
antibodies useful for veterinary application, particularly
antibodies directed to canine or feline or equine CD20, CD52,
HER2/neu, or IL-6, IL-6 receptor, for example canine CD20 or canine
CD52, together with methods of making such antibodies using
optimized immunogenic constructs and methods treatment using such
antibodies.
DETAILED DESCRIPTION OF THE INVENTION
[0012] It is to be understood that both the foregoing general
description and the following detailed description are exemplary
and explanatory only and are not restrictive of the invention, as
claimed. It must be noted that, as used herein and in the appended
claims, the singular forms include plural referents; the use of
"or" means "and/or" unless stated otherwise. Thus, for example,
reference to "a subject polypeptide" includes a plurality of such
polypeptides and reference to "the agent" includes reference to one
or more agents and equivalents thereof known to those skilled in
the art, and so forth. Moreover, it must be understood that the
invention is not limited to the particular embodiments described,
as such may, of course, vary. Further, the terminology used to
describe particular embodiments is not intended to be limiting,
since the scope of the present invention will be limited only by
its claims.
[0013] The section headings used herein are for organizational
purposes only and are not to be construed as limiting the subject
matter described. All documents, or portions of documents, cited in
this application, including but not limited to patents, patent
applications, articles, books, and treatises, are hereby expressly
incorporated by reference in their entirety for any purpose.
[0014] Unless otherwise defined, all technical and scientific terms
used herein have the same meaning as commonly understood by one of
ordinary skill in the art to which this invention belongs. Suitable
methods and materials are described below, however methods and
materials similar or equivalent to those described herein can be
used in the practice of the present invention. Thus, the materials,
methods, and examples are illustrative only and not intended to be
limiting. All publications, patent applications, patents, and other
references mentioned herein are incorporated by reference in their
entirety. In case of conflict, the present specification, including
definitions, will control.
[0015] Standard techniques may be used for recombinant DNA,
oligonucleotide synthesis, tissue culture and transfection (e.g.,
electroporation, lipofection, etc.). Enzymatic reactions and
purification techniques may be performed according to
manufacturer's specifications or as commonly accomplished in the
art or as described herein. The foregoing techniques and procedures
may be generally performed according to conventional methods well
known in the art and as described in various general and more
specific references that are cited and discussed throughout the
present specification. See e.g., Sambrook et al. Molecular Cloning:
A Laboratory Manual (2d ed., Cold Spring Harbor Laboratory Press,
Cold Spring Harbor, N.Y. (1989)).
[0016] The present invention provides methods for engineering
heterochimeric antibodies and/or fragments thereof suitable for
administration to a subject for treatment of a disease. The terms
"patient," "subject," and "individual," are used interchangeably
herein, to refer to mammals, including, but not limited to, humans,
murines, simians, felines, canines, equines, bovines, porcines,
ovines, caprines, mammalian farm and agricultural animals,
mammalian sport animals, and mammalian pets. In certain embodiments
of the invention, the subject is a companion animal, such as a dog,
cat or horse.
[0017] The heterochimeric antibody engineered thereof is the result
of the fusion of portion of the variable domain nucleotide
sequences to constant region nucleotide sequences and the
co-expression of these sequences to produce heterochimeric
recombinant antibodies. Furthermore, the invention relates to the
use of such heterochimeric antibodies antibodies and/or fragments
thereof as immunotherapeutic agents for the treatment of disease in
animals and as diagnostic agents.
[0018] Antibodies created according to the present invention offer
several advantages, such as (i) reduced immunogenicity response
upon repeated administration; (ii) increased potency mediated by an
efficient recruitment of immune system responsible for effector
functions in the targeted species; and (iii) increased
half-life.
[0019] The present invention includes generation of heterochimeric
antibodies and/or fragments thereof with the desired properties and
their use in production. The heterochimeric antibodies from the
present invention include a fragment of the variable region of an
antibody derived from a species that is different than the one of
the constant region. Thus, the heterochimeric antibodies and/or
fragments thereof retain the specificities and high affinities with
the desired effector functions in the target species.
[0020] The heterochimeric antibody of the present invention in
particular embodiments may recognize any therapeutic target
suitable for antibody therapy, for example a tumor-related antigen,
an allergy- or inflammation-related antigen, a cardiovascular
disease-related antigen, an autoimmune disease-related antigen or a
viral or bacterial infection-related antigen.
[0021] "Native antibodies" are usually glycoproteins of about
150,000 daltons, composed of two identical light chains and two
identical heavy chains. Each light chain is linked to a heavy chain
by one covalent disulfide bond, while the number of disulfide
linkages varies among the heavy chains of different immunoglobulin
isotypes. Each heavy and light chain also has regularly spaced
intrachain disulfide bridges. Each heavy chain has at one end a
variable domain (variable region) (V.sub.H) followed by a number of
constant domains (constant regions). Each light chain has a
variable domain at one end (V.sub.L) and a constant domain at its
other end; the constant domain of the light chain is aligned with
the first constant domain of the heavy chain, and the light-chain
variable domain is aligned with the variable domain of the heavy
chain.
[0022] The "light chains" of antibodies from any vertebrate species
can be assigned to one of two clearly distinct types, called kappa
and lambda.
[0023] Depending on the amino acid sequence of the constant domain
of their heavy chains, immunoglobulins can be assigned to different
classes. There are five major classes of immunoglobulins: IgA, IgD,
IgE, IgG, and IgM, and several of these may be further divided into
subclasses (isotypes), e.g., IgG1, IgG2, IgG3, IgG4, IgA, and IgA2.
The heavy-chain constant domains corresponding to the different
classes of immunoglobulins are called alpha, delta, epsilon, gamma,
and mu, respectively.
[0024] The term "variable domain" refers to the fact that certain
portions of the variable domains differ in sequence among
antibodies and are used in the binding and specificity of each
particular antibody for its particular antigen. However, the
variability is not evenly distributed throughout the variable
domains of antibodies. It is concentrated in three segments called
hypervariable regions both in the light chain and the heavy chain
variable domains. The more highly conserved portions of variable
domains are called the framework region (FR). The variable domains
of native heavy and light chains each comprise four FRs (FR1, FR2,
FR3 and FR4). The hypervariable regions in each chain are held
together in close proximity by the FRs and, with the hypervariable
regions from the other chain, contribute to the formation of the
antigen-binding site of antibodies (see Kabat et al., Sequences of
Proteins of Immunological Interest, 5th Ed. Public Health Service,
National Institutes of Health, Bethesda, Md. (1991), pages
647-669). The constant domains are not involved directly in binding
an antibody to an antigen, but exhibit various effector functions,
such as participation of the antibody in antibody-dependent
cellular toxicity.
[0025] The term "hypervariable region" when used herein refers to
the amino acid residues of an antibody which are responsible for
antigen-binding. The hypervariable region comprises amino acid
residues from a "complementarity determining region" or "CDR" in
the light chain variable domain and in the heavy chain variable
domain as defined by Kabat et al., 5th Ed. Public Health Service,
National Institutes of Health, Bethesda, Md. (1991) and/or as
defined by (Chothia and Lesk, Mol. Biol. 196:901-917 (1987) and/or
as defined as "AbM loops" by Martin, et al., Proc. Natl Acad. Sci.
USA, 86:9268-9272 (1989) and/or as defined by Lefranc et al.,
Nucleic Acids Res, 27:209-212 (1999) in the international
ImMunoGeneTics information systems database. "Framework" or "FR"
residues are those variable domain residues other than the
hypervariable region residues as herein defined.
[0026] Papain digestion of antibodies produces two identical
antigen-binding fragments, called "Fab" fragments, each with a
single antigen-binding site, and a residual "Fc" fragment, whose
name reflects its ability to readily crystallize. Pepsin treatment
yields a binding cross-linking antigen.
[0027] "Fv" as used herein, refers to the minimum antibody fragment
that contains a complete antigen-recognition and binding site. This
region consists of a dimer of one heavy chain and one light chain
variable domain.
[0028] The Fab fragment also contains the constant domain of the
light chain and the first constant domain (CH1) of the heavy chain.
Fab' fragments differ from Fab fragments by the addition of a few
residues at the carboxyl terminus of the heavy chain CH1 domain
including one or more cysteine(s) from the antibody hinge region.
Fab'-SH is the designation herein for Fab' in which the cysteine
residue(s) of the constant domains bear a free thiol group.
F(ab').sub.2 antibody fragments originally were produced as pairs
of Fab' fragments which have hinge cysteines between them. Other
chemical couplings of antibody fragments are also known.
[0029] The term "antibody" herein is used in the broadest sense and
specifically covers monoclonal antibodies (including full length
monoclonal antibodies), polyclonal antibodies, multispecific
antibodies (e.g., bispecific antibodies), and antibody fragments
exhibiting the desired biological activity. The desired biological
activity will include at least binding to a cognate antigen and may
further include complement activation and/or other effector
functions.
[0030] "Antibody fragments" or "antigen-binding moiety" comprise a
portion of a full length antibody, generally the antigen binding or
variable domain thereof. Examples of antibody fragments include
Fab, Fab', F(ab').sub.2, and Fv fragments; diabodies; linear
antibodies; single-chain antibody molecules; and multispecific
antibodies formed from antibody fragments that bind 2 or more
different antigens.
[0031] The term "monoclonal antibody" as used herein refers to an
antibody obtained from a population of substantially homogeneous
antibodies, i.e., the individual antibodies comprising the
population are identical except for possible naturally occurring
mutations that may be present in minor amounts. Monoclonal
antibodies are highly specific. For example, the monoclonal
antibodies to be used in accordance with the present invention may
be made by the hybridoma method first described by Kohler et al.,
Nature 256:495 (1975), or may be made by recombinant DNA methods.
The monoclonal antibodies may also be isolated e.g. from phage
antibody libraries.
[0032] Monoclonal antibodies are most frequently generated in mice
by administration of the "antigen" and subsequent isolation of
B-cells that make antibodies. The B-cells are then immortalized by
fusion to another, stable cell type of the same species of the B
cell to create a "hybridoma". An individual B-cell makes one
specific antibody (i.e. is clonally monospecific), which is defined
by its primary amino acid sequence and its underlying gene
sequence. As used herein, the terms "heterohybridoma" and
"heteromyeloma" refer to lymphocyte cell lines immortalized by
fusion of lymphocytes and myelomas from two different species.
[0033] Monoclonal antibodies can be initially generated, for
example, by immunizing animals with an antigen or with cells that
express the antigen. The generation of a hybridoma starts with the
immunization of mice or companion animals such as dogs.
Immunization can be performed with several types of cells in the
presence or absence of adjuvants. Cells can also be used to
identify the hybridoma cell lines with the desired properties by
ELISA, Biacore, FACS or other methodologies available to those in
the art.
[0034] Cells suitable for use in the methods of monoclonal antibody
preparation according to the present invention include: (1)
Peripheral Blood Mononuclear Cells (PBMC) or fractions of PBMC
enriched in certain type of cells collected from healthy or
diseased companion animals such as dogs, cats, or horses.
Lymphocytes are pre-incubated in some instances with factors
including factors including growth factors such as EPO, SCF,
TNF.alpha., TGF.beta., GMCSF, TPO, IL-1, IL-2, IL-3, IL-4, GCSF to
increase the expression of the antigen prior to immunization. (2)
Lymphoma cell lines or tumor cell lines established from healthy or
diseased subjects optionally pre-incubated with factors listed
above to increase the expression of the antigen prior to
immunization. (3) Cell lines derived from tissues of healthy or
diseased subjects pre-incubated in some instances with factors
listed above to increase the expression of the antigen prior to
immunization. (4) Cultured cells engineered to express an antigen
coding region or fragment thereof, such as baculovirus-infected
cells, bacterial cells, yeast cells, mammalian cells, plant cells,
fungal cells and the like. The antigen in the form of DNA, RNA,
protein, or peptide, can be included in any one of the fractions of
the cell. (5) Magnetic Proteoliposome Particles (MPLs), which are
prepared from cells expressing the antigen, such that the native
conformation of the transmenbrane receptor is maintained, have been
described previously (see e.g., Mirzabekov et al. Nat. Biotechnol.
18:649-654 (2000); Babcock et al. J Biol. Chem. 276:38433-38440
(2001); PCT Publication WO 01/49265; U.S. patent application No.
20010034432).
[0035] In certain embodiments of the invention, the generation of
monoclonal antibodies can be achieved using immunogens derived from
DNA, peptides, or proteins. Hybridomas are generated by immunizing
an animal, which can be for example, a mouse or a companion animal,
or any animal that will give a suitable antibody response. In one
aspect, immunization is performed by introducing into the animal an
antigen-encoding nucleic acid, or a protein antigen, such as canine
CD20 or CD52 or an immunogenic fragment thereof, or a nucleic acid
encoding CD20 or CD52 or an immunogenic fragment thereof. The
skilled artisan will appreciate that certain epitopes will be more
immunogenic in an animal when removed from their native
environment. Thus, a peptide corresponding to an epitope of an
antigen conjugated to a carrier such as keyhole limpet hemocyanin,
may elicit a stronger antibody response than either the peptide
alone or the epitope when part of the native protein on which it is
found. Such variations and other immunization schemes are known to
the skilled artisan are included in the immunization methods of the
invention.
[0036] The immunogen can be a plasmid carrying a nucleic acid
sequence encoding an antigen or a fragment thereof. In other
embodiments of the invention, monoclonal antibodies of the
invention can be obtained by screening a library of antibody
molecules or fragments thereof derived from immunization of
animals. Monoclonal antibodies of the invention can also be
obtained from libraries of antibodies or antibody-encoding nucleic
acids.
[0037] As used herein the term "antigen" is understood to be any
substance capable of stimulating antibody production. Also, the
term "immunogen" is understood to include any substance used to
induce an immune response.
[0038] The monoclonal antibodies herein may in some embodiments
include "chimeric" antibodies in which a portion of the heavy
and/or light chain is identical to or homologous with corresponding
sequences from antibodies derived from a particular species or
belonging to a particular antibody class or subclass, while the
remainder of the chain(s) is identical to or homologous with
corresponding sequences in antibodies from another species or
belonging to another antibody class or subclass, as well as
fragments of such antibodies, exhibiting the desired biological
activity (See e.g., U.S. Pat. No. 4,816,567; and Morrison et al.,
Proc. Natl. Acad. Sci. USA 81:6851-6855 (1984)).
[0039] "Single-chainFv" or "sFv" antibody fragments comprise the
V.sub.H and V.sub.L domains of antibody, wherein these domains are
present in a single polypeptide chain. Generally, the Fv
polypeptide further comprises a polypeptide linker between the
V.sub.H and V.sub.L domains which enables the sFv to form the
desired structure for antigen binding. For a review of sFv, see
Pluckthun in The Pharmacology of Monoclonal Antibodies, vol. 113,
Rosenburg and Moore eds. Springer-Verlag, New York, pp. 269-315
(1994).
[0040] The term "diabodies" refers to small antibody fragments with
two antigen-binding sites, which fragments comprise a heavy chain
variable domain (V.sub.H) connected to a light chain variable
domain (V.sub.L) in the same polypeptide chain (V.sub.H-V.sub.L).
By using a linker that is short to allow pairing between the two
domains on the same chain, the domains are forced to pair with the
complementary domains of another chain and create two
antigen-binding sites.
[0041] In certain aspects the present invention provides methods
for adapting antibodies to the species of an intended therapeutic
target. Generally, these methods include "mammalization" which is
defined as a method for transferring donor antigen-binding
information to a less immunogenic mammal antibody acceptor to
generate useful therapeutic treatments. More specifically, the
invention provides methods for felinization, equinization and
caninization of antibodies.
[0042] "Caninization" is defined as a method for transferring
non-canine antigen-binding information from a donor antibody to a
less immunogenic canine antibody acceptor to generate treatments
useful as therapeutics in dogs.
[0043] "Felinization" is defined as a method for transferring
non-feline antigen-binding information from a donor antibody to a
less immunogenic feline antibody acceptor to generate treatments
useful as therapeutics in cats.
[0044] "Equinization" is defined as a method for transferring
non-equine antigen-binding information from a donor antibody to a
less immunogenic equine antibody acceptor to generate treatments
useful as therapeutics in horses.
[0045] Caninized forms of non-canine antibodies provided herein are
chimeric antibodies that contain minimal sequence derived from
non-canine antibody. For the most part, caninized antibodies are
canine antibody sequences ("acceptor" or "recipient" antibody) in
which hypervariable region residues of the recipient are replaced
by hypervariable region residues from a non-canine species ("donor"
antibody) such as mouse, rat, rabbit, cat, dogs, goat, chicken,
bovine, horse, llama, camel, dromedaries, sharks, non-human
primates, human, humanized, recombinant sequence, or an engineered
sequence having the desired properties. In some instances,
framework region (FR) residues of the canine antibody are replaced
by corresponding non-canine FR residues. Furthermore, caninized
antibodies may include residues that are not found in the recipient
antibody or in the donor antibody. These modifications are made to
further refine antibody performance. The caninized antibody may
also comprise at least a portion of an immunoglobulin constant
region (Fc) of a canine antibody.
[0046] As used herein, "identity" refers to the sequence matching
between two polypeptides, molecules or between two nucleic acids.
When a position in both of the two compared sequences is occupied
by the same base or amino acid monomer subunit (for instance, if a
position in each of the two DNA molecules is occupied by adenine,
or a position in each of two polypeptides is occupied by a lysine),
then the respective molecules are identical at that position. The
"percentage identity" between two sequences is a function of the
number of matching positions shared by the two sequences divided by
the number of positions compared.times.100. Such alignment can be
provided using, for instance, the program Basic Local Alignment
Search Tool (BLAST) from the National Center for Biotechnology
Information NCBI.
[0047] In one preferred embodiment, the recombinant polypeptides,
or fragments, derivatives, or modifications thereof, are
specifically administered into a patient. In another embodiment,
the recombinant polypeptide of the invention, or fragments,
derivatives, or modifications thereof, are introduced into cells
and/or a tissue while under in vitro or ex vivo conditions, prior
to the transplantation of the cells and/or a tissue into a
mammalian organism for the purpose of treating, preventing,
reducing or otherwise lowering disease conditions or symptoms
associated or mediated by the disease.
[0048] The terms "fragment" and "region" refer to portions of a
polypeptide or nucleic acid molecule that contains at least 10%,
20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, or more of the entire
length of the reference nucleic acid molecule or polypeptide.
[0049] The terms "polynucleotide," "nucleic acid," and "nucleic
acid molecule," are used interchangeably herein to refer to
polymeric forms of nucleotides of any length. The polynucleotides
can contain deoxyribonucleotides, ribonucleotides, and/or their
analogs. Polynucleotides can have any three-dimensional structure,
and can perform any function, known or unknown. The term
polynucleotide includes single-stranded, double-stranded, and
triple helical molecules, and encompasses nucleic acids containing
nucleotide analogs or modified backbone residues or linkages, which
can be synthetic, naturally occurring, or non-naturally occurring,
and which have similar binding properties as the reference nucleic
acid.
[0050] "Oligonucleotide" refers generally to polynucleotides that
are between 5 and about 100 nucleotides of single- or
double-stranded DNA. For the purposes of this disclosure, the lower
limit of the size of an oligonucleotide is two, and there is no
upper limit to the length of an oligonucleotide. Oligonucleotides
are also known as "oligomers" or "oligos" and can be prepared by
any method known in the art including isolation from
naturally-occurring polynucleotides, enzymatic synthesis and
chemical synthesis.
[0051] The terms "polypeptide," "peptide" and "protein" are used
interchangeably herein to refer to a polymer of amino acid residues
of any length. Polypeptides can have any three-dimensional
structure, and can perform any function, known or unknown. The
terms apply to amino acid polymers in which one or more amino acid
residue is an artificial chemical mimetic of a corresponding
naturally occurring amino acid, as well as to naturally occurring
amino acid polymers and non-naturally occurring amino acid
polymers.
[0052] The term "amino acid" refers to naturally occurring and
synthetic amino acids, as well as amino acid analogs and amino acid
mimetics that function in a manner similar to the naturally
occurring amino acids. Naturally occurring amino acids are those
encoded by the genetic code, as well as those amino acids that are
later modified, e.g., hydroxyproline, y carboxyglutamate, and
O-phosphoserine. Amino acid mimetics refers to chemical compounds
that have a structure that is different from the general chemical
structure of an amino acid, but that functions in a manner similar
to a naturally occurring amino acid.
[0053] Amino acids may be referred to herein by either their
commonly known three letter symbols or by the one-letter symbols
recommended by the IUPAC-IUB Biochemical Nomenclature Commission.
Nucleotides, likewise, may be referred to by their commonly
accepted single-letter codes.
[0054] The terms "conservatively modified variants" or
"conservative variants" applies to both amino acid and nucleic acid
sequences. With respect to particular nucleic acid sequences,
conservatively modified variants refers to those nucleic acids
which encode identical or substantially identical amino acid
sequences; or for nucleic acids that do not encode an amino acid
sequence, to nucleic acids that are substantially identical. As
used herein, "substantially identical" means that two amino acid or
polynucleotide sequences differ at no more than 10% of the amino
acid or nucleotide positions, typically at no more than 5%, often
at more than 2%, and most frequently at no more than 1% of the of
the amino acid or nucleotide positions.
[0055] Because of the degeneracy of the genetic code, a large
number of functionally identical nucleic acids encode any given
protein. For instance, the codons GCA, GCC, GCG and GCU all encode
the amino acid alanine. Thus, at every position where an alanine is
specified by a codon, the codon can be altered to any of the
alternate alanine codons without altering the encoded polypeptide.
Such nucleic acid variations are "silent variations," which are one
type of conservatively modified variants. Nucleic acid sequences
encoding polypeptides described herein also encompass every
possible silent variation of the nucleic acid. The skilled artisan
will recognize that each amino acid codon in a nucleic acid (except
AUG, which is ordinarily the only codon for methionine, and TGG,
which is ordinarily the only codon for tryptophan) can be varied at
one or more positions to code for the same amino acid. Accordingly,
each silent variation of a nucleic acid that encodes a polypeptide
is implicit in each described sequence with respect to the
expression product.
[0056] "Complementarity" as applied to nucleic acids, refers to the
ability of the nucleic acid to form hydrogen bond(s) with another
polynucleotide sequence by either traditional Watson-Crick or other
non-traditional types of base pairing. In reference to the nucleic
molecules of the present invention, the binding free energy for a
nucleic acid molecule with its target or complementary sequence is
sufficient to allow the relevant function of the nucleic acid to
proceed, e.g., enzymatic nucleic acid cleavage, RNA interference,
antisense or triple helix inhibition. Determination of binding free
energies for nucleic acid molecules is well known in the art.
"Percent complementarity" refers to the percentage of contiguous
residues in a nucleic acid molecule that can form hydrogen bonds
(e.g., Watson-Crick base pairing) with another nucleic acid
molecule. "Perfectly complementary" or "100% complementarity" means
that all the contiguous nucleotides of a nucleic acid molecule will
hydrogen bond with the same number of contiguous residues in a
second nucleic acid molecule. "Substantial complementarity" and
"substantially complementary" as used herein indicate that two
nucleic acids are at least 90% complementary, typically at least
95% complementary, often at least 98% complementary, and most
frequently at least 99% complementary over a region of more than
about 15 nucleotides and more often more than about 19
nucleotides.
[0057] "Homology" is an indication that two nucleotide sequences
represent the same gene or a gene product thereof, and typically
means that that the nucleotide sequence of two or more nucleic acid
molecules are partially, substantially or completely identical.
When from the same organism, homologous polynucleotides are
representative of the same gene having the same chromosomal
location, even though there may be individual differences between
the polynucleotide sequences (such as polymorphic variants, alleles
and the like). In certain embodiments, a homolog can be found in a
non-native position in the genome, e.g. as the result of
translocation.
[0058] The term "heterologous" refers to any two or more nucleic
acid or polypeptide sequences that are not normally found in the
same relationship to each other in nature. For instance, a
heterologous nucleic acid is typically recombinantly produced,
having two or more sequences, e.g., from unrelated genes arranged
to make a new functional nucleic acid, e.g., a promoter from one
source and a coding region from another source. Similarly, a
heterologous polypeptide will often refer to two or more
subsequences that are not found in the same relationship to each
other in nature (e.g., a fusion protein).
[0059] The term "homolog" refers to a polypeptide or nucleic acid
molecule exhibiting at least 50% identity to a reference amino acid
sequence (for example, any one of the amino acid sequences
described herein) or nucleic acid sequence (for example, any one of
the nucleic acid sequences described herein). Preferably, such a
sequence is at least 55%, 57%, 60%, 65%, 68%, 70%, more preferably
80% or 85%, and most preferably 90%, 95%, 98%, or 99% identical at
the amino acid level or nucleic acid to a reference sequence.
[0060] "Similar" sequences are those which, when aligned, share
identical and similar amino acid residues, where similar residues
are conservative substitutions for corresponding amino acid
residues in an aligned reference sequence. In this regard,
conservative residues in a sequence is a residue that is physically
or functionally similar to the corresponding reference residue,
e.g., that has a similar size, shape, electric charge, chemical
properties, including the ability to form covalent or hydrogen
bonds, or the like. The "percentage similarity" between two
sequences is a function of the number of positions that contain
matching residues or conservative residues shared by the two
sequences divided by the number of positions
compared.times.100.
[0061] "Amino acid consensus sequence" as used herein refers to a
hypothetical amino acid sequence that can be generated using a
matrix of at least two, and preferably more, aligned amino acid
sequences, and allowing for gaps in the alignment, such that it is
possible to determine the most frequent amino acid residue at each
position. The consensus sequence is that sequence which comprises
the amino acids which are most frequently represented at each
position. In the event that two or more amino acids are equally
represented at a single position, the consensus sequence includes
both or all of those amino acids. In some cases, amino acid
consensus sequences correspond to a sequence or sub-sequence found
in nature. In other cases, amino acid consensus sequences are not
found in nature, but represent only theoretical sequences.
[0062] The amino acid sequence of a protein can be analyzed at
various levels. For example, conservation or variability can be
exhibited at the single residue level, multiple residue level,
multiple residues with gaps etc. Residues can exhibit conservation
of the identical residue or can be conserved at the class level.
The following eight groups each contain amino acids that are
conservative substitutions for one another: 1) Alanine (A), Glycine
(G); 2) Aspartic acid (D), Glutamic acid (E); 3) Asparagine (N),
Glutamine (Q); 4) Arginine (R), Lysine (K); 5) Isoleucine (I),
Leucine (L), Methionine (M), Valine (V); 6) Phenylalanine (F),
Tyrosine (Y), Tryptophan (W); 7) Serine (S), Threonine (T); and 8)
Cysteine (C), Methionine (M) (see, e.g., Creighton, Proteins
(1984)). Other classes are known to one of skill in the art and may
be defined using structural determinations or other data to assess
substitutability.
[0063] Regarding amino acid sequences, one of skill in the art will
recognize that individual substitutions, deletions or insertions to
a nucleic acid, peptide, polypeptide, or protein sequence which
alters, inserts or deletes a single amino acid or a small
percentage of amino acids in the encoded sequence is a
"conservatively modified variant" where the alteration results in
the substitution of an amino acid with a chemically similar amino
acid. Conservative substitution tables detailing functionally
similar amino acids are well known in the art. Such conservatively
modified variants are in addition to and do not exclude
functionally equivalent polymorphic variants, homologs, and alleles
of the invention.
[0064] As used herein, when one amino acid sequence (e.g., a first
VH or VL sequence) is aligned with one or more additional amino
acid sequences (e.g., one or more VH or VL sequences in a
database), an amino acid position in one sequence (e.g., the first
VH or VL sequence) can be compared to a "corresponding position" in
the one or more additional amino acid sequences. As used herein,
the "corresponding position" represents the equivalent position in
the sequence(s) being compared when the sequences are optimally
aligned, i.e., when the sequences are aligned to achieve the
highest percent identity or percent similarity.
[0065] As used herein, the term "antibody database" refers to a
collection of two or more antibody amino acid sequences (a
"plurality" or "multiplicity" of sequences), and typically refers
to a collection of tens, hundreds or even thousands of antibody
amino acid sequences. An antibody database can store amino acid
sequences of, for example, a collection of antibody VH regions,
antibody VL regions or both, or can store a collection of framework
sequences. In one embodiment, the antibody database is a database
comprising or consisting of germline antibody sequences. In another
embodiment, the antibody database is a database comprising or
consisting of mature antibody sequences (e.g., a Kabat database of
mature antibody sequences). In another embodiment, the antibody
database comprises or consists of sequences selected for one or
more properties. In another embodiment, the antibody database
comprises or consists of consensus sequences. In another
embodiment, the antibody database comprises or consists of similar
sequences. In yet another embodiment, the antibody database
comprises or consists of sequences from major antibody clans (Das
et al., Immunogenetics, 60:47-55 (2008); Das et al., Proc. Natl.
Ac. Sci. USA. 105:16647-16652 (2008)).
[0066] As used herein, the term "property" is a property of a
polypeptide which is desirable and/or advantageous to one of skill
in the art, e.g., in order to improve the manufacturing properties
or therapeutic efficacy of the polypeptide. In one embodiment, the
functional property is improved stability. In another embodiment,
the functional property is improved solubility. In yet another
embodiment, the functional property is non-aggregation. In still
another embodiment, the functional property is an improvement in
expression. In certain embodiments, the functional property is an
improvement in antigen binding affinity.
[0067] In the methods of the invention, the sequence of the
antibody of interest can be compared to the sequences within one or
more of a variety of different types of antibody sequence
databases. For example, in one embodiment, the antibody VH, VL or
VH and VL amino acid sequences of the database are germline
antibody VH, VL or VH and VL amino acid sequences. In another
embodiment, the antibody VH, VL or VH and VL amino acid sequences
of the database are rearranged, affinity matured antibody VH, VL or
VH and VL amino acid sequences. In yet another embodiment, the
antibody VH, VL or VH and VL amino acid sequences of the database
are pseudogene antibody VH, VL or VH and VL amino acid
sequences.
[0068] In the methods of the invention, the sequence of the
antibody to modify can be compared with all sequences within an
antibody database or, alternatively, only a selected portion of the
sequences in the database can be used for comparison purposes. That
is, the database can be limited, or constrained, to only those
sequences having a high percentage similarity or identity to the
antibody of interest. Thus, in one embodiment of the method of the
invention, the database is a constrained database in which only
those antibody VH, VL or VH and VL amino acid sequences having high
similarity to the VH, VL or VH and VL amino acid sequences are
included in the database.
[0069] The methods of the invention can be combined with other
methods known in the art for analyzing antibody structure and
antibody structure/function relationships. For example, in a one
embodiment, the methods of the invention are combined with
molecular modeling to identify additional potentially problematic
residues. Methods and software for computer modeling of antibody
structures are established in the art and can be combined with the
methods of the invention. The role of sequences can be further
determined by examining, for example, local and non-local
interactions, canonical residues, interfaces, exposure degree and
.beta.-turn propensity. Molecular modeling methods known in the art
can be applied, for example, to select "best fit" sequences if a
panel of possible sequences is under consideration.
[0070] A sequence identified to be utilized for mammalization,
caninization, felinization or equinization can be mutated using one
of several possible mutagenesis methods well established in the
art. For example, site directed mutagenesis can be used make a
particular amino acid substitution at the amino acid position of
interest. Site directed mutagenesis also can be used to create a
set of mutated sequence sin which a limited repertoire of amino
acid substitutions have been introduced at the amino acid position
of interest.
[0071] The expression "control sequences" refers to DNA sequences
necessary for the expression of an operably linked coding sequence
in a particular host organism. The control sequences that are
suitable for prokaryotes, for example, include a promoter,
optionally an operator sequence, and a ribosome binding site.
Eukaryotic cells are known to utilize promoters, polyadenylation
signals, and enhancers.
[0072] Nucleic acid is "operably linked" when it is placed into a
functional relationship with another nucleic acid sequence. For
example, DNA for a presequence or secretory leader is operably
linked to DNA for a polypeptide if it is expressed as a preprotein
that participates in the secretion of the polypeptide; a promoter
or enhancer is operably linked to a coding sequence if it affects
the transcription of the sequence; or a ribosome binding site is
operably linked to a coding sequence if it is positioned so as to
facilitate translation. Generally, "operably linked" means that the
DNA sequences being linked are contiguous, and, in the case of a
secretory leader, contiguous and in reading phase. However,
enhancers do not have to be contiguous. Linking is accomplished by
ligation at convenient restriction sites. If such sites do not
exist, the synthetic oligonucleotide adaptors or linkers are used
in accordance with conventional practice.
[0073] As used herein, the expressions "cell," "cell line," and
"cell culture" are used interchangeably and all such designations
include progeny. Thus, the words "transformants" and "transformed
cells" include the primary subject cell and cultures derived
from.
[0074] Immunogenic, as used herein, refers to antigens, (including
native antigens, fragments, mutant, and derivatives thereof, as
well as recombinant and synthetic antigens), that, when introduced
into an animal, elicit an immune response, such as a humoral or
antibody response.
[0075] As used herein, the term "not immunogenic" or
"non-immunogenic" means that an antigen, such as an antibody, or
other molecule, does not raise an antibody response of sufficient
magnitude to reduce the effectiveness of continued administration
of the antibody in the majority of treated patients for sufficient
time to achieve therapeutic efficacy.
[0076] As used herein, the term "therapeutic" encompasses the full
spectrum of treatments for a disease or disorder. A "therapeutic"
agent of the invention may act in a manner that is prophylactic or
preventive, including those that incorporate procedures designed to
target individuals that can be identified as being at risk
(pharmacogenetics); or in a manner that is ameliorative or curative
in nature; or may act to slow the rate or extent of the progression
of a disease or disorder; or may act to minimize the time required,
the occurrence or extent of any discomfort or pain, or physical
limitations associated with recuperation from a disease, disorder
or physical trauma; or may be used as an adjuvant to other
therapies and treatments.
[0077] "Treatment," as used herein, covers any administration or
application of remedies for disease in an animal, including a
human, and includes inhibiting the disease, i.e., arresting its
development; relieving the disease, i.e., causing its regression;
and eliminating the disease, i.e., causing the removal of diseased
cells or restoration of a non-diseased state. Treatment refers to
both therapeutic treatment and prophylactic or preventative
measures. Those in need of treatment include those already with the
disorder as well as those in which the disorder is to be
prevented.
[0078] A "pharmaceutical composition" or "pharmaceutically
acceptable composition" of antibodies, polypeptides, or
polynucleotides herein refers to a composition that usually
contains a pharmaceutically acceptable carrier or excipient that is
conventional in the art and which is suitable for administration
into a subject for therapeutic, diagnostic, or prophylactic
purposes. For example, compositions for oral administration can
form solutions, suspensions, tablets, pills, capsules, sustained
release formulations, oral rinses, or powders.
[0079] The term "combination therapy" refers to a therapeutic
regimen that involves the provision of at least two distinct
therapies to achieve an indicated therapeutic effect. For example,
a combination therapy may involve the administration of two or more
chemically distinct active ingredients, for example, a
chemotherapeutic agent and an antibody. Alternatively, a
combination therapy may involve the administration of an antibody
and/or one or more chemotherapeutic agents, alone or together with
the delivery of another treatment, such as radiation therapy and/or
surgery. In the context of the administration of two or more
chemically distinct active ingredients, it is understood that the
active ingredients may be administered as part of the same
composition or as different compositions. When administered as
separate compositions, the compositions comprising the different
active ingredients may be administered at the same or different
times, by the same or different routes, using the same of different
dosing regimens, all as the particular context requires and as
determined by the attending veterinarian or attending
caregiver.
[0080] The term "monotherapy" refers to a treatment regimen based
on the delivery of one therapeutically effective compound, whether
administered as a single dose or several doses over time.
[0081] "Immune conditions" are a generic name for a wide range of
diseases including arthritis, psoriasis, inflammatory bowel
disease, multiple sclerosis, myocardial infarction, stroke,
hemolytic anemia, atopic dermatitis, skin disorders, and the like,
in which the immune system or a part thereof, such as a cell of the
immune system, is abnormal or causes a disease state. Immune
conditions include primary defects in an immune cell, tissue or
organ, as well as "autoimmune conditions," in which the normal
mechanisms for preventing immune recognition of self antigens is
defective, resulting in a disease or disorder involving a
non-immune cell, tissue or organ type. Leukemias and lymphoma's are
primary immune disorders, while multiple sclerosis and lupus are
believed to be of autoimmune origin.
[0082] A multitude of therapeutic agents have been developed over
the past few decades for the treatment of various types of immune
conditions for humans and these have also been used for the
treatment of immune conditions in companion animals. The most
commonly used types of anti-immune agents include:
immunosuppressant agents (e.g., cyclosporine, thiopurine,
prednisone), and analgesic and antipyretic (e.g., aspirin,
ibuprofen, naproxen, celecoxib, nimesulide, licofelone,
omega-3-fatty acids), each of which may be administered
simultaneously, sequentially or in a common dosage regimin with
antibodies of the invention.
[0083] "Cancer" as used herein, refers to any abnormal cell or
tissue growth, e.g., a tumor, which can be malignant or
non-malignant. Cancer is characterized by uncontrolled
proliferation of cells that may or may not invade the surrounding
tissue and, hence, may or may not metastasize to new body sites.
Cancer encompasses carcinomas, which are cancers of epithelial
cells (e.g. squamous cell carcinoma, adenocarcinoma, melanomas, and
hepatomas). Cancer also encompasses sarcomas, which are tumors of
mesenchymal origin, (e.g. osteogenic sarcomas, leukemias, and
lymphomas). Cancers can involve one or more neoplastic cell type.
Cancer a generic name for a wide range of cellular malignancies
characterized by unregulated growth, lack of differentiation, and
the ability to invade local tissues and metastasize. These
neoplastic malignancies affect, with various degrees of prevalence,
every tissue and organ in the body. A multitude of therapeutic
agents have been developed over the past few decades for the
treatment of various types of cancer for humans and have been used
off-label or reformulated for the treatment of cancer in companion
animals. The most commonly used types of anti-cancer agents
include: DNA-alkylating agents (e.g., cyclophosphamide,
ifosfamide), anti-metabolites (e.g., methotrexate, a folate
antagonist, and 5-fluorouracil, a pyrimidine antagonist),
microtubule disrupters (e.g., vincristine, vinblastine,
paclitaxel), DNA intercalators (e.g., doxorubicin, daunomycin,
cisplatin), and immunosuppressant (e.g., prednisone), each of which
may be administered simultaneously, sequentially or in a common
dosage regimin with antibodies of the invention.
[0084] Antibodies (mAbs) that can be subjected to the techniques
set forth herein include monoclonal and polyclonal mAbs, and
antibody fragments such as Fab, Fab', F(ab')2, Fd, scFv, diabodies,
antibody light chains, antibody heavy chains and/or antibody
fragments derived from various sources. An antibody is obtained
from a sequence donor species. More particularly, the nucleic acid
or amino acid sequence of the variable portion of the light chain,
heavy chain or both, of the donor species antibody has specificity
for a desired antigen. The donor species is any species which was
used to generate the antibodies or antibody libraries, e.g., mouse,
rat, rabbit, cat, dogs, goat, chicken, bovine, horse, llama, camel,
dromedaries, sharks, non-human primates, human, humanized,
recombinant sequence, engineered sequence, etc. Techniques for
generating and cloning monoclonal antibodies are well known to
those skilled in the art.
[0085] After sequencing the antibody obtained from the donor
species or from a library, the variable regions (VH and VL) are
separated into discrete regions such as leader sequences,
frameworks (FRs) and CDRs using any published definition of CDRs
and frameworks (e.g., Kabat, Chothia, AbM, contact definition and
any combination thereof, and any others known to those skilled in
the art). In a particular embodiment, FRs and CDRs are identified
with reference to the Kabat definitions.
[0086] In one aspect, after determining the variable domains with
its individual framework region and CDRs from an originating
species, i.e., FR1, FR2, FR3, FR4, CDR1, CDR2, and CDR3, a set of
FR4 from the target species is selected to replace the FR4 from the
donor species.
[0087] In another aspect, a set of FR1 from the target species is
selected to replace the FR1 from the donor species.
[0088] In another aspect, one or more FR are from the target
species.
[0089] In another aspect, both the FR1 and FR4 are from the target
species.
[0090] Thus in one embodiment the antibody would have a constant
region, and FR1 and/or FR4 derived from a target species, and FR2,
FR3, CDR1, CDR2, and CDR3 derived from a donor species.
[0091] A "chimeric antibody" comprises a sequence of the constant
region or fragment thereof from a target species and the variable
domain containing the contiguous sequence
FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4 from the donor species fused to the
constant domain of the target species. FR4 regions are derived from
the J gene fragments for the light chains and for heavy chains and
can be viewed as an extension of the constant domains.
[0092] Whenever it appears herein, a numerical range such as "1 to
100" refers to each integer in the given range; e.g., "1 to 100
nucleotides" means that the nucleic acid can contain only 1
nucleotide, 2 nucleotides, 3 nucleotides, etc., up to and including
100 nucleotides.
[0093] At that point, with respect to the constant domains of light
chains, a constant domain or fragment thereof from the target
species belonging to the kappa light chain type, or the constant
domain or fragment thereof from the target species belonging to the
lambda light chain type may be fused to the light chain
heterochimeric variable domains. It is contemplated that a
heterochimeric antibodies and/or fragments thereof could comprise a
FR from a lambda variable domain fused together with a constant
domain of a kappa light chain type or a FR sequence from a kappa
variable domain fused together with a constant domain of a lambda
chain. It is also contemplated that any combination of FR from the
target species can be made with any type of light chains of the
originating species.
[0094] With respect to the constant domains of heavy chains, a
constant domain or fragment thereof of any subclass from the target
species may be fused to the heavy chain heterochimeric variable
domains.
[0095] With respect to the pairing of the heterochimeric heavy
chain and the heterochimeric light chain, any combination can be
made.
[0096] The recombinant antibody of the method disclosed herewith
can be an IgG, IgM, IgD, IgE, IgH, or IgA antibody. In some
embodiments, the antibody is an IgG antibody. More particularly,
the antibody can be an IgG.sub.1, IgG.sub.2, IgG.sub.3, or
IgG.sub.4 antibody. The donor species antibody sequence can be, for
example from a mouse, rat, rabbit, cat, dogs, goat, chicken,
bovine, horse, llama, camel, dromedaries, sharks, non-human
primates, human, humanized, recombinant sequence, consensus
sequences, pseudogene sequences, or an engineered sequence. The
target or acceptor species antibody sequence can be, for example,
from a canine antibody, a feline antibody, an equine antibody or a
human antibody.
[0097] The engineering of the recombinant antibody of the claimed
invention comprise can be created by introducing modifications,
additions or deletions to a nucleic acid encoding the antibody can
be introduced by a method comprising recombination, error-prone
PCR, shuffling, oligonucleotide-directed mutagenesis, assembly PCR,
sexual PCR mutagenesis, in vivo mutagenesis, site-specific
mutagenesis, gene reassembly, synthetic ligation reassembly or a
combination thereof.
[0098] Further envisioned within the scope of this invention is the
usage of the recombinant nucleic acids or proteins, or fragments or
derivatives thereof, for the treatment of all companion animal
diseases and/or conditions that are mediated or associated with the
onset of inflammation, as well as companion animal diseases and/or
conditions that are mediated or associated with autoimm unity. Such
diseases and/or conditions are referred to herein as inflammatory
disorders and include but are not restricted to inflammation,
autoimmune disease and immune-mediated.
[0099] In a further aspect, the invention features pharmaceutical
compositions in which antibodies of the present invention are
provided for therapeutic or prophylactic uses. The invention
features a method for treating a dog subject having a particular
antigen, e.g., one associated with disease. The method includes
administering a therapeutically effective amount of a recombinant
antibody specific for the particular antigen, with the recombinant
antibody described herein.
[0100] The amount of antibody useful to produce a therapeutic
effect can be determined by standard techniques well known to those
of ordinary skill in the art. The antibodies will generally be
provided by standard technique within a pharmaceutically acceptable
buffer, and may be administered by any desired route. The route of
administration of the antibody or antigen-binding moiety of the
invention may be oral, parenteral, by inhalation or topical. The
term parenteral as used herein includes intravenous, intramuscular,
subcutaneous, rectal, vaginal or intraperitoneal
administration.
[0101] Antibodies produced in the manner described above, or by
equivalent techniques, can be purified by a combination of affinity
and size exclusion chromatography for characterization in
functional biological assays. These assays include determination of
specificity and binding affinity as well as effector function
associated with the expressed isotype, e.g., ADCC, apoptosis, or
complement fixation. Such antibodies may be used as passive or
active therapeutic agents against a number of diseases, including B
cell lymphoma, T cell lymphoma, autoimmune diseases, inflammatory
diseases, infectious diseases, and transplantation.
[0102] In preferred embodiments of the above aspects, the antigen
is a tumor antigen, an antigen involved in an immune disorder, an
antigen involved in an autoimmune response, a receptor expressed on
a host cell or available in blood circulation or secreted by a cell
and the recombinant antibody is able to either deplete undesired
cells or to block or stimulates receptor functions, or neutralizes
active soluble products.
[0103] The antibodies (or fragments thereof) of this invention may
also be useful for treating tumors in companion animals. More
specifically, they should be useful for reducing tumor size,
inhibiting tumor growth and/or prolonging the survival time of
tumor-bearing animals. Accordingly, this invention also relates to
a method of treating tumors in a dog or other animals by
administering an effective dose. An effective dose is expected to
be in the range of about 0.05 to 100 milligrams per kilogram body
weight per day.
[0104] In a particular embodiment, the invention provides
antibodies to CD20. The canine CD20 is a non-glycosylated integral
membrane phosphoprotein expressed on the surface of almost all
normal and malignant B cells. It has four membrane spanning
hydrophobic regions and a short extracellular loop between the
third and fourth transmembrane domain.
[0105] The CD20 protein is predicted to contain domains of amino
acid sequences consisting of two extracellular domains, four
transmembrane domains, and three intracellular domains as human
CD20.
[0106] The amino acid sequence of canine CD20 shows sequence
similarities with those of human and mice. The amino acid sequences
of canine CD20 exhibit a high degree of similarity with the human
gene, suggesting a similar biological function. Despite the
sequence homology between the canine and human CD20 sequence,
Rituximab, a monoclonal antibody to the human CD20 antigen does not
react with canine B cells probably due to the lack of homology
between humans and dogs in the epitope of the extracellular domain
of CD20 recognized by Rituximab (Veterinary Journal, 2006, vol 171,
556). There are several reported versions of canine CD20. In one
embodiment, the canine CD20 is of SEQ ID NO: 67:
TABLE-US-00001
MTTPRNSMSGTLPVDPMKSPTAMYPVQKIIPKRMPSVVGPTQNFFMRESKTLGAVQI
MNGLFHIALGSLLMIHTDVYAPICITMWYPLWGGIMFIISGSLLAAADKNPRKSLVKG
KMIMNSLSLFAAISGIIFLIMDIFNITISHFFKMENLNLIKAPMPYVDIHNCDPANPSEK
NSLSIQYCGSIRSVFLGVFAVMVIFTFFQKLVTAGIVENEWKKLCSKPKSDVVVLLAA
EEKKEQPIETTEEMVELTEIASQPKKEEDIEIIPVQEEEEELEINFAEPPQEQESSPIENDS
IP
[0107] Canine antibody against the CD20 antigen expressed by normal
and malignant B lymphocytes. The antibody is produced in mammalian
cells (CHO or Per.C6) and meets manufacturing and purification
specifications. The product is a sterile, clear, colorless,
preservative free liquid concentrate for parenteral
administration.
[0108] In another embodiment, the invention provides antibodies to
CD52. The small cell-surface glycoprotein CD52, commonly called the
CAMPATH-1 antigen, is a widely distributed membrane-bound protein
occurring on a variety of cells including but not limited to
lymphocytes, monocytes, thymocytes, epithelial cells, macrophages,
peripheral blood cells, dendritic cells, eosinophils, mast cellss
and several tumor cell lines such as osteogenic tumor cells. In
some cases, CD52 or a fragment thereof may be a soluble
protein.
[0109] A variety of cells expressing the antigen CD52 are
associated with diseases such as cancers and immune conditions.
Several studies have demonstrated or disclosed that neutralization
of human CD52-expressing cells can improve tumor cell or neoplasia
either alone or in combination with other anti-cancer or
chemotherapeutic agents or treatments.
[0110] Myeloid lineage immune cell, containing a number of
membrane-bound proteins including CD52, secrete a variety of
cytokines and enzymes that result in inflammation. As some of these
substances occur in secretory vesicles that appear granular, the
process of secretion is sometimes called degranulation. Rapid
degranulation by mast cells contributes to the pathology of asthma,
anaphylaxis, and other allergic responses, while slower
degranulation by mast cells contributes to arthritis and other
types of chronic inflammation. The release of inflammatory
cytokines and enzymes by mast cells can result in tissue damage,
further attraction of mast cells, resulting in further tissue
damage.
[0111] Macrophages are white blood cells found within tissues
produced by the division of monocytes that contain a number of
membrane-bound proteins including CD52. These cells are involved in
the innate immunity and cell-mediated immunity with a role of
phagocytosis of cellular debris and pathogens and to stimulate
lymphocytes and other immune cells. Macrophages are involved in
many diseases of the immune system. Macrophages are the predominant
cells involved in creating the progressive plaque lesions of
atherosclerosis. Macrophages are believed to promote proliferation
and inflammation of cancerous cells. The arsenal of veterinary
medicine is limited when it comes to addressing immune conditions
and cancer. Most veterinary therapeutic agents have been borrowed
from human therapeutics, often with imperfect results. There is a
thus a need for improved and more specific treatments and biologic
agents for use in animals, such as companion animals. Novel and
specific treatments targeting proteins on the surface of cells
involved in animal diseases may be used to diagnose and treat such
diseases with polyclonal antibodies or fragment thereof, monoclonal
antibodies or fragment thereof, polypeptides or fragment thereof
and other agents which specifically recognize the cell surface
targets. In particular, novel antibodies and other agents disclosed
herein which specifically recognize targets on the surface of cells
that can modulate, (reduce and/or enhance), the disease-promoting
activities of cells carrying antigens such as CD20 and/or CD52. The
present invention provides antibodies and polypeptides targeting
antigens that are capable of inhibiting the disease-associated
activities of cells expressing these antigens either on the
membrane or released in blood circulation. In another aspect, the
invention provides novel compounds for use in diagnostic assays,
and for use as antigens or for selecting antibodies to antigens
such as CD20 and CD52.
[0112] The invention thus provides: heterochimeric antibodies
and/or fragments thereof that include (i) hypervariable region
sequences wholly or substantially identical to sequences found in
antibodies from a donor species; (ii) constant region sequences
wholly or substantially identical to sequences found in antibodies
from a target species which is different from the donor species;
and (iii) heavy and/or light chain variable framework sequences
which contain at least three contiguous non-CDR residues
corresponding to sequences found in antibodies from a target
species and at least three contiguous non-CDR residues
corresponding to sequences found in antibodies from a donor
species.
[0113] In certain aspects, the antibody of the invention includes
within the variable framework sequences, at least four, five, six
or more contiguous non-CDR residues corresponding to sequences
found in antibodies from a target species.
[0114] In certain aspects, the light chain variable region sequence
of the antibody of the invention includes at least four, five, six
or more contiguous non-CDR residues corresponding to sequences
found in antibodies from a donor species.
[0115] The donor species can be any species in which antibodies can
be generated, such as a rodent (e.g. mouse, rat, hamster and the
like).
[0116] The target species can be any mammal, including humans, for
which treatment is desired, e.g., in which it is desirable to
reduce the presence of neutralizing anti-antibody antibodies in an
antibody therapy. In certain embodiments, the target species is a
companion animal selected from a dog, cat, and horse. Companion
animals are generally animals that are kept as pets.
[0117] Antibodies of the invention include a framework region (FR)
(e.g. a lambda or kappa variable domain) of a donor species
antibody fused to a constant domain (kappa or lambda light chain)
from a target species antibody. In particular, the antibodies of
the invention include donor light chains that contain FR4 and/or
FR1 from a target species animal.
[0118] In certain embodiments, the antibodies of the invention
include heavy chain variable regions from a target species
antibody. In particular aspects of the invention, the heavy chain
variable region the FR1 is a target species sequence.
[0119] The antibody will be directed to and bind an antigen of the
target species, such as a dog, cat or horse antigen. The antigen
can be, for example, a tumor antigen, such as a canine, feline or
equine tumor antigen. In some aspects, antibody will also bind to
homologous antigens from other mammals, such as human antigens
including human tumor antigen. In other embodiments of the
invention, the antigen is associated with a cardiovascular disease,
an autoimmune disease, an inflammatory disease or a viral or
bacterial infection related disease. Antigens contemplated as
targets for the antibodies of the present invention include, but
are not limited to antigens that are known in the art, such as:
CD2, CD3, CD4, CD5, CD8, CD11a, CD11b, CD18, CD19, CD20, CD22,
CD23, CD25, CD26, CD28, CD29, CD11, CD33, CD34, CD38, CD40, CD40L
CD41, CD44, CD45, CD52, CD54 (ICAM-1), CD61, CD71, CD74, CD79,
CD80, CD87, CD104, CD120, CD121, CD122, CD123, CD126, CD128, CD133,
CD135, CD150, CLL-1, CD117 (c-Kit), CD152, CD153, Flk-2/Flt3, Gr-1
Ly-6, Sca-1, IGF1R, HER2/neu, EpCam, RANK-L, TRAIL-1, HGF
(hepatocyte growth factor), TNF.alpha., TNF.beta., IL-1, IL-6,
IL-8, IL-13, IL-17, C5a, TCR, adhesion molecules, the neu oncogene
product, MDR-1 (P-glycoprotein), TGFA and its receptor, EGF, PDGF,
VEGF, and their receptors, and the chemokines.
[0120] In certain embodiments antibodies of the present invention
target antigens associate with a particular disease or disorder,
such as acute inflammation, rheumatoid arthritis, transplant
rejection, asthma, allergic inflammation, restenosis, arterial
restenosis, inflammatory bowel disease, uveitis, multiple
sclerosis, psoriasis, wound healing, lupus erythematosus, allergic
rhinitis, atopic dermatitis, food allergies, diabetes mellitus,
dermatitis, thrombotic thrombocytopenic purpura, encephalitis,
leukocyte adhesion deficiency, rheumatic fever, psoriatic
arthritis, osteoarthritis, ocular inflammatory disorders,
progressive systemic sclerosis, primary biliary cirrhosis, CNS
inflammatory disorder, antigen-antibody complex mediated diseases,
autoimmune hemolytic anemia, ischemic heart disease,
atherosclerosis, post-dialysis syndrome, leukemia, acquired immune
deficiency syndrome, septic shock, lipid histiocytosis, and
cancer.
[0121] Of particular interest are antigens CD20, CD52, HER2/neu,
and IL-6, as well as the epitope recognized by mAb 231 (ATCC
HB-9401). The skilled artisan will appreciate that the antigen is
preferably isolated or derived from the target species (e.g.
canine, feline or equine), but suitable cross-reactive antibodies
can in some cases be generated by using an antigen from a xenogenic
species.
[0122] 1.1. The antibody of any of the previous embodiments wherein
the complementarity determining regions and framework regions are
defined in accordance with Kabat.
[0123] 1.2. The antibody of any of the previous embodiments wherein
the constant region of the antibody is modified to enhance a
cytotoxic effector functions selected from ADCC, antibody dependent
cellular phagocytosis (ADCP), and complement dependent cytotoxicity
(CDC).
[0124] In a further embodiment, the invention provides
[0125] 2. Antibody 2, which is an antibody to canine or feline or
equine CD20, CD52, HER2/neu, IL-6, IL-6 receptor, or the epitope
recognized by mAb 231 (ATCC HB-9401). [0126] 2.1. Antibody 2
wherein the antibody is to canine or feline or equine CD20. [0127]
2.2. Antibody 2.1 wherein the antibody is derived from or has
substantially the same hypervariable domain as an antibody raised
against an immunogenic construct comprising or expressing a peptide
containing the sequence of one or more extracellular loops of CD20.
[0128] 2.3. Any of Antibodies 2-2.2 wherein the antibody induces
apoptosis of cells expressing CD20. [0129] 2.4. Any of Antibodies
2-2.3 wherein the antibody suppresses growth of cells expressing
CD20. [0130] 2.5. Any of Antibodies 2-2.4 wherein the antibody
causes the death of cells expressing CD20 by antibody dependent
cell-mediated cytotoxicity (ADCC). [0131] 2.6. Any of Antibodies
2-2.5 wherein the antibody causes the death of cells expressing
CD20 by complement-dependent cytotoxicity (CDC). [0132] 2.7. Any of
Antibodies 2-2.6 wherein the antibody is to feline CD20, e.g., of
SEQ ID NO.:69. [0133] 2.8. Any of Antibodies 2-2.6 wherein the
antibody is to canine CD20, e.g. of SEQ ID NO.:67. [0134] 2.9.
Antibody 2.8 wherein the antibody is derived from or has
substantially the same hypervariable domain as an antibody raised
against an immunogenic construct comprising or expressing a peptide
containing a sequence selected from one or more of the following
sequences: SEQ ID NO.:67 and SEQ ID NO.:69. [0135] 2.10. Antibody
2.8 or 2.9 wherein the antibody specifically recognizes an epitope
on the extracellular loop of canine CD20, wherein the epitope
comprises or is found within a region of the CD20 comprising or
expressing a peptide containing a sequence selected from one or
more of the sequences of residues 74-84, 178-188, 154-170,
140-146,162-173, 148-159, 142-153, 148-169, 166-177, or 161-176 of
SEQ ID NO:67. [0136] 2.11. Any of Antibodies 2-2.6 wherein the
antibody is to equine CD20. [0137] 2.12. Antibody 2 wherein the
antibody is to canine or feline or equine CD52. [0138] 2.13.
Antibody 2.12 wherein the antibody is derived from or has
substantially the same hypervariable domain as an antibody raised
against an immunogenic construct comprising or expressing a peptide
containing the sequence of one or more extracellular loops of CD52.
[0139] 2.14. Any of Antibodies 2.12 or 2.13 wherein the antibody
induces apoptosis of cells expressing CD52. [0140] 2.15. Any of
Antibodies 2.12 or 2.13 wherein the antibody suppresses growth of
cells expressing CD52. [0141] 2.16. Any of Antibodies 2.12 or 2.13
wherein the antibody causes the death of cells expressing CD52 by
antibody dependent cell-mediated cytotoxicity (ADCC). [0142] 2.17.
Any of Antibodies 2.12 or 2.13 wherein the antibody causes the
death of cells expressing CD52 by complement-dependent cytotoxicity
(CDC). [0143] 2.18. Any of Antibodies 2.16-2.6 wherein the antibody
is to feline CD52. [0144] 2.19. Any of Antibodies 2-2.6 wherein the
antibody is to canine CD52. [0145] 2.20. Antibody 2.19 wherein the
antibody is derived from or has substantially the same
hypervariable domain as an antibody raised against an immunogenic
construct comprising or expressing a peptide containing a sequence
selected from one or more of the sequences of residues 4-18, 20-26,
30-39, 36-47, and/or 49-64 of SEQ ID NO:72. [0146] 2.21. Antibody
2.8 or 2.9 wherein the antibody specifically recognizes an epitope
on the extracellular loop of canine CD52, wherein the epitope
comprises or is found within a region of the CD52 selected from
residues 4-18, 20-26, 30-39, 36-47, and/or 49-64 of SEQ ID NO:72.
[0147] 2.22. Any of Antibodies 2-2.6 wherein the antibody is to
equine CD52. [0148] 2.23. Any of Antibodies 2-2.22 wherein the
antibody comprises hypervariable sequences from a donor species
antibody and constant region sequences from a target species.
[0149] 2.24. Any of Antibodies 2.23.wherein the antibody is
caninized. [0150] 2.25. Any of Antibodies 2.23 wherein the antibody
is felinized. [0151] 2.26. Any of Antibodies 2.23 wherein the
antibody is equinized. [0152] 2.27. Any of Antibodies 2.23 to 2.26
wherein the antibody is a heterochimeric antibody of any of
Antibodies 1-1.35. [0153] 2.28. Any of Antibodies 2-2.22. wherein
the antibody is monoclonal and is fully canine. [0154] 2.29. Any of
Antibodies 2-2.22 wherein the antibody is monoclonal and is fully
feline. [0155] 2.30. Any of Antibodies 2-2.22 wherein the antibody
is monoclonal and is fully equine.
[0156] The invention further provides
[0157] a. a method of treating a patient suffering from a disease
or condition characterized by the presence of abnormal cells
expressing a target antigen comprising administering a
therapeutically effective amount of an antibody binding to such
target antigen, wherein the antibody is selected from Antibody
1-1.35 or 2-2.30.
[0158] b. a method of treating a patient suffering from a disease
or condition characterized by the presence of abnormal cells
expressing CD20 comprising administering a therapeutically
effective amount of an antibody selected from Antibody 2-2.11 and
2.23-2.30.
[0159] c. Method b) wherein the patient is a dog.
[0160] d. Method c) wherein the condition to be treated is canine
lymphoma.
[0161] e. Method a) wherein the disease is selected from the group
consisting of: acute inflammation, rheumatoid arthritis, transplant
rejection, asthma, allergic inflammation, restenosis, arterial
restenosis, inflammatory bowel disease, uveitis, multiple
sclerosis, psoriasis, wound healing, lupus erythematosus, allergic
rhinitis, atopic dermatitis, food allergies, diabetes mellitus,
dermatitis, thrombotic thrombocytopenic purpura, encephalitis,
leukocyte adhesion deficiency, rheumatic fever, psoriatic
arthritis, osteoarthritis, ocular inflammatory disorders,
progressive systemic sclerosis, primary biliary cirrhosis, CNS
inflammatory disorder, antigen-antibody complex mediated diseases,
autoimmune hemolytic anemia, ischemic heart disease,
atherosclerosis, post-dialysis syndrome, leukemia, acquired immune
deficiency syndrome, septic shock, lipid histiocytosis, and
cancer.
[0162] f. Method a, b, c or d or e further comprising
administration of chemotherapy.
[0163] g. Method f wherein the chemotherapy comprises
administration of one or more agents selected from
cyclophosphamide, doxorubicin, vincristine, prednisone,
L-asparaginase, cytoxan and adriamycin.
[0164] h. Method for g wherein the chemotherapy spares or enhances
effector cells, e.g., so as to enhance or reduce interference with
ADCC effects of antibody on cancer cells.
[0165] i. Any of the foregoing methods further comprising
administration of a corticosteroid, e.g., prednisone.
[0166] j. Any of the foregoing methods further comprising
administration of radiation.
[0167] k. A method of treating a patient suffering from a disease
or condition characterized by the presence of abnormal cells
expressing CD52 comprising administering a therapeutically
effective amount of an antibody selected from Antibody
2.12-2.30.
[0168] l. Method k) wherein the patient is a dog.
[0169] m. Method 1) wherein the condition to be treated is canine
lymphoma.
[0170] n. Any of the foregoing methods comprising co-administration
of antibody to CD20 and CD52.
[0171] o. Any of the foregoing methods wherein the antibody is
administered in a method to treat or inhibit recurrence of cancer
following treatment with radiation or chemotherapy.
[0172] The invention further provides pharmaceutical compositions
comprising any of antibodies 1-1.35 or 2-2.30, e.g., for use in any
of methods a-o.
[0173] The invention further provides the use of any of antibodies
1-1.35 or 2-2.30 as pharmaceuticals, or in the manufacture of a
medicament for use in any of the methods a-o.
[0174] The invention further provides a cell line stably expressing
any of antibodies 1-1.35 or 2-2.30, for example a CHO cell line
stably expressing any of antibodies 1-1.35 or 2-2.30.
[0175] The invention further provides a vector or vectors
expressing at least one heavy chain and at least one light chain of
any of antibodies 1-1.35 or 2-2.30.
[0176] The invention further provides a method of making an
antibody comprising transforming a cell line with a vector or
vectors expressing at least one heavy chain and at least one light
chain of any of antibodies 1-1.35 or 2-2.30.
[0177] In another embodiment the invention provides a method of
diagnosing a disease or condition treatable with the antibodies of
the invention, comprising obtaining a tissue sample and measuring
binding by one of the antibodies of the invention, together with
diagnostic kits for performing such a method comprising an antibody
of the invention, e.g., any of antibodies 1-1.35 or 2-2.30.
[0178] Other features and advantages of the invention are apparent
from the following description of the preferred embodiments
thereof, and from the claims.
EXAMPLE 1
Heterochimeric Antibodies
[0179] The following EXAMPLE provides general representations of
heterochimeric antibodies, which are constructed according to
standard techniques using the sequences and general patterns
illustrated below. In the examples listed below, the CDRs are
defined using the Kabat nomenclature. Databases containing these
sequences are built from various sources such as NCBI and others
(Das et al., "Immunogenetics, 60:47-55 (2008); Das et al., Proc.
Natl. Ac. Sci. USA. 105:16647-16652 (2008)).
[0180] I. Antibody Variable Domains. Illustrated below in Table 1,
are diagrammatic representations of the heterochimerization for the
light chain antibodies, showing contiguous sequences of discrete
immunoglobulin domains.
TABLE-US-00002 TABLE 1 AVD 1:
FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4.sub.T-Lambda-C.sub.T-Lambda AVD 2:
FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4.sub.T-Kappa-C.sub.T-Lambda AVD 3:
FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4.sub.T-Lambda-C.sub.T-Kappa AVD 4:
FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4.sub.T-kappa-C.sub.T-Kappa AVD 5:
FR1.sub.T-Lambda-CDR1-FR2-CDR2-FR3-CDR3-FR4-C.sub.T-Lambda AVD 6:
FR1.sub.T-Kappa-CDR1-FR2-CDR2-FR3-CDR3-FR4-C.sub.T-Lambda AVD 7:
FR1.sub.T-Lambda-CDR1-FR2-CDR2-FR3-CDR3-FR4-C.sub.T-Kappa AVD 8:
FR1.sub.T-kappa-CDR1-FR2-CDR2-FR3-CDR3-FR4-C.sub.T-Kappa AVD 9:
FR1.sub.T-Lambda-CDR1-FR2-CDR2-FR3-CDR3-FR4.sub.T-Lambda-
C.sub.T-Lambda AVD 10:
FR1.sub.T-kappa-CDR1-FR2-CDR2-FR3-CDR3-FR4.sub.T-kappa-C.sub.T-Kap-
pa AVD = Antibody Variable Domain; T = Target species; Lambda =
lambda light chain; Kappa = kappa light chain; C = Constant domain;
FR = Framework region; CDR = Complementarity Determining
Region.
[0181] II. Antibody Heavy Chains Domains. Illustrated below in
Table 2, are diagrammatic representations of the
heterochimerization for the heavy chains, showing contiguous
sequences of discrete immunoglobulin domains. Abbreviations are as
above in EXAMPLE 1.I.
TABLE-US-00003 TABLE 2 AVD 11:
FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4.sub.T- C.sub.T AVD 12:
FR1.sub.T-CDR1-FR2-CDR2-FR3-CDR3-FR4- C.sub.T AVD 13:
FR1.sub.T-CDR1-FR2-CDR2-FR3-CDR3- FR4.sub.T-C.sub.T
[0182] III. Framework Sequences.
[0183] Exemplary framework sequences (FR4) used as a source to
construct the light chain heterochimeric antibodies and/or
fragments thereof are provided in the sequence listing as SEQ ID
NOs:1-12. In this example, sequences from canine light chains are
provided using the Kabat nomenclature.
[0184] Exemplary framework sequences (FR4) used as a source to
construct the heavy chain heterochimeric antibodies and/or
fragments thereof are provided in the sequence listing as SEQ ID
NOs:13-19. The standard abbreviations for amino acid residues are
used to list the sequences.
[0185] Exemplary framework sequences (FR1) used as a source to
construct the light chain heterochimeric antibodies and/or
fragments thereof are provided in the sequence listing as SEQ ID
NOs:20-25.
[0186] Exemplary framework sequences (FR1) used as a source to
construct the heavy chain heterochimeric antibody and/or fragments
thereof are provided in the sequence listing as SEQ ID NOs:
26-40.
EXAMPLE 2
Construction, Expression and Purification of Antibody Variants
[0187] I. Antibody Variants Derived from the Rat Anti-human CD52
Antibody.
[0188] The rat anti-human CD52 antibody was caninized according to
the present invention. The sequence of the anti-human CD52 antibody
as described in pdb 1bfo_E and pdb 1bfo_F (Campath-1G, clone YTH
34.5HL, Protein Data Bank proteins (pdb), date of deposition: May
20, 1998). Variable regions were prepared by assembling synthetic
oligonucleotides corresponding to the publically available
sequence, and cloned into pSMART with HindIII and NheI as flanking
restriction sites on the 5'- and 3'- end of the variable domains,
respectively. Assembled products were then subcloned into an
expression vector containing a promoter and the heavy chain
constant domain or containing the lambda light chain constant
domain. The entire expression cassette included the human
cytomegalovirus immediate-early (CMV) promoter, a kozak sequence
and signal peptide sequence immediately upstream of the coding
sequence and in frame with the variable region of both the light
and heavy chains to direct the resulting antibody product towards
the secretory pathway.
[0189] Antibody variants containing canine sequences were
constructed using the rat anti-human CD52 variable regions as
template. In this example, the canine sequences were compiled from
genomic sequences available at the National Center for
Biotechnology Information (NCBI) and sequences with high occurrence
were selected to construct the genes of the present example. The
CDR domains, the framework regions and the J fragments were
identified as described by Kabat and modified as listed in Table 3.
The modified expression cassettes containing the various
combinations were linked to canine constant regions listed in the
present invention. These antibody variant genes were then
transferred to expression vectors for production of recombinant
antibodies in mammalian cells.
[0190] VET111 contained the rat kappa variable domain sequence in
its entirety linked to a canine lambda constant domain of VET104.
In VET114, the rat kappa J fragment was replaced by a canine lambda
J fragment. In VET! 12, both the rat kappa J fragment and the rat
kappa FRI were replaced with canine lambda sequences. VET222
contained the rat heavy chain variable domain in its entirety
linked to the canine constant domain of VET214. In VET 224, the rat
J fragment was replaced by a canine J fragment. In VET223, both the
rat J fragment and the rat FRI were replaced with canine
sequences.
TABLE-US-00004 TABLE 3 SEQ ID Designation Structure NO: Light Chain
VET111 FR1.sub.R-VK-CDR1.sub.R-VK-FR2.sub.R-VKCDR2.sub.R-VK- 41
FR3.sub.R-VK-CDR3.sub.R-VK-FR4.sub.R-VK-C.sub.D-L VET112
FR1.sub.D-VL-CDR1.sub.R-VK-FR2.sub.R-VKCDR2.sub.R-VK- 42
FR3.sub.R-VK-CDR3.sub.R-VK-FR4.sub.D-VL-C.sub.D-L VET114
FR1.sub.R-VL-CDR1.sub.R-VK-FR2.sub.R-VK-CDR2.sub.R-VK- 43
FR3.sub.R-VK-CDR3.sub.R-VK-FR4.sub.D-VL-C.sub.D-L Heavy Chain
VET222 FR1.sub.R-VH-CDR1.sub.R-VH-FR2.sub.R-VHCDR2.sub.R-VH- 44
FR3.sub.R-VH-CDR3.sub.R-VH-FR4.sub.R-VH-C.sub.D-H VET223
FR1.sub.D-VH-CDR1.sub.R-VH-FR2.sub.R-VHCDR2.sub.R-VH- 45
FR3.sub.R-VH-CDR3.sub.R-VH-FR4.sub.D-VH-C.sub.D-H VET224
FR1.sub.R-VH-CDR1.sub.R-VH-FR2.sub.R-VHCDR2.sub.R-VH- 46
FR3.sub.R-VH-CDR3.sub.R-VH-FR4.sub.D-VH-C.sub.D-H M: Mouse; D: Dog;
FR.sub.M-VK = Murine kappa light chain (LC) FR; FR.sub.D-VL =
Canine lambda LC FR; CDR.sub.M-VK = Murine kappa LC CDR;
CDR.sub.M-VH = CDR from a murine heavy chain (HC); CD.sub.D-L or
CD.sub.D-K = Constant domain from a canine lambda or canine kappa
LC; CD.sub.D-H = Constant domain from canine HC.
[0191] II. Antibody Variants Derived from a Murine Anti-canine
Lymphoma Antibody.
[0192] The heavy and light chains of the murine anti-canine
lymphoma monoclonal antibody mab 231 were isolated from hybridoma
cells (ATCC Number: HB-9401). Briefly, total RNA was extracted from
1 million hybridoma cells using the MasterPure.TM. RNA Purification
Kit (Epicentre Biotechnology). The first-strand cDNA was
synthesized from 1 .mu.g of total RNA using SuperScript System for
RT-PCR (Invitrogen) according to the manufacturer's instructions.
The immunoglobulin heavy chain variable region (VH) and the
immunoglobulin light chain variable region (VK) were amplified by
PCR using primers described previously (O'Brien & Jones, 40:
567-592 (2001)). The PCR reactions were set as recommended by the
manufacturer (Invitrogen) The samples were denatured at 94.degree.
C. for 5 min followed by amplifications for 35 cycles (94.degree.
C. for 30 s, 55.degree. C. for 20 s, 72.degree. C. for 45 s). The
variable domains were then amplified with primers containing the
HindIII and NheI restriction sites to allow for the cloning of the
PCR product into the corresponding restriction sites of expression
vectors. Amino acid sequence of the murine anti canine lymphoma
monoclonal antibody are listed as SEQ ID NO:47 for the heavy chain
and SEQ ID NO:48 for the light chain. Antibody variants containing
canine sequences were constructed using the murine anti-canine
lymphoma antibody mab 231 variable regions as template. In this
example, the canine sequences were compiled from expressed
sequences available at the National Center for Biotechnology
Information (NCBI) and sequences with high occurrence were selected
to construct the genes of the present example. The CDR domains, the
framework regions and the J fragments were identified as described
by Kabat and modified as listed in Table 4. The modified expression
cassettes containing the various combinations were linked to canine
constant regions listed in the present invention. These composite
antibody genes were then transferred to expression vectors for
production of recombinant antibodies in mammalian cells.
[0193] The murine kappa variable domain in its entirety was either
linked to a canine lambda constant domain of VET104 or to a canine
kappa constant domain of VET105. In VET 118, the murine kappa J
fragment (or FR4) was replaced by a canine kappa J fragment. VET217
contained the murine heavy chain variable domain in its entirety
linked to the canine constant domain of VET214. In VET 218, the
murine J fragment was replaced by a canine J fragment.
TABLE-US-00005 TABLE 4 List of anti-canine lymphoma antibody
variants. Designation Structure SEQ ID NO: Light Chain VET106
FR1.sub.M-VK-CDR1.sub.M-VK-FR2.sub.M-VK-CDR2.sub.M-VK- 49
FR3.sub.M-VK-CDR3.sub.M-VKFR4.sub.M-VK-C.sub.D-L VET107
FR1.sub.M-VK-CDR1.sub.M-VK-FR2.sub.M-VK-CDR2.sub.M-VK- 50
FR3.sub.M-VK-CDR3.sub.M-VK-FR4.sub.M-VK-C.sub.D-K VET118
FR1.sub.M-VK-CDR1.sub.M-VK-FR2.sub.M-VK-CDR2.sub.M-VK- 51
FR3.sub.M-VK-CDR3.sub.M-VK-FR4.sub.D-VL-C.sub.D-L Heavy Chain
VET217 FR1.sub.M-VH-CDR1.sub.M-VH-FR2.sub.M-VH-CDR2.sub.M-VH- 52
FR3.sub.M-VH-CDR3.sub.M-VH-FR4.sub.M-VH-C.sub.D-H VET218
FR1.sub.M-VH-CDR1.sub.M-VH-FR2.sub.M-VH-CDR2.sub.M-VH- 53
FR3.sub.M-VH-CDR3.sub.M-VH-FR4.sub.D-VH-C.sub.D-H M: Mouse; D: Dog;
FR.sub.M-VK = Murine kappa LC FR; FR.sub.D-VL = FR Canine lambda LC
FR; CDR.sub.M-VK = Murine kappa LC CDR; CDR.sub.M-VH = Murine HC
CDR; CD.sub.D-L or CD.sub.D-K = Constant domain from a canine
lambda or canine kappa LC; CD.sub.D-H = Constant domain from a
canine HC.
[0194] III. Cloning of Canine Heavy and Light Chain Constant
Domains. Heavy chain and light chain sequences were cloned from
cDNA made from canine peripheral blood (PBMC) or from canine spleen
tissues. The coding regions or fragment thereof were then amplified
by PCR using the primers listed below. The PCR reactions were set
as recommended by the manufacturer (Invitrogen). The samples were
denatured at 94.degree. C. for 5 min followed by amplifications for
35 cycles (94.degree. C. for 30 s, 62.degree. C. for 20 s,
72.degree. C. for 45 s). The PCR products were first cloned into a
pUC-derived vector. The genes were then re-amplified with flanking
restriction sites for cloning into pcDNA3-derived vector
(Invitrogen). The Heavy chain was amplified with primers listed as
SEQ ID NO:57 and SEQ ID NO:58; the lambda light chain was amplified
with primers listed as SEQ ID NO:59 and SEQ ID NO:60; and the
lambda light chain was amplified with primers listed as SEQ ID
NO:61 and SEQ ID NO:62. The amino acid sequence of the heavy chain
is listed as SEQ ID NO:54 (Plasmid VET214); the amino acid sequence
of the lambda light chain is listed as SEQ ID NO:55 (Plasmid
VET104); and the amino acid sequence of the kappa light chain is
listed as SEQ ID NO:56 (Plasmid VET105).
[0195] IV. Expression, Purification and Quantitation of Antibody
Variants.
[0196] Genes were assembled from synthetic oligonucleotides and
cloned into HindIII-NheI the cloning sites of an expression vector
deriving from pcDNA3 containing a leader sequence allowing for the
secretion of the antibody molecule, a Kozak sequence, the canine
constant domain region, and a terminal codon. The light chain
variable domains were cloned either into VET104 or VET105. The
heavy chain variable domains were cloned into VET214.
[0197] These plasmids were transformed into E. coli (DH5a)
chemically competent E. coli cells (Lucigen), grown in Luria Broth
(LB) media and stocked in glycerol. Large scale plasmid DNA was
prepared using the Zyppy.TM. Plasmid Maxiprep Kit as described by
the manufacturer (Zymo Research Corp.). The antibody variants were
transiently expressed in the human embryonic kidney cell line 293F
(Invitrogen) in serum-free condition. The heavy chain (VET200
series) and light chain (VET100 series) expression vectors were
co-transfected using 293fectin (Invitrogen) and grown in
293F-FreeStyle culture medium (Invitrogen). The transfected 293
cultures expressed approximately 3-12 mg/L of recombinant antibody.
Binding assays were performed with supernatants or with recombinant
antibodies purified from supernatants.
[0198] The antibody titer was determined using a quantitative
ELISA. Plates were coated with 100 ul/well at 37.degree. C. for 1
hour with rabbit anti-dog IgG (H+L) antibody (Jackson
Immuno-Research) diluted 1:100 in carbonate buffer (100 mM
NaHCO.sub.3, 33.6 mM Na.sub.2CO.sub.3, pH 9.5). The plates were
washed three times with TBS-T (50 mM Tris, 0.14 M NaCl, 0.05%
tween-20, pH 8.0) and blocked with 200 ul/well TBS/BSA (50 mM Tris,
0.14 M NaCl, 1% BSA, pH 8.0) for 1 hour at 37.degree. C. The
standard was prepared by diluting the reference antibody (Jackson
Immuno-Research, Dog Gamma Globulin 10.0 mg) in TBS-T/BSA (TB S-T,
1% BSA) in a range of concentration from 0 to 500 ng/ml. After
washing the plates twice with TBS-T, standard/samples preparation
was added to each well and incubated at 37.degree. C. for 1 hour.
The plates were then washed 3.times. with TBS-T and incubated for 1
hour at 37.degree. C. with HRP-rabbit anti-dog IgG antibody
(Perodixase Rabbit Anti-Dog IgG (H+L) Jackson Immuno-Research)
diluted 1:20,000 in TBS-T/BSA. The plates were washed twice with
TBS-T and developed using 100 ul/well of TMB substrate. The
reaction was stopped with 1M H.sub.2SO.sub.4 and the OD was
measured at 450 nm. The standard curve was fitted using a four
parameter equation and used to calculate the antibody concentration
in the samples.
[0199] Antibodies were purified from culture supernatants using
protein A affinity chromatography. Supernatants were diluted 1:1
with Binding Buffer (Pierce) and passed over a gravity-flow column
(GE Healthcare), equilibrated with 20 resin-bed volumes of Binding
Buffer. The antibody retained on the column was washed with 15 ml
of binding buffer, eluted with low pH elution buffer (Pierce) and
collected in 1 ml fractions containing 100 ul of Binding Buffer to
neutralize the pH. Fractions with absorbance (280 nm) >0.1 were
desalted using desalting columns (Pierce). The purity of each
preparation was assessed by HPLC and was determined to be over 95%
by standard techniques. The purity of all the antibody
[0200] preparation was examined by HPLC (High Pressure Liquid
Chromatography). All the variants exhibited a similar elution
pattern consisting of a major peak detected by absorbance at 280 nm
at a position similar than the standard control. There was no
significant aggregation detected by gel filtration.
EXAMPLE 5
Binding of Antibody Variants to Cells
[0201] I. Antibody Variants to CD52 Bind Tumor Cells.
[0202] The binding of the antibody variants of the present
invention was assessed using a fluorescence-activated cell sorter
(FACS). In the present example, the antibody variants were
incubated with the CD52 positive cells and the amount of bound
antibody was assessed following incubation with a
fluorescent-labeled reporter reagent. The reporter was thereafter
measured by FACS.
[0203] Briefly, for each assay, one million cells of the human
T-cell lymphoma HUT-78 cells were resuspended in FACS buffer
(PBS+2% FBS). About 2 ug of the primary antibody were added to the
cells and the samples were incubated at 4.degree. C. for 1 hour.
The primary antibody was provided as supernatants from transfected
cells with recombinant antibody constructs or from purified
antibody preparation. The rat anti-human CD52 mAb (Serotec) was
added to the cells as a control. One ml of FACS buffer was added
and cells were spin down for 3 min at 800.times.g in Eppendorf
microcentrifuge. The cells were washed with 1 ml FACS buffer and
spin down again. The secondary antibodies such as
fluorescein-isothiocynate (FITC) conjugated goat anti-rat (Jackson
ImmunoResearch), or the FITC-conjugated goat anti-dog IgG (H+L)
(Bethyl Laboratories) were added in 100 al of FACS buffer
supplemented with 1% BSA to appropriate tubes and the tubes were
incubated at 4.degree. C. for 30 minutes. The wash steps were
repeated. The cells were then resuspended in 500 .mu.l FACS buffer
and transferred into 12.times.75 mm polystyrene test tubes. The
cells were analyzed by FACS with a FacScan cytometer using the
CellQuest software (Becton-Dickenson). Several controls were
utilized to determine the background fluorescence: (i) one tube of
cells was incubated with the FITC-conjugated secondary antibody
without the primary antibody, (ii) one tube of cells was incubated
with PBS only, and (iii) one tube was incubated with the primary
antibody without the FITC-conjugated secondary antibody.
[0204] A typical staining profile is reported in Table 5. The
results of Table 5 evidence effective binding of the chimeric
antibody (VET111 VET222) and of the antibody variants.
TABLE-US-00006 TABLE 5 Combinations of antibody variants tested by
FACS analysis for their binding to CD52 positive lymphoma cells.
Conditions Binding (%) Cells only 1.09 Cells + anti-dog IgG 4.65
Cells + anti-rat IgG 1.15 Cells + VET 111 VET 222 0.88 Cells + VET
112 VET 223 0.88 Cells + VET 114 VET 224 0.68 Cells + VET 112 VET
224 0.89 Cells + VET 114 VET 223 0.95 Cells + rat anti-human CD52
1.36 Cells + VET 111 VET 222 + anti-dog IgG 51.40 Cells + VET 112
VET 223 + anti-dog IgG 24.35 Cells + VET 114 VET 224 + anti-dog IgG
47.77 Cells + VET 112 VET 224 + anti-dog IgG 30.60 Cells + VET 114
VET 223 + anti-dog IgG 40.08 Cells + rat anti-human CD52+ anti-rat
IgG 41
[0205] II. Anti-canine Lymphoma Antibody Variants Bind Tumor Cells.
The binding of the antibody variants of the present invention were
testing on canine lymphoma cells by FACS. In the present example,
the different antibody variants were incubated with canine lymphoma
cells positive for the target antigen of mab 231 and the amount of
bound antibody was assessed following incubation with a
fluorescent-labeled reporter reagent. The reporter was thereafter
measured by FACS.
[0206] The binding assay was performed as described above. A
typical staining profile obtained with the variants is reported in
Table 6. These results evidence effective binding of the various
variants.
TABLE-US-00007 TABLE 6 Combination of antibody variants tested by
FACS analysis for their binding to cells. Conditions Binding (%)
Cells only 1.05 Cells + anti-dog IgG 4.19 Cells + anti-mouse IgG
3.13 Cells + VET 106 VET 217 1.51 Cells + VET 107 VET 217 1.25
Cells + VET 118 VET 218 1.64 Cells + mab 231 1.49 Cells + VET 106
VET 217 + anti-dog IgG 30.32 Cells + VET 107 VET 217 + anti-dog IgG
20.82 Cells + VET 118 VET218 + anti-dog IgG 27.20 Cells + mab 231 +
anti-mouse IgG 74.21
[0207] III. Anti-CD52 Antibody Variants Alter Proliferation of
Tumor Cells.
[0208] The antibody variants of the present invention were tested
for their ability to alter proliferation of lymphoma cells.
[0209] Lymphoma cells were grown in RPMI medium with FBS 10% in 5%
carbon dioxide (CO2) at 37.degree. C. Cells were seeded at 5,000
cells/well in 96-well plates in medium with 2.5% FBS. Cells were
treated with the antibody variants or the rat or dog isotype
controls (10 .mu.g/ml) and incubated for 72h at 37.degree. C. in a
CO2 incubator. Ten (10) .mu.l MTT solution was added to each well
and incubated at 37.degree. C. for 4 h according to the
manufacturer's instruction (Trevigen). Optical density (OD) was
then measured at 490 nm and data are presented as means.+-.S.D. of
three replicate measurements. The data illustrate in Table 7 that
the antibody variants had an anti-proliferative effect on lymphoma
cells similar than the rat anti-human CD52 antibody.
TABLE-US-00008 TABLE 7 Proliferation cell assay with antibody
variants and controls. Conditions Average .+-. SD Cells 0.486 .+-.
0.028 Rat Isotype 0.488 .+-. 0.043 Dog Isotype 0.479 .+-. 0.045
VET111 VET222 0.213 .+-. 0.012 VET 114 VET 224 0.246 .+-. 0.012 Rat
anti-human CD52 0.259 .+-. 0.020
EXAMPLE 6
[0210] Anti-CD52 Antibody Variants Alter Proliferation of Tumor
Cells.
[0211] The antibody variants of the present invention were tested
for their ability to alter proliferation of lymphoma cells.
[0212] Lymphoma cells were grown in ISCOVE medium with FBS 20% in
5% carbon dioxide (CO2) at 37.degree. C. Cells were seeded at 5,000
cells/well in 96-well plates in medium with 2.5% FBS. Cells were
treated with the antibody variants or the isotype control (10
.mu.g/ml) and incubated for 72 h at 37.degree. C. in a CO2
incubator. Ten (10) .mu.l MTT solution was added to each well and
incubated at 37.degree. C. for 4 h according to the manufacturer's
instruction (Trevigen). Optical density was then measured at 490
nm. Data are presented as means.+-.S.D. of three replicate
measurements. The percentage survival was expressed relative to the
non-treated controls which were defined as 100%. The data
illustrate in Table 8 that the modified antibody variants had an
anti-proliferative effect on lymphoma cells.
TABLE-US-00009 TABLE 8 Proliferation cell assay with antibody
variants and controls (PI: Proliferation Inhibition in %).
Conditions PI (%) 10 ug/ml VET 111 VET 222 Rat anti-human CD52 Rat
Isotype Dog Isotype
EXAMPLE 7
Antibody to CD20
[0213] I. Cloning of Canine and Feline CD20.
[0214] Ia. Cloning of Canine CD20 gene. The canine CD20 gene was
cloned into a mammalian expression vector and the corresponding
plasmid DNA was transfected into mammalian cells to produce a
properly folded form of the receptor. Cells expressing CD20 were
used for immunization and cell-screening based assays.
[0215] CD20 was isolated from canine peripheral blood mononuclear
cells (PBMC). Total RNA was extracted from 1 million canine
peripheral blood mononuclear cells (PBMC) using the MasterPure.TM.
RNA Purification Kit (Epicentre Biotechnology). The first-strand
cDNA was synthesized from 2 .mu.g of total RNA using SuperScript,
First-Strand Synthesis, System for RT-PCR kit (Invitrogen)
according to the manufacturer's instructions. The coding region was
amplified with primers of SEQ ID NO: 63 and SEQ ID NO: 64 and a
fragment thereof encompassing the large extracellular domain (loop)
was amplified with primers of SEQ ID NO: 65 and SEQ ID NO: 66 by
PCR. The PCR reactions were set as recommended by the manufacturer
(Invitrogen). The samples were denatured at 94.degree. C. for 5 min
followed by amplifications for 35 cycles (94.degree. C. for 30 s,
62.degree. C. for 20 s, 72.degree. C. for 45 s) and the PCR
products were cloned into pcDNA-derived vector (Invitrogen).
[0216] The amino-acid sequence of the canine CD20 isolated from
canine PBMC is listed as
[0217] SEQ ID NO 67.
[0218] Ib. Cloning of Feline CD20 Gene. The feline CD20 coding
region was isolated from feline peripheral blood mononuclear cells
(PBMC) fractionated from whole blood. Total RNA was extracted from
5 million feline peripheral blood mononuclear cells (PBMC) using
the Mini RNA Isolation Kit (Zymo Research). The first-strand cDNA
was synthesized from 2 .mu.l of total RNA using SuperScript,
First-Strand Synthesis System for RT-PCR kit according to the
manufacturer's instructions (Invitrogen). The coding region was
then amplified by PCR using the primers of SEQ ID NO:63 and SEQ ID
NO:68 using GoTaq Green Master Mix according to manufacturer's
instructions. The samples were denatured at 94.degree. C. for 5 min
followed by amplifications for 35 cycles (94.degree. C. for 30 s,
52.degree. C. for 30s, 72.degree. C. for 1 min). The PCR products
were then cloned into pJET 1.2 (Fermentas) and transformed into E.
coli strain DH5a and sequenced to verify PCR specificity.
[0219] The amino-acid sequence of the canine CD20 isolated from
feline PBMC is given as
[0220] SEQ ID NO:69.
[0221] The feline CD20 gene or fragment thereof is used for
immunization to generate antibodies. The immunogen is a plasmid
carrying the feline CD20 DNA sequence or a fragment thereof, the
CD20 protein or a fragment thereof or a cell line naturally
expressing CD20 or transfected with the CD20 gene or a fragment
thereof;
[0222] The feline CD20 gene or fragment is used to design peptides
or is expressed in cells to be used for screening assays.
[0223] Epitope prediction for canine CD20 amino acid sequence.
[0224] The epitope prediction was assessed for the amino acid
sequence of the canine CD20 isolated above by using the prediction
algorithm Emboss available online
(http://liv.bmc.uu.se/cgi-bin/emboss/antigenicc). This algorithm
scores the antigenicity potential of a given sequence. This program
predicts four potential epitopes to be exposed at the surface of
cells comprising residues 74-84, 178-188, 154-170, or 140-146 of
SEQ ID NO:69. At least six additional sequences were predicted as
potential epitopes based on hydrophilicity, flexibility,
accessibility, turns, exposed surface, and polarity of polypeptides
chains using other algorithms available on line
(http://tools.immuneepitope.org) comprising residues 162-173,
148-159, 142-153, 148-169, 166-177, 161-176 of SEQ ID NO:67.
[0225] The epitopes recognized by the anti-CD20 antibodies are
discontinuous, comprising regions from both extracellular
loops.
[0226] III. Immunization with CD20 and Generation of Murine
Monoclonal Antibodies to Canine CD20
[0227] To generate monoclonal antibodies to canine CD20, CHO-DG44
(Chinese hamster ovary cells, dihydrofolate reductase deficient
ATCC CRL-9096) and N1H:3T3 (ATCC CRL-1658) were transfected with an
expression vector encoding the full-length canine CD20 protein such
that the protein was expressed on the surface of the cells.
Magnetic Proteoliposome Particles (MPLs) containing CD20, such that
the native conformation of the transmenbrane receptor is maintained
were prepared for immunizations and panning. In brief, recombinant
canine CD20 that contained an epitope tag was solublized from a
transfected CD20-expressing cell line using the detergent CHAPSO
and the protein was captured on magentic beads via the epitope tag.
A lipid membrane was reconstituted during removal of the detergent,
such that the native membrane conformation of CD20 was maintained,
to create the CD20-MPLs.
[0228] Anti-CD20 monoclonal antibodies were generated by
immunization of mice to raise immunoglobulins specific for canine
CD20. Washed CHO-DG44 cells expressing canine CD20
(1.times.10.sup.7 cells in 100 ul) or 100 ul of CD20-MPLs
(1.times.10.sup.9 beads/ml) were used as immunogens. Mice were
immunized with antigen in Ribi adjuvant intraperitonealy three
times, then boosted twice on consecutive days. The immune response
was monitored by retro-orbital bleeds. The sera were screened by
FACS staining of CD20-expressing cells (versus untransfected
parental cells) and CD20-MPLs.
[0229] Mice with sufficient titers of anti-CD20 immunoglobulin were
used for harvesting spleens. A murine antibody library was prepared
from spleen cells of the mice and displayed on phage such that the
phage were then screened for expression of antibodies with
specificity for CD20. This combination approach is generally
described in U.S. application Ser. No. 6,092,098.
[0230] The phage display library was screened for library members
having affinity for CD20 by panning with canine CD20 incorporated
into magnetic proteoliposomes (CD20-MPL). Three rounds of panning
of the phage display library on the CD20-MPLs led to several fold
enrichment of CD20-binders as compared to background. Variable
region fragments of interest were recloned into a Fab expression
vector and the Fab retested for antigen binding against transfected
CD20-expressing cells.
[0231] IV. Anti-CD20 Antibody Leads. A lead candidate with high
affinity for the canine CD20 exhibiting efficacy is identified by
testing it in a panel of assays using methodologies available to
those in the art.
[0232] The specific binding of the newly generated anti-CD20
antibodies is assessed by ELISA and FACS with cells expressing
CD20. Since it is important to measure the relative binding
affinity of the antibodies to native CD20, live cells expressing
CD20 are used and ELISA and FACS analysis. For cell-binding assay,
CD20 expressing cells or canine lymphoma cells are washed with
phosphate-buffered saline (PBS) and seeded in well. After one hour
at room temperature to allow cell attachment to the plate surface,
the cells are washed with FBS to block non-specific binding sites
on the plates. Supernatants from cells expressing the anti-canine
CD20 are then added. After one hour incubation at room temperature,
the plates are washed with PBS. The secondary antibody is then
added and detected using standard procedures.
[0233] Alternatively, a peptide-based ELISA starting from a linear
peptide containing the residues encompassing the extracellular
domain of CD20 is designed. Additional conformational modifications
can be made to improve peptide recognition and/or epitope
presentation. The modifications may include addition of
carbohydrate, addition of residues to form a disulfide bond or to
cross-link a carrier protein.
[0234] Alternatively, whole blood from dogs is collected in sodium
heparin tubes, then incubated with the anti-CD20 antibody and the
binding of the anti-CD20 antibody to certain desirable cells are
analyzed by FACS.
[0235] Additional biophysical properties such as affinity,
thermo-stability are determined using methodologies available to
those in the art.
[0236] To assess the ability of anti-CD20 antibody to augment the
cytotoxic effects of chemotherapeutic drugs, the antibody is tested
in a chemosensitization assay using methodologies available to
those in the art.
[0237] A tumor killing assay is developed to measure the effect of
the recombinant antibody on various canine tumor cells lines.
[0238] An in vitro apoptosis assay is developed to measure the loss
of plasma membrane integrity in canine CD20 positive cells after
anti-CD20 antibody treatment by staining with Annexin V and
Propidium Iodide. This assay is used to assess the ability of
anti-CD20 antibodies to induce apoptosis.
[0239] CDC is an important effector mechanism in the removal of
tumor cells in vivo. The complement system has three parts, the
classical, lectin, and alternative pathways. The classical pathway
is activated by antibodies bound to target antigens and therefore
relevant to tumor cell removal after therapeutic antibody
treatment, while the lectin and alternative pathways target
microbes. The first step in activation of the classical pathway is
binding of the Clq component to the Fc portions of antibodies. This
triggers a proteolytic cascade that ultimately leads to target cell
death by direct disruption of the plasma membrane, or by effector
cell mediated killing through component C3b binding to receptors on
NK cells or macrophages. A CDC assay is developed to assess the
degree of complement activation and tumor killing exhibited by the
anti-CD20 antibodies. CD20 positive canine cells are incubated with
anti-CD20 antibodies and canine complement components. Cell
viability will then be detected by a fluorescent assay.
[0240] ADCC assays are conducted with fresh canine PBMC from normal
donors and diseased donors. Target cells are .sup.51Cr-labeled by
incubation with 250 .mu.Ci of .sup.51Cr for 2 h at 37.degree. C.
Then, antibodies and canine PBMC are added to the wells to assure
effector to target ratios of 50:1, 25:1, 10:1, and 2:1 with the
addition of 5,000 target cells to each well. Each condition is
performed in triplicate and the plates are incubated for 4 hours at
37.degree. C. Maximal release of .sup.51Cr from tumor cells is
established by culturing targets with detergent. Effector and
target cells are co-cultured overnight, at which time cell-free
supernatants are harvested to measure the level of .sup.51Cr
released in supernatants. The % cytotoxicity is determined using
the formula: (Experimental cpm count-Spontaneous cpm
count)/(Maximal cpm count-Spontaneous cpm count). Spontaneous
release represents the radioactivity of culture supernatants from
the target cells alone, maximal count measures the radioactivity of
supernatants from target cells lysed with detergent, and
experimental release the radioactivity measured in supernatants
from wells containing targets plus effector cells.
[0241] The anti-CD20 antibody is rapidly evaluated in lymphoma
xenograft murine models using CD20-positive canine tumor cell lines
according to methodologies available to those in the art.
[0242] V. Engineering of the Modified Anti-CD20 Antibody.
[0243] As the anti-CD20 monoclonal antibodies generated in a
non-canine mammal, most may not be suitable for repeated
administration, the corresponding chimeric or caninized antibody is
generated as described in Example 1 and tested for a panel of
properties as described in the Example above.
[0244] VI. Depletion of B Cells in Vivo Using Anti-CD20.
[0245] To ascertain the efficacy of anti-CD20 antibody in depleting
B cells in vivo, dogs are treated with single or consecutive dose
levels ranging from 0.1 mg/kg to 5 mg/kg. Dogs ranging in weight
from 2.5 to 5 kilograms are divided into three groups of two dogs
each. All animals are injected with anti-CD20 antibody produced
from mammalian cells. The three groups received antibody dosages
corresponding to 0.1 mg/kg, 0.5 mg/kg, and 5 mg/kg each day for
four (4) consecutive days by intravenous infusion; blood samples
are drawn prior to each infusion. Additional blood samples are
drawn beginning 24 hrs after the last injection (T=0) and
thereafter on days 1, 3, 7, 14 and 28.
[0246] Approximately 5 ml of whole blood from each animal is
centrifuged at 2000 RPM for 5 min. Plasma is removed for assay of
soluble anti-CD20 antibody levels. The pellet (containing
peripheral blood leukocytes and red blood cells) is resuspended in
fetal calf serum for FACS analysis.
[0247] For the labeling of leukocytes, cells are washed twice with
Hanks Balanced Salt Solution ("HBSS") and resuspended in a plasma
equivalent volume of fetal bovine serum (FBS). A Fluorescein
labeled monoclonal antibodies with specificity for the lymphocyte
surface markers of B-cell and T-cell are added to identify T and B
lymphocyte populations. Cells are incubated with fluorescent
antibodies for 30 min., washed and analyzed on a Becton Dickinson
FACScan instrument. Lymphocyte populations are initially identified
by forward versus right angle light scatter in a dot-plot bitmap
with unlabeled leucocytes. The total lymphocyte population is then
isolated by gating out all other events.
[0248] VII Treatment of Dogs.
[0249] A dog diagnosed with a condition including lymphoma,
relapsed lymphoma, leukemia, hemolytic anemia, arthritis, atopic
dermatitis is given therapy with the anti-CD20 monoclonal antibody.
The dog is infused intravenously, intraperitoneally, or
subcutaneously with 5 mg/kg of antibody, and the treatment is
repeated weekly for 4-8 weeks following the initial treatment. Two
months after the final dose, the patient shows reduced levels of
certain types of cells expressing CD20.
[0250] VIII. Treatment of Cats.
[0251] A cat diagnosed with a condition including lymphoma,
relapsed lymphoma, leukemia, hemolytic anemia, arthritis, atopic
dermatitis is given therapy with the anti-CD20 monoclonal antibody.
The cat is infused intravenously or subcutaneously with 5 mg/kg of
antibody, and the treatment is repeated weekly for 4-8 weeks
following the initial treatment. Two months after the final dose,
the patient shows reduced levels of certain types of cells
expressing CD20.
[0252] IX. Treatment of Horses.
[0253] A horse diagnosed with a condition including lymphoma,
relapsed lymphoma, leukemia, hemolytic anemia, arthritis, atopic
dermatitis is given therapy with the anti-CD20 monoclonal antibody.
The horse is infused intravenously or subcutaneously with 5 mg/kg
of antibody, and the treatment is repeated weekly for 4-8 weeks
following the initial treatment. Two months after the final dose,
the patient shows reduced levels of certain types of cells
expressing CD20.
EXAMPLE 8
Antibodies to CD52.
[0254] I. Cloning of the Canine CD52 Coding Sequence. CD52 was
isolated from canine peripheral blood mononuclear cells (PBMC).
Total RNA was extracted from 1 million canine peripheral blood
mononuclear cells (PBMC) using the MasterPure.TM. RNA Purification
Kit (Epicentre Biotechnology). The first-strand cDNA was
synthesized from 2 .mu.g of total RNA using SuperScript,
First-Strand Synthesis, System for RT-PCR kit (Invitrogen)
according to the manufacturer's instructions. The coding region or
fragment thereof was then amplified by PCR using the primers of SEQ
ID NO: 70 and SEQ ID NO: 71. The PCR reactions were set as
recommended by the manufacturer (Invitrogen). The samples are
denatured at 94.degree. C. for 5 min followed by amplifications for
35 cycles (94.degree. C. for 30 s, 62.degree. C. for 20 s,
72.degree. C. for 45 s). The PCR products are cloned into pcDNA3
(Invitrogen) and sequenced to verify PCR specificity.
[0255] The amino-acid sequence of the canine CD52 isolated from
canine PBMC is reported as
[0256] SEQ ID No: 72.
[0257] II. Cloning of the Feline CD52 Coding Sequence. The feline
CD52 gene was cloned as described above with primers designed to
amplify the canine CD52 sequence.
[0258] The amino-acid sequence of the feline CD52 isolated from
feline PBMC is as follows:
[0259] SEQ ID NO: 73.
[0260] III. Epitope Prediction for CD52. The epitope prediction was
assessed for the amino acid sequence of the canine CD52 isolated
above by using the prediction algorithm Emboss available online
(http://liv.bmc.uu.se/cgi-bin/emboss/antigenic). This algorithm
scores the antigenicity potential of a given sequence. The program
predicts at least five potential epitopes comprising residues 4-18,
20-26, 30-39, 36-47, and 49-64 of SEQ ID NO:72.
[0261] IV. Generation of Antibodies to CD52. Antibodies to CD52 are
raised using peptides encompassing CD52 amino acid sequences or
fragment thereof and using cells expressing CD52 gene or a fragment
thereof, selected, engineered and tested analogously to the
examples above.
[0262] V. Treatment of Dogs. A dog diagnosed with an immune
condition including lymphoma, relapsed lymphoma, leukemia, mast
cell tumor, hemolytic anemia, arthritis, atopic dermatitis is given
therapy with the anti-CD52 monoclonal antibody. The dog is infused
intravenously or subcutaneously with 5 mg/kg of antibody, and the
treatment is repeated weekly for 4-8 weeks following the initial
treatment. Two months after the final dose, the patient shows
reduced levels of certain types of cells expressing CD52.
[0263] VI. Treatment of Cats. A cat diagnosed with an immune
condition including lymphoma, relapsed lymphoma, leukemia, mast
cell tumor, hemolytic anemia, arthritis, atopic dermatitis is given
therapy with the anti-CD52 monoclonal antibody. The cat is infused
intravenously or subcutaneously with 5 mg/kg of antibody, and the
treatment is repeated weekly for 4-8 weeks following the initial
treatment. Two months after the final dose, the patient shows
reduced levels of certain types of cells expressing CD52.
[0264] VII. Treatment of Horses. A horse diagnosed with an immune
condition including lymphoma, relapsed lymphoma, leukemia, mast
cell tumor, hemolytic anemia, arthritis, atopic dermatitis is given
therapy with the anti-CD52 monoclonal antibody. The horse is
infused intravenously or subcutaneously with 5 mg/kg of antibody,
and the treatment is repeated weekly for 4-8 weeks following the
initial treatment. Two months after the final dose, the patient
shows reduced levels of certain types of cells expressing CD52.
[0265] Alternative combinations and variations of the examples
provided will become apparent based on this disclosure. It is not
possible to provide specific examples for all of the many possible
combinations and variations of the embodiments described, but such
combinations and variations are nevertheless intended to be within
the scope of the invention.
[0266] Summary of sequences described:
TABLE-US-00010 Canis FR4 Light Chain (1-12) FR4LC1 SEQ ID NO 1:
FGGGTHLTVL FR4LC2 SEQ ID NO 2: FGSGTPLTVL FR4LC3 SEQ ID NO 3:
FGAGTKVELIR FR4LC4 SEQ ID NO 4: FGQGTRLEVRR FR4LC5 SEQ ID NO 5:
FGSGTQLTVL FR4LC6 SEQ ID NO 6: FGRGTQLTVL FR4LC7 SEQ ID NO 7:
FGEGTQLTVL FR4LC8 SEQ ID NO 8: FGGGTKLEIK FR4LC9 SEQ ID NO 9:
FGKGTHLEIK FR4LC10 SEQ ID NO 10: FGQGTKVEIK FR4LC11 SEQ ID NO 11:
FGQGTKLEIK FR4LC12 SEQ ID NO 12: FGAGTKVEIK Canis FR4 Heavy Chain
(13-19) FR4HC1 SEQ ID NO 13: WGQGTLVTVSS FR4HC2 SEQ ID NO 14:
WGQGALVTVSS FR4HC3 SEQ ID NO 15: WGPGTSLFVSS FR4HC4 SEQ ID NO 16:
WGLGTLVTVSS FR4HC5 SEQ ID NO 17: WGPGTSLFVSS FR4HC6 SEQ ID NO 18:
WGQGTLVTVSP FR4HC7 SEQ ID NO 19: WGQGTTLTVSS Canis FR1 Light chain
(20-25) FR1LC1 SEQ ID NO 20: DIVMTQTPLSLSVSPGEPASISC FR1LC2 SEQ ID
NO 21: DIVMTQTPLSLSVSPGRPASISC FR1LC3 SEQ ID NO 22:
DIVMTQTPLSLSVSPGRTASISC FR1LC4 SEQ ID NO 23: QSVLTQPASVSGSLGQRVTISC
FR1LC5 SEQ ID NO 24: QPKASPSVTLFPPSSEELGANKATLVC FR1LC6 SEQ ID NO
25: QPKSSPLVTLFPPSSEELGANKATLVC Canis FR1 Heavy Chain (26-40)
FR1HC1 SEQ ID NO: 26 EVQLVESGGDLVKPGGSLRLSCVTS FR1HC2 SEQ ID NO: 27
EVQLVESGGNLVKPGGSLRLSCVAS FR1HC3 SEQ ID NO: 28
EVQLVESGGDLEKPGGSLRLSCVAS FR1HC4 SEQ ID NO: 29
EVQLVESGEDLVKPGGSLRLSCVAS FR1HC5 SEQ ID NO: 30
EVQLVESGGDLVKPAGSLRLSCVAS FR1HC6 SEQ ID NO: 31
EVQLVESGGDLVKPERSLRLSCVAS FR1HC7 SEQ ID NO: 32
EVQLVESGGDLVKPEGSLRLSCVAS FR1HC8 SEQ ID NO: 33
EVQLVESGGDLVKPGGTLRLSCVAS FR1HC9 SEQ ID NO: 34
EEQLVEFGGHMVNPGGSLGLSCQAS FR1HC10 SEQ ID NO: 35
EVQLVESGGDLAKPGGSLRLSCVAS FR1HC11 SEQ ID NO: 36
EVQLVESGGDLVKPEGSLRLSCVVS FR1HC12 SEQ ID NO: 37
EVQLVQSGAEVKKPGASVKVSCKTS FR1HC13 SEQ ID NO: 38
EVQLVESGGDLVKPGGSLRLSCVAS FR1HC14 SEQ ID NO: 39
EVQLVESGGDLMKPGGSLRLSCVAS FR1HC15 SEQ ID NO: 40
EVQLVESGGDLVKPGGSLRLSCVAF SEQ ID Structure Designation Protein
Sequence Light Chain SEQ ID FR1.sub.R-VK-CDR1.sub.R-VK-FR2.sub.R-
VET111 DIKMTQSPSFLSASVGDRVTLNCKASQNI NO: 41 .sub.VK CDR2
.sub.R-VK-FR3 .sub.R-VK- DKYLNWYQQKLGESPKLLIYNTNNLQTGI CDR3
.sub.R-VK FR4 .sub.R-VK-C.sub.D-L PSRFSGSGSGTDFTLTISSLQPEDVATYFCL
QHISRPRTFGTGTKLELK SEQ ID FR1.sub.D-VL-CDR1 .sub.R-VK -FR2 .sub.R-
VET112 QSVLTQPASVSGSLGQRVTISCKASQNIDK NO: 42 .sub.VK CDR2
.sub.R-VK-FR3 .sub.R-VK- YLNWYQQKLGESPKLLIYNTNNLQTGIPS CDR3
.sub.R-VK FR4 .sub.D-VL-C.sub.D-L RFSGSGSGTDFTLTISSLQPEDVATYFCLQ
HISRPRTFGGGTHLTV SEQ ID FR1.sub.R-VL-CDR1 .sub.R-VK -FR2 .sub.R-
VET114 DIKMTQSPSFLSASVGDRVTLNCKASQNI NO: 43 .sub.VK CDR2
.sub.R-VK-FR3 .sub.R-VK- DKYLNWYQQKLGESPKLLIYNTNNLQTGI CDR3
.sub.R-VK FR4 .sub.D-VL-C.sub.D-L PSRFSGSGSGTDFTLTISSLQPEDVATYFCL
QHISRPRTFGGGTHLTVL Heavy Chain SEQ ID FR1.sub.R-VH-CDR1 .sub.R-VH
-FR2 .sub.R- VET222 EVKLLESGGGLVQPGGSMRLSCAGSGFTF NO: 44 .sub.VH
CDR2 .sub.R-VH-FR3 .sub.R-VH- TDFYMNWIRQPAGKAPEWLGFIRDKAKG CDR3
.sub.R-VH FR4 .sub.R-VH-C.sub.D-H YTTEYNPSVKGRFTISRDNTQNMLYLQM
NTLRAEDTATYYCAREGHTAAPFDYWGQ GVMVTVSS SEQ ID FR1.sub.D-VH-CDR1
.sub.R-VH -FR2 R- VET223 EVQLVESGGDLVKPGGSLRLSCAGSGFTF NO: 45 VH
CDR2 .sub.R-VH-FR3 .sub.R-VH- TDFYMNWIRQPAGKAPEWLGFIRDKAKG CDR3
.sub.R-VH FR4 .sub.D-VH-C.sub.D-H YTTEYNPSVKGRFTISRDNTQNMLYLQM
NTLRAEDTATYYCAREGHTAAPFDYWGQ GTLVTVSS SEQ ID FR1.sub.R-VH-CDR1
.sub.R-VH -FR2 .sub.R- VET224 EVKLLESGGGLVQPGGSMRLSCAGSGFTF NO: 46
.sub.VH CDR2 .sub.R-VH-FR3 .sub.R-VH- TDFYMNWIRQPAGKAPEWLGFIRDKAKG
CDR3 .sub.R-VH FR4 .sub.D-VH-C.sub.D-H YTTEYNPSVKGRFTISRDNTQNMLYLQM
NTLRAEDTATYYCAREGHTAAPFDYWGQ GTLVTVSS References Sequence of mAb
231 SEQ ID EVKLVESGGGLVQPGGSLKLSCATSGFTFSDYYMYWVRQTPEKRLEW NO: 47
VAYISNGGGSTYYPDTVKGRFTISRDNAKNTLYLQMSRLKSEDTAMY
YCARHGGYYAMDYWGQGTSVTVSS SEQ ID
DIQMNQSPSSLSASLGDTITITCHASQNINVWLSWYQQKPGNIPKLLIY NO: 48
KASNLHTGVPSRFSGSGSGTGFTLTISSLQPEDIATYYCQQGQSYPLTFG GGTKLEIK SEQ ID
Structure Designation Protein Sequence Light Chain SEQ ID
FR1.sub.M-VK-CDR1 .sub.M-VK -FR2 .sub.M- VET106
DIQMNQSPSSLSASLGDTITITCHASQNI NO: 49 .sub.VK CDR2.sub.M-VK-FR3
.sub.M-VK- NVWLSWYQQKPGNIPKLLIYKASNLHT CDR3 .sub.M-VK FR4
.sub.M-VK-C.sub.D-L GVPSRFSGSGSGTGFTLTISSLQPEDIATY
YCQQGQSYPLTFGGGTKLEIKGQPKASP SVTLFPPSSEELGANKATLVCLISDFYPS
GVTVAWKADGSPITQGVETTKPSKQSN NKYAASSYLSLTPDKWKSHSSFSCLVTH
EGSTVEKKVAPAECS SEQ ID FR1.sub.M-VK-CDR1 .sub.M-VK -FR2 .sub.M-
VET107 DIQMNQSPSSLSASLGDTITITCHASQNI NO: 50 .sub.VK CDR2
.sub.M-VK-FR3 .sub.M-VK- NVWLSWYQQKPGNIPKLLIYKASNLHT CDR3 .sub.M-VK
FR4 .sub.M-VK-C.sub.D-K GVPSRFSGSGSGTGFTLTISSLQPEDIATY
YCQQGQSYPLTFGGGTKLEIKNDAQPA VYLFQPSPDQLHTGSASVVCLLNSFYPK
DINVKWKVDGVIQDTGIQESVTEQDKD STYSLSSTLTMSSTEYLSHELYSCEITHK
SLPSTLIKSFQRSECQRVD SEQ ID FR1.sub.M-VK-CDR1 .sub.M-VK -FR2 .sub.M-
VET118 DIQMNQSPSSLSASLGDTITITCHASQNI NO: 51 .sub.VK CDR2
.sub.M-VK-FR3 .sub.M-VK- NVWLSWYQQKPGNIPKLLIYKASNLHT CDR3 .sub.M-VK
FR4 .sub.D-VL-C.sub.D-L GVPSRFSGSGSGTGFTLTISSLQPEDIATY
YCQQGQSYPLTFGGGTHLTVL Heavy Chain SEQ ID FR1.sub.M-VH-CDR1
.sub.M-VH -FR2 .sub.M- VET217 EVKLVESGGGLVQPGGSLKLSCATSGFT NO: 52
.sub.VH CDR2 .sub.M-VH-FR3 .sub.M-VH- FSDYYMYWVRQTPEKRLEWVAYISNG
CDR3 .sub.M-VH FR4 .sub.M-VH-C.sub.D-H GGSTYYPDTVKGRFTISRDNAKNTLYLQ
MSRLKSEDTAMYYCARHGGYYAMDY WGQGTLVTVSS SEQ ID FR1.sub.M-VH-CDR1
.sub.M-VH -FR2 .sub.M- VET218 EVKLVESGGGLVQPGGSLKLSCATSGFT NO: 53
.sub.VH CDR2 .sub.M-VH-FR3 .sub.M-VH- FSDYYMYWVRQTPEKRLEWVAYISNG
CDR3 .sub.M-VH FR4 .sub.D-VH-C.sub.D-H GGSTYYPDTVKGRFTISRDNAKNTLYLQ
MSRLKSEDTAMYYCARHGGYYAMDY WGQGTSVTVSS Constant Plasmid Domain
Designation Sequence SEQ ID HC VET214
ASTTAPSVFPLAPSCGSTSGSTVALACLVSGYFPEP NO: 54
VTVSWNSGSLTSGVHTFPSVLQSSGLYSLSSMVT
VPSSRWPSETFTCNVAHPASKTKVDKPVPKRENG
RVPRPPDCPKCPAPEMLGGPSVFIFPPKPKDTLLIA
RTPEVTCVVVDLDPEDPEVQISWFVDGKQMQTA
KTQPREEQFNGTYRVVSVLPIGHQDWLKGKQFTC
KVNNKALPSPIERTISKARGQAHQPSVYVLPPSRE
ELSKNTVSLTCLIKDFFPPDIDVEWQSNGQQEPES
KYRTTPPQLDEDGSYFLYSKLSVDKSRWQRGDTF ICAVMHEALHNHYTQKSLSHSPGK SEQ ID
Lambda LC VET104 GQPKASPSVTLFPPSSEELGANKATLVCLISDFYPS NO: 55
GVTVAWKADGSPITQGVETTKPSKQSNNKYAASS
YLSLTPDKWKSHSSFSCLVTHEGSTVEKKVAPAE CS SEQ ID Kappa LC VET105
NDAQPAVYLFQPSPDQLHTGSASVVCLLNSFYPK NO: 56
DINVKWKVDGVIQDTGIQESVTEQDKDSTYSLSST
LTMSSTEYLSHELYSCEITHKSLPSTLIKSFQRSEC QRVD List of primers utilized
for the antibody constant domain amplification from cDNA Constant
Primer Domain Designation Primer Sequence SEQ ID NO: 57 HC HC-F
GCCTCCACCACGGCCCC SEQ ID NO: 58 HC-R TCATTTACCCGGAGAATGGG SEQ ID
NO: 59 Lambda LC L-LC-F GGTCAGCCCAAGGCCWMCC SEQ ID NO: 60 L-LC-R
CTAAGAGCACTCTGCRGGG SEQ ID NO: 61 Kappa LC K-LC-F
AATGATGCCCAGCCAGCCG SEQ ID NO: 62 K-LC-R TTAGTCCACTCTCTGACACTC
Canine CD20 Primer Region SEQ ID Designation Primer Sequence CD20
SEQ ID No: 63 CD20 FL-F 5'-TGAGATGACAACACCCAGAAA-3' SEQ ID No: 64
CD20 FL-R 5'-TTAAGGGATGCTGTCGTTTTC-3' CD20 SEQ ID No: 65 CD20 Lp-F
5'-AATATTACCATTTCCCATTTTTTTA-3' Fragment SEQ ID No: 66 CD20 Lp-R
5'-TATGCTGCCACAATATTGTATAG-3' Canine CD20 SEQ ID NO 67
MTTPRNSMSGTLPVDPMKSPTAMYPVQKIIPKRMPSVVGPTQNFFMRESKTLGAVQIMNGLF
HIALGSLLMIHTDVYAPICITMWYPLWGGIMFIISGSLLAAADKNPRKSLVKGKMIMNSLSLF
AAISGIIFLIMDIFNITISHFFKMENLNLIKAPMPYVDIHNCDPANPSEKNSLSIQYCGSIRSVFLG
VFAVMVIFTFFQKLVTAGIVENEWKKLCSKPKSDVVVLLAAEEKKEQPIETTEEMVELTEIAS
QPKKEEDIEIIPVQEEEEELEINFAEPPQEQESSPIENDSIP 10 20 30 40 50 60
MTTPRNSMSG TLPVDPMKSP TAMYPVQKII PKRMPSVVGP TQNFFMRESK TLGAVQIMNG
70 80 90 100 110 120 LFHIALGSLL MIHTDVYAPI CITMWYPLWG GIMFIISGSL
LAAADKNPRK SLVKGKMIMN 130 140 150 160 170 180 SLSLFAAISG IIFLIMDIFN
ITISHFFKME NLNLIKAPMP YVDIHNCDPA NPSEKNSLSI 190 200 210 220 230 240
QYCGSIRSVF LGVFAVMVIF TFFQKLVTAG IVENEWKKLC SKPKSDVVVL LAAEEKKEQP
250 260 270 280 290 IETTEEMVEL TEIASQPKKE EDIEIIPVQE EEEELEINFA
EPPQEQESSP IENDSIP Extracellular Domain SEQ ID NO: TDVYAPIC 74-81
SEQ ID NO: NITISHFFKMENLNLIKAP 140-187 MPYVDIHNCDPANPSEKN
SLSIQYCGSIR Epitope prediction using the Emboss Program SEQ ID NO:
HTDVYAPICIT 74-83 SEQ ID NO: LSIQYCGSIRS 178-211 SEQ ID NO:
LIKAPMPYVDIHNCDPA 154-170 SEQ ID NO: NITISHF 140-146 Epitope
prediction using tools from Immuneepitope SEQ ID NO: VDIHNCDPANPS
162-173 SEQ ID NO: KMENLNLIKAPM 148-159 SEQ ID NO: TISHFFKMENLN
142-153 SEQ ID NO: PMPYVDIHNCDP 148-169 SEQ ID NO: NCDPANPSEKNS
166-177 SEQ ID NO: YVDIHNCDPANPSEKN 161-176 Feline CD20 SEQ ID NO:
63 CD20 FLF TGAGATGACAACACCCAGAAA SEQ ID NO: 68 FCD20R
GGATCCTTAAGGAATGCTATCGTTTT The amino-acid sequence of the feline
CD20 isolated from feline PBMC is as follows: SEQ ID NO 69
MTTPRNSMSGTLPADAMKSPTAMNPVQKIIPKKMPSVVGPTQNFFMKESKPLGAVQIMNGL
FHMALGGLLMIHMEVYAPICMTVWYPLWGGIMYIISGSLLVAAEKNPRKSLVKGKMIMNSL
SLFAAISGMILLIMDIFNIAISHFFKMENLNLLKSPKPYIDIHTCQPESKPSEKNSLSIKYCDSIRS
VFLSIFAVMVVFTLFQKLVTAGIVENEWKKLCSKPKADVVVLLAAEEKKEQLVEITEEAVEL
TEVSSQPKNEEDIEIIPVQEEEEETEMNFPEPPQDQEPSLIENDSIP Canine CD52: Primer
Designation Primer Sequence
CD52 F CAACAAAGCTTGCCGCCACCATGAAGGGCTTCCTCTTCCT SEQ ID NO: 70 CD52
R CAACAGGATCCTCAGCTGAGGTAGAAGAGCT SEQ ID NO: 71 SEQ ID No 72
MKGFLFLLLTISLLVMIQIQTGVLGNSTTPRMTTKKVKSATPALSSLGGGSVLLFLANTLIQLF
YLS 10 20 30 40 50 60 MKGFLFLLLT ISLLVMIQIQ TGVLGNSTTP RMTTKKVKSA
TPALSSLGGG SVLLFLANTL IQLFYLS SEQ ID Sequence Residue Position SEQ
ID NO: GGSVLLFLANTLIQLF 49-64 SEQ ID NO: FLFLLLTISLLVMIQ 4-18 SEQ
ID NO: QTGVLGN 20-26 SEQ ID NO: KVKSATPALSSL 36-47 SEQ ID NO:
PRMTTKKVKS 30-39 feline CD52 isolated from feline PBMC is as
follows: SEQ ID NO 73:
MKGFLFLLLTISLLVMIQIQTGVLGNTTTAATTTKKPKSATPPLSSLSSGSVLLFLANILVQLFY
LS 10 20 30 40 50 60 MKGFLFLLLT ISLLVMIQIQ TGVLGNTTTA ATTTKKPKSA
TPPLSSLSSG SVLLFLANTL VQLFYLS
Sequence CWU 1
1
90110PRTCanis familiaris 1Phe Gly Gly Gly Thr His Leu Thr Val Leu 1
5 10 210PRTCanis familiaris 2Phe Gly Ser Gly Thr Pro Leu Thr Val
Leu 1 5 10 311PRTCanis familiaris 3Phe Gly Ala Gly Thr Lys Val Glu
Leu Ile Arg 1 5 10 411PRTCanis familiaris 4Phe Gly Gln Gly Thr Arg
Leu Glu Val Arg Arg 1 5 10 510PRTCanis familiaris 5Phe Gly Ser Gly
Thr Gln Leu Thr Val Leu 1 5 10 610PRTCanis familiaris 6Phe Gly Arg
Gly Thr Gln Leu Thr Val Leu 1 5 10 710PRTCanis familiaris 7Phe Gly
Glu Gly Thr Gln Leu Thr Val Leu 1 5 10 810PRTCanis familiaris 8Phe
Gly Gly Gly Thr Lys Leu Glu Ile Lys 1 5 10 910PRTCanis familiaris
9Phe Gly Lys Gly Thr His Leu Glu Ile Lys 1 5 10 1010PRTCanis
familiaris 10Phe Gly Gln Gly Thr Lys Val Glu Ile Lys 1 5 10
1110PRTCanis familiaris 11Phe Gly Gln Gly Thr Lys Leu Glu Ile Lys 1
5 10 1210PRTCanis familiaris 12Phe Gly Ala Gly Thr Lys Val Glu Ile
Lys 1 5 10 1311PRTCanis familiaris 13Trp Gly Gln Gly Thr Leu Val
Thr Val Ser Ser 1 5 10 1411PRTCanis familiaris 14Trp Gly Gln Gly
Ala Leu Val Thr Val Ser Ser 1 5 10 1511PRTCanis familiaris 15Trp
Gly Pro Gly Thr Ser Leu Phe Val Ser Ser 1 5 10 1611PRTCanis
familiaris 16Trp Gly Leu Gly Thr Leu Val Thr Val Ser Ser 1 5 10
1711PRTCanis familiaris 17Trp Gly Pro Gly Thr Ser Leu Phe Val Ser
Ser 1 5 10 1811PRTCanis familiaris 18Trp Gly Gln Gly Thr Leu Val
Thr Val Ser Pro 1 5 10 1911PRTCanis familiaris 19Trp Gly Gln Gly
Thr Thr Leu Thr Val Ser Ser 1 5 10 2023PRTCanis familiaris 20Asp
Ile Val Met Thr Gln Thr Pro Leu Ser Leu Ser Val Ser Pro Gly 1 5 10
15 Glu Pro Ala Ser Ile Ser Cys 20 2123PRTCanis familiaris 21Asp Ile
Val Met Thr Gln Thr Pro Leu Ser Leu Ser Val Ser Pro Gly 1 5 10 15
Arg Pro Ala Ser Ile Ser Cys 20 2223PRTCanis familiaris 22Asp Ile
Val Met Thr Gln Thr Pro Leu Ser Leu Ser Val Ser Pro Gly 1 5 10 15
Arg Thr Ala Ser Ile Ser Cys 20 2322PRTCanis familiaris 23Gln Ser
Val Leu Thr Gln Pro Ala Ser Val Ser Gly Ser Leu Gly Gln 1 5 10 15
Arg Val Thr Ile Ser Cys 20 2427PRTCanis familiaris 24Gln Pro Lys
Ala Ser Pro Ser Val Thr Leu Phe Pro Pro Ser Ser Glu 1 5 10 15 Glu
Leu Gly Ala Asn Lys Ala Thr Leu Val Cys 20 25 2527PRTCanis
familiaris 25Gln Pro Lys Ser Ser Pro Leu Val Thr Leu Phe Pro Pro
Ser Ser Glu 1 5 10 15 Glu Leu Gly Ala Asn Lys Ala Thr Leu Val Cys
20 25 2625PRTCanis familiaris 26Glu Val Gln Leu Val Glu Ser Gly Gly
Asp Leu Val Lys Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Val
Thr Ser 20 25 2725PRTCanis familiaris 27Glu Val Gln Leu Val Glu Ser
Gly Gly Asn Leu Val Lys Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser
Cys Val Ala Ser 20 25 2825PRTCanis familiaris 28Glu Val Gln Leu Val
Glu Ser Gly Gly Asp Leu Glu Lys Pro Gly Gly 1 5 10 15 Ser Leu Arg
Leu Ser Cys Val Ala Ser 20 25 2925PRTCanis familiaris 29Glu Val Gln
Leu Val Glu Ser Gly Glu Asp Leu Val Lys Pro Gly Gly 1 5 10 15 Ser
Leu Arg Leu Ser Cys Val Ala Ser 20 25 3025PRTCanis familiaris 30Glu
Val Gln Leu Val Glu Ser Gly Gly Asp Leu Val Lys Pro Ala Gly 1 5 10
15 Ser Leu Arg Leu Ser Cys Val Ala Ser 20 25 3125PRTCanis
familiaris 31Glu Val Gln Leu Val Glu Ser Gly Gly Asp Leu Val Lys
Pro Glu Arg 1 5 10 15 Ser Leu Arg Leu Ser Cys Val Ala Ser 20 25
3225PRTCanis familiaris 32Glu Val Gln Leu Val Glu Ser Gly Gly Asp
Leu Val Lys Pro Glu Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Val Ala
Ser 20 25 3325PRTCanis familiaris 33Glu Val Gln Leu Val Glu Ser Gly
Gly Asp Leu Val Lys Pro Gly Gly 1 5 10 15 Thr Leu Arg Leu Ser Cys
Val Ala Ser 20 25 3425PRTCanis familiaris 34Glu Glu Gln Leu Val Glu
Phe Gly Gly His Met Val Asn Pro Gly Gly 1 5 10 15 Ser Leu Gly Leu
Ser Cys Gln Ala Ser 20 25 3525PRTCanis familiaris 35Glu Val Gln Leu
Val Glu Ser Gly Gly Asp Leu Ala Lys Pro Gly Gly 1 5 10 15 Ser Leu
Arg Leu Ser Cys Val Ala Ser 20 25 3625PRTCanis familiaris 36Glu Val
Gln Leu Val Glu Ser Gly Gly Asp Leu Val Lys Pro Glu Gly 1 5 10 15
Ser Leu Arg Leu Ser Cys Val Val Ser 20 25 3725PRTCanis familiaris
37Glu Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ala 1
5 10 15 Ser Val Lys Val Ser Cys Lys Thr Ser 20 25 3825PRTCanis
familiaris 38Glu Val Gln Leu Val Glu Ser Gly Gly Asp Leu Val Lys
Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Val Ala Ser 20 25
3925PRTCanis familiaris 39Glu Val Gln Leu Val Glu Ser Gly Gly Asp
Leu Met Lys Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Val Ala
Ser 20 25 4025PRTCanis familiaris 40Glu Val Gln Leu Val Glu Ser Gly
Gly Asp Leu Val Lys Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys
Val Ala Phe 20 25 41107PRTRattus norvegicus 41Asp Ile Lys Met Thr
Gln Ser Pro Ser Phe Leu Ser Ala Ser Val Gly 1 5 10 15 Asp Arg Val
Thr Leu Asn Cys Lys Ala Ser Gln Asn Ile Asp Lys Tyr 20 25 30 Leu
Asn Trp Tyr Gln Gln Lys Leu Gly Glu Ser Pro Lys Leu Leu Ile 35 40
45 Tyr Asn Thr Asn Asn Leu Gln Thr Gly Ile Pro Ser Arg Phe Ser Gly
50 55 60 Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu
Gln Pro 65 70 75 80 Glu Asp Val Ala Thr Tyr Phe Cys Leu Gln His Ile
Ser Arg Pro Arg 85 90 95 Thr Phe Gly Thr Gly Thr Lys Leu Glu Leu
Lys 100 105 42105PRTArtificial SequenceRattus Norvegicus/Canis
Familiaris 42Gln Ser Val Leu Thr Gln Pro Ala Ser Val Ser Gly Ser
Leu Gly Gln 1 5 10 15 Arg Val Thr Ile Ser Cys Lys Ala Ser Gln Asn
Ile Asp Lys Tyr Leu 20 25 30 Asn Trp Tyr Gln Gln Lys Leu Gly Glu
Ser Pro Lys Leu Leu Ile Tyr 35 40 45 Asn Thr Asn Asn Leu Gln Thr
Gly Ile Pro Ser Arg Phe Ser Gly Ser 50 55 60 Gly Ser Gly Thr Asp
Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro Glu 65 70 75 80 Asp Val Ala
Thr Tyr Phe Cys Leu Gln His Ile Ser Arg Pro Arg Thr 85 90 95 Phe
Gly Gly Gly Thr His Leu Thr Val 100 105 43107PRTArtificial
Sequencerattus norvegicus/canis familiaris 43Asp Ile Lys Met Thr
Gln Ser Pro Ser Phe Leu Ser Ala Ser Val Gly 1 5 10 15 Asp Arg Val
Thr Leu Asn Cys Lys Ala Ser Gln Asn Ile Asp Lys Tyr 20 25 30 Leu
Asn Trp Tyr Gln Gln Lys Leu Gly Glu Ser Pro Lys Leu Leu Ile 35 40
45 Tyr Asn Thr Asn Asn Leu Gln Thr Gly Ile Pro Ser Arg Phe Ser Gly
50 55 60 Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu
Gln Pro 65 70 75 80 Glu Asp Val Ala Thr Tyr Phe Cys Leu Gln His Ile
Ser Arg Pro Arg 85 90 95 Thr Phe Gly Gly Gly Thr His Leu Thr Val
Leu 100 105 44121PRTRattus norvegicus 44Glu Val Lys Leu Leu Glu Ser
Gly Gly Gly Leu Val Gln Pro Gly Gly 1 5 10 15 Ser Met Arg Leu Ser
Cys Ala Gly Ser Gly Phe Thr Phe Thr Asp Phe 20 25 30 Tyr Met Asn
Trp Ile Arg Gln Pro Ala Gly Lys Ala Pro Glu Trp Leu 35 40 45 Gly
Phe Ile Arg Asp Lys Ala Lys Gly Tyr Thr Thr Glu Tyr Asn Pro 50 55
60 Ser Val Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Thr Gln Asn Met
65 70 75 80 Leu Tyr Leu Gln Met Asn Thr Leu Arg Ala Glu Asp Thr Ala
Thr Tyr 85 90 95 Tyr Cys Ala Arg Glu Gly His Thr Ala Ala Pro Phe
Asp Tyr Trp Gly 100 105 110 Gln Gly Val Met Val Thr Val Ser Ser 115
120 45121PRTArtificial SequenceRattus norvegicus/Canis familiaris
45Glu Val Gln Leu Val Glu Ser Gly Gly Asp Leu Val Lys Pro Gly Gly 1
5 10 15 Ser Leu Arg Leu Ser Cys Ala Gly Ser Gly Phe Thr Phe Thr Asp
Phe 20 25 30 Tyr Met Asn Trp Ile Arg Gln Pro Ala Gly Lys Ala Pro
Glu Trp Leu 35 40 45 Gly Phe Ile Arg Asp Lys Ala Lys Gly Tyr Thr
Thr Glu Tyr Asn Pro 50 55 60 Ser Val Lys Gly Arg Phe Thr Ile Ser
Arg Asp Asn Thr Gln Asn Met 65 70 75 80 Leu Tyr Leu Gln Met Asn Thr
Leu Arg Ala Glu Asp Thr Ala Thr Tyr 85 90 95 Tyr Cys Ala Arg Glu
Gly His Thr Ala Ala Pro Phe Asp Tyr Trp Gly 100 105 110 Gln Gly Thr
Leu Val Thr Val Ser Ser 115 120 46121PRTArtificial SequenceRattus
norvegicus/Canis familiaris 46Glu Val Lys Leu Leu Glu Ser Gly Gly
Gly Leu Val Gln Pro Gly Gly 1 5 10 15 Ser Met Arg Leu Ser Cys Ala
Gly Ser Gly Phe Thr Phe Thr Asp Phe 20 25 30 Tyr Met Asn Trp Ile
Arg Gln Pro Ala Gly Lys Ala Pro Glu Trp Leu 35 40 45 Gly Phe Ile
Arg Asp Lys Ala Lys Gly Tyr Thr Thr Glu Tyr Asn Pro 50 55 60 Ser
Val Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Thr Gln Asn Met 65 70
75 80 Leu Tyr Leu Gln Met Asn Thr Leu Arg Ala Glu Asp Thr Ala Thr
Tyr 85 90 95 Tyr Cys Ala Arg Glu Gly His Thr Ala Ala Pro Phe Asp
Tyr Trp Gly 100 105 110 Gln Gly Thr Leu Val Thr Val Ser Ser 115 120
47118PRTMus musculus 47Glu Val Lys Leu Val Glu Ser Gly Gly Gly Leu
Val Gln Pro Gly Gly 1 5 10 15 Ser Leu Lys Leu Ser Cys Ala Thr Ser
Gly Phe Thr Phe Ser Asp Tyr 20 25 30 Tyr Met Tyr Trp Val Arg Gln
Thr Pro Glu Lys Arg Leu Glu Trp Val 35 40 45 Ala Tyr Ile Ser Asn
Gly Gly Gly Ser Thr Tyr Tyr Pro Asp Thr Val 50 55 60 Lys Gly Arg
Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Thr Leu Tyr 65 70 75 80 Leu
Gln Met Ser Arg Leu Lys Ser Glu Asp Thr Ala Met Tyr Tyr Cys 85 90
95 Ala Arg His Gly Gly Tyr Tyr Ala Met Asp Tyr Trp Gly Gln Gly Thr
100 105 110 Ser Val Thr Val Ser Ser 115 48107PRTMus musculus 48Asp
Ile Gln Met Asn Gln Ser Pro Ser Ser Leu Ser Ala Ser Leu Gly 1 5 10
15 Asp Thr Ile Thr Ile Thr Cys His Ala Ser Gln Asn Ile Asn Val Trp
20 25 30 Leu Ser Trp Tyr Gln Gln Lys Pro Gly Asn Ile Pro Lys Leu
Leu Ile 35 40 45 Tyr Lys Ala Ser Asn Leu His Thr Gly Val Pro Ser
Arg Phe Ser Gly 50 55 60 Ser Gly Ser Gly Thr Gly Phe Thr Leu Thr
Ile Ser Ser Leu Gln Pro 65 70 75 80 Glu Asp Ile Ala Thr Tyr Tyr Cys
Gln Gln Gly Gln Ser Tyr Pro Leu 85 90 95 Thr Phe Gly Gly Gly Thr
Lys Leu Glu Ile Lys 100 105 49213PRTArtificial SequenceMus
musculus/Canis familiaris 49Asp Ile Gln Met Asn Gln Ser Pro Ser Ser
Leu Ser Ala Ser Leu Gly 1 5 10 15 Asp Thr Ile Thr Ile Thr Cys His
Ala Ser Gln Asn Ile Asn Val Trp 20 25 30 Leu Ser Trp Tyr Gln Gln
Lys Pro Gly Asn Ile Pro Lys Leu Leu Ile 35 40 45 Tyr Lys Ala Ser
Asn Leu His Thr Gly Val Pro Ser Arg Phe Ser Gly 50 55 60 Ser Gly
Ser Gly Thr Gly Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro 65 70 75 80
Glu Asp Ile Ala Thr Tyr Tyr Cys Gln Gln Gly Gln Ser Tyr Pro Leu 85
90 95 Thr Phe Gly Gly Gly Thr Lys Leu Glu Ile Lys Gly Gln Pro Lys
Ala 100 105 110 Ser Pro Ser Val Thr Leu Phe Pro Pro Ser Ser Glu Glu
Leu Gly Ala 115 120 125 Asn Lys Ala Thr Leu Val Cys Leu Ile Ser Asp
Phe Tyr Pro Ser Gly 130 135 140 Val Thr Val Ala Trp Lys Ala Asp Gly
Ser Pro Ile Thr Gln Gly Val 145 150 155 160 Glu Thr Thr Lys Pro Ser
Lys Gln Ser Asn Asn Lys Tyr Ala Ala Ser 165 170 175 Ser Tyr Leu Ser
Leu Thr Pro Asp Lys Trp Lys Ser His Ser Ser Phe 180 185 190 Ser Cys
Leu Val Thr His Glu Gly Ser Thr Val Glu Lys Lys Val Ala 195 200 205
Pro Ala Glu Cys Ser 210 50216PRTArtificial SequenceMus
muscululs/Canis familiaris 50Asp Ile Gln Met Asn Gln Ser Pro Ser
Ser Leu Ser Ala Ser Leu Gly 1 5 10 15 Asp Thr Ile Thr Ile Thr Cys
His Ala Ser Gln Asn Ile Asn Val Trp 20 25 30 Leu Ser Trp Tyr Gln
Gln Lys Pro Gly Asn Ile Pro Lys Leu Leu Ile 35 40 45 Tyr Lys Ala
Ser Asn Leu His Thr Gly Val Pro Ser Arg Phe Ser Gly 50 55 60 Ser
Gly Ser Gly Thr Gly Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro 65 70
75 80 Glu Asp Ile Ala Thr Tyr Tyr Cys Gln Gln Gly Gln Ser Tyr Pro
Leu 85 90 95 Thr Phe Gly Gly Gly Thr Lys Leu Glu Ile Lys Asn Asp
Ala Gln Pro 100 105 110 Ala Val Tyr Leu Phe Gln Pro Ser Pro Asp Gln
Leu His Thr Gly Ser 115 120 125 Ala Ser Val Val Cys Leu Leu Asn Ser
Phe Tyr Pro Lys Asp Ile Asn 130 135 140 Val Lys Trp Lys Val Asp Gly
Val Ile Gln Asp Thr Gly Ile Gln Glu 145 150 155 160 Ser Val Thr Glu
Gln Asp Lys Asp Ser Thr Tyr Ser Leu Ser Ser Thr 165 170 175 Leu Thr
Met Ser Ser Thr Glu Tyr Leu Ser His Glu Leu Tyr Ser Cys 180 185 190
Glu Ile Thr His Lys Ser Leu Pro Ser Thr Leu Ile Lys Ser Phe Gln 195
200 205 Arg Ser Glu Cys Gln Arg Val Asp 210 215 51107PRTArtificial
SequenceMus musculus/Canis familiaris 51Asp Ile Gln Met Asn Gln Ser
Pro Ser Ser Leu Ser Ala Ser Leu Gly 1 5 10 15 Asp Thr Ile Thr Ile
Thr Cys His Ala Ser Gln Asn Ile Asn Val Trp 20 25 30 Leu Ser Trp
Tyr Gln Gln Lys Pro Gly Asn Ile Pro Lys
Leu Leu Ile 35 40 45 Tyr Lys Ala Ser Asn Leu His Thr Gly Val Pro
Ser Arg Phe Ser Gly 50 55 60 Ser Gly Ser Gly Thr Gly Phe Thr Leu
Thr Ile Ser Ser Leu Gln Pro 65 70 75 80 Glu Asp Ile Ala Thr Tyr Tyr
Cys Gln Gln Gly Gln Ser Tyr Pro Leu 85 90 95 Thr Phe Gly Gly Gly
Thr His Leu Thr Val Leu 100 105 52118PRTArtificial SequenceMus
musculus/Canis familiaris 52Glu Val Lys Leu Val Glu Ser Gly Gly Gly
Leu Val Gln Pro Gly Gly 1 5 10 15 Ser Leu Lys Leu Ser Cys Ala Thr
Ser Gly Phe Thr Phe Ser Asp Tyr 20 25 30 Tyr Met Tyr Trp Val Arg
Gln Thr Pro Glu Lys Arg Leu Glu Trp Val 35 40 45 Ala Tyr Ile Ser
Asn Gly Gly Gly Ser Thr Tyr Tyr Pro Asp Thr Val 50 55 60 Lys Gly
Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Thr Leu Tyr 65 70 75 80
Leu Gln Met Ser Arg Leu Lys Ser Glu Asp Thr Ala Met Tyr Tyr Cys 85
90 95 Ala Arg His Gly Gly Tyr Tyr Ala Met Asp Tyr Trp Gly Gln Gly
Thr 100 105 110 Leu Val Thr Val Ser Ser 115 53118PRTArtificial
SequenceMus musculus/Canis familiaris 53Glu Val Lys Leu Val Glu Ser
Gly Gly Gly Leu Val Gln Pro Gly Gly 1 5 10 15 Ser Leu Lys Leu Ser
Cys Ala Thr Ser Gly Phe Thr Phe Ser Asp Tyr 20 25 30 Tyr Met Tyr
Trp Val Arg Gln Thr Pro Glu Lys Arg Leu Glu Trp Val 35 40 45 Ala
Tyr Ile Ser Asn Gly Gly Gly Ser Thr Tyr Tyr Pro Asp Thr Val 50 55
60 Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Thr Leu Tyr
65 70 75 80 Leu Gln Met Ser Arg Leu Lys Ser Glu Asp Thr Ala Met Tyr
Tyr Cys 85 90 95 Ala Arg His Gly Gly Tyr Tyr Ala Met Asp Tyr Trp
Gly Gln Gly Thr 100 105 110 Ser Val Thr Val Ser Ser 115
54335PRTCanis familiaris 54Ala Ser Thr Thr Ala Pro Ser Val Phe Pro
Leu Ala Pro Ser Cys Gly 1 5 10 15 Ser Thr Ser Gly Ser Thr Val Ala
Leu Ala Cys Leu Val Ser Gly Tyr 20 25 30 Phe Pro Glu Pro Val Thr
Val Ser Trp Asn Ser Gly Ser Leu Thr Ser 35 40 45 Gly Val His Thr
Phe Pro Ser Val Leu Gln Ser Ser Gly Leu Tyr Ser 50 55 60 Leu Ser
Ser Met Val Thr Val Pro Ser Ser Arg Trp Pro Ser Glu Thr 65 70 75 80
Phe Thr Cys Asn Val Ala His Pro Ala Ser Lys Thr Lys Val Asp Lys 85
90 95 Pro Val Pro Lys Arg Glu Asn Gly Arg Val Pro Arg Pro Pro Asp
Cys 100 105 110 Pro Lys Cys Pro Ala Pro Glu Met Leu Gly Gly Pro Ser
Val Phe Ile 115 120 125 Phe Pro Pro Lys Pro Lys Asp Thr Leu Leu Ile
Ala Arg Thr Pro Glu 130 135 140 Val Thr Cys Val Val Val Asp Leu Asp
Pro Glu Asp Pro Glu Val Gln 145 150 155 160 Ile Ser Trp Phe Val Asp
Gly Lys Gln Met Gln Thr Ala Lys Thr Gln 165 170 175 Pro Arg Glu Glu
Gln Phe Asn Gly Thr Tyr Arg Val Val Ser Val Leu 180 185 190 Pro Ile
Gly His Gln Asp Trp Leu Lys Gly Lys Gln Phe Thr Cys Lys 195 200 205
Val Asn Asn Lys Ala Leu Pro Ser Pro Ile Glu Arg Thr Ile Ser Lys 210
215 220 Ala Arg Gly Gln Ala His Gln Pro Ser Val Tyr Val Leu Pro Pro
Ser 225 230 235 240 Arg Glu Glu Leu Ser Lys Asn Thr Val Ser Leu Thr
Cys Leu Ile Lys 245 250 255 Asp Phe Phe Pro Pro Asp Ile Asp Val Glu
Trp Gln Ser Asn Gly Gln 260 265 270 Gln Glu Pro Glu Ser Lys Tyr Arg
Thr Thr Pro Pro Gln Leu Asp Glu 275 280 285 Asp Gly Ser Tyr Phe Leu
Tyr Ser Lys Leu Ser Val Asp Lys Ser Arg 290 295 300 Trp Gln Arg Gly
Asp Thr Phe Ile Cys Ala Val Met His Glu Ala Leu 305 310 315 320 His
Asn His Tyr Thr Gln Lys Ser Leu Ser His Ser Pro Gly Lys 325 330 335
55106PRTCanis familiaris 55Gly Gln Pro Lys Ala Ser Pro Ser Val Thr
Leu Phe Pro Pro Ser Ser 1 5 10 15 Glu Glu Leu Gly Ala Asn Lys Ala
Thr Leu Val Cys Leu Ile Ser Asp 20 25 30 Phe Tyr Pro Ser Gly Val
Thr Val Ala Trp Lys Ala Asp Gly Ser Pro 35 40 45 Ile Thr Gln Gly
Val Glu Thr Thr Lys Pro Ser Lys Gln Ser Asn Asn 50 55 60 Lys Tyr
Ala Ala Ser Ser Tyr Leu Ser Leu Thr Pro Asp Lys Trp Lys 65 70 75 80
Ser His Ser Ser Phe Ser Cys Leu Val Thr His Glu Gly Ser Thr Val 85
90 95 Glu Lys Lys Val Ala Pro Ala Glu Cys Ser 100 105 56109PRTCanis
familiaris 56Asn Asp Ala Gln Pro Ala Val Tyr Leu Phe Gln Pro Ser
Pro Asp Gln 1 5 10 15 Leu His Thr Gly Ser Ala Ser Val Val Cys Leu
Leu Asn Ser Phe Tyr 20 25 30 Pro Lys Asp Ile Asn Val Lys Trp Lys
Val Asp Gly Val Ile Gln Asp 35 40 45 Thr Gly Ile Gln Glu Ser Val
Thr Glu Gln Asp Lys Asp Ser Thr Tyr 50 55 60 Ser Leu Ser Ser Thr
Leu Thr Met Ser Ser Thr Glu Tyr Leu Ser His 65 70 75 80 Glu Leu Tyr
Ser Cys Glu Ile Thr His Lys Ser Leu Pro Ser Thr Leu 85 90 95 Ile
Lys Ser Phe Gln Arg Ser Glu Cys Gln Arg Val Asp 100 105
5717DNACanis familiaris 57gcctccacca cggcccc 175820DNACanis
familiaris 58tcatttaccc ggagaatggg 205919DNACanis familiaris
59ggtcagccca aggccwmcc 196019DNACanis familiaris 60ctaagagcac
tctgcrggg 196119DNACanis familiaris 61aatgatgccc agccagccg
196221DNACanis familiaris 62ttagtccact ctctgacact c 216321DNACanis
familiaris 63tgagatgaca acacccagaa a 216421DNACanis familiaris
64ttaagggatg ctgtcgtttt c 216525DNACanis familiaris 65aatattacca
tttcccattt tttta 256623DNACanis familiaris 66tatgctgcca caatattgta
tag 2367297PRTCanis familiaris 67Met Thr Thr Pro Arg Asn Ser Met
Ser Gly Thr Leu Pro Val Asp Pro 1 5 10 15 Met Lys Ser Pro Thr Ala
Met Tyr Pro Val Gln Lys Ile Ile Pro Lys 20 25 30 Arg Met Pro Ser
Val Val Gly Pro Thr Gln Asn Phe Phe Met Arg Glu 35 40 45 Ser Lys
Thr Leu Gly Ala Val Gln Ile Met Asn Gly Leu Phe His Ile 50 55 60
Ala Leu Gly Ser Leu Leu Met Ile His Thr Asp Val Tyr Ala Pro Ile 65
70 75 80 Cys Ile Thr Met Trp Tyr Pro Leu Trp Gly Gly Ile Met Phe
Ile Ile 85 90 95 Ser Gly Ser Leu Leu Ala Ala Ala Asp Lys Asn Pro
Arg Lys Ser Leu 100 105 110 Val Lys Gly Lys Met Ile Met Asn Ser Leu
Ser Leu Phe Ala Ala Ile 115 120 125 Ser Gly Ile Ile Phe Leu Ile Met
Asp Ile Phe Asn Ile Thr Ile Ser 130 135 140 His Phe Phe Lys Met Glu
Asn Leu Asn Leu Ile Lys Ala Pro Met Pro 145 150 155 160 Tyr Val Asp
Ile His Asn Cys Asp Pro Ala Asn Pro Ser Glu Lys Asn 165 170 175 Ser
Leu Ser Ile Gln Tyr Cys Gly Ser Ile Arg Ser Val Phe Leu Gly 180 185
190 Val Phe Ala Val Met Val Ile Phe Thr Phe Phe Gln Lys Leu Val Thr
195 200 205 Ala Gly Ile Val Glu Asn Glu Trp Lys Lys Leu Cys Ser Lys
Pro Lys 210 215 220 Ser Asp Val Val Val Leu Leu Ala Ala Glu Glu Lys
Lys Glu Gln Pro 225 230 235 240 Ile Glu Thr Thr Glu Glu Met Val Glu
Leu Thr Glu Ile Ala Ser Gln 245 250 255 Pro Lys Lys Glu Glu Asp Ile
Glu Ile Ile Pro Val Gln Glu Glu Glu 260 265 270 Glu Glu Leu Glu Ile
Asn Phe Ala Glu Pro Pro Gln Glu Gln Glu Ser 275 280 285 Ser Pro Ile
Glu Asn Asp Ser Ile Pro 290 295 6826PRTFelis catus 68Gly Gly Ala
Thr Cys Cys Thr Thr Ala Ala Gly Gly Ala Ala Thr Gly 1 5 10 15 Cys
Thr Ala Thr Cys Gly Thr Thr Thr Thr 20 25 69298PRTFelis catus 69Met
Thr Thr Pro Arg Asn Ser Met Ser Gly Thr Leu Pro Ala Asp Ala 1 5 10
15 Met Lys Ser Pro Thr Ala Met Asn Pro Val Gln Lys Ile Ile Pro Lys
20 25 30 Lys Met Pro Ser Val Val Gly Pro Thr Gln Asn Phe Phe Met
Lys Glu 35 40 45 Ser Lys Pro Leu Gly Ala Val Gln Ile Met Asn Gly
Leu Phe His Met 50 55 60 Ala Leu Gly Gly Leu Leu Met Ile His Met
Glu Val Tyr Ala Pro Ile 65 70 75 80 Cys Met Thr Val Trp Tyr Pro Leu
Trp Gly Gly Ile Met Tyr Ile Ile 85 90 95 Ser Gly Ser Leu Leu Val
Ala Ala Glu Lys Asn Pro Arg Lys Ser Leu 100 105 110 Val Lys Gly Lys
Met Ile Met Asn Ser Leu Ser Leu Phe Ala Ala Ile 115 120 125 Ser Gly
Met Ile Leu Leu Ile Met Asp Ile Phe Asn Ile Ala Ile Ser 130 135 140
His Phe Phe Lys Met Glu Asn Leu Asn Leu Leu Lys Ser Pro Lys Pro 145
150 155 160 Tyr Ile Asp Ile His Thr Cys Gln Pro Glu Ser Lys Pro Ser
Glu Lys 165 170 175 Asn Ser Leu Ser Ile Lys Tyr Cys Asp Ser Ile Arg
Ser Val Phe Leu 180 185 190 Ser Ile Phe Ala Val Met Val Val Phe Thr
Leu Phe Gln Lys Leu Val 195 200 205 Thr Ala Gly Ile Val Glu Asn Glu
Trp Lys Lys Leu Cys Ser Lys Pro 210 215 220 Lys Ala Asp Val Val Val
Leu Leu Ala Ala Glu Glu Lys Lys Glu Gln 225 230 235 240 Leu Val Glu
Ile Thr Glu Glu Ala Val Glu Leu Thr Glu Val Ser Ser 245 250 255 Gln
Pro Lys Asn Glu Glu Asp Ile Glu Ile Ile Pro Val Gln Glu Glu 260 265
270 Glu Glu Glu Thr Glu Met Asn Phe Pro Glu Pro Pro Gln Asp Gln Glu
275 280 285 Pro Ser Leu Ile Glu Asn Asp Ser Ile Pro 290 295
7040DNACanis familiaris 70caacaaagct tgccgccacc atgaagggct
tcctcttcct 407131DNACanis familiaris 71caacaggatc ctcagctgag
gtagaagagc t 317267PRTCanis familiaris 72Met Lys Gly Phe Leu Phe
Leu Leu Leu Thr Ile Ser Leu Leu Val Met 1 5 10 15 Ile Gln Ile Gln
Thr Gly Val Leu Gly Asn Ser Thr Thr Pro Arg Met 20 25 30 Thr Thr
Lys Lys Val Lys Ser Ala Thr Pro Ala Leu Ser Ser Leu Gly 35 40 45
Gly Gly Ser Val Leu Leu Phe Leu Ala Asn Thr Leu Ile Gln Leu Phe 50
55 60 Tyr Leu Ser 65 7367PRTFelis catus 73Met Lys Gly Phe Leu Phe
Leu Leu Leu Thr Ile Ser Leu Leu Val Met 1 5 10 15 Ile Gln Ile Gln
Thr Gly Val Leu Gly Asn Thr Thr Thr Ala Ala Thr 20 25 30 Thr Thr
Lys Lys Pro Lys Ser Ala Thr Pro Pro Leu Ser Ser Leu Ser 35 40 45
Ser Gly Ser Val Leu Leu Phe Leu Ala Asn Ile Leu Val Gln Leu Phe 50
55 60 Tyr Leu Ser 65 748PRTCanis familiaris 74Thr Asp Val Tyr Ala
Pro Ile Cys 1 5 7548PRTCanis familiaris 75Asn Ile Thr Ile Ser His
Phe Phe Lys Met Glu Asn Leu Asn Leu Ile 1 5 10 15 Lys Ala Pro Met
Pro Tyr Val Asp Ile His Asn Cys Asp Pro Ala Asn 20 25 30 Pro Ser
Glu Lys Asn Ser Leu Ser Ile Gln Tyr Cys Gly Ser Ile Arg 35 40 45
7611PRTCanis familiaris 76His Thr Asp Val Tyr Ala Pro Ile Cys Ile
Thr 1 5 10 7711PRTCanis familiaris 77Leu Ser Ile Gln Tyr Cys Gly
Ser Ile Arg Ser 1 5 10 7817PRTCanis familiaris 78Leu Ile Lys Ala
Pro Met Pro Tyr Val Asp Ile His Asn Cys Asp Pro 1 5 10 15 Ala
797PRTCanis familiaris 79Asn Ile Thr Ile Ser His Phe 1 5
8012PRTCanis familiaris 80Val Asp Ile His Asn Cys Asp Pro Ala Asn
Pro Ser 1 5 10 8112PRTCanis familiaris 81Lys Met Glu Asn Leu Asn
Leu Ile Lys Ala Pro Met 1 5 10 8212PRTCanis familiaris 82Thr Ile
Ser His Phe Phe Lys Met Glu Asn Leu Asn 1 5 10 8312PRTCanis
familiaris 83Pro Met Pro Tyr Val Asp Ile His Asn Cys Asp Pro 1 5 10
8412PRTCanis familiaris 84Asn Cys Asp Pro Ala Asn Pro Ser Glu Lys
Asn Ser 1 5 10 8516PRTCanis familiaris 85Tyr Val Asp Ile His Asn
Cys Asp Pro Ala Asn Pro Ser Glu Lys Asn 1 5 10 15 8616PRTCanis
familiaris 86Gly Gly Ser Val Leu Leu Phe Leu Ala Asn Thr Leu Ile
Gln Leu Phe 1 5 10 15 8715PRTCanis familiaris 87Phe Leu Phe Leu Leu
Leu Thr Ile Ser Leu Leu Val Met Ile Gln 1 5 10 15 887PRTCanis
familiaris 88Gln Thr Gly Val Leu Gly Asn 1 5 8912PRTCanis
familiaris 89Lys Val Lys Ser Ala Thr Pro Ala Leu Ser Ser Leu 1 5 10
9010PRTCanis familiaris 90Pro Arg Met Thr Thr Lys Lys Val Lys Ser 1
5 10
* * * * *
References