U.S. patent application number 11/828464 was filed with the patent office on 2008-02-21 for canine cd20 compositions.
This patent application is currently assigned to IDEXX Laboratories, Inc.. Invention is credited to Melissa J. Beall.
Application Number | 20080045700 11/828464 |
Document ID | / |
Family ID | 35784295 |
Filed Date | 2008-02-21 |
United States Patent
Application |
20080045700 |
Kind Code |
A1 |
Beall; Melissa J. |
February 21, 2008 |
Canine CD20 Compositions
Abstract
The invention provides canine CD20 nucleotide and protein
sequences. These compositions are useful in the diagnosis and
treatment of, e.g., CD20+ B-cell lymphoma, immune-mediated
hemolytic anemia, immune-mediated thrombocytopenia, and systemic
lupus erythematosus (SLE) in canines and felines.
Inventors: |
Beall; Melissa J.; (Cape
Elizabeth, ME) |
Correspondence
Address: |
MCDONNELL BOEHNEN HULBERT & BERGHOFF LLP
300 S. WACKER DRIVE
32ND FLOOR
CHICAGO
IL
60606
US
|
Assignee: |
IDEXX Laboratories, Inc.
|
Family ID: |
35784295 |
Appl. No.: |
11/828464 |
Filed: |
July 26, 2007 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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11138949 |
May 26, 2005 |
|
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11828464 |
Jul 26, 2007 |
|
|
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60575172 |
May 28, 2004 |
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Current U.S.
Class: |
530/387.9 |
Current CPC
Class: |
G01N 2800/104 20130101;
C07K 14/70596 20130101; C07K 16/2887 20130101; G01N 33/5052
20130101; G01N 33/564 20130101; C07K 2317/33 20130101 |
Class at
Publication: |
530/387.9 |
International
Class: |
C07K 16/18 20060101
C07K016/18 |
Claims
1. An isolated antibody or antigen binding portion thereof that
specifically binds SEQ ID NO:6 or SEQ ID NO:10.
2. The isolated antibody or antigen binding portion thereof of
claim 1, wherein the isolated antibody or antigen binding portion
thereof is a monoclonal antibody, a polyclonal antibody, or single
chain antibody.
3. The isolated antibody of claim 2, wherein the antibody is
produced by myeloma cell line ATCC PTA-6661 or ATCC PTA-6662.
4. The antigen binding portion thereof of claim 2, wherein the
antigen binding portion thereof is a Fab fragment, a F(ab').sub.2
fragment, a Fab' fragment, a F(ab).sub.2 fragment, a sFv fragment,
a single chain antibody, or a Fv fragment.
5. The isolated antibody or antigen binding portion thereof of
claim 1, wherein the antibody or antigen binding portion thereof
belongs to an antibody class selected from the group consisting of
IgG, IgM, IgA, IgD and IgE.
6. An isolated antibody or antigen binding fragment thereof that
(a) competes with a reference antibody for binding to SEQ ID NOs:6
or 10 or antigen binding fragments thereof; (b) binds to the same
epitope of SEQ ID NOs:6 or 10 or antigen binding fragments thereof
as a reference antibody; (c) binds to SEQ ID NOs:6 or 10 or antigen
binding fragments thereof with substantially the same K.sub.d as a
reference antibody; or (d) binds to SEQ ID NOs:6 or 10 or antigen
binding fragments thereof with substantially the same off rate as a
reference antibody, wherein the reference antibody is an antibody
or antigen binding fragment thereof that specifically binds to a
polypeptide of SEQ ID NOs:6 or 10 or antigen binding fragments
thereof with a binding affinity K.sub.a of 10.sup.7 l/mol or
more.
7. An isolated antibody or antigen binding fragment thereof that
competes with the isolated antibody of claim 3 for binding to SEQ
ID NOs:6 or 10 or antigen binding fragments thereof.
Description
PRIORITY
[0001] This application is a divisional application of U.S.
application Ser. No. 11/138,949, filed May 26, 2005, which claims
the benefit of U.S. Application 60/575,172 filed May 28, 2004,
which are incorporated herein by reference in their entirety.
BACKGROUND OF THE INVENTION
[0002] CD20 is a transmembrane protein that is expressed on more
than 95% of B-lymphocytes. Expression at the cell surface occurs
from the pre-B stage of development until differentiation to a
plasma cell. The protein has several functions; it serves as a
calcium channel, it is involved in intracellular signal
transduction, and it can modulate cell growth and differentiation.
In human medicine, anti-CD20 monoclonal antibody therapeutics
(e.g.: Rituxan (Rituximab) by Genentech and IDEC Pharmaceuticals)
have been successfully utilized to treat relapsed or refractory
low-grade or follicular, CD20+, B-cell non-Hodgkin's lymphoma
(NHL). Rituxan has also been used in treating immune-mediated
hemolytic anemia, immune-mediated thrombocytopenia, and systemic
lupus erythematosus (SLE). Rituxan, however, does not bind canine B
cells. See, Impellizeri et al., Vet. Cancer Society. 2003. Proceed.
23.sup.rd Ann. Conf., p. 2.
SUMMARY OF THE INVENTION
[0003] The invention provides canine CD20 nucleotide and protein
sequences. These compositions are useful in the diagnosis and
treatment of, e.g., CD20+ B-cell lymphoma, immune-mediated
hemolytic anemia, immune-mediated thrombocytopenia, and systemic
lupus erythematosus (SLE) in canines and felines.
[0004] One embodiment of the invention provides an isolated canine
CD20 protein comprising SEQ ID NO:6.
[0005] Another embodiment of the invention provides a polypeptide
comprising SEQ ID NO:6, wherein the polypeptide has one or more
amino acid substitutions at about one to about 45 positions
selected from amino acid positions 8, 9, 11, 12, 14, 15, 16, 17,
19, 21, 24, 25, 26, 27, 29, 30, 31, 32, 34, 35, 37, 43, 47, 51, 65,
73, 74, 75, 76, 77, 82, 83, 84, 94, 102, 105, 106, 108, 109, 112,
116, 118, 121, 131, 133, 134, 139, 141, 142, 143, 147, 150, 151,
153, 154, 155, 156, 157, 158, 159, 162, 163, 165, 166, 168, 170,
171, 172, 178, 180, 184, 187, 189, 192, 193, 194, 195, 196, 198,
199, 200, 201, 202, 205, 208, 218, 219, 221, 222, 223, 225, 226,
227, 228, 232, 234, 235, 240, 241, 242, 243, 244, 247, 248, 249,
251, 252, 253, 254, 259, 262, 270, 275, 277, 280, 282, 285, 288,
290, 292, 295, 296, and 297, wherein the polypeptide is not SEQ ID
NO:7, SEQ ID NO:8, or SEQ ID NO:9 and wherein the polypeptide is
isolated, purified and about 297 to about 300 amino acids long. The
polypeptide can further comprise one or more amino acid sequences
that are not canine amino acid sequences. The one or more amino
acid substitutions can be conservative amino acid substitutions.
The amino acid substitutions can be selected from the following
substitutions: TABLE-US-00001 Amino Acid Amino Acid Position
Substitution 8 V 9 N 11 P 12 F 14 A 15 E 16 A 17 T 19 G 21 L, I 24
Q, N 25 S 26 G, A 27 P 29 P, V 30 L, N 31 F, L 32 R 34 T 35 S 37 L
43 S 47 K 51 A, P 64 M 65 T 73 P 74 A, M 75 G, E 76 I 77 F 82 L, V,
M 83 S 84 V 94 Y 102 V 105 A 106 E 108 T 109 S 112 C 116 A 118 V
121 S 131 M 133 L 134 S 139 L 141 M 142 K, A 143 L 147 F 150 R 151
S, R 153 E 154 F 155 L 156 R, Q 157 T, S 158 H, S 159 T, K 162 I
163 N 165 Y 166 D, T 168 E, Q 170 S 171 K 172 S 178 P 180 T 184 Y,
N, D 187 Q 189 L 192 S 193 I 194 L 195 S 196 A 198 L 199 V 200 S
201 A 202 L 205 E 208 I 218 R 219 T, M 221 T 222 R 223 S 225 A 226
N 227 DELETE 228 I 232 S 234 G 235 N 240 T, L 241 V 242 K 243 I, M
244 K 247 V, I, A 248 I 249 G 251 S 252 G 253 T, V 254 S 259 N 262
E 270 I 275 T, A 277 T, M 280 P 282 A 285 D 288 P 290 L 292 V 295 E
296 I 297 S, A
The polypeptide comprises one or more of the following amino acid
additions: an A after amino acid 104; an E after amino acid 169,
and an E after amino acid 274.
[0006] Even another embodiment of the invention provides an
isolated polypeptide comprising SEQ ID NO:10. The polypeptide can
be present in a fusion protein.
[0007] Still another embodiment of the invention provides a
polypeptide comprising SEQ ID NO:10, wherein the polypeptide has
about one to about 12 amino acid substitutions at positions 3, 5,
6, 7, 14, 15, 17, 18, 19, 20, 21, 22, 23, 26, 27, 29, 30, 32, 35,
and 45, wherein the polypeptide is not SEQ ID NO:12, SEQ ID NO:13,
or SEQ ID NO:14, and wherein the polypeptide is isolated, purified
and is about 53 amino acids long. The amino acid substitutions can
be conservative amino acid substitutions. The amino acid
substitutions can be selected from the following substitutions:
TABLE-US-00002 Amino Acid Position Substitution 3 L 5 M 6 K, A 7 L
11 F 14 R 15 S, R 17 E 18 F 19 L 20 R, Q 21 T, S 22 H, S 23 T, K 26
I 27 N 29 Y 30 D, T 32 E, Q 34 S 35 K 36 S 42 P 44 T 45 K 48 N, Y,
D 51 Q 53 L
The polypeptide can comprise one or more amino acid sequences that
are not canine amino acid sequences. The polypeptide can comprise
an amino acid addition of E after amino acid number 33. The
polypeptide can comprise SEQ ID NO:1.
[0008] Yet another embodiment of the invention provides an antibody
or antigen binding portion thereof that specifically binds SEQ ID
NO:6 or SEQ ID NO:10. The isolated antibody or antigen binding
portion thereof can be a monoclonal antibody, a polyclonal
antibody, or single chain antibody. The antibody can be produced by
myeloma cell line ATCC PTA-6661 or ATCC PTA-6662. The antigen
binding portion thereof can be a Fab fragment, a F(ab').sub.2
fragment, or a Fv fragment.
[0009] Another embodiment of the invention provides an isolated
polynucleotide that encodes a canine CD20 protein. The
polynucleotide can comprise SEQ ID NO:5. The isolated
polynucleotide can comprise about 12 or more contiguous nucleic
acids of SEQ ID NO:5.
[0010] Even another embodiment of the invention provides an
isolated polynucleotide that encodes the extracellular domain of a
canine CD20 protein.
[0011] Yet another embodiment of the invention provides a vector
comprising a polynucleotide of the invention and a recombinant host
cell that comprises a vector of the invention.
[0012] Still another embodiment of the invention provides a method
of producing a recombinant cell that expresses a canine CD20
protein, or fragment thereof, comprising transfecting a cell with
the vector of the invention. A canine CD20 polypeptide or a
fragment thereof can be produces by expressing the polypeptide in
the recombinant host cell if the invention.
[0013] Another embodiment of the invention provides a method of
detecting a canine or feline CD20-positive B-lymphocyte. The method
comprises contacting one or more antibodies that specifically bind
to a polypeptide consisting of SEQ ID NO:6 or SEQ ID NO:10 with a
test sample under conditions that allow B-lymphocyte/antibody
complexes to form and detecting B-lymphocyte/antibody complexes.
The detection of B-lymphocyte/antibody complexes is an indication
that a canine or feline CD20-positive B-lymphocyte is present in
the sample and the absence of B-lymphocyte/antibody complexes is an
indication that a canine or feline CD20-positive B-lymphocyte is
not present in the sample. The one or more antibodies can be
monoclonal antibodies, polyclonal antibodies, or antibody
fragments. The sample can be lymph node aspirate, serum, or whole
blood.
[0014] Yet another embodiment of the invention provides a method of
treating a canine or feline for CD20-positive B-cell lymphoma,
immune-mediated hemolytic anemia, immune-mediated thrombocytopenia,
or systemic lupus erythematosus (SLE) comprising administering an
antibody of the invention to the canine or feline.
BRIEF DESCRIPTION OF THE DRAWINGS
[0015] FIG. 1 shows a portion of the nucleotide sequence for canine
CD20 from canine peripheral blood mononuclear cell cDNA (SEQ ID
NO:1).
[0016] FIG. 2 shows the translated polypeptide (SEQ ID NO:4) of SEQ
ID NO:1 aligned with comparable regions of human CD 20 (SEQ ID
NO:2) and mouse CD20 (SEQ ID NO:3).
[0017] FIG. 3 shows the full-length polynucleotide of canine CD20
(SEQ ID NO:5)
[0018] FIG. 4 shows the full-length polypeptide sequence (SEQ ID
NO:6) for canine CD20 The amino acid sequence was deduced from cDNA
sequence analysis.
[0019] FIG. 5 shows the alignment of the full length canine CD20
polypeptide (SEQ ID NO:6) with that of human (SEQ ID NO:7), mouse
(SEQ ID NO:8) and cat (SEQ ID NO:9). The canine sequence is most
similar to the feline sequence (84% identical) and less similar to
that of human (74% identical) and mouse (68% identical).
[0020] FIG. 6 is an anti-FLAG Western blot demonstrating expression
of canine CD20 in COS7 cells. Lane 1 is an empty plasmid control,
lane 2 is total cell lysate of cells transiently transfected with
canine CD20, lanes 3-8 are total cell lysates from transfected
cells immunoprecipitated with either anti-FLAG (3), anti-CD20
monoclonals (4,5), or anti-CD20 polyclonal antibodies (6-8).
[0021] FIG. 7 is an example of a COS7 cell expressing canine CD20
on the surface of the cell as detected by anti-FLAG
immunofluoresence and confocal microscopy with three-dimensional
reconstruction.
[0022] FIG. 8 shows a 53-mer polypeptide of the predominant
extracellular domain of canine CD20 (SEQ ID NO:10) that was
synthesized alone and in conjunction with a murine T-cell epitope
from ovalbumin (SEQ ID NO:11).
[0023] FIG. 9 depicts the serum titers obtained from two mice
immunized with canine CD20 as evaluated in the peptide ELISA.
[0024] FIG. 10 is a Coomassie stained SDS-PAGE gel showing the
purified IgM monoclonal antibodies that recognize the extracellular
domain of canine CD20. Monoclonal antibody F3C7 is shown in lane 1,
F7A5 is shown in lane 2, and F19 is shown in lane 3.
[0025] FIG. 11 depicts the flow cytometry results for the four
fluorescently labeled IgM monoclonal antibodies to canine CD20 when
used to label a lymph node aspirate from dogs with lymphoma.
[0026] FIG. 12 shows the flow cytometry results for the
fluorescently labeled IgM monoclonal antibody F7A5 on a lymph node
aspirate from a dog with a B-cell lymphoma. Scatter plots are also
shown for an isotype control for the CD20 antibody
(IgM-fluorescein), an anti-CD79a B-cell antibody and the
corresponding IgG1 isotype control.
[0027] FIG. 13 shows the scatter plots obtained using the colloidal
gold labeled F7A5 monoclonal antibody on a lymph node aspirate from
a normal dog as analyzed by a point of care hematology instrument.
Panel A depicts the unlabeled control and F7A5 labeled cells for
gating on small lymphocytes, while panel B depicts the same
comparison when gating on medium lymphocytes.
[0028] FIG. 14 depicts an example of how the colloidal gold labeled
F7A5 monoclonal antibody can be used on a point of care hematology
instrument to detect CD20-positive B-lymphocytes in a cat with
lymphoma.
DETAILED DESCRIPTION OF THE INVENTION
Polypeptides
[0029] A polypeptide is a polymer of three or more amino acids
covalently linked by amide bonds. A polypeptide can be
post-translationally modified. A purified polypeptide is a
polypeptide preparation that is substantially free of cellular
material, other types of polypeptides, chemical precursors,
chemicals used in synthesis of the polypeptide, or combinations
thereof. A polypeptide preparation that is substantially free of
cellular material, culture medium, chemical precursors, chemicals
used in synthesis of the polypeptide has less than about 30%, 20%,
10%, 5%, 1% or more of other polypeptides, culture medium, chemical
precursors, and/or other chemicals used in synthesis. Therefore, a
purified polypeptide is about 70%, 80%, 90%, 95%, 99% or more
pure.
[0030] Polypeptides of the invention comprise full-length canine
CD20 and fragments thereof. One embodiment of the invention
provides an isolated polypeptide comprising SEQ ID NO:6 or SEQ ID
NO:10. Another embodiment of the invention provides a polypeptide
comprising SEQ ID NO:10 and having amino acid substitutions, for
example, conservative amino acid substitutions, at one or more
positions (for example, about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11,
12, 13, 14, 15, 20, 24 or more amino acid substitutions) selected
from amino acid positions 3, 5, 6, 7, 14, 15, 17, 18, 19, 20, 21,
22, 23, 26, 27, 29, 30, 32, 35, and 45 of SEQ ID NO:10, wherein the
polypeptide is not SEQ ID NO:12 or SEQ ID NO:13, or SEQ ID NO:14
and wherein the polypeptide is isolated, purified and is about 53
amino acids long. An amino acid addition of "E" can occur after
amino acid number 33. In one embodiment of the invention SEQ ID
NO:10 has one or more substituted amino acids (for example, 1, 2,
3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 20, or 24 amino acid
substitutions) as shown in Table 1 and the polypeptide is not SEQ
ID NO:12, SEQ ID NO:13, or SEQ ID NO:14 and the polypeptide is
isolated, purified and about 53 amino acids long. TABLE-US-00003
TABLE 1 Amino Acid Position Substitution 3 L 5 M 6 K, A 7 L 11 F 14
R 15 S, R 17 E 18 F 19 L 20 R, Q 21 T, S 22 H, S 23 T, K 26 I 27 N
29 Y 30 D, T 32 E, Q 34 S 35 K 36 S 42 P 44 T 45 K 48 N, Y, D 51 Q
53 L
[0031] Another embodiment of the invention provides a polypeptide
comprising SEQ ID NO:6 having amino acid substitutions, for
example, conservative amino acid substitutions, at one or more
positions (e.g., about 1, 2, 3, 4, 5, 10, 15, 20, 25, 30, 35, 40,
45, 50, 55, 60 or more amino acid substitutions) selected from
amino acid positions 8, 9, 44, 12, 14, 15, 16, 17, 19, 21, 24, 25,
26, 27, 29, 30, 31, 32, 34, 35, 37, 43, 47, 51, 65, 73, 74, 75, 76,
77, 82, 83, 84, 94, 102, 105, 106, 108, 109, 112, 116, 118, 121,
131, 133, 134, 139, 141, 142, 143, 147, 150, 151, 153, 154, 155,
156, 157, 158, 159, 162, 163, 165, 166, 168, 170, 171, 172, 178,
180, 184, 187, 189, 192, 193, 194, 195, 196, 198, 199, 200, 201,
202, 205, 208, 218, 219, 221, 222, 223, 225, 226, 227, 228, 232,
234, 235, 240, 241, 242, 243, 244, 247, 248, 249, 251, 252, 253,
254, 259, 262, 270, 275, 277, 280, 282, 285, 288, 290, 292, 295,
296, 297 wherein the polypeptide is not SEQ ID NO:7, SEQ ID NO:8,
or SEQ ID NO:9 and wherein the polypeptide is isolated, purified,
and about 297 to about 299 amino acids long. In one embodiment of
the invention SEQ ID NO:6 has one or more (e.g., 1, 2, 3, 4, 5, 10,
15, 20, 25, 30, 35, 40, 45, 50, 55, 60 or more) substituted amino
acids as shown in Table 2 and the polypeptide is not SEQ ID NO:7,
SEQ ID NO:8, or SEQ ID NO:9 and the polypeptide is isolated,
purified and about 297 to about 300 amino acids long. Additionally,
amino acid additions can occur as follows: an A after amino acid
104; an E after amino acid 169, an E after amino acid 274 of SEQ ID
NO:6. TABLE-US-00004 TABLE 2 Amino Acid Amino Acid Position
Substitution 8 V 9 N 11 P 12 F 14 A 15 E 16 A 17 T 19 G 21 L, I 24
Q, N 25 S 26 G, A 27 P 29 P, V 30 L, N 31 F, L 32 R 34 T 35 S 37 L
43 S 47 K 51 A, P 64 M 65 T 73 P 74 A, M 75 G, E 76 I 77 F 82 L, V,
M 83 S 84 V 94 Y 102 V 105 A 106 E 108 T 109 S 112 C 116 A 118 V
121 S 131 M 133 L 134 S 139 L 141 M 142 K, A 143 L 147 F 150 R 151
S, R 153 E 154 F 155 L 156 R, Q 157 T, S 158 H, S 159 T, K 162 I
163 N 165 Y 166 D, T 168 E, Q 170 S 171 K 172 S 178 P 180 T 184 Y,
N, D 187 Q 189 L 192 S 193 I 194 L 195 S 196 A 198 L 199 V 200 S
201 A 202 L 205 E 208 I 218 R 219 T, M 221 T 222 R 223 S 225 A 226
N 227 DELETE 228 I 232 S 234 G 235 N 240 T, L 241 V 242 K 243 I, M
244 K 247 V, I, A 248 I 249 G 251 S 252 G 253 T, V 254 S 259 N 262
E 270 I 275 T, A 277 T, M 280 P 282 A 285 D 288 P 290 L 292 V 295 E
296 I 297 S, A
[0032] The basic and novel characteristics of polypeptides of the
invention that consist essentially of SEQ ID NO:6 or SEQ ID NO:10
are that they consist essentially of the sequences shown in SEQ ID
NO:6 and SEQ ID NO:10 and that they specifically bind to an
antibody, antibody fragment, or single-chain antibody of the
invention.
[0033] One of skill in the art would expect that amino acid
substations and/or additions could be made in the amino acid
sequences of SEQ ID NO:6 or SEQ ID NO:10 as described above,
wherein the amino acid sequences would retain their functional
activity. For example, a F7A5 monoclonal antibody (see Example 6),
which is specific for SEQ ID NO:10 specifically binds both feline
and canine CD20-positive B lymphocytes (see Example 7 and 8),
despite the fact that sequences of feline and canine CD20 are not
100% homologous.
[0034] An isolated polypeptide is a molecule of amino acids that is
not immediately contiguous with one or both canine flanking amino
acid sequences that the molecule is normally associated with in
nature. An isolated polypeptide is also a molecule of amino acids
that forms part of a hybrid polypeptide comprising additional
non-canine polypeptide sequences that can be, for example, a fusion
protein.
[0035] The invention also includes functionally active variants of
SEQ ID NO:6 and SEQ ID NO:10. Functionally active variants of SEQ
ID NO:6 or SEQ ID NO:10 can comprise one or more of the amino acid
substitutions as described above. In one embodiment, a functionally
active variant polypeptide includes an amino acid sequence at least
about 60% identical to a sequence shown as SEQ ID NO:6 and SEQ ID
NO:10. Preferably, the polypeptide is at least 55%, 60%, 65%, 70%,
75%, 80%, 85%, 90%, 95%, 98%, 99% or more identical to SEQ ID NO:6
or SEQ ID NO:10 and specifically binds to an antibody of the
invention. Functionally active variant polypeptides and
polypeptides of the invention also specifically bind to an
antibody, such as a monoclonal antibody, that is raised to a
polypeptide shown in SEQ ID NO:6 or SEQ ID NO:10.
[0036] Polypeptides of the invention specifically bind to an
antibody of the invention. In this context "specifically binds"
means that the polypeptide recognizes and binds to an antibody of
the invention with greater affinity than to other, non-specific
molecules. For example, an antibody raised against an antigen
(polypeptide) to which it binds more efficiently than to a
non-specific protein can be described as specifically binding to
the antigen. Binding specifically can be tested using, for example,
an enzyme-linked immunosorbant assay (ELISA), a radioimmunoassay
(RIA), or a western blot assay using methodology well known in the
art.
[0037] A polypeptide is a functionally active variant if it reacts
substantially the same as a polypeptide shown in SEQ ID NO:6 or SEQ
ID NO:10 in an assay such as an immunohistochemical assay, an
ELISA, an RIA, or a western blot assay, e.g. has 90-110% of the
specific binding activity of the original polypeptide. In one
embodiment, the assay is a competition assay wherein the
functionally active variant polypeptide is capable of reducing
binding of a polypeptide shown in SEQ ID NO:6 or SEQ ID NO:10 to a
corresponding antibody, antibody fragment, or single-chain antibody
by about 80, 95, 99, or 100%.
[0038] Functionally active variants can also comprise "polypeptide
fragments" of the invention. Polypeptide fragments comprise or
consist essentially of about 15, 20, 30, 40, 50, 100, 150, 200, or
299 amino acids of SEQ ID NO:6 or SEQ ID NO:10.
[0039] As used herein, percent identity of two amino acid sequences
(or of two nucleic acid sequences) is determined using the
algorithm of Karlin and Altschul (PNAS USA 87:2264-2268, 1990),
modified as in Karlin and Altschul, PNAS USA 90:5873-5877, 1993).
Such an algorithm is incorporated into the NBLAST and XBLAST
programs of Altschul et al. (J. Mol. Biol. 215:403-410, 1990).
BLAST nucleotide searches are performed with the NBLAST program,
score=100, wordlength=12. BLAST protein searches are performed with
the XBLAST program, score=50, wordlength=3. To obtain gapped
alignment for comparison purposes GappedBLAST is utilized as
described in Altschul et al. (Nucleic Acids Res. 25:3389-3402,
1997). When utilizing BLAST and GappedBLAST programs the default
parameters of the respective programs (e.g., XBLAST and NBLAST) are
used to obtain nucleotide sequences homologous to a nucleic acid
molecule of the invention.
[0040] Identity or identical means amino acid sequence (or nucleic
acid sequence) similarity and has an art recognized meaning.
Sequences with identity share identical or similar amino acids (or
nucleic acids). Thus, a candidate sequence sharing 85% amino acid
sequence identity with a reference sequence requires that,
following alignment of the candidate sequence with the reference
sequence, 85% of the amino acids in the candidate sequence are
identical to the corresponding amino acids in the reference
sequence, and/or constitute conservative amino acid changes.
[0041] Functionally active variants of SEQ ID NO:6 or SEQ ID NO: 10
retain substantially the same functional activity of the original
polypeptide or fragment. Naturally occurring functionally active
variants such as allelic variants and species variants and
non-naturally occurring functionally active variants are included
in the invention and can be produced by, for example, mutagenesis
techniques or by direct synthesis.
[0042] A functionally active variant differs by about, for example,
1, 2, 3, 4, 5, 10, 20, 30, 40, 50, 60 or more amino acid residues
from a polypeptide shown in SEQ ID NO:6 or SEQ ID NO:10 or a
fragment thereof. Where this comparison requires alignment the
sequences are aligned for maximum homology. The site of variation
can occur anywhere in the polypeptide, as long as activity
substantially similar to a polypeptide shown in SEQ ID NO:6 and SEQ
ID NO:10 is maintained.
[0043] Guidance concerning how to make phenotypically silent amino
acid substitutions is provided in Bowie et al., Science,
247:1306-1310 (1990), which teaches that there are two main
strategies for studying the tolerance of an amino acid sequence to
change.
[0044] The first strategy exploits the tolerance of amino acid
substitutions by natural selection during the process of evolution.
By comparing amino acid sequences in different species, the amino
acid positions which have been conserved between species can be
identified. See e.g., FIG. 5. These conserved amino acids are
likely important for protein function. In contrast, the amino acid
positions in which substitutions have been tolerated by natural
selection indicate positions which are not critical for protein
function. Thus, positions tolerating amino acid substitution can be
modified while still maintaining specific binding activity of the
polypeptide.
[0045] The second strategy uses genetic engineering to introduce
amino acid changes at specific positions of a cloned gene to
identify regions critical for protein function. For example,
site-directed mutagenesis or alanine-scanning mutagenesis (the
introduction of single alanine mutations at every residue in the
molecule) can be used (Cunningham et al., Science, 244:1081-1085
(1989)). The resulting variant molecules can then be tested for
specific binding to antibodies of the invention.
[0046] According to Bowie et al., these two strategies have
revealed that proteins are surprisingly tolerant of amino acid
substitutions. The authors further indicate which amino acid
changes are likely to be permissive at certain amino acid positions
in the protein. For example, the most buried or interior (within
the tertiary structure of the protein) amino acid residues require
nonpolar side chains, whereas few features of surface or exterior
side chains are generally conserved.
[0047] Methods of introducing a mutation into amino acids of a
protein is well known to those skilled in the art. See, e.g.,
Ausubel (ed.), Current Protocols in Molecular Biology, John Wiley
and Sons, Inc. (1994); T. Maniatis, E. F. Fritsch and J. Sambrook,
Molecular Cloning: A Laboratory Manual, Cold Spring Harbor
laboratory, Cold Spring Harbor, N.Y. (1989)). Mutations can also be
introduced using commercially available kits such as
"QuikChange.TM. Site-Directed Mutagenesis Kit" (Stratagene). The
generation of a polypeptide functionally active variant to a
polypeptide by replacing an amino acid that does not influence the
function of a polypeptide can be accomplished by one skilled in the
art.
[0048] In one embodiment of the invention, a polypeptide of the
invention is derived from a canine. A polypeptide of the invention
can be isolated from cells or tissue sources using standard protein
purification techniques. Polypeptides of the invention can also be
synthesized chemically or produced by recombinant DNA techniques.
For example, a polypeptide of the invention can be synthesized
using conventional peptide synthesizers. Additionally, a
polynucleotide encoding a polypeptide of the invention can be
introduced into an expression vector that can be expressed in a
suitable expression system using techniques well known in the art.
A variety of bacterial, yeast, plant, mammalian, and insect
expression systems are available in the art and any such expression
system can be used. Optionally, a polynucleotide encoding a
polypeptide of the invention can be translated in a cell-free
translation system.
[0049] A functionally active variant polypeptide can also be
isolated using a hybridization technique. Briefly, DNA having a
high homology to the whole or part of a nucleic acid sequence
encoding SEQ ID NO:6 or SEQ ID NO:10 is used to prepare a
functionally active polypeptide. Therefore, a polypeptide of the
invention also includes polypeptides that are functionally
equivalent to a SEQ ID NO:6 or SEQ ID NO:10 polypeptide and are
encoded by a nucleic acid molecule that hybridizes with a nucleic
acid encoding SEQ ID NO:6 or SEQ ID NO:10 or a complement thereof.
One of skill in the art can easily determine nucleic acid sequences
that encode polypeptides of the invention using readily available
codon tables. As such, these nucleic acid sequences are not
presented herein.
[0050] The stringency of hybridization for a nucleic acid encoding
a polypeptide that is a functionally active variant is, for
example, 10% formamide, 5.times.SSPE, 1.times. Denhart's solution,
and 1.times. salmon sperm DNA (low stringency conditions). More
preferable conditions are, 25% formamide, 5.times.SSPE, 1.times.
Denhart's solution, and 1.times. salmon sperm DNA (moderate
stringency conditions), and even more preferable conditions are,
50% formamide, 5.times.SSPE, 1.times. Denhart's solution, and
1.times. salmon sperm DNA (high stringency conditions). However,
several factors influence the stringency of hybridization other
than the above-described formamide concentration, and one skilled
in the art can suitably select these factors to accomplish a
similar stringency.
[0051] Nucleic acid molecules encoding a functionally active
variant polypeptide can also be isolated by a gene amplification
method such as PCR using a portion of a nucleic acid molecule DNA
encoding a polypeptide shown in SEQ ID NO:6 or SEQ ID NO: 10 as the
probe.
[0052] Polypeptides of the invention can also comprise those that
arise as a result of the existence of multiple genes, alternative
transcription events, alternative RNA splicing events, and
alternative translational and postranslational events. A
polypeptide can be expressed in systems, e.g., cultured cells,
which result in substantially the same postranslational
modifications present as when the polypeptide is expressed in a
native cell, or in systems that result in the alteration or
omission of postranslational modifications, e.g., glycosylation or
cleavage, present when expressed in a native cell.
[0053] A conservative substitution is one in which an amino acid is
substituted for another amino acid that has similar properties,
such that one skilled in the art of peptide chemistry would expect
the secondary structure and hydropathic nature of the polypeptide
to be substantially unchanged. In general, the following groups of
amino acids represent conservative changes: (1) ala, pro, gly, glu,
asp, gln, asn, ser, thr; (2) cys, ser, tyr, tlr; (3) val, ile, leu,
met, ala, phe; (4) lys, arg, his; and (5) phe, tyr, trp, his.
[0054] A polypeptide of the invention can further comprise a signal
(or leader) sequence that co-translationally or
post-translationally directs transfer of the protein. The
polypeptide can also comprise a linker or other sequence for ease
of synthesis, purification or identification of the polypeptide
(e.g., poly-His), or to enhance binding of the polypeptide to a
solid support. For example, a polypeptide can be conjugated to an
immunoglobulin Fc region or bovine serum albumin.
[0055] A polypeptide can be covalently or non-covalently linked to
an amino acid sequence to which the polypeptide is not normally
associated with in nature. Additionally, a polypeptide can be
covalently or non-covalently linked to compounds or molecules other
than amino acids. For example, a polypeptide can be linked to an
indicator reagent, an amino acid spacer, an amino acid linker, a
signal sequence, a stop transfer sequence, a transmembrane domain,
a protein purification ligand, or a combination thereof. In one
embodiment of the invention a protein purification ligand can be
one or more C amino acid residues at, for example, the amino
terminus or carboxy terminus of a polypeptide of the invention. An
amino acid spacer is a sequence of amino acids that are not usually
associated with a polypeptide of the invention in nature. An amino
acid spacer can comprise about 1, 5, 10, 20, 100, or 1,000 amino
acids.
[0056] If desired, a polypeptide can be a fusion protein, which can
also contain other amino acid sequences, such as amino acid
linkers, amino acid spacers, signal sequences, TMR stop transfer
sequences, transmembrane domains, as well as ligands useful in
protein purification, such as glutathione-5-transferase, histidine
tag, and staphylococcal protein A, or combinations thereof. A
fusion protein is two or more different amino acid sequences
operably linked to each other. A fusion protein construct can be
synthesized chemically using organic compound synthesis techniques
by joining individual polypeptide fragments together in fixed
sequence. A fusion protein construct can also be expressed by a
genetically modified host cell (such as E. coli) cultured in vitro,
which carries an introduced expression vector bearing specified
recombinant DNA sequences encoding the amino acids residues in
proper sequence. The heterologous polypeptide can be fused, for
example, to the N-terminus or C-terminus of a polypeptide of the
invention. A polypeptide of the invention can also comprise
homologous amino acid sequences, i.e., other CD20 or CD20-derived
sequences. More than one polypeptide of the invention can be
present in a fusion protein. Fragments of polypeptides of the
invention can be present in a fusion protein of the invention. A
fusion protein of the invention can comprise, e.g., one or more of
SEQ ID NO:6, SEQ ID NO:10, fragments thereof, or combinations
thereof.
[0057] Polypeptides of the invention can be in a multimeric form.
That is, a polypeptide can comprise one or more copies of SEQ ID
NO:6, SEQ ID NO:10 or a combination thereof. A multimeric
polypeptide can be a multiple antigen peptide (MAP). See e.g., Tam,
J. Immunol. Methods, 196:17-32 (1996).
[0058] Polypeptides of the invention can comprise an antigen that
is recognized by an antibody reactive against canine CD20. The
antigen can comprise one or more epitopes (i.e., antigenic
determinants). An epitope can be a linear epitope, sequential
epitope or a conformational epitope. Epitopes within a polypeptide
of the invention can be identified by several methods. See, e.g.,
U.S. Pat. No. 4,554,101; Jameson & Wolf, CABIOS 4:181-186
(1988). For example, a polypeptide of the invention can be isolated
and screened. A series of short peptides, which together span an
entire polypeptide sequence, can be prepared by proteolytic
cleavage. By starting with, for example, 100-mer polypeptide
fragments, each fragment can be tested for the presence of epitopes
recognized in an ELISA. For example, in an ELISA assay a canine
CD20 polypeptide, such as a 100 mer polypeptide fragment, is
attached to a solid support, such as the wells of a plastic
multi-well plate. A population of antibodies are labeled, added to
the solid support and allowed to bind to the unlabeled antigen,
under conditions where non-specific absorption is blocked, and any
unbound antibody and other proteins are washed away. Antibody
binding is detected by, for example, a reaction that converts a
colorless substrate into a colored reaction product. Progressively
smaller and overlapping fragments can then be tested from an
identified 100-mer to map the epitope of interest.
[0059] A polypeptide of the invention can be produced
recombinantly. A polynucleotide encoding a polypeptide of the
invention can be introduced into a recombinant expression vector,
which can be expressed in a suitable expression host cell system
using techniques well known in the art. A variety of bacterial,
yeast, plant, mammalian, and insect expression systems are
available in the art and any such expression system can be used.
Optionally, a polynucleotide encoding a polypeptide can be
translated in a cell-free translation system. A polypeptide can
also be chemically synthesized or obtained from E. ewingii
cells.
[0060] An immunogenic polypeptide of the invention can comprise an
amino acid sequence shown in, e.g., SEQ ID NO:6 or SEQ ID NO:10. An
immunogenic polypeptide can elicit antibodies or other immune
responses (e.g., T-cell responses of the immune system) that
recognize epitopes of polypeptides having SEQ ID NO:6 or SEQ ID
NO:10. An immunogenic polypeptide of the invention can also be a
fragment of a polypeptide that has an amino acid sequence shown in
SEQ ID NO:6 or SEQ ID NO:10. An immunogenic polypeptide fragment of
the invention can be about 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15,
16, 17, 18, 19, or 20 amino acids in length.
Polynucleotides
[0061] Polynucleotides of the invention contain less than an entire
canine genome and can be single- or double-stranded nucleic acids.
A polynucleotide can be RNA, DNA, cDNA, genomic DNA, chemically
synthesized RNA or DNA or combinations thereof. The polynucleotides
can be purified free of other components, such as proteins, lipids
and other polynucleotides. For example, the polynucleotide can be
50%, 75%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% pure by dry weight.
Purity can be measured by a method such as column chromatography,
polyacrylamide gel electrophoresis, or HPLC analysis. The
polynucleotides of the invention encode the polypeptides described
above. In one embodiment of the invention the polynucleotides
encode polypeptides shown in, e.g., SEQ ID NO:6 or SEQ ID NO:10 or
combinations thereof. Polynucleotides of the invention can comprise
other nucleotide sequences, such as sequences coding for linkers,
signal sequences, TMR stop transfer sequences, transmembrane
domains, or ligands useful in protein purification such as
glutathione-S-transferase, histidine tag, and staphylococcal
protein A.
[0062] The polynucleotides of the invention encode the polypeptides
described above, as well as fragments thereof. A fragment can be
about 10, 12, 15, 20, 50, 75, 100, 125, 250, 300, 400, 500, 600,
700, 800, 900, 1,000 or more polynucleotides. One of skill in the
art can obtain the polynucleotide sequence of the invention using
the disclosed polypeptide sequence and codon tables.
Polynucleotides can contain naturally occurring polynucleotides or
sequences that differ from those of any naturally occurring
sequences or polynucleotides. In one embodiment of the invention, a
polynucleotide of the invention is derived from a mammal, such as a
dog. Polynucleotides of the invention can differ from naturally
occurring nucleic acids, but still encode naturally occurring amino
acids due to the degeneracy of the genetic code. Polynucleotides of
the invention can also comprise other heterologous nucleotide
sequences, such as sequences coding for linkers, signal sequences,
amino acid spacers, heterologous signal sequences, TMR stop
transfer sequences, transmembrane domains, or ligands useful in
protein purification such as glutathione-5-transferase, histidine
tag, and staphylococcal protein A. Polynucleotides of the invention
can also comprise other homologous nucleotide sequences, i.e.,
other CD20 or CD20-derived sequences.
[0063] An isolated polynucleotide is a nucleic acid molecule that
is not immediately contiguous with one or both of the 5' and 3'
flanking sequences with which it is normally contiguous when
present in a naturally occurring genome. Therefore, an isolated
polynucleotide can be, for example, a polynucleotide that is
incorporated into a vector, such as a plasmid or viral vector, a
polynucleotide that is incorporated into the genome of a
heterologous cell (or the genome of a homologous cell, but at a
site different from that where it naturally occurs); and a
polynucleotide that exists as a separate molecule such as a
polynucleotide produced by PCR amplification, chemically synthesis,
restriction enzyme digestion, or in vitro transcription. An
isolated polynucleotide is also a nucleic acid molecule, such as a
recombinant nucleic acid molecule that forms part of hybrid
polynucleotide encoding additional polypeptide sequences that can
be used for example, in the production of a fusion protein.
[0064] A polynucleotide can also comprise one or more expression
control sequences such as promoters or enhancers, for example. A
polynucleotide of the invention can be present in a vector, such
as, for example, an expression vector. If desired, polynucleotides
can be cloned into an expression vector comprising, for example,
promoters, enhancers, or other expression control sequences that
drive expression of the polynucleotides of the invention in host
cells. The polynucleotides can be operably linked to the expression
control sequences. An expression vector can be, for example, a
plasmid, such as pBR322, pUC, or ColE1, or an adenovirus vector,
such as an adenovirus Type 2 vector or Type 5 vector. Vectors
suitable for use in the present invention include, for example,
bacterial vectors, mammalian vectors, viral vectors (such as
retroviral, adenoviral, adeno-associated viral, herpes virus,
simian virus 40 (SV40), and bovine papilloma virus vectors) and
baculoviris-derived vectors for use in insect cells.
Polynucleotides in such vectors are preferably operably linked to a
promoter, which is selected based on, e.g., the cell type in which
expression is sought.
[0065] Host cells into which vectors, such as expression vectors,
comprising polynucleotides of the invention can be introduced
include, for example, prokaryotic cells (e.g., bacterial cells) and
eukaryotic cells (e.g., yeast cells; insect cells; and mammalian
cells). Such host cells are available from a number of different
sources that are known to those skilled in the art, e.g., the
American Type Culture Collection (ATCC), Rockville, Md. Host cells
into which the polynucleotides of the invention have been
introduced, as well as their progeny, even if not identical to the
parental cells, due to mutations, are included in the
invention.
[0066] Methods for introducing polynucleotides of the invention
(e.g., vectors comprising the polynucleotides or naked
polynucleotides) into cells, either transiently or stably, are well
known in the art. For example, transformation methods using
standard CaCl.sub.2, MgCl.sub.2, or RbCl methods, protoplast fusion
methods or transfection of naked or encapsulated nucleic acids
using calcium phosphate precipitation, microinjection, viral
infection, and electroporation.
[0067] One embodiment of the invention provides methods of
producing a recombinant cell that expresses a canine CD20 protein,
or fragment thereof, comprising transfecting a cell with a vector
comprising the polynucleotide of the invention. A canine CD20
protein, or fragment thereof, can then be produced by expressing
the polypeptide in the recombinant host cell.
[0068] Isolation and purification of polypeptides produced in the
systems described above can be carried out using conventional
methods, appropriate for the particular system. For example,
preparative chromatography and immunological separations employing
antibodies, such as monoclonal or polyclonal antibodies, can be
used.
[0069] Polynucleotides can be synthesized in the laboratory, for
example, using an automatic synthesizer. An amplification method
such as PCR can be used to amplify polynucleotides from either
genomic DNA or cDNA encoding the polypeptides.
[0070] Polynucleotides and fragments thereof of the invention can
be used, for example, as probes or primers to detect the presence
of canine CD20 polynucleotides in a, such as a biological sample. A
biological sample can be, e.g., lymph node or tissue aspirate,
serum, whole blood, cellar suspension, or fluid effusion. The
ability of such probes to specifically hybridize to polynucleotide
sequences will enable them to be of use in detecting the presence
of complementary sequences in a given sample. Polynucleotide probes
of the invention can hybridize to complementary sequences in a
sample such as a biological sample, for example, lymph tissue,
thereby detecting the presence or absence of canine CD20
polynucleotides in samples. Polynucleotides from the sample can be,
for example, subjected to gel electrophoresis or other size
separation techniques or can be dot blotted without size
separation. The polynucleotide probes are preferably labeled.
Suitable labels, and methods for labeling probes are known in the
art, and include, for example, radioactive labels incorporated by
nick translation or by kinase, biotin, fluorescent probes, and
chemiluminescent probes. The polynucleotides from the sample are
then treated with the probe under hybridization conditions of
suitable stringencies.
[0071] Depending on the application, varying conditions of
hybridization can be used to achieve varying degrees of selectivity
of the probe towards the target sequence. For applications
requiring high selectivity, relatively stringent conditions can be
used, such as low salt and/or high temperature conditions, such as
provided by a salt concentration of from about 0.02 M to about 0.15
M at temperatures of from about 50.degree. C. to about 70.degree.
C. For applications requiring less selectivity, less stringent
hybridization conditions can be used. For example, salt conditions
from about 0.14 M to about 0.9 M salt, at temperatures ranging from
about 20.degree. C. to about 55.degree. C. The presence of a
hybridized complex comprising the probe and a complementary
polynucleotide from the sample indicates the presence of the
microbe or polynucleotide sequence in the sample.
Antibodies and Antibody Fragments
[0072] Antibodies, such as monoclonal and polyclonal antibodies,
that specifically bind polypeptides of the invention are part of
the invention. These antibodies can be made by using a polypeptide
or a polypeptide fragment that contains an epitope present in a
polypeptide shown in SEQ ID NO:6, SEQ ID NO:10 or SEQ ID NO:11 as
an immunogen in standard antibody production methods (see e.g.,
Kohler et al., Nature, 256:495, 1975; Ausubel et al. (1992) Current
Protocols in Molecular Biology, John Wylie and Sons, Inc. New York,
N.Y.; Harlow and Lane, Eds, (1988) Current Edition, Antibodies: A
Laboratory Manual, Cold Spring Harbor Press, N.Y.). Antibodies can
also be made using DNA immunization techniques using nucleic acid
sequence coding for polypeptides SEQ ID NO:6, SEQ ID NO:10, SEQ ID
NO:11 or fragments thereof in, e.g., standard mammalian expression
vectors. See e.g., Chambers et al., 2003 Nature Biotechnology 21:
1088-1092; Tang et al. Nature. 1992 Mar. 12; 356(6365):152-4; Barry
et al., Biotechniques. 1994 April; 16(4):616-8, 620.
[0073] An antibody is an intact immunoglobulin molecule, a fragment
of an immunoglobulin molecule, such as Fab, Fab', F(ab').sub.2,
F(ab).sub.2, Fv, sFv, or a single-chain antibody or fragments
thereof, that specifically binds to a polypeptide of the invention
(e.g., SEQ ID NO:6 or SEQ ID NO:10 and fragments thereof). Antibody
fragments retain some ability to selectively bind to the antigen
(e.g., a polypeptide of the invention) from which they are derived,
and can be made using well known methods in the art. In one
embodiment of the invention, an antibody, antibody fragment or
single-chain antibody comprises all such antibodies that
specifically bind to a polypeptide of the invention (e.g., SEQ ID
NO:6 or SEQ ID NO:10 and fragments thereof).
[0074] An antibody of the invention can be any antibody class,
including for example, IgG, IgM, IgA, IgD and IgE. An antibody or
fragment thereof binds to an epitope of a polypeptide of the
invention. An antibody can be made in vivo in suitable laboratory
animals or in vitro using recombinant DNA techniques. Means for
preparing and characterizing antibodies are well know in the art.
See, e.g., Dean, Methods Mol. Biol. 80:23-37 (1998); Dean, Methods
Mol. Biol. 32:361-79 (1994); Baileg, Methods Mol. Biol. 32:381-88
(1994); Gullick, Methods Mol. Biol. 32:389-99 (1994); Drenckchahn
et al. Methods Cell, Biol. 37:7-56 (1993); Morrison, Ann. Rev.
Immunol. 10:239-65 (1992); Wright et al. Crit. Rev. Immunol.
12:125-68 (1992). For example, polyclonal antibodies can be
produced by administering a polypeptide of the invention to an
animal, such as a human or other primate, mouse, rat, rabbit,
guinea pig, goat, pig, dog, cow, sheep, donkey, or horse. Serum
from the immunized animal is collected and the antibodies are
purified from the plasma by, for example, precipitation with
ammonium sulfate, followed by chromatography, such as affinity
chromatography. Techniques for producing and processing polyclonal
antibodies are known in the art.
[0075] "Specifically binds" or "specific for" means that the
polypeptide recognizes and binds to an antibody of the invention
with greater affinity than to other, non-specific molecules. For
example, an antibody raised against an antigen (e.g., a
polypeptide) to which it binds more efficiently than to a
non-specific protein can be described as specifically binding to
the antigen. Binding specifically can be tested using, for example,
an enzyme-linked immunosorbant assay (ELISA), a radioimmunoassay
(RIA), or a western blot assay using methodology well known in the
art.
[0076] Antibodies of the invention can be present in an antibody
fusion protein. An antibody fusion protein refers to a recombinant
molecule that comprises an antibody component and a therapeutic
agent. Examples of therapeutic agents suitable for such fusion
proteins include immunomodulators ("antibody-immunomodulator fusion
protein") and toxins ("antibody-toxin fusion protein").
[0077] Polypeptides of the invention comprise at least one epitope.
An epitope is an antigenic determinant of a polypeptide. Epitopes
within a polypeptide of the invention can be identified by several
methods. See, e.g., U.S. Pat. No. 4,554,101; Jameson & Wolf,
CABIOS 4:181-186 (1988). For example, a polypeptide of the
invention can be isolated and screened. A series of short peptides,
which together span the entire polypeptide sequence, can be
prepared by proteolytic cleavage. By starting with, for example,
100-mer polypeptide fragments, each fragment can be tested for the
presence of epitopes recognized in, for example, an enzyme-linked
immunosorbent assay (ELISA). In an ELISA assay a polypeptide, such
as a 100-mer polypeptide fragment, is attached to a solid support,
such as the wells of a plastic multi-well plate. A population of
antibodies are labeled, added to the solid support and allowed to
bind to the unlabeled antigen, under conditions where non-specific
adsorption is blocked, and any unbound antibody and other proteins
are washed away. Antibody binding is detected by, for example, a
reaction that converts a colorless indicator reagent into a colored
reaction product. Progressively smaller and overlapping fragments
can then be tested from an identified 100-mer to map the epitope of
interest.
[0078] Antigens that can be used in producing antibodies of the
invention include polypeptides and polypeptide fragments of the
invention. A polypeptide used to immunize an animal can be obtained
by standard recombinant, chemical synthetic, or purification
methods. As is well known in the art, in order to increase
immunogenicity, an antigen can be conjugated to a carrier protein.
Commonly used carriers include keyhole limpet hemocyanin (KLH),
thyroglobulin, bovine serum albumin (BSA), and tetanus toxoid. The
coupled peptide is then used to immunize an animal (e.g., a mouse,
a rat, or a rabbit). In addition to such carriers, well known
adjuvants can be administered with the antigen to facilitate
induction of a strong immune response.
[0079] Polyclonal and monoclonal antibodies can be purified, for
example, by binding to, and elution from, a matrix containing a
polypeptide or polypeptide fragment of the invention to which the
antibodies were raised. Additional methods for antibody
purification and concentration are well known in the art and can be
practiced with the antibodies of the invention. Anti-idiotype
antibodies corresponding to polypeptides of the invention are also
included in the invention, and can be produced using standard
methods.
[0080] An antibody and antigen (e.g., a polypeptide or polypeptide
fragment of the invention) specifically bind to each other if they
bind to each other with greater affinity than to other,
non-specific molecules. For example, an antibody raised against an
antigen to which it binds more efficiently than to a non-specific
protein can be described as specifically binding to the
antigen.
[0081] In one embodiment of the invention an antibody of the
invention specifically binds canine CD20. Antibodies of the
invention can be used, for example, to detect canine CD20
polypeptides in a biological sample. Antibodies of the invention
can be used in vitro or in vivo for immunodiagnosis. The antibodies
are suited for use in, for example, immunoassays in which they are
in liquid phase or bound to a solid phase carrier (e.g., a glass,
polystyrene, polypropylene, polyethylene, dextran, nylon, amylase,
natural and modified cellulose, polyacrylamide, agarose, or
magnetite carrier). The antibodies used in such immunoassays can be
detectably labeled (e.g., with an enzyme, a radioisotope, a
fluorescent compound, a colloidal metal, a chemiluminescent
compound, a phosphorescent compound, or a bioluminescent compound)
using any of several standard methods that are well known in the
art. Examples of immunoassays in which the antibodies of the
invention can be used include, e.g., competitive and
non-competitive immunoassays, which are carried out using either
direct or indirect formats. Examples of such immunoassays include
radioimmunoassays (RIA), flow cytometry, and sandwich assays (e.g.,
enzyme-linked immunosorbent assays (ELISAs)). RT-PCR assays can
also be used to quantitatively detect canine CD20. Detection of
antigens using the antibodies of the invention can be done using
immunoassays that are run in either forward, reverse, or
simultaneous modes, including immunohistochemical assays on
physiological samples. Other immunoassay formats are well known in
the art, and can be used in the invention.
[0082] Antibodies of the invention can be chimeric antibodies, for
example, humanized or caninized antibodies. A humanized antibody,
like a mouse-human chimeric antibody, can be prepared, for example,
as follows: (1) isolate the gene encoding the antibody of the
present invention from antibody-producing mouse cells; (2) replace
the constant region of the H chain of the antibody with that of the
human IgE; and (3) introduce into, for example, mouse myeloma J558L
cells (See, Neuberger et al., Nature 314:268-270 (1985)).
Alternatively, human antibodies or canine antibodies, for example,
can be prepared by immunizing mice whose immune systems have been
replaced with that of humans or canines with a polypeptide or
polypeptide fragment of the present invention.
[0083] Antibodies that specifically bind canine CD20 antigens
(e.g., CD20 polypeptides), are particularly useful for detecting
the presence of CD20 antigens in a sample, such as a lymph node or
tissue aspirate, serum, whole blood, cellular suspension, or fluid
effusion sample from a canine or feline. An immunoassay for CD20
antigen can utilize one antibody or several antibodies. Immunoassay
protocols can be based upon, for example, competition, direct
reaction, or sandwich type assays using, for example, labeled
antibody. Antibodies of the invention can be labeled with any type
of label known in the art, including, for example, fluorescent,
chemiluminescent, radioactive, enzyme, colloidal metal,
radioisotope and bioluminescent labels.
[0084] Antibodies of the invention or fragments thereof can be
bound to a support and used to detect the presence of a CD20
antigen. Supports include, for example, glass, polystyrene,
polypropylene, polyethylene, dextran, nylon, amylases, natural and
modified celluloses, polyacrylamides, agaroses and magletite.
[0085] Antibodies of the invention can also be used in
immunolocalization studies to analyze the presence and distribution
of a polypeptide of the invention during various cellular events or
physiological conditions. Antibodies can also be used to identify
molecules involved in passive immunization and to identify
molecules involved in the biosynthesis of non-protein antigens.
Identification of such molecules can be useful in vaccine
development. Antibodies of the invention, including, for example,
monoclonal antibodies and single chain antibodies, can be used to
monitor the course of amelioration of a disease. By measuring the
increase or decrease of CD20-positive cells in a test sample from
an animal, it can be determined whether a particular therapeutic
regiment aimed at ameliorating the disorder is effective.
Methods of Treatment
[0086] Antibodies of the invention can used to treat canine CD20+
B-cell lymphoma, immune-mediated hemolytic anemia, immune-mediated
thrombocytopenia, and systemic lupus erythematosus (SLE).
[0087] The invention also encompasses multimodal therapeutic
methods wherein anti-CD20 antibody administration is supplemented
with chemotherapy, or by administration of therapeutic proteins,
such as immunoconjugates and antibody fusion proteins.
[0088] In general, the dosage of administered anti-CD20 antibodies,
anti-CD20 antibody components, immunoconjugates, and fusion
proteins will vary depending upon such factors as the canine's age,
weight, sex, general medical condition and previous medical
history. Typically, it is desirable to provide the recipient with a
dosage of antibody component, immunoconjugate or fusion protein
which is in the range of from about 1 pg/kg to 10 mg/kg (amount of
agent/body weight of canine), although a lower or higher dosage
also may be administered as circumstances dictate.
[0089] Administration of antibody components, immunoconjugates or
fusion proteins to a patient can be intravenous, intraarterial,
intraperitoneal, intramuscular, subcutaneous, intrapleural,
intrathecal, by perfusion through a regional catheter, or by direct
intralesional injection. When administering therapeutic proteins by
injection, the administration can be by continuous infusion or by
single or multiple boluses.
[0090] Compositions of the invention can comprise anti-CD20
antibodies and a pharmaceutically acceptable buffer, for example,
sterile saline, sterile buffered water, propylene glycol. Methods
for preparing administrable agents, such as parenterally
administrable agents, are described in Pharmaceutical Carriers
& Formulations, Martin, Remington's Pharmaceutical Sciences,
15th Ed. (Mack Pub. Co., Easton, Pa. 1975), which is incorporated
herein by reference.
[0091] All patents, patent applications, and other scientific or
technical writings referred to anywhere herein are incorporated by
reference in their entirety. The invention illustratively described
herein suitably can be practiced in the absence of any element or
elements, limitation or limitations that are not specifically
disclosed herein. Thus, for example, in each instance herein any of
the terms "comprising", "consisting essentially of", and
"consisting of" may be replaced with either of the other two terms.
The terms and expressions which have been employed are used as
terms of description and not of limitation, and there is no
intention that in the use of such terms and expressions of
excluding any equivalents of the features shown and described or
portions thereof, but it is recognized that various modifications
are possible within the scope of the invention claimed. Thus, it
should be understood that although the present invention has been
specifically disclosed by embodiments, optional features,
modification and variation of the concepts herein disclosed may be
resorted to by those skilled in the art, and that such
modifications and variations are considered to be within the scope
of this invention as defined by the description and the appended
claims.
[0092] In addition, where features or aspects of the invention are
described in terms of Markush groups or other grouping of
alternatives, those skilled in the art will recognize that the
invention is also thereby described in terms of any individual
member or subgroup of members of the Markush group or other
group.
EXAMPLES
Example 1
[0093] Cloning Canine CD20:
[0094] The nucleotide sequence for canine CD20 has not been
published or deposited in NCBI/GenBank. Using primers deposited in
GenBank from an unpublished study using sequence tagged sites in
the canine genome (Accession: L77424, L77425), a portion of the
gene sequence for canine CD20 was amplified from canine peripheral
blood mononuclear cell (PBMC) cDNA (SEQ ID NO:1 FIG. 1).
Specifically, PBMCs were purified from 8 ml of canine whole blood
using Ficoll-Paque PLUS according to the manufacturer's
instructions (Amersham Biosciences). The PBMCs were resuspended in
Trizol (Invitrogen) and RNA was harvested as described in the
product insert. Using 2.5 .mu.g of this RNA and Thermoscript RT
(Invitrogen), canine PBMC cDNA was generated using oligo-dT primers
according to the product insert. From this gene sequence, primers
were designed for use with 5' and 3' RACE (Rapid Amplification of
cDNA Ends) using a commercially available kit (Smart RACE, BD
Biosciences--Clontech). The 5'RACE was successful using the kit
according to the manufacturer's instructions. However, the 3' RACE
product was incomplete, likely due to secondary structure in the
mRNA. The complete elucidation of the 3' sequence required the use
of a more temperature stable reverse transcriptase (Thermoscript
RT, Invitrogen). The 3' RACE cDNA was synthesized from 2.5 .mu.g of
canine RNA with 11 of Thermoscript RT at 60.degree. C. for 15
minutes, followed by incubation at 65.degree. C. for 30 minutes.
This modification to the existing RACE technique resulted in a
complete 3' sequence for canine CD20. The full length nucleotide
and translated polypeptide sequences are shown in FIGS. 3 and 4
(SEQ ID NO: 5 and 6), respectively. An alignment of the full-length
sequences for human, mouse, feline and canine CD20 is shown in FIG.
5. Canine CD20 is only 74% identical to human CD20 and 68%
identical to murine CD20.
Example 2
[0095] Expression of Canine CD20:
[0096] The full-length nucleic acid sequence for canine CD20 was
amplified from canine cDNA using a proof-reading Taq (High Fidelity
Taq, Roche) and subcloned into a commercially available expression
vector (pCMV4AFLAG; Stratagene) at the NotI/EcoRI sites using
standard molecular biology procedures. The expression plasmid was
transformed into E. coli and purified using a Maxi-Prep kit from
Qiagen. The purified plasmid was transiently transfected into COS7
cells (ATCC CRL-1651) using either Lipofectamine (Invitrogen) or
FuGene6 (Roche) according to the manufacturer's instructions. Two
days post-transfection, the cells were washed twice with phosphate
buffered saline (PBS, pH 7.2), scraped from the surface of the well
in buffer, and pelleted by centrifugation. The resulting cell
pellet was lysed in a Triton X-100 lysis buffer (50 mM Tris (pH
7.5), 150 mM NaCl, 0.5% Triton X-100, 2 mM EDTA, Protease Inhibitor
V (CalBiochem)) for 30 minutes on ice followed by centrifugation of
cell debris at 4.degree. C. for 15 minutes. For total cell lysate,
the resulting supernatant was boiled in an equal volume of SDS
sample buffer and loaded onto 4-20% SDS-PAGE gels (Pierce). For
immunoprecipitation, the lysate was incubated with either 5 ul of
anti-FLAG M2 monoclonal antibody, 2.5 ul of monoclonal anti-CD20
antibody (Biogenex MU265-UC and MU238-UC), or 5 ul of polyclonal
anti-CD20 (Santa Cruz Biotechnology sc-15361, sc-7736, or LabVision
RB-9013) overnight at 4.degree. C. with mixing. A 1:1 slurry of
Protein G sepharose (Amersham) in lysis buffer was added to the
lysate for 30 minutes at 4.degree. C. with mixing. The
antibody-bound sepharose was collected by microcentrifugation and
washed with 1 ml of lysis buffer three times. The washed sepharose
was boiled in 1.times. sample buffer before being loaded onto a
4-20% SDS-PAGE gel (Pierce). The gel was then transferred overnight
to PVDF (Millipore) for blotting.
[0097] Expression was confirmed on Western blots using an anti-FLAG
M2 monoclonal antibody (Stratagene) at 1:3000 in TBS/1% Casein
(Pierce) and chemiluminescent detection (ECL, Amersham) (FIG. 6). A
single band of approximately 40 kDa was observed for the total cell
lysate preparation. Only the anti-FLAG monoclonal antibody and the
polyclonal antisera from LabVision were successful in
immunoprecipitating canine CD20 from the total cell lysate (FIG.
6).
[0098] Cellular expression of canine CD20 was also demonstrated
using immunofluorescence with the anti-FLAG antibody. COS7 cells
transiently transfected with canine CD20, as described above, were
grown in two-well chamber slides (Nunc). At 48 hours
post-transfection, the cells were washed twice with PBS, and fixed
in 4% neutral buffered formalin for 15 minutes. Slides were washed
three times in PBS for 5 minutes each, followed by a 15 minute
incubation with 50 in M ammonium chloride in PBS. After three five
minute washes in PBS, the slides were blocked for 1 hour with PBA
(PBS, 0.1% Triton X-100, 15% normal goat serum, 1% BSA). Anti-FLAG
M2 antibody (Stratagene) was diluted 1:200 in PBA and added to the
slides upon removal of the blocking solution and incubated for 1
hour at room temperature. Slides were washed three times for 5
minutes in PBS before adding a goat anti-mouse FITC conjugated
secondary antibody (Jackson ImmunoResearch) at a 1:250 dilution in
PBA. The samples were incubated for 1 hour at room temperature and
again washed three times for five minutes in PBS. Confocal
microscopy was performed to evaluate surface expression of canine
CD20. FIG. 7 shows the results of the immunofluorescent labeling
and surface expression of canine CD20.
Example 3
[0099] Use of Canine CD20 for Peptides:
[0100] Based on the amino acid sequence of the canine CD20, and
alignment with CD20 proteins from other species, a polypeptide
sequence was identified that represents the predominant
extracellular domain of the canine CD20 protein. This 53-mer
polypeptide was synthesized alone (SEQ ID NO:10; FIG. 8) and in
conjunction with a murine T-cell epitope from ovalbumin (SEQ ID
NO:11; FIG. 8). The latter polypeptide was conjugated to KLH and
BSA and was used to immunize mice in order to generate monoclonal
antibodies to the extracellular domain of canine CD20. The former
polypeptide was used in an ELISA to screen hybridomas for a
monoclonal antibody specific to the extracellular domain of canine
CD20.
[0101] The peptide ELISA was performed by dissolving 1 mg of the
53-mer peptide (SEQ ID NO:10) in 1 ml of DMSO (Sigma). Peptide was
coated onto 96-well microtiter plates (Immunlon 4HB, Dynatech) at a
concentration of 10 ug/ml in 50 mM carbonate (pH 9) overnight at
room temperature. Plates were washed four times in PBS-T (phosphate
buffered saline (pH 7.2), 0.05% Tween-20) and blocked with 2%
Tween-20 in 100 mM Tris (pH7.4) for 2 hours at room temperature.
Plates were washed four times in PBS-T before samples were added
either neat, for hybridoma supernatants, or diluted in sample
diluent (50 mM Tris (pH 7.2), 0.05% Tween-20, 50% fetal bovine
serum), for serum samples. Plates were incubated for 1 hour at room
temperature, washed four times in PBS-T, and a 1:2500 dilution of
goat anti-mouse HRPO (Jackson ImmunoResearch) in sample diluent was
added. Following a 1 hour incubation at room temperature, plates
were washed six times in PBS-T and developed with at TMB substrate
(Moss, Inc.).
Example 4
[0102] Production of Monoclonal Antibodies to Canine CD20
[0103] Purified vector DNA (MaxiPrep Kit, Qiagen) containing the
canine CD20 gene (Example 2) was used for DNA immunization of mice
according to published protocols (Ulmer, J. B. et al. Science,
1993). Antibody titers from individual mice were evaluated ten days
after the second immunization using the peptide ELISA described in
Example 3. Positive titers were found for each DNA-immunized mouse
(FIG. 9). A third injection was performed three weeks after the
second injection and the spleen was harvested within 7-10 days for
the fusion. The spleen was fused with a mouse myeloma cell line FO
using methods well know to those skilled in the art (see
Antibodies, a Laboratory Manual, by Harlow and Lane, Cold Spring
Harbor Laboratory Press, 1988, pp 139-238). Individual monoclonal
antibody producing clones were isolated using the process of
limited dilution and screened on the peptide ELISA described in
Example 3. A total of 26 clones were isolated from the screening,
all of which produced IgM antibodies.
Example 5
[0104] Immunocytochemical Evaluation of Monoclonal Antibodies to
Canine CD20
[0105] The 26 identified clones reactive to the extracellular
domain of canine CD20 were further evaluated on smears of canine
lymph node aspirates using immunocytochemical techniques. Each
smear was outlined with an ImmEdge pen (Vector Labs), allowed to
dry, and then fixed in acetone for 3 minutes. Following a 5 minute
wash in PBS (pH 7.2), slides were treated with ammonium chloride
(50 mM in PBS) for 15 minutes at room temperature. Following a 5
minute wash in PBS, slides were blocked in PBS/NGS (PBS with 15%
normal goat serum (Vector Labs)) for 30 minutes at room
temperature. Each hybridoma supernatant was then added to the slide
and incubated for one hour at room temperature. Slides were washed
twice for 5 minutes in PBS, followed by a 30 minute incubation in a
1:200 dilution of goat anti-mouse IgM+IgG (H+ L) F(ab).sub.2 FITC
(Jackson ImmunoResearch) in PBS/NGS. Following two 5 minute washes
in PBS, slides were examined using fluorescent microscopy. Of the
26 clones initially identified, only five clearly demonstrated
immunofluoresence on the lymph node aspirates.
Example 6
[0106] Flow Cytometric Evaluation of Monoclonal Antibodies to
Canine CD20 in Canine Lymphoma
[0107] Of the five clones identified, four were propagated in 1 L
bioreactor bags (VectraCell) according to the manufacturer's
instructions using hybridoma serum-free media (Invitrogen). IgM
antibodies were purified from the culture supernatants using a
HiTrap IgM column (Amersham Biosciences) according to the product
insert (FIG. 10). Purified antibodies were conjugated to
NHS-Fluoroscein (Sigma/Fluka) by mixing a 20 molar excess of
conjugate with the antibody and allowing this to incubate for 30
minutes at room temperature. Antibodies were then purified from
unreacted NHS-Fluoroscein using a microspin desalting column (Zeba
Spin Column, Pierce) and PBS (pH 7.2). Of the four antibodies that
were fluorescently tagged, only two showed significant labeling of
lymphocytes by flow cytometry on lymph node aspirates from dogs
with lymphoma. The cell lines secreting these antibodies have been
deposited with the ATCC, Manassas Va. on Mar. 30, 2005. Strain
designations are F3C7 and F7A5, bearing ATCC Patent Deposit Numbers
PTA-6661 and PTA 6662, respectively. For flow cytometry, lymph node
aspirates were collected into 1 ml of media (Hanks Balanced Salt
Solution (HBSS), 30 mM HEPES, 2% fetal bovine serum, K.sub.3EDTA,
Pen/Strep) and stored at 4.degree. C. An aliquot of 100 ul was
blocked with 25 .mu.g of mouse gamma globulin (Jackson
ImmunoResearch) for 20 minutes on ice. The cells were then
incubated for 30 minutes on ice with approximately 60 .mu.g of
either F3C7 or F7A5. Controls included unlabeled cells, cells
labeled with a fluoroscein isotype control, cells labeled for a
standard B-cell marker (5 .mu.l anti-CD79a-RPE, DAKOCyotmation) and
an RPE isotype control (5 .mu.l IgG1-RPE, DAKOCyotmation). Cells
were washed twice with BD Stain Buffer (BD Biosciences) and fixed
in BD Cytofix for 15 minutes on ice, followed by two washes in BD
Stain Buffer. Labeled cells were analyzed on a Becton Dickinson
Flow Cytometer. Representative examples of the anti-canine CD20
monoclonal antibodies labeling B-cell lymphomas are shown in FIGS.
11 and 12.
Example 7
[0108] Use of Monoclonal Antibodies to the Extracellular Domain of
Canine CD20 in a Point of Care Hematology Instrument to Identify
B-Lymphocytes.
[0109] Monoclonal antibody F7A5 was labeled with 60 nm colloidal
gold (BBInternational). The pH of the gold was initially adjusted
to pH 9 with 100 mM K.sub.2CO.sub.3. The F7A5 monoclonal antibody
was diluted to approximately 2 mg/ml in 2 mM borate (pH 9) and was
added dropwise to the gold with stirring to a final concentration
of 12 .mu.g/ml. After 15 minutes the gold-antibody solution was
stabilized with a 1:10 dilution of 10% BSA (pH 9). After 15 minutes
of mixing the gold was centrifuged and washed three times (1% BSA,
1 mM NaCl, pH 9) and resuspended in the wash buffer to a final OD
of 5. A lymph node aspirate from the popliteal node of a healthy,
young dog was obtained and resuspended in the collection media
described in Example 6. A 100 .mu.l aliquot of this sample was
incubated with 10 .mu.l of the colloidal gold labeled F7A5
monoclonal antibody to canine CD20 for 30 minutes at room
temperature. The sample was then diluted in 0.5 mls of PBS
immediately prior to analysis. As a control, an unlabeled aliquot
of identical volume from the same aspirate was analyzed in 0.5 ml
of PBS.
[0110] Samples were analyzed on a point of care hematology
instrument (LaserCyte, IDEXX Laboratories, Inc.) under routine
conditions, substituting the standard sheath solution for PBS. See,
e.g., U.S. Pat. Publ. No. 2004/0246480. Data obtained from the
samples was analyzed using standard flow cytometry software (FCS
Express 2, De Novo Software). Initially, small lymphocytes are
gated in the forward scatter high (FSH) vs time of flight channel
(TOF). Medium lymphocytes can be identified in the extinction
integral (EXTint) channel vs. TOF. After selecting for each of
these populations, the data can be analyzed for degree of right
angle scatter, comparing the unlabeled control to the F7A5 labeled
sample. Due to the light scattering properties of colloidal gold,
cells labeled with the gold-tagged F7A5 antibody demonstrate
increased scatter in the right angle scatter channel (RAS). FIG. 13
shows that approximately 33% of small to medium lymphocytes are
identified as B-lymphocytes in this normal lymph node aspirate from
a dog. This value is consistent with the values obtained using
fluorescently labeled antibodies to canine B-cells on standard flow
cytometry as reported in the literature (D. Gibson et al., JVIM
(2004) 18:710-17).
Example 8
[0111] Use of F7A5 Monoclonal Antibody to Identify B-Lymphocytes in
a Lymph Node Aspirate from a Cat with Lymphoma
[0112] A lymph node aspirate from a cat with lymphoma was collected
and resuspended in collection media as described in Example 7. A
100 .mu.l aliquot of the sample was labeled with 10 .mu.l of the
colloidal gold labeled F7A5 monoclonal antibody for 60 minutes at
room temperature. Following labeling the sample was diluted in 1 ml
of PBS for analysis as described in Example 7. The lymphocyte
populations were determined in a manner similar to that described
for Example 7 and the gated lymphocyte population is shown in FIG.
14. Compared to the unlabeled control sample, at least 12.5% of the
gated lymphocytes are CD20 positive B-cells.
Sequence CWU 1
1
14 1 247 DNA Canis familiaris 1 gcgctctttg ctgccatttc tggaataatt
tttttgatca tggacatatt taatattacc 60 atttcccatt ttttaaaaat
ggagaatttg aatcttatta aagctcccat accatatgtt 120 gacatacaca
actgtgaccc agctaacccc tctgagaaaa actctttatc tatacaatat 180
tgtggcagca tacgatctgt tttcttgggc gtttttgctg tgatgctgat ctttgccttc
240 ttccagc 247 2 120 PRT Homo sapiens 2 Asn Ser Leu Ser Leu Phe
Ala Ala Ile Ser Gly Met Ile Leu Ser Ile 1 5 10 15 Met Asp Ile Leu
Asn Ile Lys Ile Ser His Phe Leu Lys Met Glu Ser 20 25 30 Leu Asn
Phe Ile Arg Ala His Thr Pro Tyr Ile Asn Ile Tyr Asn Cys 35 40 45
Glu Pro Ala Asn Pro Ser Glu Lys Asn Ser Pro Ser Thr Gln Tyr Cys 50
55 60 Tyr Ser Ile Gln Ser Leu Phe Leu Gly Ile Leu Ser Val Met Leu
Ile 65 70 75 80 Phe Ala Phe Phe Gln Glu Leu Val Ile Ala Gly Ile Val
Glu Asn Glu 85 90 95 Trp Lys Arg Thr Cys Ser Arg Pro Lys Ser Asn
Ile Val Leu Leu Ser 100 105 110 Ala Glu Glu Lys Lys Glu Gln Thr 115
120 3 120 PRT Mus musculus 3 Ser Ser Leu Ser Leu Phe Ala Ala Ile
Ser Gly Ile Ile Leu Ser Ile 1 5 10 15 Met Asp Ile Leu Asn Met Thr
Leu Ser His Phe Leu Lys Met Arg Arg 20 25 30 Leu Glu Leu Ile Gln
Thr Ser Lys Pro Tyr Val Asp Ile Tyr Asp Cys 35 40 45 Glu Pro Ser
Asn Ser Ser Glu Lys Asn Ser Pro Ser Thr Gln Tyr Cys 50 55 60 Asn
Ser Ile Gln Ser Val Phe Leu Gly Ile Leu Ser Ala Met Leu Ile 65 70
75 80 Ser Ala Phe Phe Gln Lys Leu Val Thr Ala Gly Ile Val Glu Asn
Glu 85 90 95 Trp Lys Arg Met Cys Thr Arg Ser Lys Ser Asn Val Val
Leu Leu Ser 100 105 110 Ala Gly Glu Lys Asn Glu Gln Thr 115 120 4
82 PRT Canis familiaris 4 Ala Leu Phe Ala Ala Ile Ser Gly Ile Ile
Phe Leu Ile Met Asp Ile 1 5 10 15 Phe Asn Ile Thr Ile Ser His Phe
Leu Lys Met Glu Asn Leu Asn Leu 20 25 30 Ile Lys Ala Pro Ile Pro
Tyr Val Asp Ile His Asn Cys Asp Pro Ala 35 40 45 Asn Pro Ser Glu
Lys Asn Ser Leu Ser Ile Gln Tyr Cys Gly Ser Ile 50 55 60 Arg Ser
Val Phe Leu Gly Val Phe Ala Val Met Leu Ile Phe Ala Phe 65 70 75 80
Phe Gln 5 984 DNA Canis familiaris 5 gggactagca ctggaagtga
actcagcagc gaacaactga atcagccact cgccctaagg 60 ccacagacac
tcaggagttc agagggtgag atgacaacac ccagaaattc aatgagtgga 120
actctcccgg tagatcctat gaaaagccct actgccatgt atcctgttca aaaaataatt
180 cccaaaagga tgccttcagt ggtgggccct acacaaaact tcttcatgag
ggaatctaag 240 acactggggg ctgtccagat tatgaatggg ctcttccaca
ttgccctagg cagcctcctg 300 atgattcaca cggatgtcta tgcgcccatc
tgtataacta tgtggtaccc tctctgggga 360 ggcattatgt tcatcatttc
tggatcactc ctggcagcag cggacaaaaa ccccaggaag 420 agtttggtca
aaggaaaaat gataatgaac tcattgagcc tctttgctgc catttctgga 480
ataatttttt tgatcatgga catatttaat attaccattt cccatttttt aaaaatggag
540 aatttgaatc ttattaaagc tcccatacca tatgttgaca tacacaactg
tgacccagct 600 aacccctctg agaaaaactc tttatctata caatattgtg
gcagcatacg atctgttttc 660 ttgggcgttt ttgctgtgat ggtgatcttt
acctttttcc agaaacttgt gacagctggc 720 attgttgaga atgaatggaa
aaaactgtgc tctaaaccta aatctgatgt agttgttctg 780 ttagctgctg
aagaaaaaaa agaacagccg attgaaacaa cagaagaaat ggttgagctg 840
actgaaatag cttcccaacc aaagaaagaa gaagacattg aaattattcc agtccaagaa
900 gaagaagagg aactggaaat aaactttgca gaacctcccc aggagcagga
atcttcacca 960 atagaaaacg acagcatccc ttaa 984 6 297 PRT Canis
familiaris 6 Met 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 Leu Lys Met Glu Asn Leu Asn Leu Ile Lys
Ala Pro Ile 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 7 297 PRT Homo sapiens 7 Met Thr Thr Pro Arg Asn Ser Val
Asn Gly Thr Phe Pro Ala Glu Pro 1 5 10 15 Met Lys Gly Pro Ile Ala
Met Gln Ser Gly Pro Lys Pro Leu Phe Arg 20 25 30 Arg Met Ser Ser
Leu Val Gly Pro Thr Gln Ser 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 Gly Leu Leu Met Ile Pro Ala Gly Ile Tyr Ala Pro Ile 65
70 75 80 Cys Val Thr Val Trp Tyr Pro Leu Trp Gly Gly Ile Met Tyr
Ile Ile 85 90 95 Ser Gly Ser Leu Leu Ala Ala Thr Glu Lys Asn Ser
Arg Lys Cys 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 Ser Ile Met
Asp Ile Leu Asn Ile Lys Ile Ser 130 135 140 His Phe Leu Lys Met Glu
Ser Leu Asn Phe Ile Arg Ala His Thr Pro 145 150 155 160 Tyr Ile Asn
Ile Tyr Asn Cys Glu Pro Ala Asn Pro Ser Glu Lys Asn 165 170 175 Ser
Pro Ser Thr Gln Tyr Cys Tyr Ser Ile Gln Ser Leu Phe Leu Gly 180 185
190 Ile Leu Ser Val Met Leu Ile Phe Ala Phe Phe Gln Glu Leu Val Ile
195 200 205 Ala Gly Ile Val Glu Asn Glu Trp Lys Arg Thr Cys Ser Arg
Pro Lys 210 215 220 Ser Asn Ile Val Leu Leu Ser Ala Glu Glu Lys Lys
Glu Gln Thr Ile 225 230 235 240 Glu Ile Lys Glu Glu Val Val Gly Leu
Thr Glu Thr Ser Ser Gln Pro 245 250 255 Lys Asn Glu Glu Asp Ile Glu
Ile Ile Pro Ile Gln Glu Glu Glu Glu 260 265 270 Glu Glu Thr Glu Thr
Asn Phe Pro Glu Pro Pro Gln Asp Gln Glu Ser 275 280 285 Ser Pro Ile
Glu Asn Asp Ser Ser Pro 290 295 8 291 PRT Mus musculus 8 Met Ser
Gly Pro Phe Pro Ala Glu Pro Thr Lys Gly Pro Leu Ala Met 1 5 10 15
Gln Pro Ala Pro Lys Val Asn Leu Lys Arg Thr Ser Ser Leu Val Gly 20
25 30 Pro Thr Gln Ser Phe Phe Met Arg Glu Ser Lys Ala Leu Gly Ala
Val 35 40 45 Gln Ile Met Asn Gly Leu Phe His Ile Thr Leu Gly Gly
Leu Leu Met 50 55 60 Ile Pro Thr Gly Val Phe Ala Pro Ile Cys Leu
Ser Val Trp Tyr Pro 65 70 75 80 Leu Trp Gly Gly Ile Met Tyr Ile Ile
Ser Gly Ser Leu Leu Ala Ala 85 90 95 Ala Ala Glu Lys Thr Ser Arg
Lys Ser Leu Val Lys Ala Lys Val Ile 100 105 110 Met Ser Ser Leu Ser
Leu Phe Ala Ala Ile Ser Gly Ile Ile Leu Ser 115 120 125 Ile Met Asp
Ile Leu Asn Met Thr Leu Ser His Phe Leu Lys Met Arg 130 135 140 Arg
Leu Glu Leu Ile Gln Thr Ser Lys Pro Tyr Val Asp Ile Tyr Asp 145 150
155 160 Cys Glu Pro Ser Asn Ser Ser Glu Lys Asn Ser Pro Ser Thr Gln
Tyr 165 170 175 Cys Asn Ser Ile Gln Ser Val Phe Leu Gly Ile Leu Ser
Ala Met Leu 180 185 190 Ile Ser Ala Phe Phe Gln Lys Leu Val Thr Ala
Gly Ile Val Glu Asn 195 200 205 Glu Trp Lys Arg Met Cys Thr Arg Ser
Lys Ser Asn Val Val Leu Leu 210 215 220 Ser Ala Gly Glu Lys Asn Glu
Gln Thr Ile Lys Met Lys Glu Glu Ile 225 230 235 240 Ile Glu Leu Ser
Gly Val Ser Ser Gln Pro Lys Asn Glu Glu Glu Ile 245 250 255 Glu Ile
Ile Pro Val Gln Glu Glu Glu Glu Glu Glu Ala Glu Ile Asn 260 265 270
Phe Pro Ala Pro Pro Gln Glu Gln Glu Ser Leu Pro Val Glu Asn Glu 275
280 285 Ile Ala Pro 290 9 298 PRT Felis catus 9 Met 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 10 53 PRT Canis
familiaris 10 Asp Ile Phe Asn Ile Thr Ile Ser His Phe Leu Lys Met
Glu Asn Leu 1 5 10 15 Asn Leu Ile Lys Ala Pro Ile Pro Tyr Val Asp
Ile His Asn Cys Asp 20 25 30 Pro Ala Asn Pro Ser Glu Lys Asn Ser
Leu Ser Ile Gln Tyr Cys Gly 35 40 45 Ser Ile Arg Ser Val 50 11 75
PRT Artificial CD20 extracellular domain peptide with T-cell
epitope (75-mer) 11 Asp Asp Leu Gln Ala Val His Ala Ala His Ala Glu
Ile Asn Glu Ala 1 5 10 15 Asp His Ile Asp Ile Asp Asp Ile Phe Asn
Ile Thr Ile Ser His Phe 20 25 30 Leu Lys Met Glu Asn Leu Asn Leu
Ile Lys Ala Pro Ile Pro Tyr Val 35 40 45 Asp Ile His Asn Cys Asp
Pro Ala Asn Pro Ser Glu Lys Asn Ser Leu 50 55 60 Ser Ile Gln Tyr
Cys Gly Ser Ile Arg Ser Val 65 70 75 12 53 PRT Homo sapiens 12 Asp
Ile Leu Asn Ile Lys Ile Ser His Phe Leu Lys Met Glu Ser Leu 1 5 10
15 Asn Phe Ile Arg Ala His Thr Pro Tyr Ile Asn Ile Tyr Asn Cys Glu
20 25 30 Pro Ala Asn Pro Ser Glu Lys Asn Ser Pro Ser Thr Gln Tyr
Cys Tyr 35 40 45 Ser Ile Gln Ser Leu 50 13 53 PRT Mus musculus 13
Asp Ile Leu Asn Met Thr Leu Ser His Phe Leu Lys Met Arg Arg Leu 1 5
10 15 Glu Leu Ile Gln Thr Ser Lys Pro Tyr Val Asp Ile Tyr Asp Cys
Glu 20 25 30 Pro Ser Asn Ser Ser Glu Lys Asn Ser Pro Ser Thr Gln
Tyr Cys Asn 35 40 45 Ser Ile Gln Ser Val 50 14 54 PRT Felis catus
14 Asp Ile Phe Asn Ile Ala Ile Ser His Phe Phe Lys Met Glu Asn Leu
1 5 10 15 Asn Leu Leu Lys Ser Pro Lys Pro Tyr Ile Asp Ile His Thr
Cys Gln 20 25 30 Pro Glu Ser Lys Pro Ser Glu Lys Asn Ser Leu Ser
Ile Lys Tyr Cys 35 40 45 Asp Ser Ile Arg Ser Val 50
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