U.S. patent application number 14/449948 was filed with the patent office on 2014-11-20 for human rhinovirus (hrv) antibodies.
The applicant listed for this patent is Theraclone Sciences, Inc.. Invention is credited to Po-Ying Chan-Hui.
Application Number | 20140341923 14/449948 |
Document ID | / |
Family ID | 46881158 |
Filed Date | 2014-11-20 |
United States Patent
Application |
20140341923 |
Kind Code |
A1 |
Chan-Hui; Po-Ying |
November 20, 2014 |
Human Rhinovirus (HRV) Antibodies
Abstract
The invention provides isolated fully human monoclonal anti-HRV
antibodies, as well as method of making and using these antibodies.
Anti-HRV antibodies of the invention prevent or treat subjects
having HRV-infections, and related diseases, including, but not
limited to, the common cold, nasopharyngitis, croup, pneumonia,
bronchiolitis, asthma, chronic obstructive pulmonary disease
(COPD), sinusitis, bacterial superinfection, and cystic
fibrosis.
Inventors: |
Chan-Hui; Po-Ying;
(Bellevue, WA) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
Theraclone Sciences, Inc. |
Seattle |
WA |
US |
|
|
Family ID: |
46881158 |
Appl. No.: |
14/449948 |
Filed: |
August 1, 2014 |
Related U.S. Patent Documents
|
|
|
|
|
|
Application
Number |
Filing Date |
Patent Number |
|
|
13596463 |
Aug 28, 2012 |
8822651 |
|
|
14449948 |
|
|
|
|
61529008 |
Aug 30, 2011 |
|
|
|
Current U.S.
Class: |
424/142.1 ;
424/216.1; 435/320.1; 435/339; 530/350; 530/388.15; 536/23.53 |
Current CPC
Class: |
C07K 2317/21 20130101;
A61P 31/20 20180101; C12N 7/00 20130101; A61P 31/16 20180101; C07K
2317/76 20130101; A61P 29/00 20180101; C07K 16/1009 20130101; C12N
2770/32734 20130101; C07K 2317/33 20130101; A61P 31/00 20180101;
A61P 37/04 20180101; A61P 31/12 20180101; A61P 11/08 20180101; C07K
14/005 20130101; A61K 39/125 20130101; A61K 45/06 20130101 |
Class at
Publication: |
424/142.1 ;
530/388.15; 536/23.53; 435/320.1; 435/339; 530/350; 424/216.1 |
International
Class: |
C07K 16/10 20060101
C07K016/10; A61K 39/125 20060101 A61K039/125; A61K 45/06 20060101
A61K045/06; C12N 7/00 20060101 C12N007/00; C07K 14/005 20060101
C07K014/005 |
Claims
1. An isolated fully human monoclonal antibody, wherein said
monoclonal antibody has the following characteristics a) binds to
an epitope in the rhinovirus capsid protein selected from the group
consisting of VP1, VP2, VP3, and VP4; b) binds to rhinovirus inside
infected cells; and c) binds to rhinovirus.
2. The antibody of claim 1, wherein the antibody binds to an
epitope comprising a portion of two or more rhinovirus capsid
proteins selected from the group consisting of VP1, VP2, VP3, and
VP4.
3. The antibody of claim 1, wherein the antibody binds to
rhinovirus serotypes from one or more clades selected from the
group consisting of clade A (major group), clade A (minor group),
clade B, and clade D.
4. The antibody of claim 1, wherein the antibody cross-neutralizes
multiple rhinovirus serotypes from the group consisting of clade A
(major group), clade A (minor group), clade B, and clade D.
5. The antibody of claim 1, wherein the antibody neutralizes at
least 40% of HRV serotypes selected from the group consisting of
HRV-12, HRV-13, HRV-16, HRV-21, HRV-23, HRV-24, HRV-28, HRV-34,
HRV-36, HRV-38, HRV-40, HRV-51, HRV-54, HRV-61, HRV-63, HRV-64,
HRV-67, HRV-74, HRV-75, HRV-76, HRV-88, HRV-89, HRV-29, HRV-31,
HRV-49, HRV-62, HRV-14, HRV-26, HRV-37, HRV-48, HRV-52, HRV-70,
HRV-83, HRV-84, HRV-86, HRV-93, HRV-08, and HRV-45.
6. The antibody of claim 1, wherein the antibody binds to at least
90% of the HRV serotypes.
7. The antibody of claim 3, wherein the antibody neutralizes the
HRV serotypes with an median IC50 value of equal to or less than
100 ng/ml.
8. The antibody of claim 1, wherein the antibody is isolated from a
B-cell from a human donor.
9. The antibody of claim 1, wherein said epitope is non-linear.
10. The antibody of claim 1, wherein the antibody comprises a
combination of complementarity determining regions (CDRs) selected
from the group consisting of: (a) a VH CDR1 region comprising the
amino acid sequence of DFYWT (SEQ ID NO: 5); a VH CDR2 region
comprising the amino acid sequence of EIDRDGATYYNPSLKS (SEQ ID NO:
6); a VH CDR3 region comprising the amino acid sequence of
RPMLRGVWGNFRSNWFDP (SEQ ID NO: 7); a VL CDR1 region comprising the
amino acid sequence of SGSSSNIGYSYVS (SEQ ID NO: 14); a VL CDR2
region comprising the amino acid sequence of ENNKRPS (SEQ ID NO:
15); and a VL CDR3 region comprising the amino acid sequence of
GTWDTRLFGGV (SEQ ID NO: 16); (b) a VH CDR1 region comprising the
amino acid sequence of DFAMH (SEQ ID NO: 21); a VH CDR2 region
comprising the amino acid sequence of SISRDGSTKYSGDSVKG (SEQ ID NO:
22); a VH CDR3 region comprising the amino acid sequence of
DSPYYLDIVGYRYFHHYGMDV (SEQ ID NO: 23); a VL CDR1 region comprising
the amino acid sequence of RASQILHSYNLA (SEQ ID NO: 30); a VL CDR2
region comprising the amino acid sequence of GAYNRAS (SEQ ID NO:
31); and a VL CDR3 region comprising the amino acid sequence of
QQYGDSPSPGLT (SEQ ID NO: 32); (c) a VH CDR1 region comprising the
amino acid sequence of QNDYHWA (SEQ ID NO: 37); a VH CDR2 region
comprising the amino acid sequence of SVHYRQKSYYSPSLKS (SEQ ID NO:
38); a VH CDR3 region comprising the amino acid sequence of
HNREDYYDSNAYFDE (SEQ ID NO: 39); a VL CDR1 region comprising the
amino acid sequence of SGDDLENTLVC (SEQ ID NO: 46); a VL CDR2
region comprising the amino acid sequence of QDSKRPS (SEQ ID NO:
47); and a VL CDR3 region comprising the amino acid sequence of
QTWHRSTAQYV (SEQ ID NO: 48); (d) a VH CDR1 region comprising the
amino acid sequence of SNDQYWA (SEQ ID NO: 53); a VH CDR2 region
comprising the amino acid sequence of SVHYRRRNYYSPSLES (SEQ ID NO:
54); a VH CDR3 region comprising the amino acid sequence of
HNWEDYYESNAYFDY (SEQ ID NO: 55); a VL CDR1 region comprising the
amino acid sequence of SGDQLENTFVC (SEQ ID NO: 62); a VL CDR2
region comprising the amino acid sequence of QGSKRPS (SEQ ID NO:
63); and a VL CDR3 region comprising the amino acid sequence of
QAWDRSTAHYV (SEQ ID NO: 64).
11. An isolated anti-HRV antibody, wherein said antibody a
combination of complementarity determining regions (CDRs) selected
from the group consisting of: (a) a VH CDR1 region comprising the
amino acid sequence of DFYWT (SEQ ID NO: 5); a VH CDR2 region
comprising the amino acid sequence of EIDRDGATYYNPSLKS (SEQ ID NO:
6); a VH CDR3 region comprising the amino acid sequence of
RPMLRGVWGNFRSNWFDP (SEQ ID NO: 7); a VL CDR1 region comprising the
amino acid sequence of SGSSSNIGYSYVS (SEQ ID NO: 14); a VL CDR2
region comprising the amino acid sequence of ENNKRPS (SEQ ID NO:
15); and a VL CDR3 region comprising the amino acid sequence of
GTWDTRLFGGV (SEQ ID NO: 16); (b) a VH CDR1 region comprising the
amino acid sequence of DFAMH (SEQ ID NO: 21); a VH CDR2 region
comprising the amino acid sequence of SISRDGSTKYSGDSVKG (SEQ ID NO:
22); a VH CDR3 region comprising the amino acid sequence of
DSPYYLDIVGYRYFHHYGMDV (SEQ ID NO: 23); a VL CDR1 region comprising
the amino acid sequence of RASQILHSYNLA (SEQ ID NO: 30); a VL CDR2
region comprising the amino acid sequence of GAYNRAS (SEQ ID NO:
31); and a VL CDR3 region comprising the amino acid sequence of
QQYGDSPSPGLT (SEQ ID NO: 32); (c) a VH CDR1 region comprising the
amino acid sequence of QNDYHWA (SEQ ID NO: 37); a VH CDR2 region
comprising the amino acid sequence of SVHYRQKSYYSPSLKS (SEQ ID NO:
38); a VH CDR3 region comprising the amino acid sequence of
HNREDYYDSNAYFDE (SEQ ID NO: 39); a VL CDR1 region comprising the
amino acid sequence of SGDDLENTLVC (SEQ ID NO: 46); a VL CDR2
region comprising the amino acid sequence of QDSKRPS (SEQ ID NO:
47); and a VL CDR3 region comprising the amino acid sequence of
QTWHRSTAQYV (SEQ ID NO: 48); and (d) a VH CDR1 region comprising
the amino acid sequence of SNDQYWA (SEQ ID NO: 53); a VH CDR2
region comprising the amino acid sequence of SVHYRRRNYYSPSLES (SEQ
ID NO: 54); a VH CDR3 region comprising the amino acid sequence of
HNWEDYYESNAYFDY (SEQ ID NO: 55); a VL CDR1 region comprising the
amino acid sequence of SGDQLENTFVC (SEQ ID NO: 62); a VL CDR2
region comprising the amino acid sequence of QGSKRPS (SEQ ID NO:
63); and a VL CDR3 region comprising the amino acid sequence of
QAWDRSTAHYV (SEQ ID NO: 64).
12. An antibody that binds the same epitope as an antibody selected
from the group consisting of the antibodies of claim 11.
13. The antibody of claim 11 further comprising, a) a heavy chain
sequence comprising the amino acid sequence of SEQ ID NO: 4 and a
light chain sequence comprising amino acid sequence SEQ ID NO: 13,
or b) a heavy chain sequence comprising the amino acid sequence of
SEQ ID NO: 20 and a light chain sequence comprising amino acid
sequence SEQ ID NO: 29, or c) a heavy chain sequence comprising the
amino acid sequence of SEQ ID NO: 36 and a light chain sequence
comprising amino acid sequence SEQ ID NO: 45, or d) a heavy chain
sequence comprising the amino acid sequence of SEQ ID NO: 52 and a
light chain sequence comprising amino acid sequence SEQ ID NO:
61.
14. A nucleic acid molecule encoding the antibody of claim 11.
15. A vector comprising the nucleic acid molecule of claim 18.
16. A cell comprising the vector of claim 15.
17. An isolated B cell clone or immortalized B-cell clone
expressing the antibody of claim 11.
18. An isolated epitope which binds to the antibody of claim
11.
19. An immunogenic polypeptide or glycopeptide comprising the
epitope of claim 18.
20. A pharmaceutical composition comprising the antibody of claim
11, and a pharmaceutically acceptable carrier.
21. The composition of claim 20, further comprising a second
therapeutic agent.
22. The composition of claim 21, wherein the second therapeutic
agent is a second antibody, an antiviral drug, an antibiotic, a
bronchodilator, a leukotriene blocker, a steroid, an
anti-inflammatory drug, or an oxygen therapy.
23. The composition of claim 21, wherein the second agent is
selected from the group consisting of: (a) a second antibody that
is specific for human rhinovirus, influenza, parainfluenza,
coronavirus, adenovirus, respiratory syncytical virus,
picornavirus, metapneumovirus, or anti-IgE antibody; (b) an
anti-viral drug selected from the group consisting of an entry
inhibitor, a fusion inhibitor, an integrase inhibitor, a nucleoside
analog, a protease inhibitor, and a reverse transcriptase
inhibitor; (c) an anti-viral drug selected from the group
consisting of Abacavir, Acicolvir, Acyclovir, Adefovir, Amantadine,
Amprenavir, Ampligen, Arbidol, Atazanavir, Atripla, Boceprevir,
Cidofovir, Combivir, Darunavir, Delavirdine, Didanosine, Docosanol,
Edoxudine, Efavirenz, Emtricitabine, Enfuvirtide, Entecavir,
Famciclovir, Fomivirsen, Fosamprenavir, Foscarnet, Fosfonet,
Ganciclovir, Ibacitabine, Imunovir, Idoxuridine, Imiquimod,
Indinavir, Inosine, Interferon (Type I, II, or III), Lamivudine,
Lopinavir, Loviride, Maraviroc, Moroxydine, Methisazone,
Nelfinavir, Nevirapine, Nexavir, Oseltamivir, Peginterferon
alpha-2a, Pencicolvir, Peramivir, Pleconaril, Podophyllotoxin,
Raltegravir, Ribavirin, Rimantadine, Ritonavir, Pyramidine,
Saquinavir, Stavudine, Tea tree oil, Tenofovir, Tenofovir
disoproxil, Tipranavir, Trifluridine, Trizivir, Tromantadine,
Truvada, Valaciclovir, Valganciclovir, Vicriviroc, Vidarabine,
Viramidine, Zalcitabine, Zanamivir, and Zidovudine; (d) an
antibiotic selected from the group consisting of an Aminoglycoside,
a Carbapenem, a Cephalosporin, a Lincosamide, a Macrolide, a
Penicillin, and a Quinolone; (e) an antibiotic selected from the
group consisting of Amikacin, Gentamicin, Kanamycin, Neomycin,
Netilmycin, Tobramycin, Paromycin, Geldanamycin, Ertapenem,
Dorpenem, Imipenem/Cilastatin, Meropenem, Cefadroxil, Cefazolin,
Cefalotin, Cefalothin, Cefalexin, Cefaclor, Ceamandole, Cefoxitin,
Cefprozil, Cefurozime, Cefixime, Cefdinir, Defditoren,
Cefoperazone, Cefotaxime, Cefazidime, Ceftibuten, Ceftizoxime,
Ceftriaxone, Cefepime, Ceftobiprole, Teicoplanin, Vancomycin,
Telavancin, Clindamycin, Lincomycin, Daptomycin, Azithromyzin,
Clarithromycin, Dirithromycin, Erythromycin, Roxithromycin,
Troleandomycin, Spectinomycin, Aztreonam, Furazolidone,
Nitofurantoin, Amoxicillin, Ampicillin, Azlocillin, Carbenicillin,
Cloxacillin, Dicloxacillin, Flucloxacillin, Mezlocillin,
Methicillin, Nafcillin, Oxacillin, Penicillin G, Penicillin V,
Piperacillin, Temocillin, Ticarcillin, Amoxicillin/clavulanate,
Ampicillin/sulbactam, Piperacillin/tazobactam,
Ticarcillin/clavulanate, Bacitracin, Colistin, Polymyxin B,
Ciprofloxacin, Enoxacin, Gatifloxacin, Levofloxacin, Lomefloxacin,
Moxifloxacin, Nalidixic acid, Norfloxacin, Ofloxacin,
Trovafloxacin, Grepafloxacin, Sparfloxacin, Temafloxacin, Mafenide,
Sulfonamidochrysoidine, Sulfacetamide, Sulfadiazine, Silver
sulfadiazine, Sulfamethizole, Sulfamethoxazole, Sulfanilimide,
Sulfasalazine, Sulfisoxazole, Trimethoprim,
Trimethoprim-Sulfamethoxazole (Co-trumoxazole), Demeclocycline,
Docycline, Minocycline, Oxytetracycline, Tetracycline, Clofazimine,
Dapsone, Capreomycin, Cycloserine, Ethambutol, Ethionamide,
Isoniazid, Pyrazinamide, Rifampicin, Rifampin, Rifabutin,
Rifapentin, Stretomycin, Arsphenamine, Choramphenicol, Fosfomycin,
Fusidic acid, Linezolid, Metonidazole, Mupirocin, Platensimycin,
Quinupristin/Dalfopristin, Rifaximin, Thamphenicol, Tigecycline,
and Tinidazole; (f) a short-acting bronchodilator or a long-acting
bronchodilator; (g) a short-acting bronchodilator comprising a
.beta.2-agonist or an anticholinergic; (h) an long-acting
bronchodilator comprising a .beta.2-agonist or a theophylline; (i)
a corticosteroid; (j) corticosteroid selected from the group
consisting of hydrocortisone, hydrocortisone acetate, cortisone
acetate, tixocortol pivalate, prednisolone, methylprednisolone,
prednisone, triamcinolone acetonide, triamcinolone alcohol,
mometasone, amcinonide, budesonide, desonide, fluocinonide,
fluocinolone acetonide, halcinonide, betamethasone, betamethasone
sodium phosphate, dexamethasone, dexamethasone sodium phosphate,
fluocortolone, hydrocortisone-17-butyrate,
hydrocortisone-17-valerate, aclometasone dipropionate,
betamethasone valerate, betamethasone dipropionate, prednicarbate,
clobetasone-17-butyrate, clobetasol-17-propionate, fluocortolone
caproate, fluocortolone pivalate, and fluprednidene acetate; (k) an
anti-inflammatory drug comprising an antihistamine or a histamine
receptor blocker; and (l) oxygen therapy comprising supplemental
oxygen gas, and wherein the arterial blood oxygen saturation of the
subject following treatment is greater than or equal to 85%.
24. A method of immunizing a subject against human rhinovirus (HRV)
infection, comprising administering to the subject the composition
of claim 20.
25. A method of preventing or treating a human rhinovirus
infection, comprising administering to a subject the composition of
claim 20.
26. The method claim 25, wherein the human rhinovirus infection
causes or exacerbates the common cold, nasopharyngitis, croup,
pneumonia, bronchiolitis, asthma, chronic obstructive pulmonary
disease (COPD), sinusitis, bacterial superinfection, or cystic
fibrosis.
27. A method of preventing or treating a human rhinovirus
(HRV)-related disease, comprising administering to a subject the
composition of claim 20.
28. The method claim 27, wherein the human rhinovirus (HRV)-related
disease is the common cold, nasopharyngitis, croup, pneumonia,
bronchiolitis, asthma, chronic obstructive pulmonary disease
(COPD), sinusitis, bacterial superinfection, or cystic
fibrosis.
29. A vaccine comprising an epitope which specifically binds to the
isolated anti-HRV antibody of claim 11.
30. A vaccine comprising the isolated anti-HRV antibody of claim
11.
Description
RELATED APPLICATIONS
[0001] This application is a continuation of U.S. patent
application U.S. Ser. No. 13/596,463, filed Aug. 28, 2012, which
claims the benefit of U.S. provisional application U.S. Ser. No.
61/529,008, filed on Aug. 30, 2011, the contents which are herein
incorporated by reference in their entirety.
INCORPORATION OF SEQUENCE LISTING
[0002] The contents of the text file named "THER019C01US
SeqList.txt," which was created on Jul. 30, 2014 and is 54 KB in
size, are hereby incorporated by reference in their entirety.
FIELD OF THE INVENTION
[0003] The present invention relates generally to prophylaxis,
therapy, diagnosis and monitoring of human rhinovirus (HRV)
infection. The invention is more specifically related to human
neutralizing monoclonal antibodies specific for HRV, such as broad
and potent neutralizing monoclonal antibodies specific for HRV and
their manufacture and use. Broad neutralization suggests that the
antibodies can neutralize HRV isolates from multiple isotypes.
BACKGROUND OF THE INVENTION
[0004] Human rhinoviruses are the most common infectious agents in
humans, worldwide. These viruses are also most commonly known as
the primary cause of the common cold. Commensurate with their role
as instigating colds, the primary route of entry for human
rhinovirus is the upper respiratory tract. These viruses travel
rapidly throughout the local population because they are
transmitted through air, e.g. via contaminated respiratory droplets
of sneezes and coughs, via contact with contaminated surfaces, and
via person-to-person contact. Infection also occurs rapidly. The
virus adheres to cell surface receptors within minutes of entering
the respiratory tract. Symptoms appear in most individuals within
days. However, the incubation time can vary from approximately 12
hours to a week.
[0005] Infection by human rhinovirus can be fatal; however, more
common symptoms include, but are not limited to sore throat, runny
nose, nasal congestion, sneezing, cough, muscle aches, fatigue,
malaise, headache, muscle weakness, and loss of appetite.
Infections frequently occur during the time of year when people
spend most time indoors and, therefore, spend most time in close
proximity to one another, e.g. from September to April. The
consequences of the human rhinovirus infection are not only
medical, but economical. Students and employees must isolate
themselves from school and colleagues to prevent spread of the
virus, which results in lost educational opportunity and
productivity.
[0006] Despite a long-felt need in the art and ongoing attempts to
cure infections caused by the human rhinovirus, including the
common cold, a need still exists for an effective treatment that
addresses the underlying cause of these illnesses by neutralizing
the virus itself.
SUMMARY OF THE INVENTION
[0007] The invention solves this long-felt need by providing
compositions and methods for preventing and treating human
rhinovirus infection.
[0008] The present invention provides a novel method for isolating
potent, broadly neutralizing monoclonal antibodies against HRV.
Peripheral Blood Mononuclear Cells (PBMCs) are obtained from a
donor selected for HRV neutralizing activity in the plasma, and
memory B cells are isolated for culture in vitro. The B cell
culture supernatants are then screened by a primary neutralization
assay in a high throughput format, and B cell cultures exhibiting
neutralizing activity are selected for rescue of monoclonal
antibodies. It is surprisingly observed that neutralizing
antibodies obtained by this method do not always exhibit epitope-
or viral-binding at levels that correlate with neutralization
activity. The method of the invention therefore allows
identification of novel antibodies with cross-isotype
neutralization properties.
[0009] Specifically, the invention provides an isolated fully human
monoclonal antibody, wherein the monoclonal antibody has the
following characteristics a) binds to an epitope on the rhinovirus
capsid protein selected from the group consisting of VP1, VP2, VP3,
and VP4; b) binds to rhinovirus inside infected cells; and c) binds
to rhinovirus. Alternatively, or in addition, the antibody binds to
an epitope comprising a portion of two or more rhinovirus capsid
proteins selected from the group consisting of VP1, VP2, VP3, and
VP4. In a preferred embodiment, the epitope is non-linear. The
antibody is isolated from a B-cell from a human donor.
[0010] In one aspect, the antibody binds to or cross-neutralizes
rhinovirus serotypes from one or more clades selected from the
group consisting of clade A (major group), clade A (minor group),
clade B, and clade D. Alternatively, the antibody binds to or
cross-neutralizes rhinovirus serotypes from two or more or three or
more clades selected from the group consisting of clade A (major
group), clade A (minor group), clade B, and clade D. In another
aspect, the antibody binds to at least 40%, 45%, 50%, 55%, 60%,
65%, 70%, 75%, 80%, 85%, 90%, 95%, or 100% of HRV serotypes
selected from the group consisting of HRV-12, HRV-13, HRV-16,
HRV-21, HRV-23, HRV-24, HRV-28, HRV-34, HRV-36, HRV-38, HRV-40,
HRV-51, HRV-54, HRV-61, HRV-63, HRV-64, HRV-67, HRV-74, HRV-75,
HRV-76, HRV-88, HRV-89, HRV-29, HRV-31, HRV-49, HRV-62, HRV-14,
HRV-26, HRV-37, HRV-48, HRV-52, HRV-70, HRV-83, HRV-84, HRV-86,
HRV-93, HRV-08, and HRV-45. Preferably, the antibody binds to at
least 90% of these HRV serotypes. Alternatively, or in addition,
the antibody neutralizes at least 40%, 45%, 50%, 55%, 60%, 65%,
70%, 75%, 80%, 85%, 90%, 95%, or 100% of HRV serotypes HRV
serotypes selected from the group consisting of HRV-12, HRV-13,
HRV-16, HRV-21, HRV-23, HRV-24, HRV-28, HRV-34, HRV-36, HRV-38,
HRV-40, HRV-51, HRV-54, HRV-61, HRV-63, HRV-64, HRV-67, HRV-74,
HRV-75, HRV-76, HRV-88, HRV-89, HRV-29, HRV-31, HRV-49, HRV-62,
HRV-14, HRV-26, HRV-37, HRV-48, HRV-52, HRV-70, HRV-83, HRV-84,
HRV-86, HRV-93, HRV-08, and HRV-45. Preferably, the antibody
neutralizes at least 40% of these HRV serotypes. Furthermore, the
antibody neutralizes the HRV serotypes with a median IC.sub.50
value of equal to or less than 100 ng/mL.
[0011] Optionally, the antibody is TCN-711 (6893_E11), TCN-716
(6362_F16), TCN-717 (6358_H17), or TCN-722 (6385_L22). The isolated
fully human monoclonal antibody that binds to or neutralizes HRV,
comprises, (a) a V.sub.H CDR1 region comprising the amino acid
sequence of DFYWT (SEQ ID NO: 5); a V.sub.H CDR2 region comprising
the amino acid sequence of EIDRDGATYYNPSLKS (SEQ ID NO: 6); a
V.sub.H CDR3 region comprising the amino acid sequence of
RPMLRGVWGNFRSNWFDP (SEQ ID NO: 7); a V.sub.L CDR1 region comprising
the amino acid sequence of SGSSSNIGYSYVS (SEQ ID NO: 14); a V.sub.L
CDR2 region comprising the amino acid sequence of ENNKRPS (SEQ ID
NO: 15); and a V.sub.L CDR3 region comprising the amino acid
sequence of GTWDTRLFGGV (SEQ ID NO: 16); (b) a V.sub.H CDR1 region
comprising the amino acid sequence of DFAMH (SEQ ID NO: 21); a
V.sub.H CDR2 region comprising the amino acid sequence of
SISRDGSTKYSGDSVKG (SEQ ID NO: 22); a V.sub.H CDR3 region comprising
the amino acid sequence of DSPYYLDIVGYRYFHHYGMDV (SEQ ID NO: 23); a
V.sub.L CDR1 region comprising the amino acid sequence of
RASQILHSYNLA (SEQ ID NO: 30); a V.sub.L CDR2 region comprising the
amino acid sequence of GAYNRAS (SEQ ID NO: 31); and a V.sub.L CDR3
region comprising the amino acid sequence of QQYGDSPSPGLT (SEQ ID
NO: 32); (c) a V.sub.H CDR1 region comprising the amino acid
sequence of QNDYHWA (SEQ ID NO: 37); a V.sub.H CDR2 region
comprising the amino acid sequence of SVHYRQKSYYSPSLKS (SEQ ID NO:
38); a V.sub.H CDR3 region comprising the amino acid sequence of
HNREDYYDSNAYFDE (SEQ ID NO: 39); a V.sub.L CDR1 region comprising
the amino acid sequence of SGDDLENTLVC (SEQ ID NO: 46); a V.sub.L
CDR2 region comprising the amino acid sequence of QDSKRPS (SEQ ID
NO: 47); and a V.sub.L CDR3 region comprising the amino acid
sequence of QTWHRSTAQYV (SEQ ID NO: 48); or (d) a V.sub.H CDR1
region comprising the amino acid sequence of SNDQYWA (SEQ ID NO:
53); a V.sub.H CDR2 region comprising the amino acid sequence of
SVHYRRRNYYSPSLES (SEQ ID NO: 54); a V.sub.H CDR3 region comprising
the amino acid sequence of HNWEDYYESNAYFDY (SEQ ID NO: 55); a
V.sub.L CDR1 region comprising the amino acid sequence of
SGDQLENTFVC (SEQ ID NO: 62); a V.sub.L CDR2 region comprising the
amino acid sequence of QGSKRPS (SEQ ID NO: 63); and a V.sub.L CDR3
region comprising the amino acid sequence of QAWDRSTAHYV (SEQ ID
NO: 64).
[0012] Alternatively, the antibody binds to the same epitope as
TCN-711 (6893_E11), TCN-716 (6362_F16), TCN-717 (6358_H17), or
TCN-722 (6385_L22). Alternatively stated, the invention provides an
antibody that binds the same epitope as an antibody comprising, (a)
a V.sub.H CDR1 region comprising the amino acid sequence of DFYWT
(SEQ ID NO: 5); a V.sub.H CDR2 region comprising the amino acid
sequence of EIDRDGATYYNPSLKS (SEQ ID NO: 6); a V.sub.H CDR3 region
comprising the amino acid sequence of RPMLRGVWGNFRSNWFDP (SEQ ID
NO: 7); a V.sub.L CDR1 region comprising the amino acid sequence of
SGSSSNIGYSYVS (SEQ ID NO: 14); a V.sub.L CDR2 region comprising the
amino acid sequence of ENNKRPS (SEQ ID NO: 15); and a V.sub.L CDR3
region comprising the amino acid sequence of GTWDTRLFGGV (SEQ ID
NO: 16); (b) a V.sub.H CDR1 region comprising the amino acid
sequence of DFAMH (SEQ ID NO: 21); a V.sub.H CDR2 region comprising
the amino acid sequence of SISRDGSTKYSGDSVKG (SEQ ID NO: 22); a
V.sub.H CDR3 region comprising the amino acid sequence of
DSPYYLDIVGYRYFHHYGMDV (SEQ ID NO: 23); a V.sub.L CDR1 region
comprising the amino acid sequence of RASQILHSYNLA (SEQ ID NO: 30);
a V.sub.L CDR2 region comprising the amino acid sequence of GAYNRAS
(SEQ ID NO: 31); and a V.sub.L CDR3 region comprising the amino
acid sequence of QQYGDSPSPGLT (SEQ ID NO: 32); (c) a V.sub.H CDR1
region comprising the amino acid sequence of QNDYHWA (SEQ ID NO:
37); a V.sub.H CDR2 region comprising the amino acid sequence of
SVHYRQKSYYSPSLKS (SEQ ID NO: 38); a V.sub.H CDR3 region comprising
the amino acid sequence of HNREDYYDSNAYFDE (SEQ ID NO: 39); a
V.sub.L CDR1 region comprising the amino acid sequence of
SGDDLENTLVC (SEQ ID NO: 46); a V.sub.L CDR2 region comprising the
amino acid sequence of QDSKRPS (SEQ ID NO: 47); and a V.sub.L CDR3
region comprising the amino acid sequence of QTWHRSTAQYV (SEQ ID
NO: 48); or (d) a V.sub.H CDR1 region comprising the amino acid
sequence of SNDQYWA (SEQ ID NO: 53); a V.sub.H CDR2 region
comprising the amino acid sequence of SVHYRRRNYYSPSLES (SEQ ID NO:
54); a V.sub.H CDR3 region comprising the amino acid sequence of
HNWEDYYESNAYFDY (SEQ ID NO: 55); a V.sub.L CDR1 region comprising
the amino acid sequence of SGDQLENTFVC (SEQ ID NO: 62); a V.sub.L
CDR2 region comprising the amino acid sequence of QGSKRPS (SEQ ID
NO: 63); and a V.sub.L CDR3 region comprising the amino acid
sequence of QAWDRSTAHYV (SEQ ID NO: 64).
[0013] The invention provides an isolated anti-HRV antibody,
wherein the antibody comprises, a V.sub.H CDR1 region comprising
the amino acid sequence of DFYWT (SEQ ID NO: 5); a V.sub.H CDR2
region comprising the amino acid sequence of EIDRDGATYYNPSLKS (SEQ
ID NO: 6); a V.sub.H CDR3 region comprising the amino acid sequence
of RPMLRGVWGNFRSNWFDP (SEQ ID NO: 7); a V.sub.L CDR1 region
comprising the amino acid sequence of SGSSSNIGYSYVS (SEQ ID NO:
14); a V.sub.L CDR2 region comprising the amino acid sequence of
ENNKRPS (SEQ ID NO: 15); and a V.sub.L CDR3 region comprising the
amino acid sequence of GTWDTRLFGGV (SEQ ID NO: 16).
[0014] The invention provides an isolated anti-HRV antibody,
wherein said antibody comprises, a V.sub.H CDR1 region comprising
the amino acid sequence of DFAMH (SEQ ID NO: 21); a V.sub.H CDR2
region comprising the amino acid sequence of SISRDGSTKYSGDSVKG (SEQ
ID NO: 22); a V.sub.H CDR3 region comprising the amino acid
sequence of DSPYYLDIVGYRYFHHYGMDV (SEQ ID NO: 23); a V.sub.L CDR1
region comprising the amino acid sequence of RASQILHSYNLA (SEQ ID
NO: 30); a V.sub.L CDR2 region comprising the amino acid sequence
of GAYNRAS (SEQ ID NO: 31); and a V.sub.L CDR3 region comprising
the amino acid sequence of QQYGDSPSPGLT (SEQ ID NO: 32).
[0015] The invention provides an isolated anti-HRV antibody,
wherein said antibody comprises, a V.sub.H CDR1 region comprising
the amino acid sequence of QNDYHWA (SEQ ID NO: 37); a V.sub.H CDR2
region comprising the amino acid sequence of SVHYRQKSYYSPSLKS (SEQ
ID NO: 38); a V.sub.H CDR3 region comprising the amino acid
sequence of HNREDYYDSNAYFDE (SEQ ID NO: 39); a V.sub.L CDR1 region
comprising the amino acid sequence of SGDDLENTLVC (SEQ ID NO: 46);
a V.sub.L CDR2 region comprising the amino acid sequence of QDSKRPS
(SEQ ID NO: 47); and a V.sub.L CDR3 region comprising the amino
acid sequence of QTWHRSTAQYV (SEQ ID NO: 48).
[0016] The invention provides an isolated anti-HRV antibody,
wherein said antibody comprises, a V.sub.H CDR1 region comprising
the amino acid sequence of SNDQYWA (SEQ ID NO: 53); a V.sub.H CDR2
region comprising the amino acid sequence of SVHYRRRNYYSPSLES (SEQ
ID NO: 54); a V.sub.H CDR3 region comprising the amino acid
sequence of HNWEDYYESNAYFDY (SEQ ID NO: 55); a V.sub.L CDR1 region
comprising the amino acid sequence of SGDQLENTFVC (SEQ ID NO: 62);
a V.sub.L CDR2 region comprising the amino acid sequence of QGSKRPS
(SEQ ID NO: 63); and a V.sub.L CDR3 region comprising the amino
acid sequence of QAWDRSTAHYV (SEQ ID NO: 64).
[0017] The invention provides an isolated monoclonal anti-HRV
antibody comprising, a) a heavy chain sequence comprising the amino
acid sequence of SEQ ID NO: 4 and a light chain sequence comprising
amino acid sequence SEQ ID NO: 13, or b) a heavy chain sequence
comprising the amino acid sequence of SEQ ID NO: 20 and a light
chain sequence comprising amino acid sequence SEQ ID NO: 29, or c)
a heavy chain sequence comprising the amino acid sequence of SEQ ID
NO: 36 and a light chain sequence comprising amino acid sequence
SEQ ID NO: 45, or d) a heavy chain sequence comprising the amino
acid sequence of SEQ ID NO: 52 and a light chain sequence
comprising amino acid sequence SEQ ID NO: 61.
[0018] The invention provides a nucleic acid molecule encoding an
antibody described herein. The invention provides a vector
comprising this nucleic acid molecule. The invention provides a
cell comprising this vector.
[0019] The invention provides an isolated B cell clone or
immortalized B-cell clone expressing an isolated monoclonal
anti-HRV antibody described herein.
[0020] The invention provides an isolated epitope which binds to an
isolated monoclonal anti-HRV antibody described herein. The
invention further provides an immunogenic polypeptide or
glycopeptide comprising this epitope.
[0021] The invention provides a composition comprising an isolated
anti-HRV antibody described herein. Moreover, the invention
provides a pharmaceutical composition comprising at least one
isolated anti-HRV antibody described herein and a pharmaceutically
acceptable carrier.
[0022] In certain embodiments, this composition or this
pharmaceutical composition further comprise a second therapeutic
agent. The second therapeutic agent is a second antibody, an
antiviral drug, an antibiotic, a bronchodilator, a leukotriene
blocker, a steroid, an antiflammatory drug, or an oxygen therapy.
The second antibody may be specific for human rhinovirus,
influenza, parainfluenza, coronavirus, adenovirus, respiratory
syncytical virus, picornavirus, metapneumovirus, or anti-IgE
antibody. If the second antibody is specific for human rhinovirus,
the antibody may be an anti-HRV antibody described herein.
[0023] The second therapeutic agent is an antiviral drug. The
anti-viral drug may be an entry inhibitor, a fusion inhibitor, an
integrase inhibitor, a nucleoside analog, a protease inhibitor, or
a reverse transcriptase inhibitor. Exemplary anti-viral drug
include, but are not limited to, Abacavir, Acicolvir, Acyclovir,
Adefovir, Amantadine, Amprenavir, Ampligen, Arbidol, Atazanavir,
Atripla, Boceprevir, Cidofovir, Combivir, Darunavir, Delavirdine,
Didanosine, Docosanol, Edoxudine, Efavirenz, Emtricitabine,
Enfuvirtide, Entecavir, Famciclovir, Fomivirsen, Fosamprenavir,
Foscarnet, Fosfonet, Ganciclovir, Ibacitabine, Imunovir,
Idoxuridine, Imiquimod, Indinavir, Inosine, Interferon (Type I, II,
or III), Interferon-alpha, Interferon-beta, Lamivudine, Lopinavir,
Loviride, Maraviroc, Moroxydine, Methisazone, Nelfinavir,
Nevirapine, Nexavir, Oseltamivir, Peginterferon alpha-2a,
Pencicolvir, Peramivir, Pleconaril, Podophyllotoxin, Raltegravir,
Ribavirin, Rimantadine, Ritonavir, Pyramidine, Saquinavir,
Stavudine, Tea tree oil, Tenofovir, Tenofovir disoproxil,
Tipranavir, Trifluridine, Trizivir, Tromantadine, Truvada,
Valaciclovir, Valganciclovir, Vicriviroc, Vidarabine, Viramidine,
Zalcitabine, Zanamivir, or Zidovudine.
[0024] The second therapeutic agent is an antibiotic. The
antibiotic may be an Aminoglycoside, a Carbapenem, a Cephalosporin,
a Lincosamide, a Macrolide, a Penicillin, or a Quinolone. Exemplary
antibiotics include, but are not limited to, Amikacin, Gentamicin,
Kanamycin, Neomycin, Netilmycin, Tobramycin, Paromycin,
Geldanamycin, Ertapenem, Dorpenem, Imipenem/Cilastatin, Meropenem,
Cefadroxil, Cefazolin, Cefalotin, Cefalothin, Cefalexin, Cefaclor,
Ceamandole, Cefoxitin, Cefprozil, Cefurozime, Cefixime, Cefdinir,
Defditoren, Cefoperazone, Cefotaxime, Cefazidime, Ceftibuten,
Ceftizoxime, Ceftriaxone, Cefepime, Ceftobiprole, Teicoplanin,
Vancomycin, Telavancin, Clindamycin, Lincomycin, Daptomycin,
Azithromyzin, Clarithromycin, Dirithromycin, Erythromycin,
Roxithromycin, Troleandomycin, Spectinomycin, Aztreonam,
Furazolidone, Nitofurantoin, Amoxicillin, Ampicillin, Azlocillin,
Carbenicillin, Cloxacillin, Dicloxacillin, Flucloxacillin,
Mezlocillin, Methicillin, Nafcillin, Oxacillin, Penicillin G,
Penicillin V, Piperacillin, Temocillin, Ticarcillin,
Amoxicillin/clavulanate, Ampicillin/sulbactam,
Piperacillin/tazobactam, Ticarcillin/clavulanate, Bacitracin,
Colistin, Polymyxin B, Ciprofloxacin, Enoxacin, Gatifloxacin,
Levofloxacin, Lomefloxacin, Moxifloxacin, Nalidixic acid,
Norfloxacin, Ofloxacin, Trovafloxacin, Grepafloxacin, Sparfloxacin,
Temafloxacin, Mafenide, Sulfonamidochrysoidine, Sulfacetamide,
Sulfadiazine, Silver sulfadiazine, Sulfamethizole,
Sulfamethoxazole, Sulfanilimide, Sulfasalazine, Sulfisoxazole,
Trimethoprim, Trimethoprim-Sulfamethoxazole (Co-trumoxazole),
Demeclocycline, Docycline, Minocycline, Oxytetracycline,
Tetracycline, Clofazimine, Dapsone, Capreomycin, Cycloserine,
Ethambutol, Ethionamide, Isoniazid, Pyrazinamide, Rifampicin,
Rifampin, Rifabutin, Rifapentin, Stretomycin, Arsphenamine,
Choramphenicol, Fosfomycin, Fusidic acid, Linezolid, Metonidazole,
Mupirocin, Platensimycin, Quinupristin/Dalfopristin, Rifaximin,
Thamphenicol, Tigecycline, Tinidazole.
[0025] The second therapeutic agent is a bronchodialator. In
certain aspects, the bronchodilator is alternatively a short- or
long-acting agent. Exemplary short-acting bronchodilators include,
but are not limited to, a .beta.2-agonist or an anticholinergic
compound. Exemplary long-acting bronchodilators include, but are
not limited to, a .beta.2-agonist or a theophylline compound.
[0026] The second therapeutic agent is a leukotriene antagonist,
inhibitor, or blocker. Leukotrienes are fatty compounds produced by
the immune system that cause the inflammation found in, for
example, the upper respiratory tract that results from genetic
predisposition (e.g., asthma or allergy), viral infection (e.g.,
bronchitis or COPD), or lifestyle and environmental factors (e.g.,
smoking, mining, or exposure to asbestos). Regardless of the cause,
leukotriene-mediated inflammation constricts airways. Accordingly,
leukotriene inhibitors are often bronchodilators. Common
leukotriene inhibitors (or modifiers) either inhibit the
5-lipoxygenase pathway (leukotriene synthase inhibitors) or
antagonize cysteinyl-leukotriene type 1 receptors (leukotriene
receptor antagonists or LTRAs). Leukotriene inhibitors, modifiers,
or antagonists involved in either pathway are contemplated.
Specifically, zileuton (Zyflo.RTM.) is a commercially-available
drug that inhibits 5-lipoxygenase. Montelukast (Singulair.RTM.) and
zafirlukast (Accolate.RTM.) are commercially-available leukotriene
inhibitors that block the activity of cysteinyl leukotrienes at the
CysLT1 receptor on target cells (e.g., bronchial smooth
muscle).
[0027] The second therapeutic agent is a steroid. In a preferred
embodiment, the steroid is a corticosteroid. Exemplary
corticosteroids include, but are not limited to, hydrocortisone,
hydrocortisone acetate, cortisone acetate, tixocortol pivalate,
prednisolone, methylprednisolone, prednisone, triamcinolone
acetonide, triamcinolone alcohol, mometasone, amcinonide,
budesonide, desonide, fluocinonide, fluocinolone acetonide,
halcinonide, betamethasone, betamethasone sodium phosphate,
dexamethasone, dexamethasone sodium phosphate, fluocortolone,
hydrocortisone-17-butyrate, hydrocortisone-17-valerate,
aclometasone dipropionate, betamethasone valerate, betamethasone
dipropionate, prednicarbate, clobetasone-17-butyrate,
clobetasol-17-propionate, fluocortolone caproate, fluocortolone
pivalate, and fluprednidene acetate.
[0028] The second therapeutic agent is an anti-inflammatory agent.
Exemplary anti-inflammatory agents include, but are not limited to,
antihistamines and histamine receptor blockers.
[0029] The second therapeutic agent is oxygen therapy. Oxygen
therapy includes, but is not limited to, supplemental oxygen gas.
In a preferred embodiment, the arterial blood oxygen saturation of
the subject following the oxygen treatment is greater than or equal
to 85%. In a more preferred embodiment, the arterial blood oxygen
saturation of the subject following the oxygen treatment is greater
than or equal to 90%.
[0030] The invention further provides a method of immunizing a
subject against human rhinovirus (HRV) infection, comprising
administering to the subject a composition or pharmaceutical
composition described herein.
[0031] The invention provides a method of preventing or treating a
human rhinovirus infection, comprising administering to a subject a
composition or pharmaceutical composition described herein. In
certain embodiments of this method, the human rhinovirus infection
causes or exacerbates the common cold, nasopharyngitis, croup,
pneumonia, bronchiolitis, asthma, chronic obstructive pulmonary
disease (COPD), sinusitis, bacterial superinfection, or cystic
fibrosis.
[0032] The invention provides a method of preventing or treating a
human rhinovirus (HRV)-related disease, comprising administering to
a subject a composition or pharmaceutical composition described
herein. In certain embodiments of this method, the human rhinovirus
(HRV)-related disease is the common cold, nasopharyngitis, croup,
pneumonia, bronchiolitis, asthma, chronic obstructive pulmonary
disease (COPD), sinusitis, bacterial superinfection, or cystic
fibrosis.
[0033] In certain embodiment of these methods, a subject in need of
immunization, prophylaxis, or treatment for HRV-infection includes
any individual who comes into frequent, routine, close, and/or
direct contact with another individual who is infected with HRV.
Moreover, a subject in need of the methods of the invention is an
individual who is at an increased risk of infection following
exposure to HRV, e.g. premature infants, those infants who do not
receive their mother's antibodies through breast milk, infants,
children, immunocompromised individuals, malnourished individuals,
those individuals with inflammatory disease (and, therefore, high
cell surface expression ICAM-1, the receptor for HRV), those
individuals without acquired immunity to HRV (e.g., no prior
exposure to HRV), and those individuals who live in areas of high
density (cities), poor nutrition, and/or poor sanitation.
Furthermore, a subject having asthma, bacterial infection within
the upper respiratory tract or chronic obstructive pulmonary
disease (COPD), is particularly susceptible to infection by HRV,
because the cells of the respiratory tract in these individuals
express ICAM-1 at higher levels. Subjects typically develop COPD
following exposure to noxious particles or gases, which most
frequently take the form of cigarette smoke. Thus, smokers have an
increased risk of infection from HRV following exposure to the
virus.
[0034] The invention provides a vaccine comprising either an
isolated anti-HRV antibody as described herein or the epitope to
which an antibody described herein binds.
[0035] The invention provides a kit comprising either an isolated
anti-HRV antibody as described herein or the epitope to which an
antibody described herein binds.
[0036] Other features and advantages of the invention will be
apparent from and are encompassed by the following detailed
description and claims.
BRIEF DESCRIPTION OF THE DRAWINGS
[0037] FIG. 1 is a graph depicting the potency of neutralization
versus human rhinovirus (HRV) serotypes. The potentcy of
neutralization is as the IC50 value on the Y-axis, which was
determined in a microneutralization assay. The cross bar in each
serotype indicates the median IC50 value. Data are representative
of two independent experiments with similar results.
[0038] FIG. 2A-B is a pair of tables indicating the relative
breadth of neutralization by TCN-717 (H17), TCN-722 (L22) and
TCN-716 (F16) antibodies. The relative breadth of neutralization of
each antibody is expressed as the % of virus serotypes neutralized.
The 22 viruses in the major group of clade A (right panel, B) are a
subset within the 38 viruses shown in the left panel (A).
Neutralization by a combination of 2 antibodies is also shown.
[0039] FIG. 3 is a graph depicting the neutralization profile of
TCN-717 (H17), TCN-722 (L22), and TCN-716 (F16) antibodies among 22
clade A major group serotypes and two clade D serotypes, as
determined by microneutralization assay. Asterisks indicate those
serotypes that were analyzed in a cytopathic effects (CPE)
assay.
[0040] FIG. 4A-B is a pair of graphs depicting the direct binding
of TCN-717 (H17) (A) and TCN-722 (L22) (B) to inactivated HRV
virions in ELISA.
[0041] FIG. 5 is a graph depicting binding of TCN-711 (E11) to four
HRV serotypes in infected HeLa cells.
DETAILED DESCRIPTION OF THE INVENTION
[0042] Human Rhinoviruses (HRVs) are small, nonenveloped viruses
that contain a single-strand RNA genome within an icosahedral
capsid. Over 100 serotypes of the virus have been identified (i.e.
approximately 101 serotypes) Rhinoviruses belong to the
Picornaviridae family.
[0043] The primary route of entry for human rhinovirus is the upper
respiratory tract, and, specifically, the nasal mucosa, mouth, and
eyes. Infection also occurs locally; confined to the upper
respiratory tract by the temperature and pH sensitivity of the
virus. The optimal temperature for rhinovirus replication is
33-35.degree. C., and, thus, the virus does not efficiently
replicate at body temperature. There is usually no gastrointestinal
involvement because the virus is unstable in such acidic
conditions. Consequently, rhinovirus does not spread from the
respiratory tract.
[0044] Rhinoviruses travel rapidly throughout the local population
because they are transmitted through air, e.g. via contaminated
respiratory droplets of sneezes and coughs, via contact with
contaminated surfaces, and via person-to-person contact. Infection
also occurs rapidly.
[0045] The virus adheres to cell surface receptors of cells within
the respiratory epithelium within minutes of entering the
respiratory tract. The major receptor for the human rhinovirus is
intercellular adhesion molecule-1 (ICAM-1). The virus uses the
ICAM-1 receptor for both attachment and for uncoating. Because some
HRV serotypes are capable of increasing the endogenous expression
of ICAM-1 within infected cells, and, therefore, increasing the
individual's susceptibility to infection, agents that inhibit
up-regulation, block translation, increase degradation, or prohibit
HRV attachment to ICAM-1 are therapeutic targets. Such targets are
contemplated for use in combination with the anti-HRV antibodies of
the invention.
[0046] Rhinoviruses are positive strand RNA viruses with a naked
nucleocapsid. Positive-sense viral RNA is similar to mRNA, and,
therefore, the single-stranded RNA genome of HRV can be immediately
translated by the host cell. As a further consequence, isolated and
purified RNA of HRV can directly cause infection, though it may be
less infectious than the whole virus particle. For this reason,
isolated and purified HRV RNA is used as an immunogen to develop
and screen for anti-HRV antibodies of the invention. Upon infection
of a cell, the HRV replicates its own genome, initially using the
machinery that is already in place to replicate and/or express
genes within the host cell's genome. The first proteins made by HRV
are enzymes, including RNA-dependent RNA polymerase, which copies
the viral RNA to form a double-stranded replicative form, which
forms the blueprints for the replication of new virions. The virion
is composed of an outer shell, also known as the capsid or
nucleocapsid, which is made of protein. The capsid protects the
contents of the core, establishes to what kind of cell the virion
can attach, and infects that cell. The virion also contains an
interior core composed of the genome, a positive, single-stranded
RNA molecule encoding the few genes required for viral reproduction
which are not present in the host cell. The virus often must supply
its own enzymes for initiating replication of its genome.
[0047] Symptoms appear in most individuals within days. However,
the incubation time can vary from approximately 12 hours to a week
depending upon the health of the subject's immune system, the
subject's genetic predisposition, and the HRV serotype. Viral
shedding can occur a few days before cold symptoms are recognized
by the patient, typically peaks on days 2-7 of the illness, and may
last as long as 3-4 weeks. What most people recognize as cold-like
symptoms are actually the local inflammatory response to the virus
in the respiratory tract, mediated by interferon, which produces
nasal discharge, nasal congestion, sneezing, and throat
irritation.
[0048] The primary HRV infection results in the production of IgA
antibodies in nasal secretions and IgG antibodies in the
bloodstream. Since these viruses do not enter the circulation, the
mucosal IgA response may be the most important for clearing the
immediate infection, and may provide immunity for 1-2 year against
that particular serotype. However, an broadly neutralizing IgG
antibody raised against an invariant epitope of all HRV serotypes
could be used as a vaccine or treatment for HRV infection, and,
this is the foundation of the present invention.
[0049] Although rhinovirus is best known as the primary cause of
the common cold, this virus also causes otitis media,
nasopharyngitis, croup, bronchiolitis, and pneumonia. The common
cold is mild and non-life-threatening in most subjects. However,
even a mild respiratory infection can become serious in an infant
or young child. Antibodies to viral serotypes develop over time.
Because they simply lack the time and experience required to
cultivate a mature immune system, the highest incidence of HRV
infection is found in infants and young children. Children may also
be more contagious than adults because they tend to have higher
virus concentrations in their mucosal secretions and experience a
longer duration of viral shedding. Thus, infants and young children
are at heightened risk for developing, and, ultimately succumbing
to, severe rhinoviral infection, e.g. nasopharyngitis, croup,
bronchiolitis, and pneumonia.
[0050] Individuals who are immune-compromised or malnourished are
also at greater risk for developing HRV infection.
Immune-compromised individuals may have an underlying medical
condition such as acquired immune deficiency syndrome, may be
taking medication to suppress their immune system following a
transplant, may have an autoimmune condition, or may be undergoing
cancer therapy such as radiation. Malnourished individuals may also
be immune-compromised, and, therefore, more susceptible to
infection by HRV.
[0051] Individuals who experience frequent, close, personal contact
with others are also at a heightened risk of exposure to HRV, and,
therefore, infection. For instance, the individual who rides public
transportation versus drives alone to work would be exposed to the
virus with increased frequency. Students who attend classes are
more susceptible during the school year than when they are on
vacation. Individuals who live in cities are more susceptible than
those who live in sparsely populated suburbs. For all of these
reasons, increased risk of exposure to and infection by HRV often
correlates with environmental factors such as poverty and
overcrowding.
[0052] Rhinovirus infection may not cause inflammatory conditions
such as asthma, but it can exacerbate its effects. Similarly, HRV
causes an upper respiratory tract, which causes a blockage of one
or more of the eustachian tubes, and, ultimately, development of
the inflammatory middle ear infection/condition, otitis media.
Binding of ICAM-1 by the rhinovirus could mediate intracellular
signaling cascades that trigger further inflammation in both of
these conditions. Specifically, ICAM-1 signaling could produce a
recruitment of inflammatory immune cells such as macrophages and
granulocytes to the upper respiratory tract (where it exacerbates
asthma), and furthermore, the middle ear (where it could exacerbate
otitis media). Thus, treatment of a subject with a composition of
the invention (which includes an anti-HRV antibody) not only
neutralizes HRV, but also eliminates the HRV-mediated inflammatory
response that exacerbates any underlying inflammatory conditions,
such as asthma, or any secondary inflammatory condition, such as
otitis media.
[0053] Rhinovirus infection can also exacerbate cystic fibrosis.
Cystic fibrosis (also known as CF or mucoviscidosis) is a recessive
genetic disease, which affects the entire body, causing progressive
disability until death. Impaired breathing is the most serious and
well-recognized symptom. Individuals with CF experience frequent
lung infections.
[0054] A preceding HRV infection can also cause bacterial
superinfection, and, therefore, sinusitis.
[0055] The present invention provides a novel method for isolating
novel broad and potent neutralizing monoclonal antibodies against
HRV. The method involves selection of a PBMC donor with high
neutralization titer of antibodies in the plasma. B cells are
screened for neutralization activity prior to rescue of antibodies.
Novel broadly neutralizing antibodies are obtained by emphasizing
neutralization as the initial screen.
[0056] Peripheral Blood Mononuclear Cells (PBMCs) were obtained
from an HRV-infected donor selected for HRV neutralizing activity
in the plasma. Memory B cells were isolated and B cell culture
supernatants were subjected to a primary screen of neutralization
assay in a high throughput format. Optionally, HRV antigen binding
assays using ELISA or like methods were also used as a screen. B
cell lysates corresponding to supernatants exhibiting neutralizing
activity were selected for rescue of monoclonal antibodies by
standard recombinant methods.
[0057] In one embodiment, the recombinant rescue of the monoclonal
antibodies involves use of a B cell culture system as described in
Weitcamp J-H et al., J. Immunol. 171:4680-4688 (2003). Any other
method for rescue of single B cells clones known in the art also
may be employed such as EBV immortalization of B cells (Traggiai
E., et al., Nat. Med. 10(8):871-875 (2004)), electrofusion
(Buchacher, A., et al., 1994. AIDS Res. Hum. Retroviruses
10:359-369), and B cell hybridoma (Karpas A. et al., Proc. Natl.
Acad. Sci. USA 98:1799-1804 (2001).
[0058] In some embodiments, monoclonal antibodies were rescued from
the B cell cultures using variable chain gene-specific RT-PCR, and
transfectant with combinations of H and L chain clones were
screened again for neutralization and HRV antigen binding
activities. mAbs with neutralization properties were selected for
further characterization.
[0059] The antibodies of the invention are able to neutralize
HRV.
[0060] Monoclonal antibodies can be produced by known procedures,
e.g., as described by R. Kennet et al. in "Monoclonal Antibodies
and Functional Cell Lines; Progress and Applications". Plenum Press
(New York), 1984. Further materials and methods applied are based
on known procedures, e.g., such as described in J. Virol.
67:6642-6647, 1993.
[0061] These antibodies can be used as prophylactic or therapeutic
agents upon appropriate formulation, or as a diagnostic tool.
[0062] A "neutralizing antibody" is one that can neutralize the
ability of that pathogen to initiate and/or perpetuate an infection
in a host and/or in target cells in vitro. The invention provides a
neutralizing monoclonal human antibody, wherein the antibody
recognizes an antigen from HRV.
[0063] Preferably an antibody according to the invention is a novel
monoclonal antibody referred to herein as TCN-711 (6893_E11),
TCN-716 (6362_F16), TCN-717 (6358_H17), or TCN-722 (6385_L22).
These antibodies were initially isolated from human samples and are
produced by the B cell cultures referred to as 6893_E11, 6362_F16,
6358_H17, or 6385_L22. These antibodies have been shown to
neutralize HRV in vitro. TCN-711 (6893_E11), TCN-716 (6362_F16),
TCN-717 (6358_H17), and TCN-722 (6385_L22) have been shown to have
broad, potent HRV neutralizing activity.
[0064] The CDRs of the antibody heavy chains are referred to as
CDRH1, CDRH2 and CDRH3, respectively. Similarly, the CDRs of the
antibody light chains are referred to as CDRL1, CDRL2 and CDRL3,
respectively. The positions of the CDR amino acids are defined
according to the IMGT numbering system as: CDR1--IMGT positions 27
to 38, CDR2--IMGT positions 56 to 65 and CDR3--IMGT positions 105
to 117. (Lefranc, M P. et al. 2003 IMGT unique numbering for
immunoglobulin and T cell receptor variable regions and Ig
superfamily V-like domains. Dev Comp Immunol. 27(1):55-77; Lefranc,
M P. 1997. Unique database numbering system for immunogenetic
analysis. Immunology Today, 18:509; Lefranc, M P. 1999. The IMGT
unique numbering for Immunoglobulins, T cell receptors and Ig-like
domains. The Immunologist, 7:132-136.)
[0065] The sequences of the antibodies were determined, including
the sequences of the variable regions of the Gamma heavy and Kappa
or Lambda light chains of the antibodies designated TCN-711
(6893_E11), TCN-716 (6362_F16), TCN-717 (6358_H17), and TCN-722
(6385_L22). In addition, the sequence of each of the
polynucleotides encoding the antibody sequences was determined.
Shown below are the polypeptide and polynucleotide sequences of the
gamma heavy chains and kappa light chains, with the signal peptides
at the N-terminus (or 5' end) and the constant regions at the
C-terminus (or 3' end) of the variable regions, which are shown in
bolded text.
[0066] TCN-711 (6893_E11) gamma heavy chain nucleotide sequence:
coding sequence (leader sequence in italics, variable region in
bold)
TABLE-US-00001 (SEQ ID NO: 1)
ATGAAACACCTGTGGTTCTTCCTCCTCCTGGCGGCAGCTCCCAGATGGGT
CCTGTCCCAGGTGCAGCTACACCAGTGGGGCACAGGAGTGTTGAAGCCTT
CGGGGACCCTGTCCCTCACCTGCGGTGTCTATGGTGGGTCCCTCACTGAT
TTCTACTGGACCTGGATCCGTCAGTCCCCCGCGAGGGGCCTGGAGTGGCT
TGGAGAAATCGATCGTGATGGGGCCACGTACTATAATCCGTCCCTAAAGA
GTCGAATCACCATTTCGATAGACACGTCCAAGAAACAATTCTCCTTGAAT
CTGCGGTCTGTGACCGCCGCGGACAGGGCTGTCTACTACTGTGCGAGGCG
CCCTATGTTACGAGGCGTTTGGGGGAATTTTCGTTCCAACTGGTTCGACC
CCTGGGGCCAGGGAACCCAGGTCACCGTCTCGAGCGCCTCCACCAAGGGC
CCATCGGTCTTCCCCCTGGCACCCTCCTCCAAGAGCACCTCTGGGGGCAC
AGCGGCCCTGGGCTGCCTGGTCAAGGACTACTTCCCCGAACCGGTGACGG
TGTCGTGGAACTCAGGCGCCCTGACCAGCGGCGTGCACACCTTCCCGGCT
GTCCTACAGTCCTCAGGACTCTACTCCCTCAGCAGCGTGGTGACCGTGCC
CTCCAGCAGCTTGGGCACCCAGACCTACATCTGCAACGTGAATCACAAGC
CCAGCAACACCAAGGTGGACAAGAGAGTTGAGCCCAAATCTTGTGACAAA
ACTCACACATGCCCACCGTGCCCAGCACCTGAACTCCTGGGGGGACCGTC
AGTCTTCCTCTTCCCCCCAAAACCCAAGGACACCCTCATGATCTCCCGGA
CCCCTGAGGTCACATGCGTGGTGGTGGACGTGAGCCACGAAGACCCTGAG
GTCAAGTTCAACTGGTACGTGGACGGCGTGGAGGTGCATAATGCCAAGAC
AAAGCCGCGGGAGGAGCAGTACAACAGCACGTACCGTGTGGTCAGCGTCC
TCACCGTCCTGCACCAGGACTGGCTGAATGGCAAGGAGTACAAGTGCAAG
GTCTCCAACAAAGCCCTCCCAGCCCCCATCGAGAAAACCATCTCCAAAGC
CAAAGGGCAGCCCCGAGAACCACAGGTGTACACCCTGCCCCCATCCCGGG
AGGAGATGACCAAGAACCAGGTCAGCCTGACCTGCCTGGTCAAAGGCTTC
TATCCCAGCGACATCGCCGTGGAGTGGGAGAGCAATGGGCAGCCGGAGAA
CAACTACAAGACCACGCCTCCCGTGCTGGACTCCGACGGCTCCTTCTTCC
TCTATAGCAAGCTCACCGTGGACAAGAGCAGGTGGCAGCAGGGGAACGTC
TTCTCATGCTCCGTGATGCATGAGGCTCTGCACAACCACTACACGCAGAA
GAGCCTCTCCCTGTCTCCGGGTAAATGA
[0067] TCN-711 (6893_E11) gamma heavy chain variable region
nucleotide sequence:
TABLE-US-00002 (SEQ ID NO: 2)
CAGGTGCAGCTACACCAGTGGGGCACAGGAGTGTTGAAGCCTTCGGGGAC
CCTGTCCCTCACCTGCGGTGTCTATGGTGGGTCCCTCACTGATTTCTACT
GGACCTGGATCCGTCAGTCCCCCGCGAGGGGCCTGGAGTGGCTTGGAGAA
ATCGATCGTGATGGGGCCACGTACTATAATCCGTCCCTAAAGAGTCGAAT
CACCATTTCGATAGACACGTCCAAGAAACAATTCTCCTTGAATCTGCGGT
CTGTGACCGCCGCGGACAGGGCTGTCTACTACTGTGCGAGGCGCCCTATG
TTACGAGGCGTTTGGGGGAATTTTCGTTCCAACTGGTTCGACCCCTGGGG
CCAGGGAACCCAGGTCACCGTCTCGAGC
[0068] TCN-711 (6893_E11) gamma heavy chain amino acid sequence:
expressed protein with leader sequence in italics and variable
region in bold.
TABLE-US-00003 (SEQ ID NO: 3)
MKHLWFFLLLAAAPRWVLSQVQLHQWGTGVLKPSGTLSLTCGVYGGSLTD
FYWTWIRQSPARGLEWLGEIDRDGATYYNPSLKSRITISIDTSKKQFSLN
LRSVTAADRAVYYCARRPMLRGVWGNFRSNWFDPWGQGTQVTVSSASTKG
PSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPA
VLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKSCDK
THTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPE
VKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCK
VSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGF
YPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNV
FSCSVMHEALHNHYTQKSLSLSPGK
[0069] TCN-711 (6893_E11) gamma heavy chain variable region amino
acid sequence: (Kabat CDRs underlined, Chothia CDRs in bold
italics)
TABLE-US-00004 (SEQ ID NO: 4) QVQLHQWGTGVLKPSGTLSLTCGVY
FYWTWIRQSPARGLEW LG YNPSLKSRITISIDTSKKQFSLNLRSVTAADRAVYY CAR
WGQGTQVTVSS
[0070] TCN-711 (6893_E11) gamma heavy chain Kabat CDRs:
TABLE-US-00005 CDR 1: (SEQ ID NO: 5) DFYWT CDR 2: (SEQ ID NO: 6)
EIDRDGATYYNPSLKS CDR 3: (SEQ ID NO: 7) RPMLRGVWGNFRSNWFDP
[0071] TCN-711 (6893_E11) gamma heavy chain Chothia CDRs:
TABLE-US-00006 CDR 1: (SEQ ID NO: 8) GGSLTD CDR 2: (SEQ ID NO: 9)
EIDRDGATY CDR 3: (SEQ ID NO: 7) RPMLRGVWGNFRSNWFDP
[0072] TCN-711 (6893_E11) lambda light chain nucleotide sequence:
coding sequence (leader sequence in italics, variable region in
bold)
TABLE-US-00007 (SEQ ID NO: 10)
ATGGCCAGCTTCCCTCTCCTCCTCACCCTTCTCATTCACTGCA
CAGGGTCCTGGGCCCAGTCTGTCTTGACGCAGCCGCCCTCAGT
GTCTGCGGCCCCAGGACAGAAGGTCTCCATCTCCTGCTCTGGA
AGCAGCTCCAACATTGGGTATAGTTATGTATCCTGGTATCAAC
AAGTCCCAGGATCAGCCCCCAAACTCCTCATCTATGAGAATAA
TAAGAGACCCTCAGGGATTCCTGACCGATTCTCGGCCTCCAAG
TCTGGCACGTCAGCCACCCTGGACATCACCGGACTCCAGACTG
GGGACGAGGCCGATTATTATTGCGGAACATGGGATACCAGGCT
GTTTGGTGGAGTGTTCGGCGGAGGGACCAAGCTGACCGTTCTA
GGTCAGCCCAAGGCTGCCCCCTCGGTCACTCTGTTCCCGCCCT
CCTCTGAGGAGCTTCAAGCCAACAAGGCCACACTGGTGTGTCT
CATAAGTGACTTCTACCCGGGAGCCGTGACAGTGGCCTGGAAG
GCAGATAGCAGCCCCGTCAAGGCGGGAGTGGAGACCACCACAC
CCTCCAAACAAAGCAACAACAAGTACGCGGCCAGCAGCTACCT
GAGCCTGACGCCTGAGCAGTGGAAGTCCCACAAAAGCTACAGC
TGCCAGGTCACGCATGAAGGGAGCACCGTGGAGAAGACAGTGG
CCCCTACAGAATGTTCATAG
[0073] TCN-711 (6893_E11) lambda light chain variable region
nucleotide sequence:
TABLE-US-00008 (SEQ ID NO: 11)
CAGTCTGTCTTGACGCAGCCGCCCTCAGTGTCTGCGGCCCCAG
GACAGAAGGTCTCCATCTCCTGCTCTGGAAGCAGCTCCAACAT
TGGGTATAGTTATGTATCCTGGTATCAACAAGTCCCAGGATCA
GCCCCCAAACTCCTCATCTATGAGAATAATAAGAGACCCTCAG
GGATTCCTGACCGATTCTCGGCCTCCAAGTCTGGCACGTCAGC
CACCCTGGACATCACCGGACTCCAGACTGGGGACGAGGCCGAT
TATTATTGCGGAACATGGGATACCAGGCTGTTTGGTGGAGTGT
TCGGCGGAGGGACCAAGCTGACCGTTCTA
[0074] TCN-711 (6893_E11) lambda light chain amino acid sequence:
expressed protein with leader sequence in italics and variable
region in bold.
TABLE-US-00009 (SEQ ID NO: 12)
MASFPLLLTLLIHCTGSWAQSVLTQPPSVSAAPGQKVSISCSG
SSSNIGYSYVSWYQQVPGSAPKLLIYENNKRPSGIPDRFSASK
SGTSATLDITGLQTGDEADYYCGTWDTRLFGGVFGGGTKLTVL
GQPKAAPSVTLFPPSSEELQANKATLVCLISDFYPGAVTVAWK
ADSSPVKAGVETTTPSKQSNNKYAASSYLSLTPEQWKSHKSYS
CQVTHEGSTVEKTVAPTECS
[0075] TCN-711 (6893_E11) lambda light chain variable region amino
acid sequence: (Kabat CDRs underlined, Chothia CDRs in bold
italics)
TABLE-US-00010 (SEQ ID NO: 13) QSVLTQPPSVSAAPGQKVSISC WYQQVPGS
APKLLIY GIPDRFSASKSGTSATLDITGLQTGDEAD YYC FGGGTKLTVL
[0076] TCN-711 (6893_E11) lambda light chain Kabat CDRs:
TABLE-US-00011 CDR 1: (SEQ ID NO: 14) SGSSSNIGYSYVS CDR 2: (SEQ ID
NO: 15) ENNKRPS CDR 3: (SEQ ID NO: 16) GTWDTRLFGGV
[0077] TCN-711 (6893_E11) lambda light chain Chothia CDRs:
TABLE-US-00012 CDR 1: (SEQ ID NO: 14) SGSSSNIGYSYVS CDR 2: (SEQ ID
NO: 15) ENNKRPS CDR 3: (SEQ ID NO: 16) GTWDTRLFGGV
[0078] TCN-716 (6362_F16) gamma heavy chain nucleotide sequence:
coding sequence (leader sequence in italics, variable region in
bold)
TABLE-US-00013 (SEQ ID NO: 17)
ATGGAGTTTGGGCTGAGCTGGGTTCTCCTTGTTGCCATTTTAAAAG
GTGCCCAGTGTGAGGTGCAACTGGTGGAGTCTGGGGGAGGCTTGGT
CCTGCCGGGGGGCTCTCTGAGACTCTCGTGTTCAGCGTCTGGATTC
ACATTGACTGACTTTGCTATGCACTGGGTCCGACAGGCTCCAGGGA
AGGGACTGGAGCTCGTCTCAAGTATTAGTCGGGATGGTTCTACTAA
ATACTCTGGAGACTCCGTGAAGGGCAGGGTCGCCATCTCCAGGGAC
AGTGTGGAGAATAAGTTGCATCTTCAGATGAGCGGTCTGAGGTCTG
CGGACACGGCTGTGTATTATTGTGTGAGAGACTCCCCCTATTATCT
TGATATTGTTGGTTATCGATACTTCCACCACTATGGAATGGACGTC
TGGGGCCAGGGGACCACGGTCACCGTCTCGAGCGCCTCCACCAAGG
GCCCATCGGTCTTCCCCCTGGCACCCTCCTCCAAGAGCACCTCTGG
GGGCACAGCGGCCCTGGGCTGCCTGGTCAAGGACTACTTCCCCGAA
CCGGTGACGGTGTCGTGGAACTCAGGCGCCCTGACCAGCGGCGTGC
ACACCTTCCCGGCTGTCCTACAGTCCTCAGGACTCTACTCCCTCAG
CAGCGTGGTGACCGTGCCCTCCAGCAGCTTGGGCACCCAGACCTAC
ATCTGCAACGTGAATCACAAGCCCAGCAACACCAAGGTGGACAAGA
GAGTTGAGCCCAAATCTTGTGACAAAACTCACACATGCCCACCGTG
CCCAGCACCTGAACTCCTGGGGGGACCGTCAGTCTTCCTCTTCCCC
CCAAAACCCAAGGACACCCTCATGATCTCCCGGACCCCTGAGGTCA
CATGCGTGGTGGTGGACGTGAGCCACGAAGACCCTGAGGTCAAGTT
CAACTGGTACGTGGACGGCGTGGAGGTGCATAATGCCAAGACAAAG
CCGCGGGAGGAGCAGTACAACAGCACGTACCGTGTGGTCAGCGTCC
TCACCGTCCTGCACCAGGACTGGCTGAATGGCAAGGAGTACAAGTG
CAAGGTCTCCAACAAAGCCCTCCCAGCCCCCATCGAGAAAACCATC
TCCAAAGCCAAAGGGCAGCCCCGAGAACCACAGGTGTACACCCTGC
CCCCATCCCGGGAGGAGATGACCAAGAACCAGGTCAGCCTGACCTG
CCTGGTCAAAGGCTTCTATCCCAGCGACATCGCCGTGGAGTGGGAG
AGCAATGGGCAGCCGGAGAACAACTACAAGACCACGCCTCCCGTGC
TGGACTCCGACGGCTCCTTCTTCCTCTATAGCAAGCTCACCGTGGA
CAAGAGCAGGTGGCAGCAGGGGAACGTCTTCTCATGCTCCGTGATG
CATGAGGCTCTGCACAACCACTACACGCAGAAGAGCCTCTCCCTGT CTCCGGGTAAATGA
[0079] TCN-716 (6362_F16) gamma heavy chain variable region
nucleotide sequence:
TABLE-US-00014 (SEQ ID NO: 18)
GAGGTGCAACTGGTGGAGTCTGGGGGAGGCTTGGTCCTGCCGGGG
GGCTCTCTGAGACTCTCGTGTTCAGCGTCTGGATTCACATTGACT
GACTTTGCTATGCACTGGGTCCGACAGGCTCCAGGGAAGGGACTG
GAGCTCGTCTCAAGTATTAGTCGGGATGGTTCTACTAAATACTCT
GGAGACTCCGTGAAGGGCAGGGTCGCCATCTCCAGGGACAGTGTG
GAGAATAAGTTGCATCTTCAGATGAGCGGTCTGAGGTCTGCGGAC
ACGGCTGTGTATTATTGTGTGAGAGACTCCCCCTATTATCTTGAT
ATTGTTGGTTATCGATACTTCCACCACTATGGAATGGACGTCTGG
GGCCAGGGGACCACGGTCACCGTCTCGAGC
[0080] TCN-716 (6362_F16) gamma heavy chain amino acid sequence:
expressed protein with leader sequence in italics and variable
region in bold.
TABLE-US-00015 (SEQ ID NO: 19)
MEFGLSWVLLVAILKGAQCEVQLVESGGGLVLPGGSLRLSCSASG
FTLTDFAMHWVRQAPGKGLELVSSISRDGSTKYSGDSVKGRVAIS
RDSVENKLHLQMSGLRSADTAVYYCVRDSPYYLDIVGYRYFHHYG
MDVWGQGTTVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKD
YFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSL
GTQTYICNVNHKPSNTKVDKRVEPKSCDKTHTCPPCPAPELLGGP
SVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVE
VHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKAL
PAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFY
PSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQ
QGNVFSCSVMHEALHNHYTQKSLSLSPGK
[0081] TCN-716 (6362_F16) gamma heavy chain variable region amino
acid sequence: (Kabat CDRs underlined, Chothia CDRs in bold
italics)
TABLE-US-00016 (SEQ ID NO: 20) EVQLVESGGGLVLPGGSLRLSCSAS
FAMHWVRQAPGKG LELVS SGDSVKGRVAISRDSVENKLHLQMSGLRS ADTAVYYCVR
WGQGTTVTVSS
[0082] TCN-716 (6362_F16) gamma heavy chain Kabat CDRs:
TABLE-US-00017 CDR 1: (SEQ ID NO: 21) DFAMH CDR 2: (SEQ ID NO: 22)
SISRDGSTKYSGDSVKG CDR 3: (SEQ ID NO: 23) DSPYYLDIVGYRYFHHYGMDV
[0083] TCN-716 (6362_F16) gamma heavy chain Chothia CDRs:
TABLE-US-00018 CDR 1: (SEQ ID NO: 24) GFTLTD CDR 2: (SEQ ID NO: 25)
SISRDGSTKY CDR 3: (SEQ ID NO: 23) DSPYYLDIVGYRYFHHYGMDV
[0084] TCN-716 (6362_F16) kappa light chain nucleotide sequence:
coding sequence (leader sequence in italics, variable region in
bold)
TABLE-US-00019 (SEQ ID NO: 26)
ATGGAAACCCCAGCTCAGCTTCTCTTCCTCCTGCTACTCTGGCTCCC
AGATACCACCGGAGAGATTGTGTTGACGCAGTCGCCAGGCACCCTGT
CTTTGTCTCCAGGGGACAGAGTCACCCTCTCCTGCAGGGCCAGTCAA
ATTCTTCACAGCTATAATTTAGCCTGGTATCAGCACAGACCTGGCCA
GGCTCCCAGGCTCCTCATTTATGGTGCATATAACAGGGCCAGTGTGG
GCATCCCAGACAGGTTCAGTGGCAGGTCTGGGGCAGACTTCACCCTC
ACCATCGGCAGACTGCAGCGTGACGATTTTGCAGTTTATTACTGTCA
ACAGTATGGTGACTCACCATCACCAGGCCTCACTTTCGGCGGAGGAA
CCAAACTGGAGTTCAAACGTACGGTGGCTGCACCATCTGTCTTCATC
TTCCCGCCATCTGATGAGCAGTTGAAATCTGGAACTGCCTCTGTTGT
GTGCCTGCTGAATAACTTCTATCCCAGAGAGGCCAAAGTACAGTGGA
AGGTGGATAACGCCCTCCAATCGGGTAACTCCCAGGAGAGTGTCACA
GAGCAGGACAGCAAGGACAGCACCTACAGCCTCAGCAGCACCCTGAC
GCTGAGCAAAGCAGACTACGAGAAACACAAAGTCTACGCCTGCGAAG
TCACCCATCAGGGCCTGAGCTCGCCCGTCACAAAGAGCTTCAACAG GGGAGAGTGTTAG
[0085] TCN-716 (6362_F16) kappa light chain variable region
nucleotide sequence:
TABLE-US-00020 (SEQ ID NO: 27)
GAGATTGTGTTGACGCAGTCGCCAGGCACCCTGTCTTTGTCTCCAG
GGGACAGAGTCACCCTCTCCTGCAGGGCCAGTCAAATTCTTCACAG
CTATAATTTAGCCTGGTATCAGCACAGACCTGGCCAGGCTCCCAGG
CTCCTCATTTATGGTGCATATAACAGGGCCAGTGGCATCCCAGACA
GGTTCAGTGGCAGTGGGTCTGGGGCAGACTTCACCCTCACCATCGG
CAGACTGCAGCGTGACGATTTTGCAGTTTATTACTGTCAACAGTAT
GGTGACTCACCATCACCAGGCCTCACTTTCGGCGGAGGAACCAAAC TGGAGTTCAAA
[0086] TCN-716 (6362_F16) kappa light chain amino acid sequence:
expressed protein with leader sequence in italics and variable
region in bold.
TABLE-US-00021 (SEQ ID NO: 28)
METPAQLLFLLLLWLPDTTGEIVLTQSPGTLSLSPGDRVTLSCRA
SQILHSYNLAWYQHRPGQAPRLLIYGAYNRASGIPDRFSGSGSGA
DFTLTIGRLQRDDFAVYYCQQYGDSPSPGLTFGGGTKLEFKRTVA
APSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQS
GNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGL SSPVTKSFNRGEC
[0087] TCN-716 (6362_F16) kappa light chain variable region amino
acid sequence: (Kabat CDRs underlined, Chothia CDRs in bold
italics)
TABLE-US-00022 (SEQ ID NO: 29) EIVLTQSPGTLSLSPGDRVTLSC WYQHRPGQ
APRLLIY GIPDRFSGSGSGADFTLTIGRLQRDDFAVY YC GGGTKLEFK
[0088] TCN-716 (6362_F16) kappa light chain Kabat CDRs:
TABLE-US-00023 CDR 1: (SEQ ID NO: 30) RASQILHSYNLA CDR 2: (SEQ ID
NO: 31) GAYNRAS CDR 3: (SEQ ID NO: 32) QQYGDSPSPGLT
[0089] TCN-716 (6362_F16) kappa light chain Chothia CDRs:
TABLE-US-00024 CDR 1: (SEQ ID NO: 30) RASQILHSYNLA CDR 2: (SEQ ID
NO: 31) GAYNRAS CDR 3: (SEQ ID NO: 32) QQYGDSPSPGLT
[0090] TCN-717 (6358_H17) gamma heavy chain nucleotide sequence:
coding sequence (leader sequence in italics, variable region in
bold)
TABLE-US-00025 (SEQ ID NO: 33)
ATGAAACACCTGTGGTTCTTCCTCCTACTGATGGCGGCTCCCAGATG
GGTCCTGTCCCAGCTGCAACTGCTTGAGTCGGGCCCAAGACTGGTGA
AGGCTTCGGAGACCCTGTCACTCACCTGCAGTGTCCCTATGGGCTCC
ATCCTCCAAAATGATTATCATTGGGCCTGGGTCCGCCAGCCCCCAGG
GAGGGGCCTGGAGTGGATTGGGAGTGTTCACTATAGACAAAAATCCT
ACTACAGCCCGTCCCTCAAGAGCCGAGTCTTCATGTCCGTAGACACG
TCCAGAGACCAGTTCTCCCTAAAACTCTTCTCTCTGGCCGCCGCGGA
CACGGCCGTATATTATTGTGCGAGACATAATCGGGAAGATTATTATG
ACAGTAATGCCTACTTTGACGAGTGGGGCCTGGGAGCTCGGATCACC
GTCTCGAGCGCCTCCACCAAGGGCCCATCGGTCTTCCCCCTGGCACC
CTCCTCCAAGAGCACCTCTGGGGGCACAGCGGCCCTGGGCTGCCTGG
TCAAGGACTACTTCCCCGAACCGGTGACGGTGTCGTGGAACTCAGGC
GCCCTGACCAGCGGCGTGCACACCTTCCCGGCTGTCCTACAGTCCTC
AGGACTCTACTCCCTCAGCAGCGTGGTGACCGTGCCCTCCAGCAGCT
TGGGCACCCAGACCTACATCTGCAACGTGAATCACAAGCCCAGCAAC
ACCAAGGTGGACAAGAGAGTTGAGCCCAAATCTTGTGACAAAACTCA
CACATGCCCACCGTGCCCAGCACCTGAACTCCTGGGGGGACCGTCAG
TCTTCCTCTTCCCCCCAAAACCCAAGGACACCCTCATGATCTCCCGG
ACCCCTGAGGTCACATGCGTGGTGGTGGACGTGAGCCACGAAGACCC
TGAGGTCAAGTTCAACTGGTACGTGGACGGCGTGGAGGTGCATAATG
CCAAGACAAAGCCGCGGGAGGAGCAGTACAACAGCACGTACCGTGTG
GTCAGCGTCCTCACCGTCCTGCACCAGGACTGGCTGAATGGCAAGGA
GTACAAGTGCAAGGTCTCCAACAAAGCCCTCCCAGCCCCCATCGAGA
AAACCATCTCCAAAGCCAAAGGGCAGCCCCGAGAACCACAGGTGTAC
ACCCTGCCCCCATCCCGGGAGGAGATGACCAAGAACCAGGTCAGCCT
GACCTGCCTGGTCAAAGGCTTCTATCCCAGCGACATCGCCGTGGAGT
GGGAGAGCAATGGGCAGCCGGAGAACAACTACAAGACCACGCCTCCC
GTGCTGGACTCCGACGGCTCCTTCTTCCTCTATAGCAAGCTCACCGT
GGACAAGAGCAGGTGGCAGCAGGGGAACGTCTTCTCATGCTCCGTGA
TGCATGAGGCTCTGCACAACCACTACACGCAGAAGAGCCTCTCCCTG TCTCCGGGTAAATGA
[0091] TCN-717 (6358_H17) gamma heavy chain variable region
nucleotide sequence:
TABLE-US-00026 (SEQ ID NO: 34)
CAGCTGCAACTGCTTGAGTCGGGCCCAAGACTGGTGAAGGCTTCGG
AGACCCTGTCACTCACCTGCAGTGTCCCTATGGGCTCCATCCTCCA
AAATGATTATCATTGGGCCTGGGTCCGCCAGCCCCCAGGGAGGGGC
CTGGAGTGGATTGGGAGTGTTCACTATAGACAAAAATCCTACTACA
GCCCGTCCCTCAAGAGCCGAGTCTTCATGTCCGTAGACACGTCCAG
AGACCAGTTCTCCCTAAAACTCTTCTCTCTGGCCGCCGCGGACACG
GCCGTATATTATTGTGCGAGACATAATCGGGAAGATTATTATGACAG
TAATGCCTACTTTGACGAGTGGGGCCTGGGAGCTCGGATCACCGTCT CGAGC
[0092] TCN-717 (6358_H17) gamma heavy chain amino acid sequence:
expressed protein with leader sequence in italics and variable
region in bold.
TABLE-US-00027 (SEQ ID NO: 35)
MKHLWFFLLLMAAPRWVLSQLQLLESGPRLVKASETLSLTCSVPMGS
ILQNDYHWAWVRQPPGRGLEWIGSVHYRQKSYYSPSLKSRVFMSVDT
SRDQFSLKLFSLAAADTAVYYCARHNREDYYDSNAYFDEWGLGARIT
VSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSG
ALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSN
TKVDKRVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISR
TPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRV
VSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVY
TLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPP
VLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSL SPGK
[0093] TCN-717 (6358_H17) gamma heavy chain variable region amino
acid sequence: (Kabat CDRs underlined, Chothia CDRs in bold
italics)
TABLE-US-00028 (SEQ ID NO: 36) QLQLLESGPRLVKASETLSLTCSVP
YHWAWVRQPPG RGLEWIG YSPSLKSRVFMSVDTSRDQFSLKLFSLA AADTAVYYCAR
WGLGARITVSS
[0094] TCN-717 (6358_H17) gamma heavy chain Kabat CDRs:
TABLE-US-00029 CDR 1: (SEQ ID NO: 37) QNDYHWA CDR 2: (SEQ ID NO:
38) SVHYRQKSYYSPSLKS CDR 3: (SEQ ID NO: 39) HNREDYYDSNAYFDE
[0095] TCN-717 (6358_H17) gamma heavy chain Chothia CDRs:
TABLE-US-00030 CDR 1: (SEQ ID NO: 40) MGSILQND CDR 2: (SEQ ID NO:
41) SVHYRQKSY CDR 3: (SEQ ID NO: 39) HNREDYYDSNAYFDE
[0096] TCN-717 (6358_H17) lambda light chain nucleotide sequence:
coding sequence (leader sequence in italics, variable region in
bold)
TABLE-US-00031 (SEQ ID NO: 42)
ATGGCCAGCTTCCCTCTCCTCCTCGGCGTCCTTGCTTACTGCACA
GGGTCGGGGGCCTCCTATGAGTTGTCTCAGCCACCCTCAGTGTCC
GTGTTCCCGGGACAGACAGCAAGCATCACCTGTTCTGGAGATGAC
TTGGAAAACACCCTTGTTTGTTGGTATCAACAAAAGTCAGGGCAGT
CCCCTGTGTTGGTCGTCTATCAAGATTCCAAGCGGCCCTCAGGGAT
CCCTGAGCGATTCTCTGGCTCCAGAGTTAAAGACACAGCCACTCTG
ACCATCAGCGGGACGCAGGCTTTCGATGAGGCTGACTATTATTGTC
AGACGTGGCACAGGTCCACCGCCCAGTATGTCTTCGGACCTGGGAC
CAAGGTCACCGTTCTAGGTCAGCCCAAGGCTGCCCCCTCGGTCACT
CTGTTCCCGCCCTCCTCTGAGGAGCTTCAAGCCAACAAGGCCACAC
TGGTGTGTCTCATAAGTGACTTCTACCCGGGAGCCGTGACAGTGG
CCTGGAAGGCAGATAGCAGCCCCGTCAAGGCGGGAGTGGAGACCA
CCACACCCTCCAAACAAAGCAACAACAAGTACGCGGCCAGCAGCT
ACCTGAGCCTGACGCCTGAGCAGTGGAAGTCCCACAAAAGCTACA
GCTGCCAGGTCACGCATGAAGGGAGCACCGTGGAGAAGACAGTGG
CCCCTACAGAATGTTCATAG
[0097] TCN-717 (6358_H17) lambda light chain variable region
nucleotide sequence:
TABLE-US-00032 (SEQ ID NO: 43)
TCCTATGAGTTGTCTCAGCCACCCTCAGTGTCCGTGTTCCCGGGAC
AGACAGCAAGCATCACCTGTTCTGGAGATGACTTGGAAAACACCCT
TGTTTGTTGGTATCAACAAAAGTCAGGGCAGTCCCCTGTGTTGGTC
GTCTATCAAGATTCCAAGCGGCCCTCAGGGATCCCTGAGCGATTCT
CTGGCTCCAGAGTTAAAGACACAGCCACTCTGACCATCAGCGGGAC
GCAGGCTTTCGATGAGGCTGACTATTATTGTCAGACGTGGCACAGG
TCCACCGCCCAGTATGTCTTCGGACCTGGGACCAAGGTCACCGTTC TA
[0098] TCN-717 (6358_H17) lambda light chain amino acid sequence:
expressed protein with leader sequence in italics and variable
region in bold.
TABLE-US-00033 (SEQ ID NO: 44)
MASFPLLLGVLAYCTGSGASYELSQPPSVSVFPGQTASITCSGD
DLENTLVCWYQQKSGQSPVLVVYQDSKRPSGIPERFSGSRVKDT
ATLTISGTQAFDEADYYCQTWHRSTAQYVFGPGTKVTVLGQPKA
APSVTLFPPSSEELQANKATLVCLISDFYPGAVTVAWKADSSPV
KAGVETTTPSKQSNNKYAASSYLSLTPEQWKSHKSYSCQVTHEG STVEKTVAPTECS
[0099] TCN-717 (6358_H17) lambda light chain variable region amino
acid sequence: (Kabat CDRs underlined, Chothia CDRs in bold
italics)
TABLE-US-00034 (SEQ ID NO: 45) SYELSQPPSVSVFPGQTASITC
WYQQKSGQSPVLVVY GIPERFSGSRVKDTATLTISGTQAFDEADYYC FGPGTKVTVL
[0100] TCN-717 (6358_H17) lambda light chain Kabat CDRs:
TABLE-US-00035 CDR 1: (SEQ ID NO: 46) SGDDLENTLVC CDR 2: (SEQ ID
NO: 47) QDSKRPS CDR 3: (SEQ ID NO: 48) QTWHRSTAQYV
[0101] TCN-717 (6358_H17) lambda light chain Chothia CDRs:
TABLE-US-00036 CDR 1: (SEQ ID NO: 46) SGDDLENTLVC CDR 2: (SEQ ID
NO: 47) QDSKRPS CDR 3: (SEQ ID NO: 48) QTWHRSTAQYV
[0102] TCN-722 (6385_L22) gamma heavy chain nucleotide sequence:
coding sequence (leader sequence in italics, variable region in
bold)
TABLE-US-00037 (SEQ ID NO: 49)
ATGAAACACCTGTGGTTCTTCCTCCTGCTGGTGGCGGCTCCCAGATGGGT
CCTGTCCCAGTTGCAGCTGCTTGAGTCGGGCCCAGGACTGGTGAAGCCTT
CGGAGACCCTTTCACTCACCTGCAGTGTCTCTGGGGACTCCCTCCTCAGT
AATGATCAATACTGGGCCTGGGTCCGCCAGCCCCCAGGGAGGGGCCTGGA
GTGGATTGGGAGTGTTCACTATAGACGACGAAACTACTACAGCCCGTCCC
TGGAGAGCCGGATCTTCATGTCAGTAGACACGTCCAGAAACGAGTTCTCC
TTAAAAGTTTTCTCTGTGACGGCCGCGGACACGGCCGTGTATTATTGTGC
GAGACACAATTGGGAAGATTATTATGAGAGTAATGCCTACTTTGACTACT
GGGGCCTGGGAACCCGGATCACCGTCTCGAGCGCCTCCACCAAGGGCCCA
TCGGTCTTCCCCCTGGCACCCTCCTCCAAGAGCACCTCTGGGGGCACAGC
GGCCCTGGGCTGCCTGGTCAAGGACTACTTCCCCGAACCGGTGACGGTGT
CGTGGAACTCAGGCGCCCTGACCAGCGGCGTGCACACCTTCCCGGCTGTC
CTACAGTCCTCAGGACTCTACTCCCTCAGCAGCGTGGTGACCGTGCCCTC
CAGCAGCTTGGGCACCCAGACCTACATCTGCAACGTGAATCACAAGCCCA
GCAACACCAAGGTGGACAAGAGAGTTGAGCCCAAATCTTGTGACAAAACT
CACACATGCCCACCGTGCCCAGCACCTGAACTCCTGGGGGGACCGTCAGT
CTTCCTCTTCCCCCCAAAACCCAAGGACACCCTCATGATCTCCCGGACCC
CTGAGGTCACATGCGTGGTGGTGGACGTGAGCCACGAAGACCCTGAGGTC
AAGTTCAACTGGTACGTGGACGGCGTGGAGGTGCATAATGCCAAGACAAA
GCCGCGGGAGGAGCAGTACAACAGCACGTACCGTGTGGTCAGCGTCCTCA
CCGTCCTGCACCAGGACTGGCTGAATGGCAAGGAGTACAAGTGCAAGGTC
TCCAACAAAGCCCTCCCAGCCCCCATCGAGAAAACCATCTCCAAAGCCAA
AGGGCAGCCCCGAGAACCACAGGTGTACACCCTGCCCCCATCCCGGGAGG
AGATGACCAAGAACCAGGTCAGCCTGACCTGCCTGGTCAAAGGCTTCTAT
CCCAGCGACATCGCCGTGGAGTGGGAGAGCAATGGGCAGCCGGAGAACAA
CTACAAGACCACGCCTCCCGTGCTGGACTCCGACGGCTCCTTCTTCCTCT
ATAGCAAGCTCACCGTGGACAAGAGCAGGTGGCAGCAGGGGAACGTCTTC
TCATGCTCCGTGATGCATGAGGCTCTGCACAACCACTACACGCAGAAGAG
CCTCTCCCTGTCTCCGGGTAAATGA
[0103] TCN-722 (6385_L22) gamma heavy chain variable region
nucleotide sequence:
TABLE-US-00038 (SEQ ID NO: 50)
CAGTTGCAGCTGCTTGAGTCGGGCCCAGGACTGGTGAAGCCTTCGGAGAC
CCTTTCACTCACCTGCAGTGTCTCTGGGGACTCCCTCCTCAGTAATGATC
AATACTGGGCCTGGGTCCGCCAGCCCCCAGGGAGGGGCCTGGAGTGGATT
GGGAGTGTTCACTATAGACGACGAAACTACTACAGCCCGTCCCTGGAGAG
CCGGATCTTCATGTCAGTAGACACGTCCAGAAACGAGTTCTCCTTAAAAG
TTTTCTCTGTGACGGCCGCGGACACGGCCGTGTATTATTGTGCGAGACAC
AATTGGGAAGATTATTATGAGAGTAATGCCTACTTTGACTACTGGGGCCT
GGGAACCCGGATCACCGTCTCGAGC
[0104] TCN-722 (6385_L22) gamma heavy chain amino acid sequence:
expressed protein with leader sequence in italics and variable
region in bold.
TABLE-US-00039 (SEQ ID NO: 51)
MKHLWFFLLLVAAPRWVLSQLQLLESGPGLVKPSETLSLTCSVSGDSLLS
NDQYWAWVRQPPGRGLEWIGSVHYRRRNYYSPSLESRIFMSVDTSRNEFS
LKVFSVTAADTAVYYCARHNWEDYYESNAYFDYWGLGTRITVSSASTKGP
SVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAV
LQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKSCDKT
HTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEV
KFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKV
SNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFY
PSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVF
SCSVMHEALHNHYTQKSLSLSPGK
[0105] TCN-722 (6385_L22) gamma heavy chain variable region amino
acid sequence: (Kabat CDRs underlined, Chothia CDRs in bold
italics)
TABLE-US-00040 (SEQ ID NO: 52) QLQLLESGPGLVKPSETLSLTCSVS
QYWAWVRQPPGRGLEW IG YSPSLESRIFMSVDTSRNEFSLKVFSVTAADTAVYYCAR
WGLGTRITVSS
[0106] TCN-722 (6385_L22) gamma heavy chain Kabat CDRs:
TABLE-US-00041 CDR 1: (SEQ ID NO: 53) SNDQYWA CDR 2: (SEQ ID NO:
54) SVHYRRRNYYSPSLES CDR 3: (SEQ ID NO: 55) HNWEDYYESNAYFDY
[0107] TCN-722 (6385_L22) gamma heavy chain Chothia CDRs:
TABLE-US-00042 CDR 1: (SEQ ID NO: 56) GDSLLSND CDR 2: (SEQ ID NO:
57) SVHYRRRNY CDR 3: (SEQ ID NO: 55) HNWEDYYESNAYFDY
[0108] TCN-722 (6385_L22) lambda light chain nucleotide sequence:
coding sequence (leader sequence in italics, variable region in
bold)
TABLE-US-00043 (SEQ ID NO: 58)
ATGGCCAGCTTCCCTCTCTTCCTCGGCGTCCTTGCTTACTGCACAGGATC
GGGGGCCTCCTTTGACTTGACTCAGCCACCCTCAGTGTCCGTGTCCCCAG
GACAGACCGCAACCATCACCTGTTCTGGAGATCAATTGGAAAATACCTTT
GTTTGCTGGTATCAACAGAGGTCAGGCCAGGCCCCTGTGTTGGTCATCTA
TCAAGGTTCCAAGCGGCCCTCAGGGATCCCTGAGCGATTCTCTGGCTCCA
GGTCTGGGAACACAGCCACTCTGACCATCAGCAGGACCCAGGCTTTGGAT
GAGGCTGACTATTACTGTCAGGCGTGGGACAGGTCCACCGCCCACTATGT
CTTCGGACCTGGGACCAAGGTCACCGTTCTAGGTCAGCCCAAGGCTGCCC
CCTCGGTCACTCTGTTCCCGCCCTCCTCTGAGGAGCTTCAAGCCAACAAG
GCCACACTGGTGTGTCTCATAAGTGACTTCTACCCGGGAGCCGTGACAGT
GGCCTGGAAGGCAGATAGCAGCCCCGTCAAGGCGGGAGTGGAGACCACCA
CACCCTCCAAACAAAGCAACAACAAGTACGCGGCCAGCAGCTACCTGAGC
CTGACGCCTGAGCAGTGGAAGTCCCACAAAAGCTACAGCTGCCAGGTCAC
GCATGAAGGGAGCACCGTGGAGAAGACAGTGGCCCCTACAGAATGTTCAT AG
[0109] TCN-722 (6385_L22) lambda light chain variable region
nucleotide sequence:
TABLE-US-00044 (SEQ ID NO: 59)
TCCTTTGACTTGACTCAGCCACCCTCAGTGTCCGTGTCCCCAGGACAGAC
CGCAACCATCACCTGTTCTGGAGATCAATTGGAAAATACCTTTGTTTGCT
GGTATCAACAGAGGTCAGGCCAGGCCCCTGTGTTGGTCATCTATCAAGGT
TCCAAGCGGCCCTCAGGGATCCCTGAGCGATTCTCTGGCTCCAGGTCTGG
GAACACAGCCACTCTGACCATCAGCAGGACCCAGGCTTTGGATGAGGCTG
ACTATTACTGTCAGGCGTGGGACAGGTCCACCGCCCACTATGTCTTCGGA
CCTGGGACCAAGGTCACCGTTCTA
[0110] TCN-722 (6385_L22) lambda light chain amino acid sequence:
expressed protein with leader sequence in italics and variable
region in bold.
TABLE-US-00045 (SEQ ID NO: 60)
MASFPLFLGVLAYCTGSGASFDLTQPPSVSVSPGQTATITCSGDQLENTF
VCWYQQRSGQAPVLVIYQGSKRPSGIPERFSGSRSGNTATLTISRTQALD
EADYYCQAWDRSTAHYVFGPGTKVTVLGQPKAAPSVTLFPPSSEELQANK
ATLVCLISDFYPGAVTVAWKADSSPVKAGVETTTPSKQSNNKYAASSYLS
LTPEQWKSHKSYSCQVTHEGSTVEKTVAPTECS
[0111] TCN-722 (6385_L22) lambda light chain variable region amino
acid sequence: (Kabat CDRs underlined, Chothia CDRs in bold
italics)
TABLE-US-00046 (SEQ ID NO: 61) SFDLTQPPSVSVSPGQTATITC
WYQQRSGQAPVLVIY GIPERFSGSRSGNTATLTISRTQALDEADYYC FGPGTKVTVL
[0112] TCN-722 (6385_L22) lambda light chain Kabat CDRs:
TABLE-US-00047 CDR 1: (SEQ ID NO: 62) SGDQLENTFVC CDR 2: (SEQ ID
NO: 63) QGSKRPS CDR 3: (SEQ ID NO: 64) QAWDRSTAHYV
[0113] TCN-722 (6385_L22) lambda light chain Chothia CDRs:
TABLE-US-00048 CDR 1: (SEQ ID NO: 62) SGDQLENTFVC CDR 2: (SEQ ID
NO: 63) QGSKRPS CDR 3: (SEQ ID NO: 64) QAWDRSTAHYV
[0114] The TCN-711 (6893_E11) antibody includes a heavy chain
variable region (SEQ ID NO: 4), encoded by the nucleic acid
sequence shown in SEQ ID NO: 2, and a light chain variable region
(SEQ ID NO: 13) encoded by the nucleic acid sequence shown in SEQ
ID NO:
[0115] The heavy chain CDRs of the TCN-711 (6893_E11) antibody have
the following sequences per Kabat definition: CDR 1: DFYWT (SEQ ID
NO: 5), CDR 2: EIDRDGATYYNPSLKS (SEQ ID NO: 6) and CDR 3:
RPMLRGVWGNFRSNWFDP (SEQ ID NO: 7). The light chain CDRs of the
TCN-711 (6893_E11) antibody have the following sequences per Kabat
definition: CDR 1: SGSSSNIGYSYVS (SEQ ID NO: 14), CDR 2: ENNKRPS
(SEQ ID NO: 15), and CDR 3: GTWDTRLFGGV (SEQ ID NO: 16).
[0116] The heavy chain CDRs of the TCN-711 (6893_E11) antibody have
the following sequences per Chothia definition: CDR 1: GGSLTD (SEQ
ID NO: 8), CDR 2: EIDRDGATY (SEQ ID NO: 9), and CDR 3:
RPMLRGVWGNFRSNWFDP (SEQ ID NO: 7). The light chain CDRs of the
TCN-711 (6893_E11) antibody have the following sequences per
Chothia definition: CDR 1: SGSSSNIGYSYVS (SEQ ID NO: 14), CDR 2:
ENNKRPS (SEQ ID NO: 15), and CDR 3: GTWDTRLFGGV (SEQ ID NO:
16).
[0117] The TCN-716 (6362_F16) antibody includes a heavy chain
variable region (SEQ ID NO: 20), encoded by the nucleic acid
sequence shown in SEQ ID NO: 18, and a light chain variable region
(SEQ ID NO: 29) encoded by the nucleic acid sequence shown in SEQ
ID NO: 27.
[0118] The heavy chain CDRs of the TCN-716 (6362_F16) antibody have
the following sequences per Kabat definition: CDR 1: DFAMH (SEQ ID
NO: 21), CDR 2: SISRDGSTKYSGDSVKG (SEQ ID NO: 22), and CDR 3:
DSPYYLDIVGYRYFHHYGMDV (SEQ ID NO: 23). The light chain CDRs of the
TCN-716 (6362_F16) antibody have the following sequences per Kabat
definition: CDR 1: RASQILHSYNLA (SEQ ID NO: 30), CDR 2: GAYNRAS
(SEQ ID NO: 31), CDR 3: QQYGDSPSPGLT (SEQ ID NO: 32).
[0119] The heavy chain CDRs of the TCN-716 (6362_F16) antibody have
the following sequences per Chothia definition: CDR 1: GFTLTD (SEQ
ID NO: 24), CDR 2: SISRDGSTKY (SEQ ID NO: 25), and CDR 3:
DSPYYLDIVGYRYFHHYGMDV (SEQ ID NO: 23). The light chain CDRs of the
TCN-716 (6362_F16) antibody have the following sequences per
Chothia definition: CDR 1: RASQILHSYNLA (SEQ ID NO: 30), CDR 2:
GAYNRAS (SEQ ID NO: 31), CDR 3: QQYGDSPSPGLT (SEQ ID NO: 32).
[0120] The TCN-717 (6358_H17) antibody includes a heavy chain
variable region (SEQ ID NO: 36), encoded by the nucleic acid
sequence shown in SEQ ID NO: 34, and a light chain variable region
(SEQ ID NO: 45) encoded by the nucleic acid sequence shown in SEQ
ID NO: 43.
[0121] The heavy chain CDRs of the TCN-717 (6358_H17) antibody have
the following sequences per Kabat definition: CDR 1: QNDYHWA (SEQ
ID NO: 37), CDR 2: SVHYRQKSYYSPSLKS (SEQ ID NO: 38), and CDR 3:
HNREDYYDSNAYFDE (SEQ ID NO: 39). The light chain CDRs of the
TCN-717 (6358_H17) antibody have the following sequences per Kabat
definition: CDR 1: SGDDLENTLVC (SEQ ID NO: 46), CDR 2: QDSKRPS (SEQ
ID NO: 47), and CDR 3: QTWHRSTAQYV (SEQ ID NO: 48).
[0122] The heavy chain CDRs of the TCN-717 (6358_H17) antibody have
the following sequences per Chothia definition: CDR 1: MGSILQND
(SEQ ID NO: 40), CDR 2: SVHYRQKSY (SEQ ID NO: 41), and CDR 3:
HNREDYYDSNAYFDE (SEQ ID NO: 39). The light chain CDRs of the
TCN-717 (6358_H17) antibody have the following sequences per
Chothia definition: CDR 1: SGDDLENTLVC (SEQ ID NO: 46), CDR 2:
QDSKRPS (SEQ ID NO: 47), and CDR 3: QTWHRSTAQYV (SEQ ID NO:
48).
[0123] The TCN-722 (6385_L22) antibody includes a heavy chain
variable region (SEQ ID NO: 52), encoded by the nucleic acid
sequence shown in SEQ ID NO: 50, and a light chain variable region
(SEQ ID NO: 61) encoded by the nucleic acid sequence shown in SEQ
ID NO: 59.
[0124] The heavy chain CDRs of the TCN-722 (6385_L22) antibody have
the following sequences per Kabat definition: CDR 1: SNDQYWA (SEQ
ID NO: 53), CDR 2: SVHYRRRNYYSPSLES (SEQ ID NO: 54), and CDR 3:
HNWEDYYESNAYFDY (SEQ ID NO: 55). The light chain CDRs of the
TCN-722 (6385_L22) antibody have the following sequences per Kabat
definition: CDR 1: SGDQLENTFVC (SEQ ID NO: 62), CDR 2: QGSKRPS (SEQ
ID NO: 63), and CDR 3: QAWDRSTAHYV (SEQ ID NO: 64).
[0125] The heavy chain CDRs of the TCN-722 (6385_L22) antibody have
the following sequences per Chothia definition: CDR 1: GDSLLSND
(SEQ ID NO: 56), CDR 2: SVHYRRRNY (SEQ ID NO: 57), and CDR 3:
HNWEDYYESNAYFDY (SEQ ID NO: 55). The light chain CDRs of the
TCN-722 (6385_L22) antibody have the following sequences per
Chothia definition: CDR 1: SGDQLENTFVC (SEQ ID NO: 62), CDR 2:
QGSKRPS (SEQ ID NO: 63), and CDR 3: QAWDRSTAHYV (SEQ ID NO:
64).
[0126] In one aspect, an antibody according to the invention
contains a heavy chain having the amino acid sequence of SEQ ID
NOs: 3, 19, 35, or 51, and a light chain having the amino acid
sequence of SEQ ID NOs: 12, 28, 44, or 60. Alternatively, an
antibody according to the invention contains a heavy chain variable
region having the amino acid sequence of SEQ ID NOs: 4, 20, 36, or
52, and a light chain variable region having the amino acid
sequence of SEQ ID NOs: 13, 29, 45, or 61.
[0127] In another aspect, an antibody according to the invention
contains a heavy chain having the amino acid sequence encoded by
the nucleic acid sequence of SEQ ID NOs: 1, 17, 33, or 49, and a
light chain having the amino acid sequence encoded by the nucleic
acid sequence of SEQ ID NOs: 10, 26, 42, or 58. Alternatively, an
antibody according to the invention contains a heavy chain variable
region having the amino acid sequence encoded by the nucleic acid
sequence of SEQ ID NOs: 2, 18, 34, or 50 and a light chain variable
region having the amino acid sequence encoded by the nucleic acid
sequence of SEQ ID NOs: 11, 27, 43, or 59. Furthermore, an antibody
according to the invention contains a heavy chain having the amino
acid sequence encoded by a nucleic acid sequence of SEQ ID NOs: 2,
18, 34, or 50, which contains a silent or degenerate mutation, and
a light chain having the amino acid sequence encoded by the nucleic
acid sequence of SEQ ID NOs: 11, 27, 43, or 59, which contains a
silent or degenerate mutation. Silent and degenerate mutations
alter the nucleic acid sequence, but do not alter the resultant
amino acid sequence.
[0128] Preferably the three heavy chain CDRs include an amino acid
sequence of at least 90%, 92%, 95%, 97%, 98%, 99%, or more
identical to the amino acid sequences of SEQ ID NOs: 5, 6, 7, 21,
22, 23, 37, 38, 39, 53, 54, or 55 (as determined by the Kabat
method) or 8, 9, 7, 24, 25, 23, 40, 41, 39, 56, 57, or 55 (as
determined by the Chothia method) and a light chain with three CDRs
that include an amino acid sequence of at least 90%, 92%, 95%, 97%,
98%, 99%, or more identical to the amino acid sequence of 14, 15,
16, 30, 31, 32, 46, 47, 48, 62, 63, or 64 (as determined by the
Kabat or Chothia method).
[0129] The heavy chain of the anti-HRV monoclonal antibody is
derived from a germ line variable (V) gene such as, for example,
the IGHV4-34*01, IGHV4-34*02, IGHV3-64*05, IGHV3-64*03, IGHV4-39*07
germline genes.
[0130] The anti-HRV antibodies of the invention include a variable
heavy chain (V.sub.H) region encoded by human IGHV4-34*01,
IGHV4-34*02, IGHV3-64*05, IGHV3-64*03, IGHV4-39*07 germline gene
sequences. The germline IGHV4-34*01, IGHV4-34*02, IGHV3-64*05,
IGHV3-64*03, IGHV4-39*07 gene sequences are shown, e.g., in
Accession numbers AB019439, M99684, M77301, M77298, X92259,
AM940222, AM940222. The anti-HRV antibodies of the invention
include a V.sub.H region that is encoded by a nucleic acid sequence
that is at least 80% homologous to the IGHV4-34*01, IGHV4-34*02,
IGHV3-64*05, IGHV3-64*03, IGHV4-39*07 germline gene sequences.
Preferably, the nucleic acid sequence is at least 90%, 95%, 96%,
97% homologous to the IGHV4-34*01, IGHV4-34*02, IGHV3-64*05,
IGHV3-64*03, IGHV4-39*07 germline gene sequences, and more
preferably, at least 98%, 99% homologous to the IGHV4-34*01,
IGHV4-34*02, IGHV3-64*05, IGHV3-64*03, IGHV4-39*07 germline gene
sequences. The V.sub.H region of the anti-HRV antibody is at least
80% homologous to the amino acid sequence of the V.sub.H region
encoded by the IGHV4-34*01, IGHV4-34*02, IGHV3-64*05, IGHV3-64*03,
IGHV4-39*07 V.sub.H germline gene sequences. Preferably, the amino
acid sequence of V.sub.H region of the anti-HRV antibody is at
least 90%, 95%, 96%, 97% homologous to the amino acid sequence
encoded by the IGHV4-34*01, IGHV4-34*02, IGHV3-64*05, IGHV3-64*03,
IGHV4-39*07 germline gene sequences, and more preferably, at least
98%, 99% homologous to the sequence encoded by the IGHV4-34*01,
IGHV4-34*02, IGHV3-64*05, IGHV3-64*03, IGHV4-39*07 germline gene
sequences.
[0131] The light chain of the anti-HRV monoclonal antibody is
derived from a germ line variable (V) gene such as, for example,
the IGLV1-51*02, IGLV1-51*01, IGKV3-20*01, IGLV3-1*01 germline
genes.
[0132] The anti-HRV antibodies of the invention also include a
variable light chain (V.sub.L) region encoded by human IGLV1-51*02,
IGLV1-51*01, IGKV3-20*01, IGLV3-1*01 germline gene sequences. The
human IGLV1-51*02, IGLV1-51*01, IGKV3-20*01, IGLV3-1*01 V.sub.L
germline gene sequences are shown, e.g., Accession numbers M30446,
Z73661, X12686, X57826.
[0133] The anti-HRV antibodies include a V.sub.L region that is
encoded by a nucleic acid sequence that is at least 80% homologous
to the IGLV1-51*02, IGLV1-51*01, IGKV3-20*01, IGLV3-1*01 germline
gene sequences. Preferably, the nucleic acid sequence is at least
90%, 95%, 96%, 97% homologous to the IGLV1-51*02, IGLV1-51*01,
IGKV3-20*01, IGLV3-1*01 germline gene sequences, and more
preferably, at least 98%, 99% homologous to the IGLV1-51*02,
IGLV1-51*01, IGKV3-20*01, IGLV3-1*01 germline gene sequences. The
V.sub.L region of the anti-HRV antibody is at least 80% homologous
to the amino acid sequence of the V.sub.L region encoded the
IGLV1-51*02, IGLV1-51*01, IGKV3-20*01, IGLV3-1*01 germline gene
sequences. Preferably, the amino acid sequence of V.sub.L region of
the anti-HRV antibody is at least 90%, 95%, 96%, 97% homologous to
the amino acid sequence encoded by the IGLV1-51*02, IGLV1-51*01,
IGKV3-20*01, IGLV3-1*01 germline gene sequences, and more
preferably, at least 98%, 99% homologous to the sequence encoded by
the IGLV1-51*02, IGLV1-51*01, IGKV3-20*01, IGLV3-1*01 germline gene
sequences.
[0134] Monoclonal and recombinant antibodies are particularly
useful in identification and purification of the individual
polypeptides or other antigens against which they are directed. The
antibodies of the invention have additional utility in that they
may be employed as reagents in immunoassays, radioimmunoassays
(RIA) or enzyme-linked immunosorbent assays (ELISA). In these
applications, the antibodies can be labeled with an
analytically-detectable reagent such as a radioisotope, a
fluorescent molecule or an enzyme. The antibodies may also be used
for the molecular identification and characterization (epitope
mapping) of antigens.
[0135] As mentioned above, the antibodies of the invention can be
used to map the epitopes to which they bind. Applicants have
discovered that antibodies TCN-711 (6893_E11), TCN-716 (6362_F16),
TCN-717 (6358_H17), and TCN-722 (6385_L22) neutralize HRV. Although
the Applicant does not wish to be bound by this theory, it is
postulated that the antibodies TCN-711 (6893_E11), TCN-716
(6362_F16), TCN-717 (6358_H17), and TCN-722 (6385_L22) bind to one
or more conformational epitopes formed by HRV-encoded proteins.
[0136] The epitopes recognized by these antibodies may have a
number of uses. The epitopes and mimotopes in purified or synthetic
form can be used to raise immune responses (i.e. as a vaccine, or
for the production of antibodies for other uses) or for screening
patient serum for antibodies that immunoreact with the epitopes or
mimotopes. Preferably, such an epitope or mimotope, or antigen
comprising such an epitope or mimotope is used as a vaccine for
raising an immune response. The antibodies of the invention can
also be used in a method to monitor the quality of vaccines in
particular to check that the antigen in a vaccine contains the
correct immunogenic epitope in the correct conformation.
[0137] The epitopes may also be useful in screening for ligands
that bind to said epitopes. Such ligands preferably block the
epitopes and thus prevent infection. Such ligands are encompassed
within the scope of the invention.
[0138] Standard techniques of molecular biology may be used to
prepare DNA sequences coding for the antibodies or fragments of the
antibodies of the present invention. Desired DNA sequences may be
synthesized completely or in part using oligonucleotide synthesis
techniques. Site-directed mutagenesis and polymerase chain reaction
(PCR) techniques may be used as appropriate.
[0139] Any suitable host cell/vector system may be used for
expression of the DNA sequences encoding the antibody molecules of
the present invention or fragments thereof. Bacterial, for example
E. coli, and other microbial systems may be used, in part, for
expression of antibody fragments such as Fab and F(ab').sub.2
fragments, and especially Fv fragments and single chain antibody
fragments, for example, single chain Fvs. Eukaryotic, e.g.
mammalian, host cell expression systems may be used for production
of larger antibody molecules, including complete antibody
molecules. Suitable mammalian host cells include CHO, HEK293T,
PER.C6, myeloma or hybridoma cells.
[0140] The present invention also provides a process for the
production of an antibody molecule according to the present
invention comprising culturing a host cell comprising a vector of
the present invention under conditions suitable for leading to
expression of protein from DNA encoding the antibody molecule of
the present invention, and isolating the antibody molecule. The
antibody molecule may comprise only a heavy or light chain
polypeptide, in which case only a heavy chain or light chain
polypeptide coding sequence needs to be used to transfect the host
cells. For production of products comprising both heavy and light
chains, the cell line may be transfected with two vectors, a first
vector encoding a light chain polypeptide and a second vector
encoding a heavy chain polypeptide. Alternatively, a single vector
may be used, the vector including sequences encoding light chain
and heavy chain polypeptides.
[0141] Alternatively, antibodies according to the invention may be
produced by i) expressing a nucleic acid sequence according to the
invention in a cell, and ii) isolating the expressed antibody
product. Additionally, the method may include iii) purifying the
antibody. Transformed B cells are screened for those producing
antibodies of the desired antigen specificity, and individual B
cell clones can then be produced from the positive cells. The
screening step may be carried out by ELISA, by staining of tissues
or cells (including transfected cells), a neutralization assay or
one of a number of other methods known in the art for identifying
desired antigen specificity. The assay may select on the basis of
simple antigen recognition, or may select on the additional basis
of a desired function e.g. to select neutralizing antibodies rather
than just antigen-binding antibodies, to select antibodies that can
change characteristics of targeted cells, such as their signaling
cascades, their shape, their growth rate, their capability of
influencing other cells, their response to the influence by other
cells or by other reagents or by a change in conditions, their
differentiation status, etc.
[0142] The cloning step for separating individual clones from the
mixture of positive cells may be carried out using limiting
dilution, micromanipulation, single cell deposition by cell sorting
or another method known in the art. Preferably the cloning is
carried out using limiting dilution.
[0143] The immortalized B cell clones of the invention can be used
in various ways e.g. as a source of monoclonal antibodies, as a
source of nucleic acid (DNA or mRNA) encoding a monoclonal antibody
of interest, for research, etc.
[0144] Unless otherwise defined, scientific and technical terms
used in connection with the present invention shall have the
meanings that are commonly understood by those of ordinary skill in
the art. Further, unless otherwise required by context, singular
terms shall include pluralities and plural terms shall include the
singular. Generally, nomenclatures utilized in connection with, and
techniques of, cell and tissue culture, molecular biology, and
protein and oligo- or polynucleotide chemistry and hybridization
described herein are those well known and commonly used in the art.
Standard techniques are used for recombinant DNA, oligonucleotide
synthesis, and tissue culture and transformation (e.g.,
electroporation, lipofection). Enzymatic reactions and purification
techniques are performed according to manufacturer's specifications
or as commonly accomplished in the art or as described herein. The
practice of the present invention will employ, unless indicated
specifically to the contrary, conventional methods of virology,
immunology, microbiology, molecular biology and recombinant DNA
techniques within the skill of the art, many of which are described
below for the purpose of illustration. Such techniques are
explained fully in the literature. See, e.g., Sambrook, et al.
Molecular Cloning: A Laboratory Manual (2nd Edition, 1989);
Maniatis et al. Molecular Cloning: A Laboratory Manual (1982); DNA
Cloning: A Practical Approach, vol. I & II (D. Glover, ed.);
Oligonucleotide Synthesis (N. Gait, ed., 1984); Nucleic Acid
Hybridization (B. Hames & S. Higgins, eds., 1985);
Transcription and Translation (B. Hames & S. Higgins, eds.,
1984); Animal Cell Culture (R. Freshney, ed., 1986); Perbal, A
Practical Guide to Molecular Cloning (1984). The nomenclatures
utilized in connection with, and the laboratory procedures and
techniques of, analytical chemistry, synthetic organic chemistry,
and medicinal and pharmaceutical chemistry described herein are
those well known and commonly used in the art. Standard techniques
are used for chemical syntheses, chemical analyses, pharmaceutical
preparation, formulation, and delivery, and treatment of
patients.
[0145] The following definitions are useful in understanding the
present invention.
[0146] The term "antibody" (Ab) as used herein includes monoclonal
antibodies, polyclonal antibodies, multispecific antibodies (e.g.,
bispecific antibodies), and antibody fragments, as long as they
exhibit the desired biological activity. The term "immunoglobulin"
(Ig) is used interchangeably with "antibody" herein.
[0147] A "neutralizing antibody" may inhibit the entry of HRV
virus.
[0148] By "broad and potent neutralizing antibodies" are meant
antibodies that neutralize more than one HRV virus species (from
diverse clades and different strains within a clade) in a
neutralization assay. A broad neutralizing antibody may neutralize
at least 2, 3, 4, 5, 6, 7, 8, 9 or more different strains or
serotypes of HRV, the strains belonging to the same or different
clades. A broad neutralizing antibody may neutralize multiple HRV
serotypes belonging to at least 2, 3, or 4, different clades.
Preferably, the half-maximal inhibitory concentration of the
monoclonal antibody may be equal to or less than about 100 ng/ml to
neutralize about 50% of the input virus in the neutralization
assay. However, the half-maximal inhibitory concentration of the
monoclonal antibody may be equal to or less than about 100 ng/ml,
90 ng/ml, 80 ng/ml, 70 ng/ml, 60 ng/ml, 50 ng/ml, 40 ng/ml, 30
ng/ml, 20 ng/ml, 10 ng/ml, 1 ng/ml, or any concentration in between
to neutralize about 50% of the input virus in the neutralization
assay.
[0149] An "isolated antibody" is one that has been separated and/or
recovered from a component of its natural environment. Contaminant
components of its natural environment are materials that would
interfere with diagnostic or therapeutic uses for the antibody, and
may include enzymes, hormones, and other proteinaceous or
nonproteinaceous solutes. In preferred embodiments, the antibody is
purified: (1) to greater than 95% by weight of antibody as
determined by the Lowry method, and most preferably more than 99%
by weight; (2) to a degree sufficient to obtain at least 15
residues of N-terminal or internal amino acid sequence by use of a
spinning cup sequenator; or (3) to homogeneity by SDS-PAGE under
reducing or non-reducing conditions using Coomassie blue or,
preferably, silver stain. Isolated antibody includes the antibody
in situ within recombinant cells since at least one component of
the antibody's natural environment will not be present. Ordinarily,
however, isolated antibody will be prepared by at least one
purification step.
[0150] The basic four-chain antibody unit is a heterotetrameric
glycoprotein composed of two identical light (L) chains and two
identical heavy (H) chains. An IgM antibody consists of 5 basic
heterotetramer units along with an additional polypeptide called J
chain, and therefore contain 10 antigen binding sites, while
secreted IgA antibodies can polymerize to form polyvalent
assemblages comprising 2-5 of the basic 4-chain units along with J
chain. In the case of IgGs, the 4-chain unit is generally about
150,000 daltons. Each L chain is linked to an H chain by one
covalent disulfide bond, while the two H chains are linked to each
other by one or more disulfide bonds depending on the H chain
isotype. Each H and L chain also has regularly spaced intrachain
disulfide bridges. Each H chain has at the N-terminus, a variable
region (V.sub.H) followed by three constant domains (C.sub.H) for
each of the .alpha. and .gamma. chains and four C.sub.H domains for
.mu. and .epsilon. isotypes. Each L chain has at the N-terminus, a
variable region (V.sub.L) followed by a constant domain (C.sub.L)
at its other end. The V.sub.L is aligned with the V.sub.H and the
C.sub.L is aligned with the first constant domain of the heavy
chain (C.sub.H1). Particular amino acid residues are believed to
form an interface between the light chain and heavy chain variable
regions. The pairing of a V.sub.H and V.sub.L together forms a
single antigen-binding site. For the structure and properties of
the different classes of antibodies, see, e.g., Basic and Clinical
Immunology, 8th edition, Daniel P. Stites, Abba I. Terr and
Tristram G. Parslow (eds.), Appleton & Lange, Norwalk, Conn.,
1994, page 71, and Chapter 6.
[0151] The L chain from any vertebrate species can be assigned to
one of two clearly distinct types, called kappa (K) and lambda (X),
based on the amino acid sequences of their constant domains
(C.sub.L). Depending on the amino acid sequence of the constant
domain of their heavy chains (C.sub.H), immunoglobulins can be
assigned to different classes or isotypes. There are five classes
of immunoglobulins: IgA, IgD, IgE, IgG, and IgM, having heavy
chains designated alpha (.alpha.), delta (.delta.), epsilon
(.epsilon.), gamma (.gamma.) and mu (.mu.), respectively. The
.gamma. and .alpha. classes are further divided into subclasses on
the basis of relatively minor differences in C.sub.H sequence and
function, e.g., humans express the following subclasses: IgG1,
IgG2, IgG3, IgG4, IgA1, and IgA2.
[0152] The term "variable" refers to the fact that certain segments
of the V domains differ extensively in sequence among antibodies.
The V domain mediates antigen binding and defines specificity of a
particular antibody for its particular antigen. However, the
variability is not evenly distributed across the 110-amino acid
span of the variable regions. Instead, the V regions consist of
relatively invariant stretches called framework regions (FRs) of
15-30 amino acids separated by shorter regions of extreme
variability called "hypervariable regions" that are each 9-12 amino
acids long. The variable regions of native heavy and light chains
each comprise four FRs, largely adopting a .beta.-sheet
configuration, connected by three hypervariable regions, which form
loops connecting, and in some cases forming part of, the
.beta.-sheet structure. 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)). 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 cytotoxicity (ADCC).
[0153] The term "hypervariable region" when used herein refers to
the amino acid residues of an antibody that are responsible for
antigen binding. The hypervariable region generally comprises amino
acid residues from a "complementarity determining region" or "CDR"
(e.g., around about residues 24-34 (L1), 50-56 (L2) and 89-97 (L3)
in the V.sub.L, and around about 31-35 (H1), 50-65 (H2) and 95-102
(H3) in the V.sub.H when numbered in accordance with the Kabat
numbering system; Kabat et al., Sequences of Proteins of
Immunological Interest, 5th Ed. Public Health Service, National
Institutes of Health, Bethesda, Md. (1991)); and/or those residues
from a "hypervariable loop" (e.g., residues 24-34 (L1), 50-56 (L2)
and 89-97 (L3) in the V.sub.L, and 26-32 (H1), 52-56 (H2) and
95-101 (H3) in the V.sub.H when numbered in accordance with the
Chothia numbering system; Chothia and Lesk, J. Mol. Biol.
196:901-917 (1987)); and/or those residues from a "hypervariable
loop"/CDR (e.g., residues 27-38 (L1), 56-65 (L2) and 105-120 (L3)
in the V.sub.L, and 27-38 (H1), 56-65 (H2) and 105-120 (H3) in the
V.sub.H when numbered in accordance with the IMGT numbering system;
Lefranc, M. P. et al. Nucl. Acids Res. 27:209-212 (1999), Ruiz, M.
e al. Nucl. Acids Res. 28:219-221 (2000)). Optionally the antibody
has symmetrical insertions at one or more of the following points
28, 36 (L1), 63, 74-75 (L2) and 123 (L3) in the V.sub.L, and 28, 36
(H1), 63, 74-75 (H2) and 123 (H3) in the V.sub.H when numbered in
accordance with AHo; Honneger, A. and Plunkthun, A. J. Mol. Biol.
309:657-670 (2001)).
[0154] By "germline nucleic acid residue" is meant the nucleic acid
residue that naturally occurs in a germline gene encoding a
constant or variable region. "Germline gene" is the DNA found in a
germ cell (i.e., a cell destined to become an egg or in the sperm).
A "germline mutation" refers to a heritable change in a particular
DNA that has occurred in a germ cell or the zygote at the
single-cell stage, and when transmitted to offspring, such a
mutation is incorporated in every cell of the body. A germline
mutation is in contrast to a somatic mutation which is acquired in
a single body cell. In some cases, nucleotides in a germline DNA
sequence encoding for a variable region are mutated (i.e., a
somatic mutation) and replaced with a different nucleotide.
[0155] 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, being directed against a single
antigenic site. Furthermore, in contrast to polyclonal antibody
preparations that include different antibodies directed against
different determinants (epitopes), each monoclonal antibody is
directed against a single determinant on the antigen. In addition
to their specificity, the monoclonal antibodies are advantageous in
that they may be synthesized uncontaminated by other antibodies.
The modifier "monoclonal" is not to be construed as requiring
production of the antibody by any particular method. For example,
the monoclonal antibodies useful in the present invention may be
prepared by the hybridoma methodology first described by Kohler et
al., Nature, 256:495 (1975), or may be made using recombinant DNA
methods in bacterial, eukaryotic animal or plant cells (see, e.g.,
U.S. Pat. No. 4,816,567). The "monoclonal antibodies" may also be
isolated from phage antibody libraries using the techniques
described in Clackson et al., Nature, 352:624-628 (1991) and Marks
et al., J. Mol. Biol., 222:581-597 (1991), for example.
[0156] In some aspects, the alternative EBV immortalization method
described in WO2004/076677 is used. Using this method, B-cells
producing the antibody of the invention can be transformed with EBV
in the presence of a polyclonal B cell activator. Transformation
with EBV is a standard technique and can easily be adapted to
include polyclonal B cell activators. Additional stimulants of
cellular growth and differentiation may be added during the
transformation step to further enhance the efficiency. These
stimulants may be cytokines such as IL-2 and IL-15. In a
particularly preferred aspect, IL-2 is added during the
immortalization step to further improve the efficiency of
immortalization, but its use is not essential.
[0157] The monoclonal antibodies herein include "chimeric"
antibodies in which a portion of the heavy and/or light chain is
identical with or homologous to corresponding sequences in
antibodies derived from a particular species or belonging to a
particular antibody class or subclass, while the remainder of the
chain(s) is identical with or homologous to corresponding sequences
in antibodies derived from another species or belonging to another
antibody class or subclass, as well as fragments of such
antibodies, so long as they exhibit the desired biological activity
(see U.S. Pat. No. 4,816,567; and Morrison et al., Proc. Natl.
Acad. Sci. USA, 81:6851-6855 (1984)). The present invention
provides variable region antigen-binding sequences derived from
human antibodies. Accordingly, chimeric antibodies of primary
interest herein include antibodies having one or more human antigen
binding sequences (e.g., CDRs) and containing one or more sequences
derived from a non-human antibody, e.g., an FR or C region
sequence. In addition, chimeric antibodies of primary interest
herein include those comprising a human variable region antigen
binding sequence of one antibody class or subclass and another
sequence, e.g., FR or C region sequence, derived from another
antibody class or subclass. Chimeric antibodies of interest herein
also include those containing variable region antigen-binding
sequences related to those described herein or derived from a
different species, such as a non-human primate (e.g., Old World
Monkey, Ape, etc). Chimeric antibodies also include primatized and
humanized antibodies.
[0158] Furthermore, chimeric antibodies may comprise residues that
are not found in the recipient antibody or in the donor antibody.
These modifications are made to further refine antibody
performance. For further details, see Jones et al., Nature
321:522-525 (1986); Riechmann et al., Nature 332:323-329 (1988);
and Presta, Curr. Op. Struct. Biol. 2:593-596 (1992).
[0159] A "humanized antibody" is generally considered to be a human
antibody that has one or more amino acid residues introduced into
it from a source that is non-human. These non-human amino acid
residues are often referred to as "import" residues, which are
typically taken from an "import" variable region. Humanization is
traditionally performed following the method of Winter and
co-workers (Jones et al., Nature, 321:522-525 (1986); Reichmann et
al., Nature, 332:323-327 (1988); Verhoeyen et al., Science,
239:1534-1536 (1988)), by substituting import hypervariable region
sequences for the corresponding sequences of a human antibody.
Accordingly, such "humanized" antibodies are chimeric antibodies
(U.S. Pat. No. 4,816,567), wherein substantially less than an
intact human variable region has been substituted by the
corresponding sequence from a non-human species.
[0160] A "human antibody" is an antibody containing only sequences
present in an antibody naturally produced by a human. However, as
used herein, human antibodies may comprise residues or
modifications not found in a naturally occurring human antibody,
including those modifications and variant sequences described
herein. These are typically made to further refine or enhance
antibody performance.
[0161] An "intact" antibody is one that comprises an
antigen-binding site as well as a C.sub.L and at least heavy chain
constant domains, C.sub.H1, C.sub.H2 and C.sub.H3. The constant
domains may be native sequence constant domains (e.g., human native
sequence constant domains) or amino acid sequence variant thereof.
Preferably, the intact antibody has one or more effector
functions.
[0162] An "antibody fragment" comprises a portion of an intact
antibody, preferably the antigen binding or variable region of the
intact antibody. Examples of antibody fragments include Fab, Fab',
F(ab').sub.2, and Fv fragments; diabodies; linear antibodies (see
U.S. Pat. No. 5,641,870; Zapata et al., Protein Eng. 8(10):
1057-1062 [1995]); single-chain antibody molecules; and
multispecific antibodies formed from antibody fragments.
[0163] The phrase "functional fragment or analog" of an antibody is
a compound having qualitative biological activity in common with a
full-length antibody. For example, a functional fragment or analog
of an anti-IgE antibody is one that can bind to an IgE
immunoglobulin in such a manner so as to prevent or substantially
reduce the ability of such molecule from having the ability to bind
to the high affinity receptor, Fc.sub..epsilon.RI.
[0164] Papain digestion of antibodies produces two identical
antigen-binding fragments, called "Fab" fragments, and a residual
"Fc" fragment, a designation reflecting the ability to crystallize
readily. The Fab fragment consists of an entire L chain along with
the variable region domain of the H chain (V.sub.H), and the first
constant domain of one heavy chain (C.sub.H1). Each Fab fragment is
monovalent with respect to antigen binding, i.e., it has a single
antigen-binding site. Pepsin treatment of an antibody yields a
single large F(ab').sub.2 fragment that roughly corresponds to two
disulfide linked Fab fragments having divalent antigen-binding
activity and is still capable of cross-linking antigen. Fab'
fragments differ from Fab fragments by having additional few
residues at the carboxy terminus of the C.sub.H1 domain including
one or more cysteines 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 that
have hinge cysteines between them. Other chemical couplings of
antibody fragments are also known.
[0165] The "Fc" fragment comprises the carboxy-terminal portions of
both H chains held together by disulfides. The effector functions
of antibodies are determined by sequences in the Fc region, which
region is also the part recognized by Fc receptors (FcR) found on
certain types of cells.
[0166] "Fv" is the minimum antibody fragment that contains a
complete antigen-recognition and -binding site. This fragment
consists of a dimer of one heavy- and one light-chain variable
region domain in tight, non-covalent association. From the folding
of these two domains emanate six hypervariable loops (three loops
each from the H and L chain) that contribute the amino acid
residues for antigen binding and confer antigen binding specificity
to the antibody. However, even a single variable region (or half of
an Fv comprising only three CDRs specific for an antigen) has the
ability to recognize and bind antigen, although at a lower affinity
than the entire binding site.
[0167] "Single-chain Fv" also abbreviated as "sFv" or "scFv" are
antibody fragments that comprise the V.sub.H and V.sub.L antibody
domains connected into a single polypeptide chain. Preferably, the
sFv polypeptide further comprises a polypeptide linker between the
V.sub.H and V.sub.L domains that 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); Borrebaeck 1995, infra.
[0168] The term "diabodies" refers to small antibody fragments
prepared by constructing sFv fragments (see preceding paragraph)
with short linkers (about 5-10 residues) between the V.sub.H and
V.sub.L domains such that inter-chain but not intra-chain pairing
of the V domains is achieved, resulting in a bivalent fragment,
i.e., fragment having two antigen-binding sites. Bispecific
diabodies are heterodimers of two "crossover" sFv fragments in
which the V.sub.H and V.sub.L domains of the two antibodies are
present on different polypeptide chains. Diabodies are described
more fully in, for example, EP 404,097; WO 93/11161; and Hollinger
et al., Proc. Natl. Acad. Sci. USA, 90:6444-6448 (1993).
[0169] Domain antibodies (dAbs), which can be produced in fully
human form, are the smallest known antigen-binding fragments of
antibodies, ranging from 11 kDa to 15 kDa. dAbs are the robust
variable regions of the heavy and light chains of immunoglobulins
(VH and VL respectively). They are highly expressed in microbial
cell culture, show favorable biophysical properties including
solubility and temperature stability, and are well suited to
selection and affinity maturation by in vitro selection systems
such as phage display. dAbs are bioactive as monomers and, owing to
their small size and inherent stability, can be formatted into
larger molecules to create drugs with prolonged serum half-lives or
other pharmacological activities. Examples of this technology have
been described in WO9425591 for antibodies derived from Camelidae
heavy chain Ig, as well in US20030130496 describing the isolation
of single domain fully human antibodies from phage libraries.
[0170] As used herein, an antibody that "internalizes" is one that
is taken up by (i.e., enters) the cell upon binding to an antigen
on a mammalian cell (e.g., a cell surface polypeptide or receptor).
The internalizing antibody will of course include antibody
fragments, human or chimeric antibody, and antibody conjugates. For
certain therapeutic applications, internalization in vivo is
contemplated. The number of antibody molecules internalized will be
sufficient or adequate to kill a cell or inhibit its growth,
especially an infected cell. Depending on the potency of the
antibody or antibody conjugate, in some instances, the uptake of a
single antibody molecule into the cell is sufficient to kill the
target cell to which the antibody binds. For example, certain
toxins are highly potent in killing such that internalization of
one molecule of the toxin conjugated to the antibody is sufficient
to kill the infected cell.
[0171] As used herein, an antibody is said to be "immunospecific,"
"specific for" or to "specifically bind" an antigen if it reacts at
a detectable level with the antigen, preferably with an affinity
constant, K.sub.a, of greater than or equal to about 10.sup.4
M.sup.-1, or greater than or equal to about 10.sup.5 M.sup.-1,
greater than or equal to about 10.sup.6 M.sup.-1, greater than or
equal to about 10.sup.7 M.sup.-1, or greater than or equal to
10.sup.8 M.sup.-1. Affinity of an antibody for its cognate antigen
is also commonly expressed as a dissociation constant K.sub.D, and
in certain embodiments, HRV antibody specifically binds to an HRV
polypeptide if it binds with a K.sub.D of less than or equal to
10.sup.-4 M, less than or equal to about 10.sup.-5 M, less than or
equal to about 10.sup.-6 M, less than or equal to 10.sup.-7 M, or
less than or equal to 10.sup.-8 M. Affinities of antibodies can be
readily determined using conventional techniques, for example,
those described by Scatchard et al. (Ann. N.Y. Acad. Sci. USA
51:660 (1949)).
[0172] Binding properties of an antibody to antigens, cells or
tissues thereof may generally be determined and assessed using
immunodetection methods including, for example,
immunofluorescence-based assays, such as immuno-histochemistry
(IHC) and/or fluorescence-activated cell sorting (FACS).
[0173] An antibody having a "biological characteristic" of a
designated antibody is one that possesses one or more of the
biological characteristics of that antibody which distinguish it
from other antibodies. For example, in certain embodiments, an
antibody with a biological characteristic of a designated antibody
will bind the same epitope as that bound by the designated antibody
and/or have a common effector function as the designated
antibody.
[0174] The term "antagonist" antibody is used in the broadest
sense, and includes an antibody that partially or fully blocks,
inhibits, or neutralizes a biological activity of an epitope,
polypeptide, or cell that it specifically binds. Methods for
identifying antagonist antibodies may comprise contacting a
polypeptide or cell specifically bound by a candidate antagonist
antibody with the candidate antagonist antibody and measuring a
detectable change in one or more biological activities normally
associated with the polypeptide or cell.
[0175] An "antibody that inhibits the growth of infected cells" or
a "growth inhibitory" antibody is one that binds to and results in
measurable growth inhibition of infected cells expressing or
capable of expressing an HRV epitope bound by an antibody.
Preferred growth inhibitory antibodies inhibit growth of infected
cells by greater than 20%, preferably from about 20% to about 50%,
and even more preferably, by greater than 50% (e.g., from about 50%
to about 100%) as compared to the appropriate control, the control
typically being infected cells not treated with the antibody being
tested. Growth inhibition can be measured at an antibody
concentration of about 0.1 to 30 .mu.g/ml or about 0.5 nM to 200 nM
in cell culture, where the growth inhibition is determined 1-10
days after exposure of the infected cells to the antibody. Growth
inhibition of infected cells in vivo can be determined in various
ways known in the art.
[0176] The antibody is growth inhibitory in vivo if administration
of the antibody at about 1 .mu.g/kg to about 100 mg/kg body weight
results in reduction the percent of infected cells or total number
of infected cells within about 5 days to 3 months from the first
administration of the antibody, preferably within about 5 to 30
days.
[0177] An antibody that "induces apoptosis" is one which induces
programmed cell death as determined by binding of annexin V,
fragmentation of DNA, cell shrinkage, dilation of endoplasmic
reticulum, cell fragmentation, and/or formation of membrane
vesicles (called apoptotic bodies). Preferably the cell is an
infected cell. Various methods are available for evaluating the
cellular events associated with apoptosis. For example,
phosphatidyl serine (PS) translocation can be measured by annexin
binding; DNA fragmentation can be evaluated through DNA laddering;
and nuclear/chromatin condensation along with DNA fragmentation can
be evaluated by any increase in hypodiploid cells. Preferably, the
antibody that induces apoptosis is one that results in about 2 to
50 fold, preferably about 5 to 50 fold, and most preferably about
10 to 50 fold, induction of annexin binding relative to untreated
cell in an annexin binding assay.
[0178] Antibody "effector functions" refer to those biological
activities attributable to the Fc region (a native sequence Fc
region or amino acid sequence variant Fc region) of an antibody,
and vary with the antibody isotype. Examples of antibody effector
functions include: C1q binding and complement dependent
cytotoxicity; Fc receptor binding; antibody-dependent cell-mediated
cytotoxicity (ADCC); phagocytosis; down regulation of cell surface
receptors (e.g., B cell receptor); and B cell activation.
[0179] "Antibody-dependent cell-mediated cytotoxicity" or "ADCC"
refers to a form of cytotoxicity in which secreted Ig bound to Fc
receptors (FcRs) present on certain cytotoxic cells (e.g., Natural
Killer (NK) cells, neutrophils, and macrophages) enable these
cytotoxic effector cells to bind specifically to an antigen-bearing
target cell and subsequently kill the target cell with cytotoxins.
The antibodies "arm" the cytotoxic cells and are required for such
killing. The primary cells for mediating ADCC, NK cells, express
Fc.gamma.RIII only, whereas monocytes express Fc.gamma.RI,
Fc.gamma.RII and Fc.gamma.RIII. FcR expression on hematopoietic
cells is summarized in Table 4 on page 464 of Ravetch and Kinet,
Annu. Rev. Immunol 9:457-92 (1991). To assess ADCC activity of a
molecule of interest, an in vitro ADCC assay, such as that
described in U.S. Pat. No. 5,500,362 or U.S. Pat. No. 5,821,337 may
be performed. Useful effector cells for such assays include
peripheral blood mononuclear cells (PBMC) and Natural Killer (NK)
cells.
[0180] Alternatively, or additionally, ADCC activity of the
molecule of interest may be assessed in vivo, e.g., in a animal
model such as that disclosed in Clynes et al., Proc. Natl. Acad.
Sci. (USA) 95:652-656 (1998).
[0181] "Fc receptor" or "FcR" describes a receptor that binds to
the Fc region of an antibody. In certain embodiments, the FcR is a
native sequence human FcR. Moreover, a preferred FcR is one that
binds an IgG antibody (a gamma receptor) and includes receptors of
the Fc.gamma.RI, Fc.gamma.RII, and Fc.gamma.RIII subclasses,
including allelic variants and alternatively spliced forms of these
receptors. FC.gamma.RII receptors include Fc.gamma.RIIA (an
"activating receptor") and Fc.gamma.RIIB (an "inhibiting
receptor"), which have similar amino acid sequences that differ
primarily in the cytoplasmic domains thereof. Activating receptor
Fc.gamma.RIIA contains an immunoreceptor tyrosine-based activation
motif (ITAM) in its cytoplasmic domain Inhibiting receptor
Fc.gamma.RIIB contains an immunoreceptor tyrosine-based inhibition
motif (ITIM) in its cytoplasmic domain (see review M. in Daeron,
Annu Rev. Immunol. 15:203-234 (1997)). FcRs are reviewed in Ravetch
and Kinet, Annu Rev. Immunol 9:457-92 (1991); Capel et al.,
Immunomethods 4:25-34 (1994); and de Haas et al., J. Lab. Clin.
Med. 126:330-41 (1995). Other FcRs, including those to be
identified in the future, are encompassed by the term "FcR" herein.
The term also includes the neonatal receptor, FcRn, which is
responsible for the transfer of maternal IgGs to the fetus (Guyer
et al., J. Immunol. 117:587 (1976) and Kim et al., J. Immunol.
24:249 (1994)).
[0182] "Human effector cells" are leukocytes that express one or
more FcRs and perform effector functions. Preferably, the cells
express at least Fc.gamma.RIII and perform ADCC effector function.
Examples of human leukocytes that mediate ADCC include PBMC, NK
cells, monocytes, cytotoxic T cells and neutrophils; with PBMCs and
NK cells being preferred. The effector cells may be isolated from a
native source, e.g., from blood.
[0183] "Complement dependent cytotoxicity" or "CDC" refers to the
lysis of a target cell in the presence of complement. Activation of
the classical complement pathway is initiated by the binding of the
first component of the complement system (C1q) to antibodies (of
the appropriate subclass) that are bound to their cognate antigen.
To assess complement activation, a CDC assay, e.g., as described in
Gazzano-Santoro et al., J. Immunol. Methods 202:163 (1996), may be
performed.
[0184] A "mammal" for purposes of treating an infection, refers to
any mammal, including humans, domestic and farm animals, and zoo,
sports, or pet animals, such as dogs, cats, cattle, horses, sheep,
pigs, goats, rabbits, etc. Preferably, the mammal is human.
[0185] "Treating" or "treatment" or "alleviation" refers to both
therapeutic treatment and prophylactic or preventative measures;
wherein the object is to prevent or slow down (lessen) the targeted
pathologic condition or disorder. Those in need of treatment
include those already with the disorder as well as those prone to
have the disorder or those in whom the disorder is to be prevented.
A subject or mammal is successfully "treated" for an infection if,
after receiving a therapeutic amount of an antibody according to
the methods of the present invention, the patient shows observable
and/or measurable reduction in or absence of one or more of the
following: reduction in the number of infected cells or absence of
the infected cells; reduction in the percent of total cells that
are infected; and/or relief to some extent, one or more of the
symptoms associated with the specific infection; reduced morbidity
and mortality, and improvement in quality of life issues. The above
parameters for assessing successful treatment and improvement in
the disease are readily measurable by routine procedures familiar
to a physician.
[0186] The term "therapeutically effective amount" refers to an
amount of an antibody or a drug effective to "treat" a disease or
disorder in a subject or mammal. See preceding definition of
"treating."
[0187] "Chronic" administration refers to administration of the
agent(s) in a continuous mode as opposed to an acute mode, so as to
maintain the initial therapeutic effect (activity) for an extended
period of time. "Intermittent" administration is treatment that is
not consecutively done without interruption, but rather is cyclic
in nature.
[0188] Administration "in combination with" one or more further
therapeutic agents includes simultaneous (concurrent) and
consecutive administration in any order.
[0189] "Carriers" as used herein include pharmaceutically
acceptable carriers, excipients, or stabilizers that are nontoxic
to the cell or mammal being exposed thereto at the dosages and
concentrations employed. Often the physiologically acceptable
carrier is an aqueous pH buffered solution. Examples of
physiologically acceptable carriers include buffers such as
phosphate, citrate, and other organic acids; antioxidants including
ascorbic acid; low molecular weight (less than about 10 residues)
polypeptide; proteins, such as serum albumin, gelatin, or
immunoglobulins; hydrophilic polymers such as polyvinylpyrrolidone;
amino acids such as glycine, glutamine, asparagine, arginine or
lysine; monosaccharides, disaccharides, and other carbohydrates
including glucose, mannose, or dextrins; chelating agents such as
EDTA; sugar alcohols such as mannitol or sorbitol; salt-forming
counterions such as sodium; and/or nonionic surfactants such as
TWEEN.TM. polyethylene glycol (PEG), and PLURONICS.TM..
[0190] The term "cytotoxic agent" as used herein refers to a
substance that inhibits or prevents the function of cells and/or
causes destruction of cells. The term is intended to include
radioactive isotopes (e.g., At.sup.211, I.sup.131, I.sup.125,
Y.sup.90, Re.sup.186, Re.sup.188, sm.sup.153, Bi.sup.212, P.sup.32
and radioactive isotopes of Lu), chemotherapeutic agents e.g.,
methotrexate, adriamicin, vinca alkaloids (vincristine,
vinblastine, etoposide), doxorubicin, melphalan, mitomycin C,
chlorambucil, daunorubicin or other intercalating agents, enzymes
and fragments thereof such as nucleolytic enzymes, antibiotics, and
toxins such as small molecule toxins or enzymatically active toxins
of bacterial, fungal, plant or animal origin, including fragments
and/or variants thereof, and the various antitumor or anticancer
agents disclosed below. Other cytotoxic agents are described
below.
[0191] A "growth inhibitory agent" when used herein refers to a
compound or composition which inhibits growth of a cell, either in
vitro or in vivo. Examples of growth inhibitory agents include
agents that block cell cycle progression, such as agents that
induce G1 arrest and M-phase arrest. Classical M-phase blockers
include the vinca alkaloids (vincristine, vinorelbine and
vinblastine), taxanes, and topoisomerase II inhibitors such as
doxorubicin, epirubicin, daunorubicin, etoposide, and bleomycin.
Those agents that arrest G1 also spill over into S-phase arrest,
for example, DNA alkylating agents such as tamoxifen, prednisone,
dacarbazine, mechlorethamine, cisplatin, methotrexate,
5-fluorouracil, and ara-C. Further information can be found in The
Molecular Basis of Cancer, Mendelsohn and Israel, eds., Chapter 1,
entitled "Cell cycle regulation, oncogenes, and antineoplastic
drugs" by Murakami et al. (W B Saunders: Philadelphia, 1995),
especially p. 13. The taxanes (paclitaxel and docetaxel) are
anticancer drugs both derived from the yew tree. Docetaxel
(TAXOTERE.TM., Rhone-Poulenc Rorer), derived from the European yew,
is a semisynthetic analogue of paclitaxel (TAXOL.RTM.,
Bristol-Myers Squibb). Paclitaxel and docetaxel promote the
assembly of microtubules from tubulin dimers and stabilize
microtubules by preventing depolymerization, which results in the
inhibition of mitosis in cells.
[0192] "Label" as used herein refers to a detectable compound or
composition that is conjugated directly or indirectly to the
antibody so as to generate a "labeled" antibody. The label may be
detectable by itself (e.g., radioisotope labels or fluorescent
labels) or, in the case of an enzymatic label, may catalyze
chemical alteration of a substrate compound or composition that is
detectable.
[0193] The term "epitope tagged" as used herein refers to a
chimeric polypeptide comprising a polypeptide fused to a "tag
polypeptide." The tag polypeptide has enough residues to provide an
epitope against which an antibody can be made, yet is short enough
such that it does not interfere with activity of the polypeptide to
which it is fused. The tag polypeptide is also preferably fairly
unique so that the antibody does not substantially cross-react with
other epitopes. Suitable tag polypeptides generally have at least
six amino acid residues and usually between about 8 and 50 amino
acid residues (preferably, between about 10 and 20 amino acid
residues).
[0194] A "small molecule" is defined herein to have a molecular
weight below about 500 Daltons.
[0195] The terms "nucleic acid" and "polynucleotide" are used
interchangeably herein to refer to single- or double-stranded RNA,
DNA, or mixed polymers. Polynucleotides may include genomic
sequences, extra-genomic and plasmid sequences, and smaller
engineered gene segments that express, or may be adapted to express
polypeptides.
[0196] An "isolated nucleic acid" is a nucleic acid that is
substantially separated from other genome DNA sequences as well as
proteins or complexes such as ribosomes and polymerases, which
naturally accompany a native sequence. The term embraces a nucleic
acid sequence that has been removed from its naturally occurring
environment, and includes recombinant or cloned DNA isolates and
chemically synthesized analogues or analogues biologically
synthesized by heterologous systems. A substantially pure nucleic
acid includes isolated forms of the nucleic acid. Of course, this
refers to the nucleic acid as originally isolated and does not
exclude genes or sequences later added to the isolated nucleic acid
by the hand of man.
[0197] The term "polypeptide" is used in its conventional meaning,
i.e., as a sequence of amino acids. The polypeptides are not
limited to a specific length of the product. Peptides,
oligopeptides, and proteins are included within the definition of
polypeptide, and such terms may be used interchangeably herein
unless specifically indicated otherwise. This term also does not
refer to or exclude post-expression modifications of the
polypeptide, for example, glycosylations, acetylations,
phosphorylations and the like, as well as other modifications known
in the art, both naturally occurring and non-naturally occurring. A
polypeptide may be an entire protein, or a subsequence thereof.
Particular polypeptides of interest in the context of this
invention are amino acid subsequences comprising CDRs and being
capable of binding an antigen or HRV-infected cell.
[0198] An "isolated polypeptide" is one that has been identified
and separated and/or recovered from a component of its natural
environment. In preferred embodiments, the isolated polypeptide
will be purified (1) to greater than 95% by weight of polypeptide
as determined by the Lowry method, and most preferably more than
99% by weight, (2) to a degree sufficient to obtain at least 15
residues of N-terminal or internal amino acid sequence by use of a
spinning cup sequenator, or (3) to homogeneity by SDS-PAGE under
reducing or non-reducing conditions using Coomassie blue or,
preferably, silver stain. Isolated polypeptide includes the
polypeptide in situ within recombinant cells since at least one
component of the polypeptide's natural environment will not be
present. Ordinarily, however, isolated polypeptide will be prepared
by at least one purification step.
[0199] A "native sequence" polynucleotide is one that has the same
nucleotide sequence as a polynucleotide derived from nature. A
"native sequence" polypeptide is one that has the same amino acid
sequence as a polypeptide (e.g., antibody) derived from nature
(e.g., from any species). Such native sequence polynucleotides and
polypeptides can be isolated from nature or can be produced by
recombinant or synthetic means.
[0200] A polynucleotide "variant," as the term is used herein, is a
polynucleotide that typically differs from a polynucleotide
specifically disclosed herein in one or more substitutions,
deletions, additions and/or insertions. Such variants may be
naturally occurring or may be synthetically generated, for example,
by modifying one or more of the polynucleotide sequences of the
invention and evaluating one or more biological activities of the
encoded polypeptide as described herein and/or using any of a
number of techniques well known in the art.
[0201] A polypeptide "variant," as the term is used herein, is a
polypeptide that typically differs from a polypeptide specifically
disclosed herein in one or more substitutions, deletions, additions
and/or insertions. Such variants may be naturally occurring or may
be synthetically generated, for example, by modifying one or more
of the above polypeptide sequences of the invention and evaluating
one or more biological activities of the polypeptide as described
herein and/or using any of a number of techniques well known in the
art.
[0202] Modifications may be made in the structure of the
polynucleotides and polypeptides of the present invention and still
obtain a functional molecule that encodes a variant or derivative
polypeptide with desirable characteristics. When it is desired to
alter the amino acid sequence of a polypeptide to create an
equivalent, or even an improved, variant or portion of a
polypeptide of the invention, one skilled in the art will typically
change one or more of the codons of the encoding DNA sequence.
[0203] For example, certain amino acids may be substituted for
other amino acids in a protein structure without appreciable loss
of its ability to bind other polypeptides (e.g., antigens) or
cells. Since it is the binding capacity and nature of a protein
that defines that protein's biological functional activity, certain
amino acid sequence substitutions can be made in a protein
sequence, and, of course, it's underlying DNA coding sequence, and
nevertheless obtain a protein with like properties. It is thus
contemplated that various changes may be made in the peptide
sequences of the disclosed compositions, or corresponding DNA
sequences that encode said peptides without appreciable loss of
their biological utility or activity.
[0204] In many instances, a polypeptide variant will contain one or
more conservative substitutions. 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.
[0205] In making such changes, the hydropathic index of amino acids
may be considered. The importance of the hydropathic amino acid
index in conferring interactive biologic function on a protein is
generally understood in the art (Kyte and Doolittle, 1982). It is
accepted that the relative hydropathic character of the amino acid
contributes to the secondary structure of the resultant protein,
which in turn defines the interaction of the protein with other
molecules, for example, enzymes, substrates, receptors, DNA,
antibodies, antigens, and the like. Each amino acid has been
assigned a hydropathic index on the basis of its hydrophobicity and
charge characteristics (Kyte and Doolittle, 1982). These values
are: isoleucine (+4.5); valine (+4.2); leucine (+3.8);
phenylalanine (+2.8); cysteine/cystine (+2.5); methionine (+1.9);
alanine (+1.8); glycine (-0.4); threonine (-0.7); serine (-0.8);
tryptophan (-0.9); tyrosine (-1.3); proline (-1.6); histidine
(-3.2); glutamate (-3.5); glutamine (-3.5); aspartate (-3.5);
asparagine (-3.5); lysine (-3.9); and arginine (-4.5).
[0206] It is known in the art that certain amino acids may be
substituted by other amino acids having a similar hydropathic index
or score and still result in a protein with similar biological
activity, i.e. still obtain a biological functionally equivalent
protein. In making such changes, the substitution of amino acids
whose hydropathic indices are within .+-.2 is preferred, those
within .+-.1 are particularly preferred, and those within .+-.0.5
are even more particularly preferred. It is also understood in the
art that the substitution of like amino acids can be made
effectively on the basis of hydrophilicity. U.S. Pat. No. 4,554,101
states that the greatest local average hydrophilicity of a protein,
as governed by the hydrophilicity of its adjacent amino acids,
correlates with a biological property of the protein.
[0207] As detailed in U.S. Pat. No. 4,554,101, the following
hydrophilicity values have been assigned to amino acid residues:
arginine (+3.0); lysine (+3.0); aspartate (+3.0.+-.1); glutamate
(+3.0.+-.1); serine (+0.3); asparagine (+0.2); glutamine (+0.2);
glycine (0); threonine (-0.4); proline (-0.5.+-.1); alanine (-0.5);
histidine (-0.5); cysteine (-1.0); methionine (-1.3); valine
(-1.5); leucine (-1.8); isoleucine (-1.8); tyrosine (-2.3);
phenylalanine (-2.5); tryptophan (-3.4). It is understood that an
amino acid can be substituted for another having a similar
hydrophilicity value and still obtain a biologically equivalent,
and in particular, an immunologically equivalent protein. In such
changes, the substitution of amino acids whose hydrophilicity
values are within .+-.2 is preferred, those within .+-.1 are
particularly preferred, and those within .+-.0.5 are even more
particularly preferred.
[0208] As outlined above, amino acid substitutions are generally
therefore based on the relative similarity of the amino acid
side-chain substituents, for example, their hydrophobicity,
hydrophilicity, charge, size, and the like. Exemplary substitutions
that take various of the foregoing characteristics into
consideration are well known to those of skill in the art and
include: arginine and lysine; glutamate and aspartate; serine and
threonine; glutamine and asparagine; and valine, leucine and
isoleucine.
[0209] Amino acid substitutions may further be made on the basis of
similarity in polarity, charge, solubility, hydrophobicity,
hydrophilicity and/or the amphipathic nature of the residues. For
example, negatively charged amino acids include aspartic acid and
glutamic acid; positively charged amino acids include lysine and
arginine; and amino acids with uncharged polar head groups having
similar hydrophilicity values include leucine, isoleucine and
valine; glycine and alanine; asparagine and glutamine; and serine,
threonine, phenylalanine and tyrosine. Other groups of amino acids
that may represent conservative changes include: (1) ala, pro, gly,
glu, asp, gln, asn, ser, thr; (2) cys, ser, tyr, thr; (3) val, ile,
leu, met, ala, phe; (4) lys, arg, his; and (5) phe, tyr, trp, his.
A variant may also, or alternatively, contain nonconservative
changes. In a preferred embodiment, variant polypeptides differ
from a native sequence by substitution, deletion or addition of
five amino acids or fewer. Variants may also (or alternatively) be
modified by, for example, the deletion or addition of amino acids
that have minimal influence on the immunogenicity, secondary
structure and hydropathic nature of the polypeptide.
[0210] Polypeptides may comprise a signal (or leader) sequence at
the N-terminal end of the protein, which co-translationally or
post-translationally directs transfer of the protein. The
polypeptide may also be conjugated to 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 may be
conjugated to an immunoglobulin Fc region.
[0211] When comparing polynucleotide and polypeptide sequences, two
sequences are said to be "identical" if the sequence of nucleotides
or amino acids in the two sequences is the same when aligned for
maximum correspondence, as described below. Comparisons between two
sequences are typically performed by comparing the sequences over a
comparison window to identify and compare local regions of sequence
similarity. A "comparison window" as used herein, refers to a
segment of at least about 20 contiguous positions, usually 30 to
about 75, 40 to about 50, in which a sequence may be compared to a
reference sequence of the same number of contiguous positions after
the two sequences are optimally aligned.
[0212] Optimal alignment of sequences for comparison may be
conducted using the Megalign program in the Lasergene suite of
bioinformatics software (DNASTAR, Inc., Madison, Wis.), using
default parameters. This program embodies several alignment schemes
described in the following references: Dayhoff, M. O. (1978) A
model of evolutionary change in proteins--Matrices for detecting
distant relationships. In Dayhoff, M. O. (ed.) Atlas of Protein
Sequence and Structure, National Biomedical Research Foundation,
Washington DC Vol. 5, Suppl. 3, pp. 345-358; Hein J. (1990) Unified
Approach to Alignment and Phylogenes pp. 626-645 Methods in
Enzymology vol. 183, Academic Press, Inc., San Diego, Calif.;
Higgins, D. G. and Sharp, P. M. (1989) CABIOS 5:151-153; Myers, E.
W. and Muller W. (1988) CABIOS 4:11-17; Robinson, E. D. (1971)
Comb. Theon 11:105; Santou, N. Nes, M. (1987) Mol. Biol. Evol.
4:406-425; Sneath, P. H. A. and Sokal, R. R. (1973) Numerical
Taxonomy--the Principles and Practice of Numerical Taxonomy,
Freeman Press, San Francisco, Calif.; Wilbur, W. J. and Lipman, D.
J. (1983) Proc. Natl. Acad., Sci. USA 80:726-730.
[0213] Alternatively, optimal alignment of sequences for comparison
may be conducted by the local identity algorithm of Smith and
Waterman (1981) Add. APL. Math 2:482, by the identity alignment
algorithm of Needleman and Wunsch (1970) J. Mol. Biol. 48:443, by
the search for similarity methods of Pearson and Lipman (1988)
Proc. Natl. Acad. Sci. USA 85: 2444, by computerized
implementations of these algorithms (GAP, BESTFIT, BLAST, FASTA,
and TFASTA in the Wisconsin Genetics Software Package, Genetics
Computer Group (GCG), 575 Science Dr., Madison, Wis.), or by
inspection.
[0214] One preferred example of algorithms that are suitable for
determining percent sequence identity and sequence similarity are
the BLAST and BLAST 2.0 algorithms, which are described in Altschul
et al. (1977) Nucl. Acids Res. 25:3389-3402 and Altschul et al.
(1990) J. Mol. Biol. 215:403-410, respectively. BLAST and BLAST 2.0
can be used, for example with the parameters described herein, to
determine percent sequence identity for the polynucleotides and
polypeptides of the invention. Software for performing BLAST
analyses is publicly available through the National Center for
Biotechnology Information.
[0215] In one illustrative example, cumulative scores can be
calculated using, for nucleotide sequences, the parameters M
(reward score for a pair of matching residues; always >0) and N
(penalty score for mismatching residues; always <0). Extension
of the word hits in each direction are halted when: the cumulative
alignment score falls off by the quantity X from its maximum
achieved value; the cumulative score goes to zero or below, due to
the accumulation of one or more negative-scoring residue
alignments; or the end of either sequence is reached. The BLAST
algorithm parameters W, T and X determine the sensitivity and speed
of the alignment. The BLASTN program (for nucleotide sequences)
uses as defaults a wordlength (W) of 11, and expectation (E) of 10,
and the BLOSUM62 scoring matrix (see Henikoff and Henikoff (1989)
Proc. Natl. Acad. Sci. USA 89:10915) alignments, (B) of 50,
expectation (E) of 10, M=5, N=-4 and a comparison of both
strands.
[0216] For amino acid sequences, a scoring matrix can be used to
calculate the cumulative score. Extension of the word hits in each
direction are halted when: the cumulative alignment score falls off
by the quantity X from its maximum achieved value; the cumulative
score goes to zero or below, due to the accumulation of one or more
negative-scoring residue alignments; or the end of either sequence
is reached. The BLAST algorithm parameters W, T and X determine the
sensitivity and speed of the alignment.
[0217] In one approach, the "percentage of sequence identity" is
determined by comparing two optimally aligned sequences over a
window of comparison of at least 20 positions, wherein the portion
of the polynucleotide or polypeptide sequence in the comparison
window may comprise additions or deletions (i.e., gaps) of 20
percent or less, usually 5 to 15 percent, or 10 to 12 percent, as
compared to the reference sequences (which does not comprise
additions or deletions) for optimal alignment of the two sequences.
The percentage is calculated by determining the number of positions
at which the identical nucleic acid bases or amino acid residues
occur in both sequences to yield the number of matched positions,
dividing the number of matched positions by the total number of
positions in the reference sequence (i.e., the window size) and
multiplying the results by 100 to yield the percentage of sequence
identity.
[0218] "Homology" refers to the percentage of residues in the
polynucleotide or polypeptide sequence variant that are identical
to the non-variant sequence after aligning the sequences and
introducing gaps, if necessary, to achieve the maximum percent
homology. In particular embodiments, polynucleotide and polypeptide
variants have at least 70%, at least 75%, at least 80%, at least
90%, at least 95%, at least 98%, or at least 99% polynucleotide or
polypeptide homology with a polynucleotide or polypeptide described
herein.
[0219] "Vector" includes shuttle and expression vectors. Typically,
the plasmid construct will also include an origin of replication
(e.g., the ColE1 origin of replication) and a selectable marker
(e.g., ampicillin or tetracycline resistance), for replication and
selection, respectively, of the plasmids in bacteria. An
"expression vector" refers to a vector that contains the necessary
control sequences or regulatory elements for expression of the
antibodies including antibody fragment of the invention, in
bacterial or eukaryotic cells. Suitable vectors are disclosed
below. As used in this specification and the appended claims, the
singular forms "a," "an" and "the" include plural references unless
the content clearly dictates otherwise.
[0220] The invention also includes nucleic acid sequences encoding
part or all of the light and heavy chains and CDRs of the present
invention. Due to redundancy of the genetic code, variants of these
sequences will exist that encode the same amino acid sequences.
[0221] Variant antibodies are also included within the scope of the
invention. Thus, variants of the sequences recited in the
application are also included within the scope of the invention.
Further variants of the antibody sequences having improved affinity
may be obtained using methods known in the art and are included
within the scope of the invention. For example, amino acid
substitutions may be used to obtain antibodies with further
improved affinity. Alternatively, codon optimization of the
nucleotide sequence may be used to improve the efficiency of
translation in expression systems for the production of the
antibody.
[0222] Preferably, such variant antibody sequences will share 70%
or more (i.e. 80, 85, 90, 95, 97, 98, 99% or more) sequence
identity with the sequences recited in the application. Preferably
such sequence identity is calculated with regard to the full length
of the reference sequence (i.e. the sequence recited in the
application). Preferably, percentage identity, as referred to
herein, is as determined using BLAST version 2.1.3 using the
default parameters specified by the NCBI (the National Center for
Biotechnology Information; http://www.ncbi.nlm.nih.gov/) [Blosum 62
matrix; gap open penalty=11 and gap extension penalty=1].
[0223] Further included within the scope of the invention are
vectors such as expression vectors, comprising a nucleic acid
sequence according to the invention. Cells transformed with such
vectors are also included within the scope of the invention.
[0224] As will be understood by the skilled artisan, general
description of antibodies herein and methods of preparing and using
the same also apply to individual antibody polypeptide constituents
and antibody fragments.
[0225] The antibodies of the present invention may be polyclonal or
monoclonal antibodies. However, in preferred embodiments, they are
monoclonal. In particular embodiments, antibodies of the present
invention are human antibodies. Methods of producing polyclonal and
monoclonal antibodies are known in the art and described generally,
e.g., in U.S. Pat. No. 6,824,780.
[0226] Typically, the antibodies of the present invention are
produced recombinantly, using vectors and methods available in the
art, as described further below. Human antibodies may also be
generated by in vitro activated B cells (see U.S. Pat. Nos.
5,567,610 and 5,229,275).
[0227] Human antibodies may also be produced in transgenic animals
(e.g., mice) that are capable of producing a full repertoire of
human antibodies in the absence of endogenous immunoglobulin
production. For example, it has been described that the homozygous
deletion of the antibody heavy-chain joining region (J.sub.H) gene
in chimeric and germ-line mutant mice results in complete
inhibition of endogenous antibody production. Transfer of the human
germ-line immunoglobulin gene array into such germ-line mutant mice
results in the production of human antibodies upon antigen
challenge. See, e.g., Jakobovits et al., Proc. Natl. Acad. Sci.
USA, 90:2551 (1993); Jakobovits et al., Nature, 362:255-258 (1993);
Bruggemann et al., Year in Immuno., 7:33 (1993); U.S. Pat. Nos.
5,545,806, 5,569,825, 5,591,669 (all of GenPharm); U.S. Pat. No.
5,545,807; and WO 97/17852. Such animals may be genetically
engineered to produce human antibodies comprising a polypeptide of
the present invention.
[0228] In certain embodiments, antibodies of the present invention
are chimeric antibodies that comprise sequences derived from both
human and non-human sources. In particular embodiments, these
chimeric antibodies are humanized or Primatized.TM.. In practice,
humanized antibodies are typically human antibodies in which some
hypervariable region residues and possibly some FR residues are
substituted by residues from analogous sites in rodent
antibodies.
[0229] In the context of the present invention, chimeric antibodies
also include human antibodies wherein the human hypervariable
region or one or more CDRs are retained, but one or more other
regions of sequence have been replaced by corresponding sequences
from a non-human animal.
[0230] The choice of non-human sequences, both light and heavy, to
be used in making the chimeric antibodies is important to reduce
antigenicity and human anti-non-human antibody responses when the
antibody is intended for human therapeutic use. It is further
important that chimeric antibodies retain high binding affinity for
the antigen and other favorable biological properties. To achieve
this goal, according to a preferred method, chimeric antibodies are
prepared by a process of analysis of the parental sequences and
various conceptual chimeric products using three-dimensional models
of the parental human and non-human sequences. Three-dimensional
immunoglobulin models are commonly available and are familiar to
those skilled in the art. Computer programs are available which
illustrate and display probable three-dimensional conformational
structures of selected candidate immunoglobulin sequences.
[0231] Inspection of these displays permits analysis of the likely
role of the residues in the functioning of the candidate
immunoglobulin sequence, i.e., the analysis of residues that
influence the ability of the candidate immunoglobulin to bind its
antigen. In this way, FR residues can be selected and combined from
the recipient and import sequences so that the desired antibody
characteristic, such as increased affinity for the target
antigen(s), is achieved. In general, the hypervariable region
residues are directly and most substantially involved in
influencing antigen binding.
[0232] As noted above, antibodies (or immunoglobulins) can be
divided into five different classes, based on differences in the
amino acid sequences in the constant region of the heavy chains.
All immunoglobulins within a given class have very similar heavy
chain constant regions. These differences can be detected by
sequence studies or more commonly by serological means (i.e. by the
use of antibodies directed to these differences). Antibodies, or
fragments thereof, of the present invention may be any class, and
may, therefore, have a gamma, mu, alpha, delta, or epsilon heavy
chain. A gamma chain may be gamma 1, gamma 2, gamma 3, or gamma 4;
and an alpha chain may be alpha 1 or alpha 2.
[0233] In a preferred embodiment, an antibody of the present
invention, or fragment thereof, is an IgG. IgG is considered the
most versatile immunoglobulin, because it is capable of carrying
out all of the functions of immunoglobulin molecules. IgG is the
major Ig in serum, and the only class of Ig that crosses the
placenta. IgG also fixes complement, although the IgG4 subclass
does not. Macrophages, monocytes, PMN's and some lymphocytes have
Fc receptors for the Fc region of IgG. Not all subclasses bind
equally well: IgG2 and IgG4 do not bind to Fc receptors. A
consequence of binding to the Fc receptors on PMN's, monocytes and
macrophages is that the cell can now internalize the antigen
better. IgG is an opsonin that enhances phagocytosis. Binding of
IgG to Fc receptors on other types of cells results in the
activation of other functions. Antibodies of the present invention
may be of any IgG subclass.
[0234] In another preferred embodiment, an antibody, or fragment
thereof, of the present invention is an IgE. IgE is the least
common serum Ig since it binds very tightly to Fc receptors on
basophils and mast cells even before interacting with antigen. As a
consequence of its binding to basophils and mast cells, IgE is
involved in allergic reactions. Binding of the allergen to the IgE
on the cells results in the release of various pharmacological
mediators that result in allergic symptoms. IgE also plays a role
in parasitic helminth diseases. Eosinophils have Fc receptors for
IgE and binding of eosinophils to IgE-coated helminths results in
killing of the parasite. IgE does not fix complement.
[0235] In various embodiments, antibodies of the present invention,
and fragments thereof, comprise a variable light chain that is
either kappa or lambda. The lambda chain may be any of subtype,
including, e.g., lambda 1, lambda 2, lambda 3, and lambda 4.
[0236] As noted above, the present invention further provides
antibody fragments comprising a polypeptide of the present
invention. In certain circumstances there are advantages of using
antibody fragments, rather than whole antibodies. For example, the
smaller size of the fragments allows for rapid clearance, and may
lead to improved access to certain tissues, such as solid tumors.
Examples of antibody fragments include: Fab, Fab', F(ab').sub.2 and
Fv fragments; diabodies; linear antibodies; single-chain
antibodies; and multispecific antibodies formed from antibody
fragments.
[0237] Various techniques have been developed for the production of
antibody fragments. Traditionally, these fragments were derived via
proteolytic digestion of intact antibodies (see, e.g., Morimoto et
al., Journal of Biochemical and Biophysical Methods 24:107-117
(1992); and Brennan et al., Science, 229:81 (1985)). However, these
fragments can now be produced directly by recombinant host cells.
Fab, Fv and ScFv antibody fragments can all be expressed in and
secreted from E. coli, thus allowing the facile production of large
amounts of these fragments. Fab'-SH fragments can be directly
recovered from E. coli and chemically coupled to form F(ab').sub.2
fragments (Carter et al., Bio/Technology 10:163-167 (1992)).
According to another approach, F(ab').sub.2 fragments can be
isolated directly from recombinant host cell culture. Fab and
F(ab').sub.2 fragment with increased in vivo half-life comprising a
salvage receptor binding epitope residues are described in U.S.
Pat. No. 5,869,046. Other techniques for the production of antibody
fragments will be apparent to the skilled practitioner.
[0238] In other embodiments, the antibody of choice is a single
chain Fv fragment (scFv). See WO 93/16185; U.S. Pat. Nos.
5,571,894; and 5,587,458. Fv and sFv are the only species with
intact combining sites that are devoid of constant regions. Thus,
they are suitable for reduced nonspecific binding during in vivo
use. sFv fusion proteins may be constructed to yield fusion of an
effector protein at either the amino or the carboxy terminus of an
sFv. See Antibody Engineering, ed. Borrebaeck, supra. The antibody
fragment may also be a "linear antibody", e.g., as described in
U.S. Pat. No. 5,641,870 for example. Such linear antibody fragments
may be monospecific or bispecific.
[0239] In certain embodiments, antibodies of the present invention
are bispecific or multi-specific. Bispecific antibodies are
antibodies that have binding specificities for at least two
different epitopes. Exemplary bispecific antibodies may bind to two
different epitopes of a single antigen. Other such antibodies may
combine a first antigen binding site with a binding site for a
second antigen. Alternatively, an anti-HRV arm may be combined with
an arm that binds to a triggering molecule on a leukocyte, such as
a T-cell receptor molecule (e.g., CD3), or Fc receptors for IgG
(Fc.gamma.R), such as Fc.gamma.RI (CD64), Fc.gamma.RII (CD32) and
Fc.gamma.RIII (CD16), so as to focus and localize cellular defense
mechanisms to the infected cell. Bispecific antibodies may also be
used to localize cytotoxic agents to infected cells. These
antibodies possess an HRV-binding arm and an arm that binds the
cytotoxic agent (e.g., saporin, anti-interferon-.alpha., vinca
alkaloid, ricin A chain, methotrexate or radioactive isotope
hapten). Bispecific antibodies can be prepared as full length
antibodies or antibody fragments (e.g., F(ab').sub.2 bispecific
antibodies). WO 96/16673 describes a bispecific
anti-ErbB2/anti-Fc.gamma.RIII antibody and U.S. Pat. No. 5,837,234
discloses a bispecific anti-ErbB2/anti-Fc.gamma.RI antibody. A
bispecific anti-ErbB2/Fc.alpha. antibody is shown in WO98/02463.
U.S. Pat. No. 5,821,337 teaches a bispecific anti-ErbB2/anti-CD3
antibody.
[0240] Methods for making bispecific antibodies are known in the
art. Traditional production of full length bispecific antibodies is
based on the co-expression of two immunoglobulin heavy chain-light
chain pairs, where the two chains have different specificities
(Millstein et al., Nature, 305:537-539 (1983)). Because of the
random assortment of immunoglobulin heavy and light chains, these
hybridomas (quadromas) produce a potential mixture of ten different
antibody molecules, of which only one has the correct bispecific
structure. Purification of the correct molecule, which is usually
done by affinity chromatography steps, is rather cumbersome, and
the product yields are low. Similar procedures are disclosed in WO
93/08829, and in Traunecker et al., EMBO J., 10:3655-3659
(1991).
[0241] According to a different approach, antibody variable regions
with the desired binding specificities (antibody-antigen combining
sites) are fused to immunoglobulin constant domain sequences.
Preferably, the fusion is with an Ig heavy chain constant domain,
comprising at least part of the hinge, C.sub.H2, and C.sub.H3
regions. It is preferred to have the first heavy-chain constant
region (C.sub.H1) containing the site necessary for light chain
bonding, present in at least one of the fusions. DNAs encoding the
immunoglobulin heavy chain fusions and, if desired, the
immunoglobulin light chain, are inserted into separate expression
vectors, and are co-transfected into a suitable host cell. This
provides for greater flexibility in adjusting the mutual
proportions of the three polypeptide fragments in embodiments when
unequal ratios of the three polypeptide chains used in the
construction provide the optimum yield of the desired bispecific
antibody. It is, however, possible to insert the coding sequences
for two or all three polypeptide chains into a single expression
vector when the expression of at least two polypeptide chains in
equal ratios results in high yields or when the ratios have no
significant affect on the yield of the desired chain
combination.
[0242] In a preferred embodiment of this approach, the bispecific
antibodies are composed of a hybrid immunoglobulin heavy chain with
a first binding specificity in one arm, and a hybrid immunoglobulin
heavy chain-light chain pair (providing a second binding
specificity) in the other arm. It was found that this asymmetric
structure facilitates the separation of the desired bispecific
compound from unwanted immunoglobulin chain combinations, as the
presence of an immunoglobulin light chain in only one half of the
bispecific molecule provides for a facile way of separation. This
approach is disclosed in WO 94/04690. For further details of
generating bispecific antibodies see, for example, Suresh et al.,
Methods in Enzymology, 121:210 (1986).
[0243] According to another approach described in U.S. Pat. No.
5,731,168, the interface between a pair of antibody molecules can
be engineered to maximize the percentage of heterodimers that are
recovered from recombinant cell culture. The preferred interface
comprises at least a part of the C.sub.H3 domain. In this method,
one or more small amino acid side chains from the interface of the
first antibody molecule are replaced with larger side chains (e.g.,
tyrosine or tryptophan). Compensatory "cavities" of identical or
similar size to the large side chain(s) are created on the
interface of the second antibody molecule by replacing large amino
acid side chains with smaller ones (e.g., alanine or threonine).
This provides a mechanism for increasing the yield of the
heterodimer over other unwanted end-products such as
homodimers.
[0244] Bispecific antibodies include cross-linked or
"heteroconjugate" antibodies. For example, one of the antibodies in
the heteroconjugate can be coupled to avidin, the other to biotin.
Such antibodies have, for example, been proposed to target immune
system cells to unwanted cells (U.S. Pat. No. 4,676,980), and for
treatment of HRV infection (WO 91/00360, WO 92/200373, and EP
03089). Heteroconjugate antibodies may be made using any convenient
cross-linking methods. Suitable cross-linking agents are well known
in the art, and are disclosed in U.S. Pat. No. 4,676,980, along
with a number of cross-linking techniques.
[0245] Techniques for generating bispecific antibodies from
antibody fragments have also been described in the literature. For
example, bispecific antibodies can be prepared using chemical
linkage. Brennan et al., Science, 229: 81 (1985) describe a
procedure wherein intact antibodies are proteolytically cleaved to
generate F(ab).sub.2 fragments. These fragments are reduced in the
presence of the dithiol complexing agent, sodium arsenite, to
stabilize vicinal dithiols and prevent intermolecular disulfide
formation. The Fab' fragments generated are then converted to
thionitrobenzoate (TNB) derivatives. One of the Fab'-TNB
derivatives is then reconverted to the Fab'-thiol by reduction with
mercaptoethylamine and is mixed with an equimolar amount of the
other Fab'-TNB derivative to form the bispecific antibody. The
bispecific antibodies produced can be used as agents for the
selective immobilization of enzymes.
[0246] Recent progress has facilitated the direct recovery of
Fab'-SH fragments from E. coli, which can be chemically coupled to
form bispecific antibodies. Shalaby et al., J. Exp. Med., 175:
217-225 (1992) describe the production of a humanized bispecific
antibody F(ab').sub.2 molecule. Each Fab' fragment was separately
secreted from E. coli and subjected to directed chemical coupling
in vitro to form the bispecific antibody. The bispecific antibody
thus formed was able to bind to cells overexpressing the ErbB2
receptor and normal human T cells, as well as trigger the lytic
activity of human cytotoxic lymphocytes against human breast tumor
targets.
[0247] Various techniques for making and isolating bispecific
antibody fragments directly from recombinant cell culture have also
been described. For example, bispecific antibodies have been
produced using leucine zippers. Kostelny et al., J. Immunol.,
148(5):1547-1553 (1992). The leucine zipper peptides from the Fos
and Jun proteins were linked to the Fab' portions of two different
antibodies by gene fusion. The antibody homodimers were reduced at
the hinge region to form monomers and then re-oxidized to form the
antibody heterodimers. This method can also be utilized for the
production of antibody homodimers. The "diabody" technology
described by Hollinger et al., Proc. Natl. Acad. Sci. USA,
90:6444-6448 (1993) has provided an alternative mechanism for
making bispecific antibody fragments. The fragments comprise a
V.sub.H connected to a V.sub.L by a linker that is too short to
allow pairing between the two domains on the same chain.
Accordingly, the V.sub.H and V.sub.L domains of one fragment are
forced to pair with the complementary V.sub.L and V.sub.H domains
of another fragment, thereby forming two antigen-binding sites.
Another strategy for making bispecific antibody fragments by the
use of single-chain Fv (sFv) dimers has also been reported. See
Gruber et al., J. Immunol., 152:5368 (1994).
[0248] Antibodies with more than two valencies are contemplated.
For example, trispecific antibodies can be prepared. Tutt et al.,
J. Immunol. 147: 60 (1991). A multivalent antibody may be
internalized (and/or catabolized) faster than a bivalent antibody
by a cell expressing an antigen to which the antibodies bind. The
antibodies of the present invention can be multivalent antibodies
with three or more antigen binding sites (e.g., tetravalent
antibodies), which can be readily produced by recombinant
expression of nucleic acid encoding the polypeptide chains of the
antibody. The multivalent antibody can comprise a dimerization
domain and three or more antigen binding sites. The preferred
dimerization domain comprises (or consists of) an Fc region or a
hinge region. In this scenario, the antibody will comprise an Fc
region and three or more antigen binding sites amino-terminal to
the Fc region. The preferred multivalent antibody herein comprises
(or consists of) three to about eight, but preferably four, antigen
binding sites. The multivalent antibody comprises at least one
polypeptide chain (and preferably two polypeptide chains), wherein
the polypeptide chain(s) comprise two or more variable regions. For
instance, the polypeptide chain(s) may comprise
VD1-(X1).sub.n-VD2-(X2).sub.n-Fc, wherein VD1 is a first variable
region, VD2 is a second variable region, Fc is one polypeptide
chain of an Fc region, X1 and X2 represent an amino acid or
polypeptide, and n is 0 or 1. For instance, the polypeptide
chain(s) may comprise: VH-CH1-flexible linker-VH-CH1-Fc region
chain; or VH-CH1-VH-CH1-Fc region chain. The multivalent antibody
herein preferably further comprises at least two (and preferably
four) light chain variable region polypeptides. The multivalent
antibody herein may, for instance, comprise from about two to about
eight light chain variable region polypeptides. The light chain
variable region polypeptides contemplated here comprise a light
chain variable region and, optionally, further comprise a C.sub.L
domain.
[0249] Antibodies of the invention further include single chain
antibodies. In particular embodiments, antibodies of the invention
are internalizing antibodies.
[0250] Amino acid sequence modification(s) of the antibodies
described herein are contemplated. For example, it may be desirable
to improve the binding affinity and/or other biological properties
of the antibody. Amino acid sequence variants of the antibody may
be prepared by introducing appropriate nucleotide changes into a
polynucleotide that encodes the antibody, or a chain thereof, or by
peptide synthesis. Such modifications include, for example,
deletions from, and/or insertions into and/or substitutions of,
residues within the amino acid sequences of the antibody. Any
combination of deletion, insertion, and substitution may be made to
arrive at the final antibody, provided that the final construct
possesses the desired characteristics. The amino acid changes also
may alter post-translational processes of the antibody, such as
changing the number or position of glycosylation sites. Any of the
variations and modifications described above for polypeptides of
the present invention may be included in antibodies of the present
invention.
[0251] A useful method for identification of certain residues or
regions of an antibody that are preferred locations for mutagenesis
is called "alanine scanning mutagenesis" as described by Cunningham
and Wells in Science, 244:1081-1085 (1989). Here, a residue or
group of target residues are identified (e.g., charged residues
such as arg, asp, his, lys, and glu) and replaced by a neutral or
negatively charged amino acid (most preferably alanine or
polyalanine) to affect the interaction of the amino acids with PSCA
antigen. Those amino acid locations demonstrating functional
sensitivity to the substitutions then are refined by introducing
further or other variants at, or for, the sites of substitution.
Thus, while the site for introducing an amino acid sequence
variation is predetermined, the nature of the mutation per se need
not be predetermined. For example, to analyze the performance of a
mutation at a given site, ala scanning or random mutagenesis is
conducted at the target codon or region and the expressed
anti-antibody variants are screened for the desired activity.
[0252] Amino acid sequence insertions include amino- and/or
carboxyl-terminal fusions ranging in length from one residue to
polypeptides containing a hundred or more residues, as well as
intrasequence insertions of single or multiple amino acid residues.
Examples of terminal insertions include an antibody with an
N-terminal methionyl residue or the antibody fused to a cytotoxic
polypeptide. Other insertional variants of an antibody include the
fusion to the N- or C-terminus of the antibody to an enzyme (e.g.,
for ADEPT) or a polypeptide that increases the serum half-life of
the antibody.
[0253] Another type of variant is an amino acid substitution
variant. These variants have at least one amino acid residue in the
antibody molecule replaced by a different residue. The sites of
greatest interest for substitutional mutagenesis include the
hypervariable regions, but FR alterations are also contemplated.
Conservative and non-conservative substitutions are
contemplated.
[0254] Substantial modifications in the biological properties of
the antibody are accomplished by selecting substitutions that
differ significantly in their effect on maintaining (a) the
structure of the polypeptide backbone in the area of the
substitution, for example, as a sheet or helical conformation, (b)
the charge or hydrophobicity of the molecule at the target site, or
(c) the bulk of the side chain.
[0255] Any cysteine residue not involved in maintaining the proper
conformation of the antibody also may be substituted, generally
with serine, to improve the oxidative stability of the molecule and
prevent aberrant crosslinking Conversely, cysteine bond(s) may be
added to the antibody to improve its stability (particularly where
the antibody is an antibody fragment such as an Fv fragment).
[0256] One type of substitutional variant involves substituting one
or more hypervariable region residues of a parent antibody.
Generally, the resulting variant(s) selected for further
development will have improved biological properties relative to
the parent antibody from which they are generated. A convenient way
for generating such substitutional variants involves affinity
maturation using phage display. Briefly, several hypervariable
region sites (e.g., 6-7 sites) are mutated to generate all possible
amino substitutions at each site. The antibody variants thus
generated are displayed in a monovalent fashion from filamentous
phage particles as fusions to the gene III product of M13 packaged
within each particle. The phage-displayed variants are then
screened for their biological activity (e.g., binding affinity) as
herein disclosed. In order to identify candidate hypervariable
region sites for modification, alanine scanning mutagenesis can be
performed to identify hypervariable region residues contributing
significantly to antigen binding. Alternatively, or additionally,
it may be beneficial to analyze a crystal structure of the
antigen-antibody complex to identify contact points between the
antibody and an antigen or infected cell. Such contact residues and
neighboring residues are candidates for substitution according to
the techniques elaborated herein. Once such variants are generated,
the panel of variants is subjected to screening as described herein
and antibodies with superior properties in one or more relevant
assays may be selected for further development.
[0257] Another type of amino acid variant of the antibody alters
the original glycosylation pattern of the antibody. By altering is
meant deleting one or more carbohydrate moieties found in the
antibody, and/or adding one or more glycosylation sites that are
not present in the antibody.
[0258] Glycosylation of antibodies is typically either N-linked or
O-linked. N-linked refers to the attachment of the carbohydrate
moiety to the side chain of an asparagine residue. The tripeptide
sequences asparagine-X-serine and asparagine-X-threonine, where X
is any amino acid except proline, are the recognition sequences for
enzymatic attachment of the carbohydrate moiety to the asparagine
side chain. Thus, the presence of either of these tripeptide
sequences in a polypeptide creates a potential glycosylation site.
O-linked glycosylation refers to the attachment of one of the
sugars N-aceylgalactosamine, galactose, or xylose to a hydroxyamino
acid, most commonly serine or threonine, although 5-hydroxyproline
or 5-hydroxylysine may also be used. Addition of glycosylation
sites to the antibody is conveniently accomplished by altering the
amino acid sequence such that it contains one or more of the
above-described tripeptide sequences (for N-linked glycosylation
sites). The alteration may also be made by the addition of, or
substitution by, one or more serine or threonine residues to the
sequence of the original antibody (for O-linked glycosylation
sites).
[0259] The antibody of the invention is modified with respect to
effector function, e.g., so as to enhance antigen-dependent
cell-mediated cyotoxicity (ADCC) and/or complement dependent
cytotoxicity (CDC) of the antibody. This may be achieved by
introducing one or more amino acid substitutions in an Fc region of
the antibody. Alternatively or additionally, cysteine residue(s)
may be introduced in the Fc region, thereby allowing interchain
disulfide bond formation in this region. The homodimeric antibody
thus generated may have improved internalization capability and/or
increased complement-mediated cell killing and antibody-dependent
cellular cytotoxicity (ADCC). See Caron et al., J. Exp Med.
176:1191-1195 (1992) and Shopes, B. J. Immunol. 148:2918-2922
(1992). Homodimeric antibodies with enhanced anti-infection
activity may also be prepared using heterobifunctional
cross-linkers as described in Wolff et al., Cancer Research
53:2560-2565 (1993). Alternatively, an antibody can be engineered
which has dual Fc regions and may thereby have enhanced complement
lysis and ADCC capabilities. See Stevenson et al., Anti-Cancer Drug
Design 3:219-230 (1989). To increase the serum half-life of the
antibody, one may incorporate a salvage receptor binding epitope
into the antibody (especially an antibody fragment) as described in
U.S. Pat. No. 5,739,277, for example. As used herein, the term
"salvage receptor binding epitope" refers to an epitope of the Fc
region of an IgG molecule (e.g., IgG.sub.1, IgG.sub.2, IgG.sub.3,
or IgG.sub.4) that is responsible for increasing the in vivo serum
half-life of the IgG molecule.
[0260] Antibodies of the present invention may also be modified to
include an epitope tag or label, e.g., for use in purification or
diagnostic applications. The invention also pertains to therapy
with immunoconjugates comprising an antibody conjugated to an
anti-cancer agent such as a cytotoxic agent or a growth inhibitory
agent. Chemotherapeutic agents useful in the generation of such
immunoconjugates have been described above.
[0261] Conjugates of an antibody and one or more small molecule
toxins, such as a calicheamicin, maytansinoids, a trichothene, and
CC1065, and the derivatives of these toxins that have toxin
activity, are also contemplated herein.
[0262] In one preferred embodiment, an antibody (full length or
fragments) of the invention is conjugated to one or more
maytansinoid molecules. Maytansinoids are mitototic inhibitors that
act by inhibiting tubulin polymerization. Maytansine was first
isolated from the east African shrub Maytenus serrata (U.S. Pat.
No. 3,896,111). Subsequently, it was discovered that certain
microbes also produce maytansinoids, such as maytansinol and C-3
maytansinol esters (U.S. Pat. No. 4,151,042). Synthetic maytansinol
and derivatives and analogues thereof are disclosed, for example,
in U.S. Pat. Nos. 4,137,230; 4,248,870; 4,256,746; 4,260,608;
4,265,814; 4,294,757; 4,307,016; 4,308,268; 4,308,269; 4,309,428;
4,313,946; 4,315,929; 4,317,821; 4,322,348; 4,331,598; 4,361,650;
4,364,866; 4,424,219; 4,450,254; 4,362,663; and 4,371,533.
[0263] In an attempt to improve their therapeutic index, maytansine
and maytansinoids have been conjugated to antibodies specifically
binding to tumor cell antigens. Immunoconjugates containing
maytansinoids and their therapeutic use are disclosed, for example,
in U.S. Pat. Nos. 5,208,020, 5,416,064 and European Patent EP 0 425
235 B1. Liu et al., Proc. Natl. Acad. Sci. USA 93:8618-8623 (1996)
described immunoconjugates comprising a maytansinoid designated DM1
linked to the monoclonal antibody C242 directed against human
colorectal cancer. The conjugate was found to be highly cytotoxic
towards cultured colon cancer cells, and showed antitumor activity
in an in vivo tumor growth assay.
[0264] Antibody-maytansinoid conjugates are prepared by chemically
linking an antibody to a maytansinoid molecule without
significantly diminishing the biological activity of either the
antibody or the maytansinoid molecule. An average of 3-4
maytansinoid molecules conjugated per antibody molecule has shown
efficacy in enhancing cytotoxicity of target cells without
negatively affecting the function or solubility of the antibody,
although even one molecule of toxin/antibody would be expected to
enhance cytotoxicity over the use of naked antibody. Maytansinoids
are well known in the art and can be synthesized by known
techniques or isolated from natural sources. Suitable maytansinoids
are disclosed, for example, in U.S. Pat. No. 5,208,020 and in the
other patents and nonpatent publications referred to hereinabove.
Preferred maytansinoids are maytansinol and maytansinol analogues
modified in the aromatic ring or at other positions of the
maytansinol molecule, such as various maytansinol esters.
[0265] There are many linking groups known in the art for making
antibody conjugates, including, for example, those disclosed in
U.S. Pat. No. 5,208,020 or EP Patent 0 425 235 B1, and Chari et
al., Cancer Research 52: 127-131 (1992). The linking groups include
disufide groups, thioether groups, acid labile groups, photolabile
groups, peptidase labile groups, or esterase labile groups, as
disclosed in the above-identified patents, disulfide and thioether
groups being preferred.
[0266] Immunoconjugates may be made using a variety of bifunctional
protein coupling agents such as
N-succinimidyl-3-(2-pyridyldithio)propionate (SPDP),
succinimidyl-4-(N-maleimidomethyl)cyclohexane-1-carboxylate,
iminothiolane (IT), bifunctional derivatives of imidoesters (such
as dimethyl adipimidate HCL), active esters (such as disuccinimidyl
suberate), aldehydes (such as glutareldehyde), bis-azido compounds
(such as bis(p-azidobenzoyl)hexanediamine), bis-diazonium
derivatives (such as bis-(p-diazoniumbenzoyl)-ethylenediamine),
diisocyanates (such as toluene 2,6-diisocyanate), and bis-active
fluorine compounds (such as 1,5-difluoro-2,4-dinitrobenzene).
Particularly preferred coupling agents include
N-succinimidyl-3-(2-pyridyldithio)propionate (SPDP) (Carlsson et
al., Biochem. J. 173:723-737 [1978]) and
N-succinimidyl-4-(2-pyridylthio)pentanoate (SPP) to provide for a
disulfide linkage. For example, a ricin immunotoxin can be prepared
as described in Vitetta et al., Science 238: 1098 (1987).
Carbon-14-labeled 1-isothiocyanatobenzyl-3-methyldiethylene
triaminepentaacetic acid (MX-DTPA) is an exemplary chelating agent
for conjugation of radionucleotide to the antibody. See WO94/11026.
The linker may be a "cleavable linker" facilitating release of the
cytotoxic drug in the cell. For example, an acid-labile linker,
Cancer Research 52: 127-131 (1992); U.S. Pat. No. 5,208,020) may be
used.
[0267] Another immunoconjugate of interest comprises an antibody
conjugated to one or more calicheamicin molecules. The
calicheamicin family of antibiotics is capable of producing
double-stranded DNA breaks at sub-picomolar concentrations. For the
preparation of conjugates of the calicheamicin family, see U.S.
Pat. Nos. 5,712,374, 5,714,586, 5,739,116, 5,767,285, 5,770,701,
5,770,710, 5,773,001, 5,877,296 (all to American Cyanamid Company).
Another drug that the antibody can be conjugated is QFA which is an
antifolate. Both calicheamicin and QFA have intracellular sites of
action and do not readily cross the plasma membrane. Therefore,
cellular uptake of these agents through antibody mediated
internalization greatly enhances their cytotoxic effects.
[0268] Examples of other agents that can be conjugated to the
antibodies of the invention include BCNU, streptozoicin,
vincristine and 5-fluorouracil, the family of agents known
collectively LL-E33288 complex described in U.S. Pat. Nos.
5,053,394, 5,770,710, as well as esperamicins (U.S. Pat. No.
5,877,296).
[0269] Enzymatically active toxins and fragments thereof that can
be used include, e.g., diphtheria A chain, nonbinding active
fragments of diphtheria toxin, exotoxin A chain (from Pseudomonas
aeruginosa), ricin A chain, abrin A chain, modeccin A chain,
alpha-sarcin, Aleurites fordii proteins, dianthin proteins,
Phytolaca americana proteins (PAPI, PAPII, and PAP-S), momordica
charantia inhibitor, curcin, crotin, sapaonaria officinalis
inhibitor, gelonin, mitogellin, restrictocin, phenomycin, enomycin
and the tricothecenes. See, for example, WO 93/21232.
[0270] The present invention further includes an immunoconjugate
formed between an antibody and a compound with nucleolytic activity
(e.g., a ribonuclease or a DNA endonuclease such as a
deoxyribonuclease; DNase).
[0271] For selective destruction of infected cells, the antibody
includes a highly radioactive atom. A variety of radioactive
isotopes are available for the production of radioconjugated
anti-PSCA antibodies. Examples include At.sup.211, I.sup.131,
I.sup.125, Y.sup.90, Re.sup.186, Re.sup.188, sm.sup.153,
Bi.sup.212, P.sup.32, Pb.sup.212 and radioactive isotopes of Lu.
When the conjugate is used for diagnosis, it may comprise a
radioactive atom for scintigraphic studies, for example tc.sup.99m
or I.sup.123, or a spin label for nuclear magnetic resonance (NMR)
imaging (also known as Magnetic Resonance Imaging, MRI), such as
iodine-123, iodine-131, indium-111, fluorine-19, carbon-13,
nitrogen-15, oxygen-17, gadolinium, manganese or iron.
[0272] The radio- or other label is incorporated in the conjugate
in known ways. For example, the peptide may be biosynthesized or
may be synthesized by chemical amino acid synthesis using suitable
amino acid precursors involving, for example, fluorine-19 in place
of hydrogen. Labels such as tc.sup.99m or I.sup.123, Re.sup.186,
Re.sup.188 and In.sup.111 can be attached via a cysteine residue in
the peptide. Yttrium-90 can be attached via a lysine residue. The
IODOGEN method (Fraker et al. (1978) Biochem. Biophys. Res. Commun.
80: 49-57 can be used to incorporate iodine-123. "Monoclonal
Antibodies in Immunoscintigraphy" (Chatal, CRC Press 1989)
describes other methods in detail.
[0273] Alternatively, a fusion protein comprising the antibody and
cytotoxic agent is made, e.g., by recombinant techniques or peptide
synthesis. The length of DNA may comprise respective regions
encoding the two portions of the conjugate either adjacent one
another or separated by a region encoding a linker peptide which
does not destroy the desired properties of the conjugate.
[0274] The antibodies of the present invention are also used in
antibody dependent enzyme mediated prodrug therapy (ADET) by
conjugating the antibody to a prodrug-activating enzyme which
converts a prodrug (e.g., a peptidyl chemotherapeutic agent, see
WO81/01145) to an active anti-cancer drug (see, e.g., WO 88/07378
and U.S. Pat. No. 4,975,278).
[0275] The enzyme component of the immunoconjugate useful for ADEPT
includes any enzyme capable of acting on a prodrug in such a way so
as to covert it into its more active, cytotoxic form. Enzymes that
are useful in the method of this invention include, but are not
limited to, alkaline phosphatase useful for converting
phosphate-containing prodrugs into free drugs; arylsulfatase useful
for converting sulfate-containing prodrugs into free drugs;
cytosine deaminase useful for converting non-toxic 5-fluorocytosine
into the anti-cancer drug, 5-fluorouracil; proteases, such as
serratia protease, thermolysin, subtilisin, carboxypeptidases and
cathepsins (such as cathepsins B and L), that are useful for
converting peptide-containing prodrugs into free drugs;
D-alanylcarboxypeptidases, useful for converting prodrugs that
contain D-amino acid substituents; carbohydrate-cleaving enzymes
such as .beta.-galactosidase and neuraminidase useful for
converting glycosylated prodrugs into free drugs; .beta.-lactamase
useful for converting drugs derivatized with .beta.-lactams into
free drugs; and penicillin amidases, such as penicillin V amidase
or penicillin G amidase, useful for converting drugs derivatized at
their amine nitrogens with phenoxyacetyl or phenylacetyl groups,
respectively, into free drugs. Alternatively, antibodies with
enzymatic activity, also known in the art as "abzymes", can be used
to convert the prodrugs of the invention into free active drugs
(see, e.g., Massey, Nature 328: 457-458 (1987)). Antibody-abzyme
conjugates can be prepared as described herein for delivery of the
abzyme to an infected cell population.
[0276] The enzymes of this invention can be covalently bound to the
antibodies by techniques well known in the art such as the use of
the heterobifunctional crosslinking reagents discussed above.
Alternatively, fusion proteins comprising at least the antigen
binding region of an antibody of the invention linked to at least a
functionally active portion of an enzyme of the invention can be
constructed using recombinant DNA techniques well known in the art
(see, e.g., Neuberger et al., Nature, 312: 604-608 (1984).
[0277] Other modifications of the antibody are contemplated herein.
For example, the antibody may be linked to one of a variety of
nonproteinaceous polymers, e.g., polyethylene glycol, polypropylene
glycol, polyoxyalkylenes, or copolymers of polyethylene glycol and
polypropylene glycol. The antibody also may be entrapped in
microcapsules prepared, for example, by coacervation techniques or
by interfacial polymerization (for example, hydroxymethylcellulose
or gelatin-microcapsules and poly-(methylmethacylate)microcapsules,
respectively), in colloidal drug delivery systems (for example,
liposomes, albumin microspheres, microemulsions, nano-particles and
nanocapsules), or in macroemulsions. Such techniques are disclosed
in Remington's Pharmaceutical Sciences, 16th edition, Oslo, A.,
Ed., (1980).
[0278] The antibodies disclosed herein are also formulated as
immunoliposomes. A "liposome" is a small vesicle composed of
various types of lipids, phospholipids and/or surfactant that is
useful for delivery of a drug to a mammal. The components of the
liposome are commonly arranged in a bilayer formation, similar to
the lipid arrangement of biological membranes. Liposomes containing
the antibody are prepared by methods known in the art, such as
described in Epstein et al., Proc. Natl. Acad. Sci. USA, 82:3688
(1985); Hwang et al., Proc. Natl Acad. Sci. USA, 77:4030 (1980);
U.S. Pat. Nos. 4,485,045 and 4,544,545; and WO97/38731 published
Oct. 23, 1997. Liposomes with enhanced circulation time are
disclosed in U.S. Pat. No. 5,013,556.
[0279] Particularly useful liposomes can be generated by the
reverse phase evaporation method with a lipid composition
comprising phosphatidylcholine, cholesterol and PEG-derivatized
phosphatidylethanolamine (PEG-PE). Liposomes are extruded through
filters of defined pore size to yield liposomes with the desired a
diameter. Fab' fragments of the antibody of the present invention
can be conjugated to the liposomes as described in Martin et al.,
J. Biol. Chem. 257: 286-288 (1982) via a disulfide interchange
reaction. A chemotherapeutic agent is optionally contained within
the liposome. See Gabizon et al., J. National Cancer Inst.
81(19)1484 (1989).
[0280] Antibodies of the present invention, or fragments thereof,
may possess any of a variety of biological or functional
characteristics. In certain embodiments, these antibodies are HRV
protein specific antibodies, indicating that they specifically bind
to or preferentially bind to HRV as compared to a normal control
cell.
[0281] In particular embodiments, an antibody of the present
invention is an antagonist antibody, which partially or fully
blocks or inhibits a biological activity of a polypeptide or cell
to which it specifically or preferentially binds. In other
embodiments, an antibody of the present invention is a growth
inhibitory antibody, which partially or fully blocks or inhibits
the growth of an infected cell to which it binds. In another
embodiment, an antibody of the present invention induces apoptosis.
In yet another embodiment, an antibody of the present invention
induces or promotes antibody-dependent cell-mediated cytotoxicity
or complement dependent cytotoxicity.
[0282] HRV-expressing cells or virus described above are used to
screen the biological sample obtained from a patient infected with
HRV for the presence of antibodies that preferentially bind to the
cell expressing HRV polypeptides using standard biological
techniques. For example, in certain embodiments, the antibodies may
be labeled, and the presence of label associated with the cell
detected, e.g., using FMAT or FACs analysis. In particular
embodiments, the biological sample is blood, serum, plasma,
bronchial lavage, or saliva. Methods of the present invention may
be practiced using high throughput techniques.
[0283] Identified human antibodies may then be characterized
further. For example the particular conformational epitopes with in
the HRV polypeptides that are necessary or sufficient for binding
of the antibody may be determined, e.g., using site-directed
mutagenesis of expressed HRV polypeptides. These methods may be
readily adapted to identify human antibodies that bind any protein
expressed on a cell surface. Furthermore, these methods may be
adapted to determine binding of the antibody to the virus itself,
as opposed to a cell expressing a recombinant HRV protein or
infected with the virus.
[0284] Polynucleotide sequences encoding the antibodies, variable
regions thereof, or antigen-binding fragments thereof may be
subcloned into expression vectors for the recombinant production of
human anti-HRV antibodies. In one embodiment, this is accomplished
by obtaining mononuclear cells from the patient from the serum
containing the identified HRV antibody was obtained; producing B
cell clones from the mononuclear cells; inducing the B cells to
become antibody-producing plasma cells; and screening the
supernatants produced by the plasma cells to determine if it
contains the HRV antibody. Once a B cell clone that produces an HRV
antibody is identified, reverse-transcription polymerase chain
reaction (RT-PCR) is performed to clone the DNAs encoding the
variable regions or portions thereof of the HRV antibody. These
sequences are then subcloned into expression vectors suitable for
the recombinant production of human HRV antibodies. The binding
specificity may be confirmed by determining the recombinant
antibody's ability to bind cells expressing HRV polypeptide.
[0285] In particular embodiments of the methods described herein, B
cells isolated from peripheral blood or lymph nodes are sorted,
e.g., based on their being CD19 positive, and plated, e.g., as low
as a single cell specificity per well, e.g., in 96, 384, or 1536
well configurations. The cells are induced to differentiate into
antibody-producing cells, e.g., plasma cells, and the culture
supernatants are harvested and tested for binding to cells
expressing the infectious agent polypeptide on their surface using,
e.g., FMAT or FACS analysis. Positive wells are then subjected to
whole well RT-PCR to amplify heavy and light chain variable regions
of the IgG molecule expressed by the clonal daughter plasma cells.
The resulting PCR products encoding the heavy and light chain
variable regions, or portions thereof, are subcloned into human
antibody expression vectors for recombinant expression. The
resulting recombinant antibodies are then tested to confirm their
original binding specificity and may be further tested for
pan-specificity across various strains of isolates of the
infectious agent.
[0286] Thus, in one embodiment, a method of identifying HRV
antibodies is practiced as follows. First, full length or
approximately full length HRV cDNAs are transfected into a cell
line for expression of HRV polypeptides. Secondly, individual human
plasma or sera samples are tested for antibodies that bind the
cell-expressed HRV polypeptides. And lastly, MAbs derived from
plasma- or serum-positive individuals are characterized for binding
to the same cell-expressed HRV polypeptides. Further definition of
the fine specificities of the MAbs can be performed at this
point.
[0287] Polynucleotides that encode the HRV antibodies or portions
thereof of the present invention may be isolated from cells
expressing HRV antibodies, according to methods available in the
art and described herein, including amplification by polymerase
chain reaction using primers specific for conserved regions of
human antibody polypeptides. For example, light chain and heavy
chain variable regions may be cloned from the B cell according to
molecular biology techniques described in WO 92/02551; U.S. Pat.
No. 5,627,052; or Babcook et al., Proc. Natl. Acad. Sci. USA
93:7843-48 (1996). In certain embodiments, polynucleotides encoding
all or a region of both the heavy and light chain variable regions
of the IgG molecule expressed by the clonal daughter plasma cells
expressing the HRV antibody are subcloned and sequenced. The
sequence of the encoded polypeptide may be readily determined from
the polynucleotide sequence.
[0288] Isolated polynucleotides encoding a polypeptide of the
present invention may be subcloned into an expression vector to
recombinantly produce antibodies and polypeptides of the present
invention, using procedures known in the art and described
herein.
[0289] Binding properties of an antibody (or fragment thereof) to
HRV polypeptides or HRV infected cells or tissues may generally be
determined and assessed using immunodetection methods including,
for example, immunofluorescence-based assays, such as
immuno-histochemistry (IHC) and/or fluorescence-activated cell
sorting (FACS). Immunoassay methods may include controls and
procedures to determine whether antibodies bind specifically to HRV
polypeptides from one or more specific clades or strains of HRV,
and do not recognize or cross-react with normal control cells.
[0290] Following pre-screening of serum to identify patients that
produce antibodies to an infectious agent or polypeptide thereof,
e.g., HRV, the methods of the present invention typically include
the isolation or purification of B cells from a biological sample
previously obtained from a patient or subject. The patient or
subject may be currently or previously diagnosed with or suspect or
having a particular disease or infection, or the patient or subject
may be considered free or a particular disease or infection.
Typically, the patient or subject is a mammal and, in particular
embodiments, a human. The biological sample may be any sample that
contains B cells, including but not limited to, lymph node or lymph
node tissue, pleural effusions, peripheral blood, ascites, tumor
tissue, or cerebrospinal fluid (CSF). In various embodiments, B
cells are isolated from different types of biological samples, such
as a biological sample affected by a particular disease or
infection. However, it is understood that any biological sample
comprising B cells may be used for any of the embodiments of the
present invention.
[0291] Once isolated, the B cells are induced to produce
antibodies, e.g., by culturing the B cells under conditions that
support B cell proliferation or development into a plasmacyte,
plasmablast, or plasma cell. The antibodies are then screened,
typically using high throughput techniques, to identify an antibody
that specifically binds to a target antigen, e.g., a particular
tissue, cell, infectious agent, or polypeptide. In certain
embodiments, the specific antigen, e.g., cell surface polypeptide
bound by the antibody is not known, while in other embodiments, the
antigen specifically bound by the antibody is known.
[0292] According to the present invention, B cells may be isolated
from a biological sample, e.g., a tumor, tissue, peripheral blood
or lymph node sample, by any means known and available in the art.
B cells are typically sorted by FACS based on the presence on their
surface of a B cell-specific marker, e.g., CD19, CD138, and/or
surface IgG. However, other methods known in the art may be
employed, such as, e.g., column purification using CD19 magnetic
beads or IgG-specific magnetic beads, followed by elution from the
column. However, magnetic isolation of B cells utilizing any marker
may result in loss of certain B cells. Therefore, in certain
embodiments, the isolated cells are not sorted but, instead,
phicol-purified mononuclear cells isolated from tumor are directly
plated to the appropriate or desired number of specificities per
well.
[0293] In order to identify B cells that produce an infectious
agent-specific antibody, the B cells are typically plated at low
density (e.g., a single cell specificity per well, 1-10 cells per
well, 10-100 cells per well, 1-100 cells per well, less than 10
cells per well, or less than 100 cells per well) in multi-well or
microtiter plates, e.g., in 96, 384, or 1536 well configurations.
When the B cells are initially plated at a density greater than one
cell per well, then the methods of the present invention may
include the step of subsequently diluting cells in a well
identified as producing an antigen-specific antibody, until a
single cell specificity per well is achieved, thereby facilitating
the identification of the B cell that produces the antigen-specific
antibody. Cell supernatants or a portion thereof and/or cells may
be frozen and stored for future testing and later recovery of
antibody polynucleotides.
[0294] In certain embodiments, the B cells are cultured under
conditions that favor the production of antibodies by the B cells.
For example, the B cells may be cultured under conditions favorable
for B cell proliferation and differentiation to yield
antibody-producing plasmablast, plasmacytes, or plasma cells. In
particular embodiments, the B cells are cultured in the presence of
a B cell mitogen, such as lipopolysaccharide (LPS) or CD40 ligand.
In one specific embodiment, B cells are differentiated to
antibody-producing cells by culturing them with feed cells and/or
other B cell activators, such as CD40 ligand.
[0295] Cell culture supernatants or antibodies obtained therefrom
may be tested for their ability to bind to a target antigen, using
routine methods available in the art, including those described
herein. In particular embodiments, culture supernatants are tested
for the presence of antibodies that bind to a target antigen using
high-throughput methods. For example, B cells may be cultured in
multi-well microtiter dishes, such that robotic plate handlers may
be used to simultaneously sample multiple cell supernatants and
test for the presence of antibodies that bind to a target antigen.
In particular embodiments, antigens are bound to beads, e.g.,
paramagnetic or latex beads) to facilitate the capture of
antibody/antigen complexes. In other embodiments, antigens and
antibodies are fluorescently labeled (with different labels) and
FACS analysis is performed to identify the presence of antibodies
that bind to target antigen. In one embodiment, antibody binding is
determined using FMAT.TM. analysis and instrumentation (Applied
Biosystems, Foster City, Calif.). FMAT.TM. is a fluorescence
macro-confocal platform for high-throughput screening, which
mix-and-read, non-radioactive assays using live cells or beads.
[0296] In the context of comparing the binding of an antibody to a
particular target antigen (e.g., a biological sample such as
infected tissue or cells, or infectious agents) as compared to a
control sample (e.g., a biological sample such as uninfected cells,
or a different infectious agent), in various embodiments, the
antibody is considered to preferentially bind a particular target
antigen if at least two-fold, at least three-fold, at least
five-fold, or at least ten-fold more antibody binds to the
particular target antigen as compared to the amount that binds a
control sample.
[0297] Polynucleotides encoding antibody chains, variable regions
thereof, or fragments thereof, may be isolated from cells utilizing
any means available in the art. In one embodiment, polynucleotides
are isolated using polymerase chain reaction (PCR), e.g., reverse
transcription-PCR (RT-PCR) using oligonucleotide primers that
specifically bind to heavy or light chain encoding polynucleotide
sequences or complements thereof using routine procedures available
in the art. In one embodiment, positive wells are subjected to
whole well RT-PCR to amplify the heavy and light chain variable
regions of the IgG molecule expressed by the clonal daughter plasma
cells. These PCR products may be sequenced.
[0298] The resulting PCR products encoding the heavy and light
chain variable regions or portions thereof are then subcloned into
human antibody expression vectors and recombinantly expressed
according to routine procedures in the art (see, e.g., U.S. Pat.
No. 7,112,439). The nucleic acid molecules encoding a
tumor-specific antibody or fragment thereof, as described herein,
may be propagated and expressed according to any of a variety of
well-known procedures for nucleic acid excision, ligation,
transformation, and transfection. Thus, in certain embodiments
expression of an antibody fragment may be preferred in a
prokaryotic host cell, such as Escherichia coli (see, e.g.,
Pluckthun et al., Methods Enzymol. 178:497-515 (1989)). In certain
other embodiments, expression of the antibody or an antigen-binding
fragment thereof may be preferred in a eukaryotic host cell,
including yeast (e.g., Saccharomyces cerevisiae,
Schizosaccharomyces pombe, and Pichia pastoris); animal cells
(including mammalian cells); or plant cells. Examples of suitable
animal cells include, but are not limited to, myeloma, COS, CHO, or
hybridoma cells. Examples of plant cells include tobacco, corn,
soybean, and rice cells. By methods known to those having ordinary
skill in the art and based on the present disclosure, a nucleic
acid vector may be designed for expressing foreign sequences in a
particular host system, and then polynucleotide sequences encoding
the tumor-specific antibody (or fragment thereof) may be inserted.
The regulatory elements will vary according to the particular
host.
[0299] One or more replicable expression vectors containing a
polynucleotide encoding a variable and/or constant region may be
prepared and used to transform an appropriate cell line, for
example, a non-producing myeloma cell line, such as a mouse NSO
line or a bacterium, such as E. coli, in which production of the
antibody will occur. In order to obtain efficient transcription and
translation, the polynucleotide sequence in each vector should
include appropriate regulatory sequences, particularly a promoter
and leader sequence operatively linked to the variable region
sequence. Particular methods for producing antibodies in this way
are generally well known and routinely used. For example, molecular
biology procedures are described by Sambrook et al. (Molecular
Cloning, A Laboratory Manual, 2nd ed., Cold Spring Harbor
Laboratory, New York, 1989; see also Sambrook et al., 3rd ed., Cold
Spring Harbor Laboratory, New York, (2001)). While not required, in
certain embodiments, regions of polynucleotides encoding the
recombinant antibodies may be sequenced. DNA sequencing can be
performed as described in Sanger et al. (Proc. Natl. Acad. Sci. USA
74:5463 (1977)) and the Amersham International plc sequencing
handbook and including improvements thereto.
[0300] In particular embodiments, the resulting recombinant
antibodies or fragments thereof are then tested to confirm their
original specificity and may be further tested for pan-specificity,
e.g., with related infectious agents. In particular embodiments, an
antibody identified or produced according to methods described
herein is tested for cell killing via antibody dependent cellular
cytotoxicity (ADCC) or apoptosis, and/or well as its ability to
internalize.
[0301] The present invention, in other aspects, provides
polynucleotide compositions. In preferred embodiments, these
polynucleotides encode a polypeptide of the invention, e.g., a
region of a variable chain of an antibody that binds to HRV.
Polynucleotides of the invention are single-stranded (coding or
antisense) or double-stranded DNA (genomic, cDNA or synthetic) or
RNA molecules. RNA molecules include, but are not limited to, HnRNA
molecules, which contain introns and correspond to a DNA molecule
in a one-to-one manner, and mRNA molecules, which do not contain
introns. Alternatively, or in addition, coding or non-coding
sequences are present within a polynucleotide of the present
invention. Also alternatively, or in addition, a polynucleotide is
linked to other molecules and/or support materials of the
invention. Polynucleotides of the invention are used, e.g., in
hybridization assays to detect the presence of an HRV antibody in a
biological sample, and in the recombinant production of
polypeptides of the invention. Further, the invention includes all
polynucleotides that encode any polypeptide of the present
invention.
[0302] In other related embodiments, the invention provides
polynucleotide variants having substantial identity to the
sequences of SEQ ID NOs: 1, 2, 10, 11, 17, 18, 26, 27, 33, 34, 42,
43, 49, 50, 58, 59, for example those comprising at least 70%
sequence identity, preferably at least 75%, 80%, 85%, 90%, 95%,
96%, 97%, 98%, or 99% or higher, sequence identity compared to a
polynucleotide sequence of this invention, as determined using the
methods described herein, (e.g., BLAST analysis using standard
parameters). One skilled in this art will recognize that these
values can be appropriately adjusted to determine corresponding
identity of proteins encoded by two nucleotide sequences by taking
into account codon degeneracy, amino acid similarity, reading frame
positioning, and the like.
[0303] Typically, polynucleotide variants contain one or more
substitutions, additions, deletions and/or insertions, preferably
such that the immunogenic binding properties of the polypeptide
encoded by the variant polynucleotide is not substantially
diminished relative to a polypeptide encoded by a polynucleotide
sequence specifically set forth herein.
[0304] In additional embodiments, the present invention provides
polynucleotide fragments comprising various lengths of contiguous
stretches of sequence identical to or complementary to one or more
of the sequences disclosed herein. For example, polynucleotides are
provided by this invention that comprise at least about 10, 15, 20,
30, 40, 50, 75, 100, 150, 200, 300, 400, 500 or 1000 or more
contiguous nucleotides of one or more of the sequences disclosed
herein as well as all intermediate lengths there between. As used
herein, the term "intermediate lengths" is meant to describe any
length between the quoted values, such as 16, 17, 18, 19, etc.; 21,
22, 23, etc.; 30, 31, 32, etc.; 50, 51, 52, 53, etc.; 100, 101,
102, 103, etc.; 150, 151, 152, 153, etc.; including all integers
through 200-500; 500-1,000, and the like.
[0305] In another embodiment of the invention, polynucleotide
compositions are provided that are capable of hybridizing under
moderate to high stringency conditions to a polynucleotide sequence
provided herein, or a fragment thereof, or a complementary sequence
thereof. Hybridization techniques are well known in the art of
molecular biology. For purposes of illustration, suitable
moderately stringent conditions for testing the hybridization of a
polynucleotide of this invention with other polynucleotides include
prewashing in a solution of 5.times.SSC, 0.5% SDS, 1.0 mM EDTA (pH
8.0); hybridizing at 50.degree. C.-60.degree. C., 5.times.SSC,
overnight; followed by washing twice at 65.degree. C. for 20
minutes with each of 2.times., 0.5.times. and 0.2.times.SSC
containing 0.1% SDS. One skilled in the art will understand that
the stringency of hybridization can be readily manipulated, such as
by altering the salt content of the hybridization solution and/or
the temperature at which the hybridization is performed. For
example, in another embodiment, suitable highly stringent
hybridization conditions include those described above, with the
exception that the temperature of hybridization is increased, e.g.,
to 60-65.degree. C. or 65-70.degree. C.
[0306] In preferred embodiments, the polypeptide encoded by the
polynucleotide variant or fragment has the same binding specificity
(i.e., specifically or preferentially binds to the same epitope or
HRV strain) as the polypeptide encoded by the native
polynucleotide. In certain preferred embodiments, the
polynucleotides described above, e.g., polynucleotide variants,
fragments and hybridizing sequences, encode polypeptides that have
a level of binding activity of at least about 50%, preferably at
least about 70%, and more preferably at least about 90% of that for
a polypeptide sequence specifically set forth herein.
[0307] The polynucleotides of the present invention, or fragments
thereof, regardless of the length of the coding sequence itself,
may be combined with other DNA sequences, such as promoters,
polyadenylation signals, additional restriction enzyme sites,
multiple cloning sites, other coding segments, and the like, such
that their overall length may vary considerably. A nucleic acid
fragment of almost any length is employed, with the total length
preferably being limited by the ease of preparation and use in the
intended recombinant DNA protocol. For example, illustrative
polynucleotide segments with total lengths of about 10,000, about
5000, about 3000, about 2,000, about 1,000, about 500, about 200,
about 100, about 50 base pairs in length, and the like, (including
all intermediate lengths) are included in many implementations of
this invention.
[0308] It will be appreciated by those of ordinary skill in the art
that, as a result of the degeneracy of the genetic code, there are
multiple nucleotide sequences that encode a polypeptide as
described herein. Some of these polynucleotides bear minimal
homology to the nucleotide sequence of any native gene.
Nonetheless, polynucleotides that encode a polypeptide of the
present invention but which vary due to differences in codon usage
are specifically contemplated by the invention. Further, alleles of
the genes including the polynucleotide sequences provided herein
are within the scope of the invention. Alleles are endogenous genes
that are altered as a result of one or more mutations, such as
deletions, additions and/or substitutions of nucleotides. The
resulting mRNA and protein may, but need not, have an altered
structure or function. Alleles may be identified using standard
techniques (such as hybridization, amplification and/or database
sequence comparison).
[0309] In certain embodiments of the present invention, mutagenesis
of the disclosed polynucleotide sequences is performed in order to
alter one or more properties of the encoded polypeptide, such as
its binding specificity or binding strength. Techniques for
mutagenesis are well-known in the art, and are widely used to
create variants of both polypeptides and polynucleotides. A
mutagenesis approach, such as site-specific mutagenesis, is
employed for the preparation of variants and/or derivatives of the
polypeptides described herein. By this approach, specific
modifications in a polypeptide sequence are made through
mutagenesis of the underlying polynucleotides that encode them.
These techniques provides a straightforward approach to prepare and
test sequence variants, for example, incorporating one or more of
the foregoing considerations, by introducing one or more nucleotide
sequence changes into the polynucleotide.
[0310] Site-specific mutagenesis allows the production of mutants
through the use of specific oligonucleotide sequences include the
nucleotide sequence of the desired mutation, as well as a
sufficient number of adjacent nucleotides, to provide a primer
sequence of sufficient size and sequence complexity to form a
stable duplex on both sides of the deletion junction being
traversed. Mutations are employed in a selected polynucleotide
sequence to improve, alter, decrease, modify, or otherwise change
the properties of the polynucleotide itself, and/or alter the
properties, activity, composition, stability, or primary sequence
of the encoded polypeptide.
[0311] In other embodiments of the present invention, the
polynucleotide sequences provided herein are used as probes or
primers for nucleic acid hybridization, e.g., as PCR primers. The
ability of such nucleic acid probes to specifically hybridize to a
sequence of interest enables them to detect the presence of
complementary sequences in a given sample. However, other uses are
also encompassed by the invention, such as the use of the sequence
information for the preparation of mutant species primers, or
primers for use in preparing other genetic constructions. As such,
nucleic acid segments of the invention that include a sequence
region of at least about a 15-nucleotide long contiguous sequence
that has the same sequence as, or is complementary to, a 15
nucleotide long contiguous sequence disclosed herein is
particularly useful. Longer contiguous identical or complementary
sequences, e.g., those of about 20, 30, 40, 50, 100, 200, 500, 1000
(including all intermediate lengths) including full length
sequences, and all lengths in between, are also used in certain
embodiments.
[0312] Polynucleotide molecules having sequence regions consisting
of contiguous nucleotide stretches of 10-14, 15-20, 30, 50, or even
of 100-200 nucleotides or so (including intermediate lengths as
well), identical or complementary to a polynucleotide sequence
disclosed herein, are particularly contemplated as hybridization
probes for use in, e.g., Southern and Northern blotting, and/or
primers for use in, e.g., polymerase chain reaction (PCR). The
total size of fragment, as well as the size of the complementary
stretch (es), ultimately depends on the intended use or application
of the particular nucleic acid segment. Smaller fragments are
generally used in hybridization embodiments, wherein the length of
the contiguous complementary region may be varied, such as between
about 15 and about 100 nucleotides, but larger contiguous
complementarity stretches may be used, according to the length
complementary sequences one wishes to detect.
[0313] The use of a hybridization probe of about 15-25 nucleotides
in length allows the formation of a duplex molecule that is both
stable and selective. Molecules having contiguous complementary
sequences over stretches greater than 12 bases in length are
generally preferred, though, in order to increase stability and
selectivity of the hybrid, and thereby improve the quality and
degree of specific hybrid molecules obtained. Nucleic acid
molecules having gene-complementary stretches of 15 to 25
contiguous nucleotides, or even longer where desired, are generally
preferred.
[0314] Hybridization probes are selected from any portion of any of
the sequences disclosed herein. All that is required is to review
the sequences set forth herein, or to any continuous portion of the
sequences, from about 15-25 nucleotides in length up to and
including the full length sequence, that one wishes to utilize as a
probe or primer. The choice of probe and primer sequences is
governed by various factors. For example, one may wish to employ
primers from towards the termini of the total sequence.
[0315] Polynucleotide of the present invention, or fragments or
variants thereof, are readily prepared by, for example, directly
synthesizing the fragment by chemical means, as is commonly
practiced using an automated oligonucleotide synthesizer. Also,
fragments are obtained by application of nucleic acid reproduction
technology, such as the PCR.TM. technology of U.S. Pat. No.
4,683,202, by introducing selected sequences into recombinant
vectors for recombinant production, and by other recombinant DNA
techniques generally known to those of skill in the art of
molecular biology.
[0316] The invention provides vectors and host cells comprising a
nucleic acid of the present invention, as well as recombinant
techniques for the production of a polypeptide of the present
invention. Vectors of the invention include those capable of
replication in any type of cell or organism, including, e.g.,
plasmids, phage, cosmids, and mini chromosomes. In various
embodiments, vectors comprising a polynucleotide of the present
invention are vectors suitable for propagation or replication of
the polynucleotide, or vectors suitable for expressing a
polypeptide of the present invention. Such vectors are known in the
art and commercially available.
[0317] Polynucleotides of the present invention are synthesized,
whole or in parts that are then combined, and inserted into a
vector using routine molecular and cell biology techniques,
including, e.g., subcloning the polynucleotide into a linearized
vector using appropriate restriction sites and restriction enzymes.
Polynucleotides of the present invention are amplified by
polymerase chain reaction using oligonucleotide primers
complementary to each strand of the polynucleotide. These primers
also include restriction enzyme cleavage sites to facilitate
subcloning into a vector. The replicable vector components
generally include, but are not limited to, one or more of the
following: a signal sequence, an origin of replication, and one or
more marker or selectable genes.
[0318] In order to express a polypeptide of the present invention,
the nucleotide sequences encoding the polypeptide, or functional
equivalents, are inserted into an appropriate expression vector,
i.e., a vector that contains the necessary elements for the
transcription and translation of the inserted coding sequence.
Methods well known to those skilled in the art are used to
construct expression vectors containing sequences encoding a
polypeptide of interest and appropriate transcriptional and
translational control elements. These methods include in vitro
recombinant DNA techniques, synthetic techniques, and in vivo
genetic recombination. Such techniques are described, for example,
in Sambrook, J., et al. (1989) Molecular Cloning, A Laboratory
Manual, Cold Spring Harbor Press, Plainview, N.Y., and Ausubel, F.
M. et al. (1989) Current Protocols in Molecular Biology, John Wiley
& Sons, New York. N.Y.
[0319] A variety of expression vector/host systems are utilized to
contain and express polynucleotide sequences. These include, but
are not limited to, microorganisms such as bacteria transformed
with recombinant bacteriophage, plasmid, or cosmid DNA expression
vectors; yeast transformed with yeast expression vectors; insect
cell systems infected with virus expression vectors (e.g.,
baculovirus); plant cell systems transformed with virus expression
vectors (e.g., cauliflower mosaic virus, CaMV; tobacco mosaic
virus, TMV) or with bacterial expression vectors (e.g., Ti or
pBR322 plasmids); or animal cell systems.
[0320] Within one embodiment, the variable regions of a gene
expressing a monoclonal antibody of interest are amplified from a
hybridoma cell using nucleotide primers. These primers are
synthesized by one of ordinary skill in the art, or may be
purchased from commercially available sources (see, e.g.,
Stratagene (La Jolla, Calif.), which sells primers for amplifying
mouse and human variable regions. The primers are used to amplify
heavy or light chain variable regions, which are then inserted into
vectors such as ImmunoZAP.TM. H or ImmunoZAP.TM. L (Stratagene),
respectively. These vectors are then introduced into E. coli,
yeast, or mammalian-based systems for expression. Large amounts of
a single-chain protein containing a fusion of the V.sub.H and
V.sub.L domains are produced using these methods (see Bird et al.,
Science 242:423-426 (1988)).
[0321] The "control elements" or "regulatory sequences" present in
an expression vector are those non-translated regions of the
vector, e.g., enhancers, promoters, 5' and 3' untranslated regions,
that interact with host cellular proteins to carry out
transcription and translation. Such elements may vary in their
strength and specificity. Depending on the vector system and host
utilized, any number of suitable transcription and translation
elements, including constitutive and inducible promoters, is
used.
[0322] Examples of promoters suitable for use with prokaryotic
hosts include the phoa promoter, .beta.-lactamase and lactose
promoter systems, alkaline phosphatase promoter, a tryptophan (trp)
promoter system, and hybrid promoters such as the tac promoter.
However, other known bacterial promoters are suitable. Promoters
for use in bacterial systems also usually contain a Shine-Dalgarno
sequence operably linked to the DNA encoding the polypeptide.
Inducible promoters such as the hybrid lacZ promoter of the
PBLUESCRIPT phagemid (Stratagene, La Jolla, Calif.) or PSPORT1
plasmid (Gibco BRL, Gaithersburg, Md.) and the like are used.
[0323] A variety of promoter sequences are known for eukaryotes and
any are used according to the present invention. Virtually all
eukaryotic genes have an AT-rich region located approximately 25 to
30 bases upstream from the site where transcription is initiated.
Another sequence found 70 to 80 bases upstream from the start of
transcription of many genes is a CNCAAT region where N may be any
nucleotide. At the 3' end of most eukaryotic genes is an AATAAA
sequence that may be the signal for addition of the poly A tail to
the 3' end of the coding sequence. All of these sequences are
suitably inserted into eukaryotic expression vectors.
[0324] In mammalian cell systems, promoters from mammalian genes or
from mammalian viruses are generally preferred. Polypeptide
expression from vectors in mammalian host cells are controlled, for
example, by promoters obtained from the genomes of viruses such as
polyoma virus, fowlpox virus, adenovirus (e.g., Adenovirus 2),
bovine papilloma virus, avian sarcoma virus, cytomegalovirus (CMV),
a retrovirus, hepatitis-B virus and most preferably Simian Virus 40
(SV40), from heterologous mammalian promoters, e.g., the actin
promoter or an immunoglobulin promoter, and from heat-shock
promoters, provided such promoters are compatible with the host
cell systems. If it is necessary to generate a cell line that
contains multiple copies of the sequence encoding a polypeptide,
vectors based on SV40 or EBV may be advantageously used with an
appropriate selectable marker. One example of a suitable expression
vector is pcDNA-3.1 (Invitrogen, Carlsbad, Calif.), which includes
a CMV promoter.
[0325] A number of viral-based expression systems are available for
mammalian expression of polypeptides. For example, in cases where
an adenovirus is used as an expression vector, sequences encoding a
polypeptide of interest may be ligated into an adenovirus
transcription/translation complex consisting of the late promoter
and tripartite leader sequence. Insertion in a non-essential E1 or
E3 region of the viral genome may be used to obtain a viable virus
that is capable of expressing the polypeptide in infected host
cells (Logan, J. and Shenk, T. (1984) Proc. Natl. Acad. Sci.
81:3655-3659). In addition, transcription enhancers, such as the
Rous sarcoma virus (RSV) enhancer, may be used to increase
expression in mammalian host cells.
[0326] In bacterial systems, any of a number of expression vectors
is selected depending upon the use intended for the expressed
polypeptide. For example, when large quantities are desired,
vectors that direct high level expression of fusion proteins that
are readily purified are used. Such vectors include, but are not
limited to, the multifunctional E. coli cloning and expression
vectors such as BLUESCRIPT (Stratagene), in which the sequence
encoding the polypeptide of interest may be ligated into the vector
in frame with sequences for the amino-terminal Met and the
subsequent 7 residues of .beta.-galactosidase, so that a hybrid
protein is produced; pIN vectors (Van Heeke, G. and S. M. Schuster
(1989) J. Biol. Chem. 264:5503-5509); and the like. pGEX Vectors
(Promega, Madison, Wis.) are also used to express foreign
polypeptides as fusion proteins with glutathione S-transferase
(GST). In general, such fusion proteins are soluble and can easily
be purified from lysed cells by adsorption to glutathione-agarose
beads followed by elution in the presence of free glutathione.
Proteins made in such systems are designed to include heparin,
thrombin, or factor XA protease cleavage sites so that the cloned
polypeptide of interest can be released from the GST moiety at
will.
[0327] In the yeast, Saccharomyces cerevisiae, a number of vectors
containing constitutive or inducible promoters such as alpha
factor, alcohol oxidase, and PGH are used. Examples of other
suitable promoter sequences for use with yeast hosts include the
promoters for 3-phosphoglycerate kinase or other glycolytic
enzymes, such as enolase, glyceraldehyde-3-phosphate dehydrogcnase,
hexokinase, pyruvate decarboxylase, phosphofructokinase,
glucose-6-phosphate isomerase, 3-phosphoglycerate mutase, pyruvate
kinase, triosephosphate isomerase, phosphoglucose isomerase, and
glucokinase. For reviews, see Ausubel et al. (supra) and Grant et
al. (1987) Methods Enzymol. 153:516-544. Other yeast promoters that
are inducible promoters having the additional advantage of
transcription controlled by growth conditions include the promoter
regions for alcohol dehydrogenase 2, isocytochrome C, acid
phosphatase, degradative enzymes associated with nitrogen
metabolism, metallothionein, glyceraldehyde-3-phosphate
dehydrogenase, and enzymes responsible for maltose and galactose
utilization. Suitable vectors and promoters for use in yeast
expression are further described in EP 73,657. Yeast enhancers also
are advantageously used with yeast promoters.
[0328] In cases where plant expression vectors are used, the
expression of sequences encoding polypeptides are driven by any of
a number of promoters. For example, viral promoters such as the 35S
and 19S promoters of CaMV are used alone or in combination with the
omega leader sequence from TMV (Takamatsu, N. (1987) EMBO J.
6:307-311. Alternatively, plant promoters such as the small subunit
of RUBISCO or heat shock promoters are used (Coruzzi, G. et al.
(1984) EMBO J. 3:1671-1680; Broglie, R. et al. (1984) Science
224:838-843; and Winter, J., et al. (1991) Results Probl. Cell
Differ. 17:85-105). These constructs can be introduced into plant
cells by direct DNA transformation or pathogen-mediated
transfection. Such techniques are described in a number of
generally available reviews (see, e.g., Hobbs, S. or Murry, L. E.
in McGraw Hill Yearbook of Science and Technology (1992) McGraw
Hill, New York, N.Y.; pp. 191-196).
[0329] An insect system is also used to express a polypeptide of
interest. For example, in one such system, Autographa californica
nuclear polyhedrosis virus (AcNPV) is used as a vector to express
foreign genes in Spodoptera frugiperda cells or in Trichoplusia
larvae. The sequences encoding the polypeptide are cloned into a
non-essential region of the virus, such as the polyhedrin gene, and
placed under control of the polyhedrin promoter. Successful
insertion of the polypeptide-encoding sequence renders the
polyhedrin gene inactive and produce recombinant virus lacking coat
protein. The recombinant viruses are then used to infect, for
example, S. frugiperda cells or Trichoplusia larvae, in which the
polypeptide of interest is expressed (Engelhard, E. K. et al.
(1994) Proc. Natl. Acad. Sci. 91:3224-3227).
[0330] Specific initiation signals are also used to achieve more
efficient translation of sequences encoding a polypeptide of
interest. Such signals include the ATG initiation codon and
adjacent sequences. In cases where sequences encoding the
polypeptide, its initiation codon, and upstream sequences are
inserted into the appropriate expression vector, no additional
transcriptional or translational control signals may be needed.
However, in cases where only coding sequence, or a portion thereof,
is inserted, exogenous translational control signals including the
ATG initiation codon are provided. Furthermore, the initiation
codon is in the correct reading frame to ensure correct translation
of the inserted polynucleotide. Exogenous translational elements
and initiation codons are of various origins, both natural and
synthetic.
[0331] Transcription of a DNA encoding a polypeptide of the
invention is often increased by inserting an enhancer sequence into
the vector. Many enhancer sequences are known, including, e.g.,
those identified in genes encoding globin, elastase, albumin,
.alpha.-fetoprotein, and insulin. Typically, however, an enhancer
from a eukaryotic cell virus is used. Examples include the SV40
enhancer on the late side of the replication origin (bp 100-270),
the cytomegalovirus early promoter enhancer, the polyoma enhancer
on the late side of the replication origin, and adenovirus
enhancers. See also Yaniv, Nature 297:17-18 (1982) on enhancing
elements for activation of eukaryotic promoters. The enhancer is
spliced into the vector at a position 5' or 3' to the
polypeptide-encoding sequence, but is preferably located at a site
5' from the promoter.
[0332] Expression vectors used in eukaryotic host cells (yeast,
fungi, insect, plant, animal, human, or nucleated cells from other
multicellular organisms) typically also contain sequences necessary
for the termination of transcription and for stabilizing the mRNA.
Such sequences are commonly available from the 5' and, occasionally
3', untranslated regions of eukaryotic or viral DNAs or cDNAs.
These regions contain nucleotide segments transcribed as
polyadenylated fragments in the untranslated portion of the mRNA
encoding anti-PSCA antibody. One useful transcription termination
component is the bovine growth hormone polyadenylation region. See
WO94/11026 and the expression vector disclosed therein.
[0333] Suitable host cells for cloning or expressing the DNA in the
vectors herein are the prokaryote, yeast, plant or higher eukaryote
cells described above. Examples of suitable prokaryotes for this
purpose include eubacteria, such as Gram-negative or Gram-positive
organisms, for example, Enterobacteriaceae such as Escherichia,
e.g., E. coli, Enterobacter, Erwinia, Klebsiella, Proteus,
Salmonella, e.g., Salmonella typhimurium, Serratia, e.g., Serratia
marcescans, and Shigella, as well as Bacilli such as B. subtilis
and B. licheniformis (e.g., B. licheniformis 41P disclosed in DD
266,710 published 12 Apr. 1989), Pseudomonas such as P. aeruginosa,
and Streptomyces. One preferred E. coli cloning host is E. coli 294
(ATCC 31,446), although other strains such as E. coli B, E. coli
X1776 (ATCC 31,537), and E. coli W3110 (ATCC 27,325) are suitable.
These examples are illustrative rather than limiting.
[0334] Saccharomyces cerevisiae, or common baker's yeast, is the
most commonly used among lower eukaryotic host microorganisms.
However, a number of other genera, species, and strains are
commonly available and used herein, such as Schizosaccharomyces
pombe; Kluyveromyces hosts such as, e.g., K lactis, K. fragilis
(ATCC 12,424), K. bulgaricus (ATCC 16,045), K wickeramii (ATCC
24,178), K. waltii (ATCC 56,500), K. drosophilarum (ATCC 36,906),
K. thermotolerans, and K. marxianus; yarrowia (EP 402,226); Pichia
pastoris. (EP 183,070); Candida; Trichoderma reesia (EP 244,234);
Neurospora crassa; Schwanniomyces such as Schwanniomyces
occidentalis; and filamentous fungi such as, e.g., Neurospora,
Penicillium, Tolypocladium, and Aspergillus hosts such as A.
nidulans and A. niger.
[0335] In certain embodiments, a host cell strain is chosen for its
ability to modulate the expression of the inserted sequences or to
process the expressed protein in the desired fashion. Such
modifications of the polypeptide include, but are not limited to,
acetylation, carboxylation. glycosylation, phosphorylation,
lipidation, and acylation. Post-translational processing that
cleaves a "prepro" form of the protein is also used to facilitate
correct insertion, folding and/or function. Different host cells
such as CHO, COS, HeLa, MDCK, HEK293, and WI38, which have specific
cellular machinery and characteristic mechanisms for such
post-translational activities, are chosen to ensure the correct
modification and processing of the foreign protein.
[0336] Methods and reagents specifically adapted for the expression
of antibodies or fragments thereof are also known and available in
the art, including those described, e.g., in U.S. Pat. Nos.
4,816,567 and 6,331,415. In various embodiments, antibody heavy and
light chains, or fragments thereof, are expressed from the same or
separate expression vectors. In one embodiment, both chains are
expressed in the same cell, thereby facilitating the formation of a
functional antibody or fragment thereof.
[0337] Full length antibody, antibody fragments, and antibody
fusion proteins are produced in bacteria, in particular when
glycosylation and Fc effector function are not needed, such as when
the therapeutic antibody is conjugated to a cytotoxic agent (e.g.,
a toxin) and the immunoconjugate by itself shows effectiveness in
infected cell destruction. For expression of antibody fragments and
polypeptides in bacteria, see, e.g., U.S. Pat. Nos. 5,648,237,
5,789,199, and 5,840,523, which describes translation initiation
region (TIR) and signal sequences for optimizing expression and
secretion. After expression, the antibody is isolated from the E.
coli cell paste in a soluble fraction and can be purified through,
e.g., a protein A or G column depending on the isotype. Final
purification can be carried out using a process similar to that
used for purifying antibody expressed e.g., in CHO cells.
[0338] Suitable host cells for the expression of glycosylated
polypeptides and antibodies are derived from multicellular
organisms. Examples of invertebrate cells include plant and insect
cells. Numerous baculoviral strains and variants and corresponding
permissive insect host cells from hosts such as Spodoptera
frugiperda (caterpillar), Aedes aegypti (mosquito), Aede albopicius
(mosquito), Drosophila melanogaster (fruitfly), and Bombyx mori
have been identified. A variety of viral strains for transfection
are publicly available, e.g., the L-1 variant of Autographa
californica NPV and the Bm-5 strain of Bombyx mori NPV, and such
viruses are used as the virus herein according to the present
invention, particularly for transfection of Spodoptera frugiperda
cells. Plant cell cultures of cotton, corn, potato, soybean,
petunia, tomato, and tobacco are also utilized as hosts.
[0339] Methods of propagation of antibody polypeptides and
fragments thereof in vertebrate cells in culture (tissue culture)
are encompassed by the invention. Examples of mammalian host cell
lines used in the methods of the invention are monkey kidney CV1
line transformed by SV40 (COS-7, ATCC CRL 1651); human embryonic
kidney line (293 or 293 cells subcloned for growth in suspension
culture, Graham et al., J. Gen Virol. 36:59 (1977)); baby hamster
kidney cells (BHK, ATCC CCL 10); Chinese hamster ovary cells/-DHFR
(CHO, Urlaub et al., Proc. Natl. Acad. Sci. USA 77:4216 (1980));
mouse sertoli cells (TM4, Mather, Biol. Reprod. 23:243-251 (1980));
monkey kidney cells (CV1 ATCC CCL 70); African green monkey kidney
cells (VERO-76, ATCC CRL-1587); human cervical carcinoma cells
(HELA, ATCC CCL 2); canine kidney cells (MDCK, ATCC CCL 34);
buffalo rat liver cells (BRL 3A, ATCC CRL 1442); human lung cells
(W138, ATCC CCL 75); human liver cells (Hep G2, HB 8065); mouse
mammary tumor (MMT 060562, ATCC CCL51); TR1 cells (Mather et al.,
Annals N.Y. Acad. Sci. 383:44-68 (1982)); MRC 5 cells; FS4 cells;
and a human hepatoma line (Hep G2).
[0340] Host cells are transformed with the above-described
expression or cloning vectors for polypeptide production and
cultured in conventional nutrient media modified as appropriate for
inducing promoters, selecting transformants, or amplifying the
genes encoding the desired sequences.
[0341] For long-term, high-yield production of recombinant
proteins, stable expression is generally preferred. For example,
cell lines that stably express a polynucleotide of interest are
transformed using expression vectors that contain viral origins of
replication and/or endogenous expression elements and a selectable
marker gene on the same or on a separate vector. Following the
introduction of the vector, cells are allowed to grow for 1-2 days
in an enriched media before they are switched to selective media.
The purpose of the selectable marker is to confer resistance to
selection, and its presence allows growth and recovery of cells
that successfully express the introduced sequences. Resistant
clones of stably transformed cells are proliferated using tissue
culture techniques appropriate to the cell type.
[0342] A plurality of selection systems are used to recover
transformed cell lines. These include, but are not limited to, the
herpes simplex virus thymidine kinase (Wigler, M. et al. (1977)
Cell 11:223-32) and adenine phosphoribosyltransferase (Lowy, I. et
al. (1990) Cell 22:817-23) genes that are employed in tk.sup.- or
aprt.sup.- cells, respectively. Also, antimetabolite, antibiotic or
herbicide resistance is used as the basis for selection; for
example, dhfr, which confers resistance to methotrexate (Wigler, M.
et al. (1980) Proc. Natl. Acad. Sci. 77:3567-70); npt, which
confers resistance to the aminoglycosides, neomycin and G-418
(Colbere-Garapin, F. et al. (1981) J. Mol. Biol. 150:1-14); and als
or pat, which confer resistance to chlorsulfuron and
phosphinotricin acetyltransferase, respectively (Murry, supra).
Additional selectable genes have been described. For example, trpB
allows cells to utilize indole in place of tryptophan, and hisD
allows cells to utilize histinol in place of histidine (Hartman, S.
C. and R. C. Mulligan (1988) Proc. Natl. Acad. Sci. 85:8047-51).
The use of visible markers has gained popularity with such markers
as anthocyanins, beta-glucuronidase and its substrate GUS, and
luciferase and its substrate luciferin, being widely used not only
to identify transformants, but also to quantify the amount of
transient or stable protein expression attributable to a specific
vector system (Rhodes, C. A. et al. (1995) Methods Mol. Biol.
55:121-131).
[0343] Although the presence/absence of marker gene expression
suggests that the gene of interest is also present, its presence
and expression is confirmed. For example, if the sequence encoding
a polypeptide is inserted within a marker gene sequence,
recombinant cells containing sequences are identified by the
absence of marker gene function. Alternatively, a marker gene is
placed in tandem with a polypeptide-encoding sequence under the
control of a single promoter. Expression of the marker gene in
response to induction or selection usually indicates expression of
the tandem gene as well.
[0344] Alternatively, host cells that contain and express a desired
polynucleotide sequence are identified by a variety of procedures
known to those of skill in the art. These procedures include, but
are not limited to, DNA-DNA or DNA-RNA hybridizations and protein
bioassay or immunoassay techniques which include, for example,
membrane, solution, or chip based technologies for the detection
and/or quantification of nucleic acid or protein.
[0345] A variety of protocols for detecting and measuring the
expression of polynucleotide-encoded products, using either
polyclonal or monoclonal antibodies specific for the product are
known in the art. Nonlimiting examples include enzyme-linked
immunosorbent assay (ELISA), radioimmunoassay (RIA), and
fluorescence activated cell sorting (FACS). A two-site,
monoclonal-based immunoassay utilizing monoclonal antibodies
reactive to two non-interfering epitopes on a given polypeptide is
preferred for some applications, but a competitive binding assay
may also be employed. These and other assays are described, among
other places, in Hampton, R. et al. (1990; Serological Methods, a
Laboratory Manual, APS Press, St Paul. Minn.) and Maddox, D. E. et
al. (1983; J. Exp. Med. 158:1211-1216).
[0346] Various labels and conjugation techniques are known by those
skilled in the art and are used in various nucleic acid and amino
acid assays. Means for producing labeled hybridization or PCR
probes for detecting sequences related to polynucleotides include
oligolabeling, nick translation, end-labeling or PCR amplification
using a labeled nucleotide. Alternatively, the sequences, or any
portions thereof are cloned into a vector for the production of an
mRNA probe. Such vectors are known in the art, are commercially
available, and are used to synthesize RNA probes in vitro by
addition of an appropriate RNA polymerase such as T7, T3, or SP6
and labeled nucleotides. These procedures are conducted using a
variety of commercially available kits. Suitable reporter molecules
or labels, which are used include, but are not limited to,
radionucleotides, enzymes, fluorescent, chemiluminescent, or
chromogenic agents as well as substrates, cofactors, inhibitors,
magnetic particles, and the like.
[0347] The polypeptide produced by a recombinant cell is secreted
or contained intracellularly depending on the sequence and/or the
vector used. Expression vectors containing polynucleotides of the
invention are designed to contain signal sequences that direct
secretion of the encoded polypeptide through a prokaryotic or
eukaryotic cell membrane.
[0348] In certain embodiments, a polypeptide of the invention is
produced as a fusion polypeptide further including a polypeptide
domain that facilitates purification of soluble proteins. Such
purification-facilitating domains include, but are not limited to,
metal chelating peptides such as histidine-tryptophan modules that
allow purification on immobilized metals, protein A domains that
allow purification on immobilized immunoglobulin, and the domain
utilized in the FLAGS extension/affinity purification system
(Amgen, Seattle, Wash.). The inclusion of cleavable linker
sequences such as those specific for Factor XA or enterokinase
(Invitrogen. San Diego, Calif.) between the purification domain and
the encoded polypeptide are used to facilitate purification. An
exemplary expression vector provides for expression of a fusion
protein containing a polypeptide of interest and a nucleic acid
encoding 6 histidine residues preceding a thioredoxin or an
enterokinase cleavage site. The histidine residues facilitate
purification on IMIAC (immobilized metal ion affinity
chromatography) as described in Porath, J. et al. (1992, Prot. Exp.
Purif. 3:263-281) while the enterokinase cleavage site provides a
means for purifying the desired polypeptide from the fusion
protein. A discussion of vectors used for producing fusion proteins
is provided in Kroll, D. J. et al. (1993; DNA Cell Biol.
12:441-453).
[0349] In certain embodiments, a polypeptide of the present
invention is fused with a heterologous polypeptide, which may be a
signal sequence, or other polypeptide having a specific cleavage
site at the N-terminus of the mature protein or polypeptide. The
heterologous signal sequence selected preferably is one that is
recognized and processed (i.e., cleaved by a signal peptidase) by
the host cell. For prokaryotic host cells, the signal sequence is
selected, for example, from the group of the alkaline phosphatase,
penicillinase, 1pp, or heat-stable enterotoxin II leaders. For
yeast secretion, the signal sequence is selected from, e.g., the
yeast invertase leader, a factor leader (including Saccharomyces
and Kluyveromyces a factor leaders), or acid phosphatase leader,
the C. albicans glucoamylase leader, or the signal described in WO
90/13646. In mammalian cell expression, mammalian signal sequences
as well as viral secretory leaders, for example, the herpes simplex
gD signal, are available.
[0350] When using recombinant techniques, the polypeptide or
antibody is produced intracellularly, in the periplasmic space, or
directly secreted into the medium. If the polypeptide or antibody
is produced intracellularly, as a first step, the particulate
debris, either host cells or lysed fragments, are removed, for
example, by centrifugation or ultrafiltration. Carter et al.,
Bio/Technology 10:163-167 (1992) describe a procedure for isolating
antibodies that are secreted to the periplasmic space of E. coli.
Briefly, cell paste is thawed in the presence of sodium acetate (pH
3.5), EDTA, and phenylmethylsulfonylfluoride (PMSF) over about 30
min. Cell debris is removed by centrifugation. Where the
polypeptide or antibody is secreted into the medium, supernatants
from such expression systems are generally first concentrated using
a commercially available protein concentration filter, for example,
an Amicon or Millipore Pellicon ultrafiltration unit. Optionally, a
protease inhibitor such as PMSF is included in any of the foregoing
steps to inhibit proteolysis and antibiotics are included to
prevent the growth of adventitious contaminants.
[0351] The polypeptide or antibody composition prepared from the
cells are purified using, for example, hydroxylapatite
chromatography, gel electrophoresis, dialysis, and affinity
chromatography, with affinity chromatography being the preferred
purification technique. The suitability of protein A as an affinity
ligand depends on the species and isotype of any immunoglobulin Fc
domain that is present in the polypeptide or antibody. Protein A is
used to purify antibodies or fragments thereof that are based on
human .gamma..sub.1, .gamma..sub.2, or .gamma..sub.4 heavy chains
(Lindmark et al., J. Immunol. Meth. 62:1-13 (1983)). Protein G is
recommended for all mouse isotypes and for human .gamma..sub.3
(Guss et al., EMBO J. 5:15671575 (1986)). The matrix to which the
affinity ligand is attached is most often agarose, but other
matrices are available. Mechanically stable matrices such as
controlled pore glass or poly(styrenedivinyl)benzene allow for
faster flow rates and shorter processing times than can be achieved
with agarose. Where the polypeptide or antibody comprises a
C.sub.H3 domain, the Bakerbond ABX.TM. resin (J. T. Baker,
Phillipsburg, N.J.) is useful for purification. Other techniques
for protein purification such as fractionation on an ion-exchange
column, ethanol precipitation, Reverse Phase HPLC, chromatography
on silica, chromatography on heparin SEPHAROSE.TM. chromatography
on an anion or cation exchange resin (such as a polyaspartic acid
column), chromatofocusing, SDS-PAGE, and ammonium sulfate
precipitation are also available depending on the polypeptide or
antibody to be recovered.
[0352] Following any preliminary purification step(s), the mixture
comprising the polypeptide or antibody of interest and contaminants
are subjected to low pH hydrophobic interaction chromatography
using an elution buffer at a pH between about 2.5-4.5, preferably
performed at low salt concentrations (e.g., from about 0-0.25M
salt).
[0353] The invention further includes pharmaceutical formulations
including a polypeptide, antibody, or modulator of the present
invention, at a desired degree of purity, and a pharmaceutically
acceptable carrier, excipient, or stabilizer (Remingion's
Pharmaceutical Sciences 16th edition, Osol, A. Ed. (1980)). In
certain embodiments, pharmaceutical formulations are prepared to
enhance the stability of the polypeptide or antibody during
storage, e.g., in the form of lyophilized formulations or aqueous
solutions.
[0354] Acceptable carriers, excipients, or stabilizers are nontoxic
to recipients at the dosages and concentrations employed, and
include, e.g., buffers such as acetate, Tris, phosphate, citrate,
and other organic acids; antioxidants including ascorbic acid and
methionine; preservatives (such as octadecyldimethylbenzyl ammonium
chloride; hexamethonium chloride; benzalkonium chloride,
benzethonium chloride; phenol, butyl or benzyl alcohol; alkyl
parabens such as methyl or propyl paraben; catechol; resorcinol;
cyclohexanol; 3-pentanol; and m-cresol); low molecular weight (less
than about 10 residues) polypeptides; proteins, such as serum
albumin, gelatin, or immunoglobulins; hydrophilic polymers such as
polyvinylpyrrolidone; amino acids such as glycine, glutamine,
asparagine, histidine, arginine, or lysine; monosaccharides,
disaccharides, and other carbohydrates including glucose, mannose,
or dextrins; chelating agents such as EDTA; tonicifiers such as
trehalose and sodium chloride; sugars such as sucrose, mannitol,
trehalose or sorbitol; surfactant such as polysorbate; salt-forming
counter-ions such as sodium; metal complexes (e.g. Zn-protein
complexes); and/or non-ionic surfactants such as TWEEN.TM.,
PLURONICS.TM. or polyethylene glycol (PEG). In certain embodiments,
the therapeutic formulation preferably comprises the polypeptide or
antibody at a concentration of between 5-200 mg/ml, preferably
between 10-100 mg/ml.
[0355] The formulations herein also contain one or more additional
therapeutic agents suitable for the treatment of the particular
indication, e.g., infection being treated, or to prevent undesired
side-effects. Preferably, the additional therapeutic agent has an
activity complementary to the polypeptide or antibody of the resent
invention, and the two do not adversely affect each other. For
example, in addition to the polypeptide or antibody of the
invention, an additional or second antibody, anti-viral agent,
anti-infective agent and/or cardioprotectant is added to the
formulation. Such molecules are suitably present in the
pharmaceutical formulation in amounts that are effective for the
purpose intended.
[0356] The active ingredients, e.g., polypeptides and antibodies of
the invention and other therapeutic agents, are also entrapped in
microcapsules prepared, for example, by coacervation techniques or
by interfacial polymerization, for example, hydroxymethylcellulose
or gelatin-microcapsules and polymethylmethacylate) microcapsules,
respectively, in colloidal drug delivery systems (for example,
liposomes, albumin microspheres, microemulsions, nano-particles and
nanocapsules) or in macroemulsions. Such techniques are disclosed
in Remingion's Pharmaceutical Sciences 16th edition, Osol, A. Ed.
(1980).
[0357] Sustained-release preparations are prepared. Suitable
examples of sustained-release preparations include, but are not
limited to, semi-permeable matrices of solid hydrophobic polymers
containing the antibody, which matrices are in the form of shaped
articles, e.g., films, or microcapsules. Nonlimiting examples of
sustained-release matrices include polyesters, hydrogels (for
example, poly(2-hydroxyethyl-methacrylate), or poly(vinylalcohol)),
polylactides (U.S. Pat. No. 3,773,919), copolymers of L-glutamic
acid and .gamma. ethyl-L-glutamate, non-degradable ethylene-vinyl
acetate, degradable lactic acid-glycolic acid copolymers such as
the LUPRON DEPOT.TM. (injectable microspheres composed of lactic
acid-glycolic acid copolymer and leuprolide acetate), and
poly-D-(-)-3-hydroxyburyric acid.
[0358] Formulations to be used for in vivo administration are
preferably sterile. This is readily accomplished by filtration
through sterile filtration membranes.
[0359] Antibodies of the invention can be coupled to a drug for
delivery to a treatment site or coupled to a detectable label to
facilitate imaging of a site comprising cells of interest, such as
cells infected with HRV. Methods for coupling antibodies to drugs
and detectable labels are well known in the art, as are methods for
imaging using detectable labels. Labeled antibodies may be employed
in a wide variety of assays, employing a wide variety of labels.
Detection of the formation of an antibody-antigen complex between
an antibody of the invention and an epitope of interest (an HRV
epitope) can be facilitated by attaching a detectable substance to
the antibody. Suitable detection means include the use of labels
such as radionucleotides, enzymes, coenzymes, fluorescers,
chemiluminescers, chromogens, enzyme substrates or co-factors,
enzyme inhibitors, prosthetic group complexes, free radicals,
particles, dyes, and the like. Examples of suitable enzymes include
horseradish peroxidase, alkaline phosphatase, .beta.-galactosidase,
or acetylcholinesterase; examples of suitable prosthetic group
complexes include streptavidin/biotin and avidin/biotin; examples
of suitable fluorescent materials include umbelliferone,
fluorescein, fluorescein isothiocyanate, rhodamine,
dichlorotriazinylamine fluorescein, dansyl chloride or
phycoerythrin; an example of a luminescent material is luminol;
examples of bioluminescent materials include luciferase, luciferin,
and aequorin; and examples of suitable radioactive material include
.sup.125I, .sup.131I, .sup.35S, or .sup.3H. Such labeled reagents
may be used in a variety of well-known assays, such as
radioimmunoassays, enzyme immunoassays, e.g., ELISA, fluorescent
immunoassays, and the like.
[0360] The antibodies are tagged with such labels by known methods.
For instance, coupling agents such as aldehydes, carbodiimides,
dimaleimide, imidates, succinimides, bid-diazotized benzadine and
the like are used to tag the antibodies with the above-described
fluorescent, chemiluminescent, and enzyme labels. An enzyme is
typically combined with an antibody using bridging molecules such
as carbodiimides, periodate, diisocyanates, glutaraldehyde and the
like. Various labeling techniques are described in Morrison,
Methods in Enzymology 32b, 103 (1974), Syvanen et al., J. Biol.
Chem. 284, 3762 (1973) and Bolton and Hunter, Biochem J. 133,
529(1973).
[0361] An antibody according to the invention may be conjugated to
a therapeutic moiety such as a cytotoxin, a therapeutic agent, or a
radioactive metal ion or radioisotope. Examples of radioisotopes
include, but are not limited to, I-131, I-123, I-125, Y-90, Re-188,
Re-186, At-211, Cu-67, Bi-212, Bi-213, Pd-109, Tc-99, In-111, and
the like. Such antibody conjugates can be used for modifying a
given biological response; the drug moiety is not to be construed
as limited to classical chemical therapeutic agents. For example,
the drug moiety may be a protein or polypeptide possessing a
desired biological activity. Such proteins may include, for
example, a toxin such as abrin, ricin A, pseudomonas exotoxin, or
diphtheria toxin.
[0362] Techniques for conjugating such therapeutic moiety to
antibodies are well known. See, for example, Amon et al. (1985)
"Monoclonal Antibodies for Immunotargeting of Drugs in Cancer
Therapy," in Monoclonal Antibodies and Cancer Therapy, ed. Reisfeld
et al. (Alan R. Liss, Inc.), pp. 243-256; ed. Hellstrom et al.
(1987) "Antibodies for Drug Delivery," in Controlled Drug Delivery,
ed. Robinson et al. (2d ed; Marcel Dekker, Inc.), pp. 623-653;
Thorpe (1985) "Antibody Carriers of Cytotoxic Agents in Cancer
Therapy: A Review," in Monoclonal Antibodies '84: Biological and
Clinical Applications, ed. Pinchera et al. pp. 475-506 (Editrice
Kurtis, Milano, Italy, 1985); "Analysis, Results, and Future
Prospective of the Therapeutic Use of Radiolabeled Antibody in
Cancer Therapy," in Monoclonal Antibodies for Cancer Detection and
Therapy, ed. Baldwin et al. (Academic Press, New York, 1985), pp.
303-316; and Thorpe et al. (1982) Immunol. Rev. 62:119-158.
[0363] Diagnostic methods generally involve contacting a biological
sample obtained from a patient, such as, e.g., blood, serum,
saliva, urine, sputum, a cell swab sample, or a tissue biopsy, with
an HRV antibody and determining whether the antibody preferentially
binds to the sample as compared to a control sample or
predetermined cut-off value, thereby indicating the presence of
infected cells. In particular embodiments, at least two-fold,
three-fold, or five-fold more HRV antibody binds to an infected
cell as compared to an appropriate control normal cell or tissue
sample. A pre-determined cut-off value is determined, e.g., by
averaging the amount of HRV antibody that binds to several
different appropriate control samples under the same conditions
used to perform the diagnostic assay of the biological sample being
tested.
[0364] Bound antibody is detected using procedures described herein
and known in the art. In certain embodiments, diagnostic methods of
the invention are practiced using HRV antibodies that are
conjugated to a detectable label, e.g., a fluorophore, to
facilitate detection of bound antibody. However, they are also
practiced using methods of secondary detection of the HRV antibody.
These include, for example, RIA, ELISA, precipitation,
agglutination, complement fixation and immuno-fluorescence.
[0365] HRV antibodies of the present invention are capable of
differentiating between patients with and patients without an HRV
infection, and determining whether or not a patient has an
infection, using the representative assays provided herein.
According to one method, a biological sample is obtained from a
patient suspected of having or known to have HRV infection. In
preferred embodiments, the biological sample includes cells from
the patient. The sample is contacted with an HRV antibody, e.g.,
for a time and under conditions sufficient to allow the HRV
antibody to bind to infected cells present in the sample. For
instance, the sample is contacted with an HRV antibody for 10
seconds, 30 seconds, 1 minute, 5 minutes, 10 minutes, 30 minutes, 1
hour, 6 hours, 12 hours, 24 hours, 3 days or any point in between.
The amount of bound HRV antibody is determined and compared to a
control value, which may be, e.g., a pre-determined value or a
value determined from normal tissue sample. An increased amount of
antibody bound to the patient sample as compared to the control
sample is indicative of the presence of infected cells in the
patient sample.
[0366] In a related method, a biological sample obtained from a
patient is contacted with an HRV antibody for a time and under
conditions sufficient to allow the antibody to bind to infected
cells. Bound antibody is then detected, and the presence of bound
antibody indicates that the sample contains infected cells. This
embodiment is particularly useful when the HRV antibody does not
bind normal cells at a detectable level.
[0367] Different HRV antibodies possess different binding and
specificity characteristics. Depending upon these characteristics,
particular HRV antibodies are used to detect the presence of one or
more strains of HRV. For example, certain antibodies bind
specifically to only one or several strains of HRV, whereas others
bind to all or a majority of different strains of HRV. Antibodies
specific for only one strain of HRV are used to identify the strain
of an infection.
[0368] In certain embodiments, antibodies that bind to an infected
cell preferably generate a signal indicating the presence of an
infection in at least about 20% of patients with the infection
being detected, more preferably at least about 30% of patients.
Alternatively, or in addition, the antibody generates a negative
signal indicating the absence of the infection in at least about
90% of individuals without the infection being detected. Each
antibody satisfies the above criteria; however, antibodies of the
present invention are used in combination to improve
sensitivity.
[0369] The present invention also includes kits useful in
performing diagnostic and prognostic assays using the antibodies of
the present invention. Kits of the invention include a suitable
container comprising an HRV antibody of the invention in either
labeled or unlabeled form. In addition, when the antibody is
supplied in a labeled form suitable for an indirect binding assay,
the kit further includes reagents for performing the appropriate
indirect assay. For example, the kit includes one or more suitable
containers including enzyme substrates or derivatizing agents,
depending on the nature of the label. Control samples and/or
instructions are also included.
[0370] Passive immunization has proven to be an effective and safe
strategy for the prevention and treatment of viral diseases. (See
Keller et al., Clin. Microbiol. Rev. 13:602-14 (2000); Casadevall,
Nat. Biotechnol. 20:114 (2002); Shibata et al., Nat. Med. 5:204-10
(1999); and Igarashi et al., Nat. Med. 5:211-16 (1999), each of
which are incorporated herein by reference)). Passive immunization
using human monoclonal antibodies provides an immediate treatment
strategy for emergency prophylaxis and treatment of HRV.
[0371] HRV antibodies and fragments thereof, and therapeutic
compositions, of the invention specifically bind or preferentially
bind to infected cells, as compared to normal control uninfected
cells and tissue. Thus, these HRV antibodies are used to
selectively target infected cells or tissues in a patient,
biological sample, or cell population. In light of the
infection-specific binding properties of these antibodies, the
present invention provides methods of regulating (e.g., inhibiting)
the growth of infected cells, methods of killing infected cells,
and methods of inducing apoptosis of infected cells. These methods
include contacting an infected cell with an HRV antibody of the
invention. These methods are practiced in vitro, ex vivo, and in
vivo.
[0372] In various embodiments, antibodies of the invention are
intrinsically therapeutically active. Alternatively, or in
addition, antibodies of the invention are conjugated to a cytotoxic
agent or growth inhibitory agent, e.g., a radioisotope or toxin
that is used in treating infected cells bound or contacted by the
antibody.
[0373] Subjects at risk for HRV-related diseases or disorders
include patients who have come into contact with an infected person
or who have been exposed to HRV in some other way. Administration
of a prophylactic agent can occur prior to the manifestation of
symptoms characteristic of HRV-related disease or disorder, such
that a disease or disorder is prevented or, alternatively, delayed
in its progression.
[0374] Methods for preventing an increase in HRV virus titer, virus
replication, virus proliferation or an amount of an HRV viral
protein in a subject are further provided. In one embodiment, a
method includes administering to the subject an amount of an HRV
antibody effective to prevent an increase in HRV titer, virus
replication or an amount of an HRV protein of one or more HRV
strains or isolates in the subject.
[0375] For in vivo treatment of human and non-human patients, the
patient is usually administered or provided a pharmaceutical
formulation including an HRV antibody of the invention. When used
for in vivo therapy, the antibodies of the invention are
administered to the patient in therapeutically effective amounts
(i.e., amounts that eliminate or reduce the patient's viral
burden). The antibodies are administered to a human patient, in
accord with known methods, such as intravenous administration,
e.g., as a bolus or by continuous infusion over a period of time,
by intramuscular, intraperitoneal, intracerobrospinal,
subcutaneous, intra-articular, intrasynovial, intrathecal, oral,
topical, or inhalation routes. The antibodies may be administered
parenterally, when possible, at the target cell site, or
intravenously. Intravenous or subcutaneous administration of the
antibody is preferred in certain embodiments. Therapeutic
compositions of the invention are administered to a patient or
subject systemically, parenterally, or locally.
[0376] For parenteral administration, the antibodies are formulated
in a unit dosage injectable form (solution, suspension, emulsion)
in association with a pharmaceutically acceptable, parenteral
vehicle. Examples of such vehicles are water, saline, Ringer's
solution, dextrose solution, and 5% human serum albumin. Nonaqueous
vehicles such as fixed oils and ethyl oleate are also used.
Liposomes are used as carriers. The vehicle contains minor amounts
of additives such as substances that enhance isotonicity and
chemical stability, e.g., buffers and preservatives. The antibodies
are typically formulated in such vehicles at concentrations of
about 1 mg/ml to 10 mg/ml.
[0377] The dose and dosage regimen depends upon a variety of
factors readily determined by a physician, such as the nature of
the infection and the characteristics of the particular cytotoxic
agent or growth inhibitory agent conjugated to the antibody (when
used), e.g., its therapeutic index, the patient, and the patient's
history. Generally, a therapeutically effective amount of an
antibody is administered to a patient. In particular embodiments,
the amount of antibody administered is in the range of about 0.1
mg/kg to about 50 mg/kg of patient body weight. Depending on the
type and severity of the infection, about 0.1 mg/kg to about 50
mg/kg body weight (e.g., about 0.1-15 mg/kg/dose) of antibody is an
initial candidate dosage for administration to the patient,
whether, for example, by one or more separate administrations, or
by continuous infusion. The progress of this therapy is readily
monitored by conventional methods and assays and based on criteria
known to the physician or other persons of skill in the art.
[0378] In one particular embodiment, an immunoconjugate including
the antibody conjugated with a cytotoxic agent is administered to
the patient. Preferably, the immunoconjugate is internalized by the
cell, resulting in increased therapeutic efficacy of the
immunoconjugate in killing the cell to which it binds. In one
embodiment, the cytotoxic agent targets or interferes with the
nucleic acid in the infected cell. Examples of such cytotoxic
agents are described above and include, but are not limited to,
maytansinoids, calicheamicins, ribonucleases and DNA
endonucleases.
[0379] Other therapeutic regimens are combined with the
administration of the HRV antibody of the present invention. The
combined administration includes co-administration, using separate
formulations or a single pharmaceutical formulation, and
consecutive administration in either order, wherein preferably
there is a time period while both (or all) active agents
simultaneously exert their biological activities. Preferably such
combined therapy results in a synergistic therapeutic effect.
[0380] In certain embodiments, it is desirable to combine
administration of an antibody of the invention with another
antibody directed against another antigen associated with the
infectious agent.
[0381] Aside from administration of the antibody protein to the
patient, the invention provides methods of administration of the
antibody by gene therapy. Such administration of nucleic acid
encoding the antibody is encompassed by the expression
"administering a therapeutically effective amount of an antibody".
See, for example, PCT Patent Application Publication WO96/07321
concerning the use of gene therapy to generate intracellular
antibodies.
[0382] In another embodiment, anti-HRV antibodies of the invention
are used to determine the structure of bound antigen, e.g.,
conformational epitopes, the structure of which is then used to
develop a vaccine having or mimicking this structure, e.g., through
chemical modeling and SAR methods. Such a vaccine could then be
used to prevent HRV infection.
[0383] The following examples are included to demonstrate preferred
embodiments of the invention. It should be appreciated by those of
skill in the art that the techniques disclosed in the examples
which follow represent techniques discovered by the inventor to
function well in the practice of the invention, and thus can be
considered to constitute preferred modes for its practice. However,
those of skill in the art should, in light of the present
disclosure, appreciate that many changes can be made in the
specific embodiments which are disclosed and still obtain a like or
similar result without departing from the spirit and scope of the
invention.
EXAMPLES
Example 1
Isolation and Characterization of Cross-Serotype Neutralizing
Monoclonal Antibodies Against Rhinovirus
[0384] IgG expressing memory B cells were isolated from a healthy
individual by negative depletion of other peripheral blood
mononuclear cells (PBMC) on magnetic beads. Memory B cells were
activated at near clonal density in 384-well microplates in the
presence of cytokines and feeder cells that promote polyclonal B
cell activation. Supernatants of B cell culture wells containing
secreted antibodies were screened for neutralization against 2
serotypes of rhinovirus (HRV) in cytopathic effect (CPE) assay.
Variable regions of the IgG heavy and light chains from the B cell
clones that neutralized both serotypes were rescued by RT-PCR and
the sequences were determined by 454 pyrosequencing (also known as
deep sequencing). The sequences from an individual B cell clone
were then compared with those from other B cell clones to identify
clonally related antibodies also known "sister" mAbs. These
clonally related sister clones are likely derived from the same
precursor B cell. The variable regions were synthesized as DNA and
cloned in expression vectors with the appropriate IgG1, Ig.kappa.
or Ig.lamda. constant domain. Monoclonal antibodies were
reconstituted by transient transfection in HEK293 cells followed by
purification from serum-free culture supernatants.
[0385] Purified monoclonal antibodies were analyzed in a titration
series of concentrations for neutralization activity against a
panel of 38 HRV serotypes that include 22 major group viruses and 4
minor group viruses in clade A, 2 viruses in clade D, and 10
viruses in clade B in microneutralization or CPE assay. The IC50
values determined for three monoclonal antibodies, TCN-717(or H17),
TCN-722 (or L22) and TCN-716 (or F16) against each virus that was
neutralized are shown in FIG. 1. FIG. 2 shows the % serotypes in
the panel of 38 viruses and in the subset of 22 viruses from the
clade A major group that were neutralized by each of the three
monoclonal antibodies or by two antibodies in combination. The
percent (%) serotypes neutralized represents the relative breadth
of neutralization by these antibodies. From this panel of 38 HRV
serotypes, TCN-717, TCN-722 or TCN-716 neutralized only viruses
belonging to the clade A major group and clade D. FIG. 3 shows the
neutralization profile of each antibody for 26 clade A serotypes
and two clade D serotypes. Although TCN-717 and TCN-722 are
clonally related by sequence, their neutralization profiles reveal
differences in the fine specificities. TCN-717 and TCN-722 were
further analyzed for direct binding to intact inactivated virus
immobilized on plastic by ELISA. The direct binding of TCN-717 to
HRV-28 and HRV-36 and binding of TCN-722 to HRV-28, HRV-34 and
HRV-36 are shown in FIG. 4.
Example 2
Isolation and Characterization of Cross-Serotype Non-Neutralizing
Monoclonal Antibodies Against Rhinovirus
[0386] Memory B cells were isolated from a healthy individual
different from the one described in Example 1 using similar method.
B cell culture supernatants were screened for cross-serotype
binding reactivity by capturing human IgG on microarray glass
slides and incubating with inactivated virus of ten different
serotypes separately. Variable regions of the IgG heavy and light
chains from a B cell clone, TCN-711 (or E11), that bound to nine
serotypes were rescued by RT-PCR and the sequences were determined
by deep sequencing. The variable regions were synthesized as DNA
and cloned in an expression vector with IgG1 constant domain and
another one with Ig.lamda. constant domain. Monoclonal antibodies
were reconstituted by transient transfection in HEK293 cells
followed by purification from serum-free culture supernatants.
[0387] Purified TCN-711 was analyzed in a titration series of
concentrations for binding activity against a panel of 38 HRV
serotypes that include 22 major group viruses and 4 minor group
viruses in clade A, 2 viruses in clade D, and 10 viruses in clade
B. The panel of serotypes tested is the same as used in Example 1.
To detect binding activity of TCN-711, HeLa cells were infected
with individual HRV serotype overnight. After washing to remove
free virus, infected cells were fixed, permeabilized one day later
and incubated with TCN-711 at 2 .mu.g/ml for one hour. Bound
TCN-711 was detected by incubation with Alexafluor-647 conjugated
anti-human Fc and visualized by imaging on an InCell Analyzer.
Table 1 shows the specific binding of TCN-711 to 35 HRV serotypes
that include viruses from clade A major and minor groups, clade B
and clade D, which represents 92% of the serotypes in the panel of
38 viruses. The binding of TCN-711 to four serotypes in a titration
series of concentrations was shown in FIG. 5. Half-maximal binding
of TCN-711 was detected from 1 to 10 ng/ml.
TABLE-US-00049 TABLE 1 Binding profile of TCN-711 to a panel of HRV
serotypes. Binding of Serotype Clade TCN-711 HRV-12 A/major gp +
HRV-13 + HRV-16 + HRV-21 + HRV-23 + HRV-24 + HRV-28 + HRV-34 +
HRV-36 + HRV-38 + HRV-40 + HRV-51 + HRV-54 + HRV-61 + HRV-63 +
HRV-64 - HRV-67 + HRV-74 + HRV-75 + HRV-76 + HRV-88 + HRV-89 +
HRV-29 A/minor gp + HRV-31 + HRV-49 + HRV-62 + HRV-14 B + HRV-26 -
HRV-37 + HRV-48 - HRV-52 + HRV-70 + HRV-83 + HRV-84 + HRV-86 +
HRV-93 + HRV-08 D + HRV-45 +
OTHER EMBODIMENTS
[0388] Although specific embodiments of the invention have been
described herein for purposes of illustration, various
modifications may be made without deviating from the spirit and
scope of the invention. Accordingly, the invention is not limited
except as by the appended claims.
[0389] While the invention has been described in conjunction with
the detailed description thereof, the foregoing description is
intended to illustrate and not limit the scope of the invention,
which is defined by the scope of the appended claims. Other
aspects, advantages, and modifications are within the scope of the
following claims.
[0390] The patent and scientific literature referred to herein
establishes the knowledge that is available to those with skill in
the art. All United States patents and published or unpublished
United States patent applications cited herein are incorporated by
reference. All published foreign patents and patent applications
cited herein are hereby incorporated by reference. Genbank and NCBI
submissions indicated by accession number cited herein are hereby
incorporated by reference. All other published references,
documents, manuscripts and scientific literature cited herein are
hereby incorporated by reference.
[0391] While this invention has been particularly shown and
described with references to preferred embodiments thereof, it will
be understood by those skilled in the art that various changes in
form and details may be made therein without departing from the
scope of the invention encompassed by the appended claims.
Sequence CWU 1
1
6411428DNAHomo sapiens 1atgaaacacc tgtggttctt cctcctcctg gcggcagctc
ccagatgggt cctgtcccag 60gtgcagctac accagtgggg cacaggagtg ttgaagcctt
cggggaccct gtccctcacc 120tgcggtgtct atggtgggtc cctcactgat
ttctactgga cctggatccg tcagtccccc 180gcgaggggcc tggagtggct
tggagaaatc gatcgtgatg gggccacgta ctataatccg 240tccctaaaga
gtcgaatcac catttcgata gacacgtcca agaaacaatt ctccttgaat
300ctgcggtctg tgaccgccgc ggacagggct gtctactact gtgcgaggcg
ccctatgtta 360cgaggcgttt gggggaattt tcgttccaac tggttcgacc
cctggggcca gggaacccag 420gtcaccgtct cgagcgcctc caccaagggc
ccatcggtct tccccctggc accctcctcc 480aagagcacct ctgggggcac
agcggccctg ggctgcctgg tcaaggacta cttccccgaa 540ccggtgacgg
tgtcgtggaa ctcaggcgcc ctgaccagcg gcgtgcacac cttcccggct
600gtcctacagt cctcaggact ctactccctc agcagcgtgg tgaccgtgcc
ctccagcagc 660ttgggcaccc agacctacat ctgcaacgtg aatcacaagc
ccagcaacac caaggtggac 720aagagagttg agcccaaatc ttgtgacaaa
actcacacat gcccaccgtg cccagcacct 780gaactcctgg ggggaccgtc
agtcttcctc ttccccccaa aacccaagga caccctcatg 840atctcccgga
cccctgaggt cacatgcgtg gtggtggacg tgagccacga agaccctgag
900gtcaagttca actggtacgt ggacggcgtg gaggtgcata atgccaagac
aaagccgcgg 960gaggagcagt acaacagcac gtaccgtgtg gtcagcgtcc
tcaccgtcct gcaccaggac 1020tggctgaatg gcaaggagta caagtgcaag
gtctccaaca aagccctccc agcccccatc 1080gagaaaacca tctccaaagc
caaagggcag ccccgagaac cacaggtgta caccctgccc 1140ccatcccggg
aggagatgac caagaaccag gtcagcctga cctgcctggt caaaggcttc
1200tatcccagcg acatcgccgt ggagtgggag agcaatgggc agccggagaa
caactacaag 1260accacgcctc ccgtgctgga ctccgacggc tccttcttcc
tctatagcaa gctcaccgtg 1320gacaagagca ggtggcagca ggggaacgtc
ttctcatgct ccgtgatgca tgaggctctg 1380cacaaccact acacgcagaa
gagcctctcc ctgtctccgg gtaaatga 14282378DNAHomo sapiens 2caggtgcagc
tacaccagtg gggcacagga gtgttgaagc cttcggggac cctgtccctc 60acctgcggtg
tctatggtgg gtccctcact gatttctact ggacctggat ccgtcagtcc
120cccgcgaggg gcctggagtg gcttggagaa atcgatcgtg atggggccac
gtactataat 180ccgtccctaa agagtcgaat caccatttcg atagacacgt
ccaagaaaca attctccttg 240aatctgcggt ctgtgaccgc cgcggacagg
gctgtctact actgtgcgag gcgccctatg 300ttacgaggcg tttgggggaa
ttttcgttcc aactggttcg acccctgggg ccagggaacc 360caggtcaccg tctcgagc
3783475PRTHomo sapiens 3Met Lys His Leu Trp Phe Phe Leu Leu Leu Ala
Ala Ala Pro Arg Trp 1 5 10 15 Val Leu Ser Gln Val Gln Leu His Gln
Trp Gly Thr Gly Val Leu Lys 20 25 30 Pro Ser Gly Thr Leu Ser Leu
Thr Cys Gly Val Tyr Gly Gly Ser Leu 35 40 45 Thr Asp Phe Tyr Trp
Thr Trp Ile Arg Gln Ser Pro Ala Arg Gly Leu 50 55 60 Glu Trp Leu
Gly Glu Ile Asp Arg Asp Gly Ala Thr Tyr Tyr Asn Pro 65 70 75 80 Ser
Leu Lys Ser Arg Ile Thr Ile Ser Ile Asp Thr Ser Lys Lys Gln 85 90
95 Phe Ser Leu Asn Leu Arg Ser Val Thr Ala Ala Asp Arg Ala Val Tyr
100 105 110 Tyr Cys Ala Arg Arg Pro Met Leu Arg Gly Val Trp Gly Asn
Phe Arg 115 120 125 Ser Asn Trp Phe Asp Pro Trp Gly Gln Gly Thr Gln
Val Thr Val Ser 130 135 140 Ser Ala Ser Thr Lys Gly Pro Ser Val Phe
Pro Leu Ala Pro Ser Ser 145 150 155 160 Lys Ser Thr Ser Gly Gly Thr
Ala Ala Leu Gly Cys Leu Val Lys Asp 165 170 175 Tyr Phe Pro Glu Pro
Val Thr Val Ser Trp Asn Ser Gly Ala Leu Thr 180 185 190 Ser Gly Val
His Thr Phe Pro Ala Val Leu Gln Ser Ser Gly Leu Tyr 195 200 205 Ser
Leu Ser Ser Val Val Thr Val Pro Ser Ser Ser Leu Gly Thr Gln 210 215
220 Thr Tyr Ile Cys Asn Val Asn His Lys Pro Ser Asn Thr Lys Val Asp
225 230 235 240 Lys Arg Val Glu Pro Lys Ser Cys Asp Lys Thr His Thr
Cys Pro Pro 245 250 255 Cys Pro Ala Pro Glu Leu Leu Gly Gly Pro Ser
Val Phe Leu Phe Pro 260 265 270 Pro Lys Pro Lys Asp Thr Leu Met Ile
Ser Arg Thr Pro Glu Val Thr 275 280 285 Cys Val Val Val Asp Val Ser
His Glu Asp Pro Glu Val Lys Phe Asn 290 295 300 Trp Tyr Val Asp Gly
Val Glu Val His Asn Ala Lys Thr Lys Pro Arg 305 310 315 320 Glu Glu
Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val 325 330 335
Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser 340
345 350 Asn Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala
Lys 355 360 365 Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro
Ser Arg Glu 370 375 380 Glu Met Thr Lys Asn Gln Val Ser Leu Thr Cys
Leu Val Lys Gly Phe 385 390 395 400 Tyr Pro Ser Asp Ile Ala Val Glu
Trp Glu Ser Asn Gly Gln Pro Glu 405 410 415 Asn Asn Tyr Lys Thr Thr
Pro Pro Val Leu Asp Ser Asp Gly Ser Phe 420 425 430 Phe Leu Tyr Ser
Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly 435 440 445 Asn Val
Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn His Tyr 450 455 460
Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly Lys 465 470 475 4126PRTHomo
sapiens 4Gln Val Gln Leu His Gln Trp Gly Thr Gly Val Leu Lys Pro
Ser Gly 1 5 10 15 Thr Leu Ser Leu Thr Cys Gly Val Tyr Gly Gly Ser
Leu Thr Asp Phe 20 25 30 Tyr Trp Thr Trp Ile Arg Gln Ser Pro Ala
Arg Gly Leu Glu Trp Leu 35 40 45 Gly Glu Ile Asp Arg Asp Gly Ala
Thr Tyr Tyr Asn Pro Ser Leu Lys 50 55 60 Ser Arg Ile Thr Ile Ser
Ile Asp Thr Ser Lys Lys Gln Phe Ser Leu 65 70 75 80 Asn Leu Arg Ser
Val Thr Ala Ala Asp Arg Ala Val Tyr Tyr Cys Ala 85 90 95 Arg Arg
Pro Met Leu Arg Gly Val Trp Gly Asn Phe Arg Ser Asn Trp 100 105 110
Phe Asp Pro Trp Gly Gln Gly Thr Gln Val Thr Val Ser Ser 115 120 125
55PRTHomo sapiens 5Asp Phe Tyr Trp Thr 1 5 616PRTHomo sapiens 6Glu
Ile Asp Arg Asp Gly Ala Thr Tyr Tyr Asn Pro Ser Leu Lys Ser 1 5 10
15 718PRTHomo sapiens 7Arg Pro Met Leu Arg Gly Val Trp Gly Asn Phe
Arg Ser Asn Trp Phe 1 5 10 15 Asp Pro 86PRTHomo sapiens 8Gly Gly
Ser Leu Thr Asp 1 5 99PRTHomo sapiens 9Glu Ile Asp Arg Asp Gly Ala
Thr Tyr 1 5 10708DNAHomo sapiens 10atggccagct tccctctcct cctcaccctt
ctcattcact gcacagggtc ctgggcccag 60tctgtcttga cgcagccgcc ctcagtgtct
gcggccccag gacagaaggt ctccatctcc 120tgctctggaa gcagctccaa
cattgggtat agttatgtat cctggtatca acaagtccca 180ggatcagccc
ccaaactcct catctatgag aataataaga gaccctcagg gattcctgac
240cgattctcgg cctccaagtc tggcacgtca gccaccctgg acatcaccgg
actccagact 300ggggacgagg ccgattatta ttgcggaaca tgggatacca
ggctgtttgg tggagtgttc 360ggcggaggga ccaagctgac cgttctaggt
cagcccaagg ctgccccctc ggtcactctg 420ttcccgccct cctctgagga
gcttcaagcc aacaaggcca cactggtgtg tctcataagt 480gacttctacc
cgggagccgt gacagtggcc tggaaggcag atagcagccc cgtcaaggcg
540ggagtggaga ccaccacacc ctccaaacaa agcaacaaca agtacgcggc
cagcagctac 600ctgagcctga cgcctgagca gtggaagtcc cacaaaagct
acagctgcca ggtcacgcat 660gaagggagca ccgtggagaa gacagtggcc
cctacagaat gttcatag 70811330DNAHomo sapiens 11cagtctgtct tgacgcagcc
gccctcagtg tctgcggccc caggacagaa ggtctccatc 60tcctgctctg gaagcagctc
caacattggg tatagttatg tatcctggta tcaacaagtc 120ccaggatcag
cccccaaact cctcatctat gagaataata agagaccctc agggattcct
180gaccgattct cggcctccaa gtctggcacg tcagccaccc tggacatcac
cggactccag 240actggggacg aggccgatta ttattgcgga acatgggata
ccaggctgtt tggtggagtg 300ttcggcggag ggaccaagct gaccgttcta
33012235PRTHomo sapiens 12Met Ala Ser Phe Pro Leu Leu Leu Thr Leu
Leu Ile His Cys Thr Gly 1 5 10 15 Ser Trp Ala Gln Ser Val Leu Thr
Gln Pro Pro Ser Val Ser Ala Ala 20 25 30 Pro Gly Gln Lys Val Ser
Ile Ser Cys Ser Gly Ser Ser Ser Asn Ile 35 40 45 Gly Tyr Ser Tyr
Val Ser Trp Tyr Gln Gln Val Pro Gly Ser Ala Pro 50 55 60 Lys Leu
Leu Ile Tyr Glu Asn Asn Lys Arg Pro Ser Gly Ile Pro Asp 65 70 75 80
Arg Phe Ser Ala Ser Lys Ser Gly Thr Ser Ala Thr Leu Asp Ile Thr 85
90 95 Gly Leu Gln Thr Gly Asp Glu Ala Asp Tyr Tyr Cys Gly Thr Trp
Asp 100 105 110 Thr Arg Leu Phe Gly Gly Val Phe Gly Gly Gly Thr Lys
Leu Thr Val 115 120 125 Leu Gly Gln Pro Lys Ala Ala Pro Ser Val Thr
Leu Phe Pro Pro Ser 130 135 140 Ser Glu Glu Leu Gln Ala Asn Lys Ala
Thr Leu Val Cys Leu Ile Ser 145 150 155 160 Asp Phe Tyr Pro Gly Ala
Val Thr Val Ala Trp Lys Ala Asp Ser Ser 165 170 175 Pro Val Lys Ala
Gly Val Glu Thr Thr Thr Pro Ser Lys Gln Ser Asn 180 185 190 Asn Lys
Tyr Ala Ala Ser Ser Tyr Leu Ser Leu Thr Pro Glu Gln Trp 195 200 205
Lys Ser His Lys Ser Tyr Ser Cys Gln Val Thr His Glu Gly Ser Thr 210
215 220 Val Glu Lys Thr Val Ala Pro Thr Glu Cys Ser 225 230 235
13110PRTHomo sapiens 13Gln Ser Val Leu Thr Gln Pro Pro Ser Val Ser
Ala Ala Pro Gly Gln 1 5 10 15 Lys Val Ser Ile Ser Cys Ser Gly Ser
Ser Ser Asn Ile Gly Tyr Ser 20 25 30 Tyr Val Ser Trp Tyr Gln Gln
Val Pro Gly Ser Ala Pro Lys Leu Leu 35 40 45 Ile Tyr Glu Asn Asn
Lys Arg Pro Ser Gly Ile Pro Asp Arg Phe Ser 50 55 60 Ala Ser Lys
Ser Gly Thr Ser Ala Thr Leu Asp Ile Thr Gly Leu Gln 65 70 75 80 Thr
Gly Asp Glu Ala Asp Tyr Tyr Cys Gly Thr Trp Asp Thr Arg Leu 85 90
95 Phe Gly Gly Val Phe Gly Gly Gly Thr Lys Leu Thr Val Leu 100 105
110 1413PRTHomo sapiens 14Ser Gly Ser Ser Ser Asn Ile Gly Tyr Ser
Tyr Val Ser 1 5 10 157PRTHomo sapiens 15Glu Asn Asn Lys Arg Pro Ser
1 5 1611PRTHomo sapiens 16Gly Thr Trp Asp Thr Arg Leu Phe Gly Gly
Val 1 5 10 171440DNAHomo sapiens 17atggagtttg ggctgagctg ggttctcctt
gttgccattt taaaaggtgc ccagtgtgag 60gtgcaactgg tggagtctgg gggaggcttg
gtcctgccgg ggggctctct gagactctcg 120tgttcagcgt ctggattcac
attgactgac tttgctatgc actgggtccg acaggctcca 180gggaagggac
tggagctcgt ctcaagtatt agtcgggatg gttctactaa atactctgga
240gactccgtga agggcagggt cgccatctcc agggacagtg tggagaataa
gttgcatctt 300cagatgagcg gtctgaggtc tgcggacacg gctgtgtatt
attgtgtgag agactccccc 360tattatcttg atattgttgg ttatcgatac
ttccaccact atggaatgga cgtctggggc 420caggggacca cggtcaccgt
ctcgagcgcc tccaccaagg gcccatcggt cttccccctg 480gcaccctcct
ccaagagcac ctctgggggc acagcggccc tgggctgcct ggtcaaggac
540tacttccccg aaccggtgac ggtgtcgtgg aactcaggcg ccctgaccag
cggcgtgcac 600accttcccgg ctgtcctaca gtcctcagga ctctactccc
tcagcagcgt ggtgaccgtg 660ccctccagca gcttgggcac ccagacctac
atctgcaacg tgaatcacaa gcccagcaac 720accaaggtgg acaagagagt
tgagcccaaa tcttgtgaca aaactcacac atgcccaccg 780tgcccagcac
ctgaactcct ggggggaccg tcagtcttcc tcttcccccc aaaacccaag
840gacaccctca tgatctcccg gacccctgag gtcacatgcg tggtggtgga
cgtgagccac 900gaagaccctg aggtcaagtt caactggtac gtggacggcg
tggaggtgca taatgccaag 960acaaagccgc gggaggagca gtacaacagc
acgtaccgtg tggtcagcgt cctcaccgtc 1020ctgcaccagg actggctgaa
tggcaaggag tacaagtgca aggtctccaa caaagccctc 1080ccagccccca
tcgagaaaac catctccaaa gccaaagggc agccccgaga accacaggtg
1140tacaccctgc ccccatcccg ggaggagatg accaagaacc aggtcagcct
gacctgcctg 1200gtcaaaggct tctatcccag cgacatcgcc gtggagtggg
agagcaatgg gcagccggag 1260aacaactaca agaccacgcc tcccgtgctg
gactccgacg gctccttctt cctctatagc 1320aagctcaccg tggacaagag
caggtggcag caggggaacg tcttctcatg ctccgtgatg 1380catgaggctc
tgcacaacca ctacacgcag aagagcctct ccctgtctcc gggtaaatga
144018390DNAHomo sapiens 18gaggtgcaac tggtggagtc tgggggaggc
ttggtcctgc cggggggctc tctgagactc 60tcgtgttcag cgtctggatt cacattgact
gactttgcta tgcactgggt ccgacaggct 120ccagggaagg gactggagct
cgtctcaagt attagtcggg atggttctac taaatactct 180ggagactccg
tgaagggcag ggtcgccatc tccagggaca gtgtggagaa taagttgcat
240cttcagatga gcggtctgag gtctgcggac acggctgtgt attattgtgt
gagagactcc 300ccctattatc ttgatattgt tggttatcga tacttccacc
actatggaat ggacgtctgg 360ggccagggga ccacggtcac cgtctcgagc
39019479PRTHomo sapiens 19Met Glu Phe Gly Leu Ser Trp Val Leu Leu
Val Ala Ile Leu Lys Gly 1 5 10 15 Ala Gln Cys Glu Val Gln Leu Val
Glu Ser Gly Gly Gly Leu Val Leu 20 25 30 Pro Gly Gly Ser Leu Arg
Leu Ser Cys Ser Ala Ser Gly Phe Thr Leu 35 40 45 Thr Asp Phe Ala
Met His Trp Val Arg Gln Ala Pro Gly Lys Gly Leu 50 55 60 Glu Leu
Val Ser Ser Ile Ser Arg Asp Gly Ser Thr Lys Tyr Ser Gly 65 70 75 80
Asp Ser Val Lys Gly Arg Val Ala Ile Ser Arg Asp Ser Val Glu Asn 85
90 95 Lys Leu His Leu Gln Met Ser Gly Leu Arg Ser Ala Asp Thr Ala
Val 100 105 110 Tyr Tyr Cys Val Arg Asp Ser Pro Tyr Tyr Leu Asp Ile
Val Gly Tyr 115 120 125 Arg Tyr Phe His His Tyr Gly Met Asp Val Trp
Gly Gln Gly Thr Thr 130 135 140 Val Thr Val Ser Ser Ala Ser Thr Lys
Gly Pro Ser Val Phe Pro Leu 145 150 155 160 Ala Pro Ser Ser Lys Ser
Thr Ser Gly Gly Thr Ala Ala Leu Gly Cys 165 170 175 Leu Val Lys Asp
Tyr Phe Pro Glu Pro Val Thr Val Ser Trp Asn Ser 180 185 190 Gly Ala
Leu Thr Ser Gly Val His Thr Phe Pro Ala Val Leu Gln Ser 195 200 205
Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro Ser Ser Ser 210
215 220 Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn His Lys Pro Ser
Asn 225 230 235 240 Thr Lys Val Asp Lys Arg Val Glu Pro Lys Ser Cys
Asp Lys Thr His 245 250 255 Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu
Leu Gly Gly Pro Ser Val 260 265 270 Phe Leu Phe Pro Pro Lys Pro Lys
Asp Thr Leu Met Ile Ser Arg Thr 275 280 285 Pro Glu Val Thr Cys Val
Val Val Asp Val Ser His Glu Asp Pro Glu 290 295 300 Val Lys Phe Asn
Trp Tyr Val Asp Gly Val Glu Val His Asn Ala Lys 305 310 315 320 Thr
Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser 325 330
335 Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys
340 345 350 Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu Lys
Thr Ile 355 360 365 Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val
Tyr Thr Leu Pro 370 375 380 Pro Ser Arg Glu Glu Met Thr Lys Asn Gln
Val Ser Leu Thr Cys Leu 385 390 395 400 Val Lys Gly Phe Tyr Pro Ser
Asp Ile Ala Val Glu Trp Glu Ser Asn 405 410 415 Gly Gln Pro Glu Asn
Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser 420 425 430 Asp Gly Ser
Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg 435 440 445 Trp
Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met His Glu Ala Leu 450 455
460 His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly Lys 465
470 475 20130PRTHomo sapiens 20Glu Val Gln Leu Val Glu Ser
Gly Gly Gly Leu Val Leu Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser
Cys Ser Ala Ser Gly Phe Thr Leu Thr Asp Phe 20 25 30 Ala Met His
Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Leu Val 35 40 45 Ser
Ser Ile Ser Arg Asp Gly Ser Thr Lys Tyr Ser Gly Asp Ser Val 50 55
60 Lys Gly Arg Val Ala Ile Ser Arg Asp Ser Val Glu Asn Lys Leu His
65 70 75 80 Leu Gln Met Ser Gly Leu Arg Ser Ala Asp Thr Ala Val Tyr
Tyr Cys 85 90 95 Val Arg Asp Ser Pro Tyr Tyr Leu Asp Ile Val Gly
Tyr Arg Tyr Phe 100 105 110 His His Tyr Gly Met Asp Val Trp Gly Gln
Gly Thr Thr Val Thr Val 115 120 125 Ser Ser 130 215PRTHomo sapiens
21Asp Phe Ala Met His 1 5 2217PRTHomo sapiens 22Ser Ile Ser Arg Asp
Gly Ser Thr Lys Tyr Ser Gly Asp Ser Val Lys 1 5 10 15 Gly
2321PRTHomo sapiens 23Asp Ser Pro Tyr Tyr Leu Asp Ile Val Gly Tyr
Arg Tyr Phe His His 1 5 10 15 Tyr Gly Met Asp Val 20 246PRTHomo
sapiens 24Gly Phe Thr Leu Thr Asp 1 5 2510PRTHomo sapiens 25Ser Ile
Ser Arg Asp Gly Ser Thr Lys Tyr 1 5 10 26717DNAHomo sapiens
26atggaaaccc cagctcagct tctcttcctc ctgctactct ggctcccaga taccaccgga
60gagattgtgt tgacgcagtc gccaggcacc ctgtctttgt ctccagggga cagagtcacc
120ctctcctgca gggccagtca aattcttcac agctataatt tagcctggta
tcagcacaga 180cctggccagg ctcccaggct cctcatttat ggtgcatata
acagggccag tggcatccca 240gacaggttca gtggcagtgg gtctggggca
gacttcaccc tcaccatcgg cagactgcag 300cgtgacgatt ttgcagttta
ttactgtcaa cagtatggtg actcaccatc accaggcctc 360actttcggcg
gaggaaccaa actggagttc aaacgtacgg tggctgcacc atctgtcttc
420atcttcccgc catctgatga gcagttgaaa tctggaactg cctctgttgt
gtgcctgctg 480aataacttct atcccagaga ggccaaagta cagtggaagg
tggataacgc cctccaatcg 540ggtaactccc aggagagtgt cacagagcag
gacagcaagg acagcaccta cagcctcagc 600agcaccctga cgctgagcaa
agcagactac gagaaacaca aagtctacgc ctgcgaagtc 660acccatcagg
gcctgagctc gcccgtcaca aagagcttca acaggggaga gtgttag 71727333DNAHomo
sapiens 27gagattgtgt tgacgcagtc gccaggcacc ctgtctttgt ctccagggga
cagagtcacc 60ctctcctgca gggccagtca aattcttcac agctataatt tagcctggta
tcagcacaga 120cctggccagg ctcccaggct cctcatttat ggtgcatata
acagggccag tggcatccca 180gacaggttca gtggcagtgg gtctggggca
gacttcaccc tcaccatcgg cagactgcag 240cgtgacgatt ttgcagttta
ttactgtcaa cagtatggtg actcaccatc accaggcctc 300actttcggcg
gaggaaccaa actggagttc aaa 33328238PRTHomo sapiens 28Met Glu Thr Pro
Ala Gln Leu Leu Phe Leu Leu Leu Leu Trp Leu Pro 1 5 10 15 Asp Thr
Thr Gly Glu Ile Val Leu Thr Gln Ser Pro Gly Thr Leu Ser 20 25 30
Leu Ser Pro Gly Asp Arg Val Thr Leu Ser Cys Arg Ala Ser Gln Ile 35
40 45 Leu His Ser Tyr Asn Leu Ala Trp Tyr Gln His Arg Pro Gly Gln
Ala 50 55 60 Pro Arg Leu Leu Ile Tyr Gly Ala Tyr Asn Arg Ala Ser
Gly Ile Pro 65 70 75 80 Asp Arg Phe Ser Gly Ser Gly Ser Gly Ala Asp
Phe Thr Leu Thr Ile 85 90 95 Gly Arg Leu Gln Arg Asp Asp Phe Ala
Val Tyr Tyr Cys Gln Gln Tyr 100 105 110 Gly Asp Ser Pro Ser Pro Gly
Leu Thr Phe Gly Gly Gly Thr Lys Leu 115 120 125 Glu Phe Lys Arg Thr
Val Ala Ala Pro Ser Val Phe Ile Phe Pro Pro 130 135 140 Ser Asp Glu
Gln Leu Lys Ser Gly Thr Ala Ser Val Val Cys Leu Leu 145 150 155 160
Asn Asn Phe Tyr Pro Arg Glu Ala Lys Val Gln Trp Lys Val Asp Asn 165
170 175 Ala Leu Gln Ser Gly Asn Ser Gln Glu Ser Val Thr Glu Gln Asp
Ser 180 185 190 Lys Asp Ser Thr Tyr Ser Leu Ser Ser Thr Leu Thr Leu
Ser Lys Ala 195 200 205 Asp Tyr Glu Lys His Lys Val Tyr Ala Cys Glu
Val Thr His Gln Gly 210 215 220 Leu Ser Ser Pro Val Thr Lys Ser Phe
Asn Arg Gly Glu Cys 225 230 235 29111PRTHomo sapiens 29Glu Ile Val
Leu Thr Gln Ser Pro Gly Thr Leu Ser Leu Ser Pro Gly 1 5 10 15 Asp
Arg Val Thr Leu Ser Cys Arg Ala Ser Gln Ile Leu His Ser Tyr 20 25
30 Asn Leu Ala Trp Tyr Gln His Arg Pro Gly Gln Ala Pro Arg Leu Leu
35 40 45 Ile Tyr Gly Ala Tyr Asn Arg Ala Ser Gly Ile Pro Asp Arg
Phe Ser 50 55 60 Gly Ser Gly Ser Gly Ala Asp Phe Thr Leu Thr Ile
Gly Arg Leu Gln 65 70 75 80 Arg Asp Asp Phe Ala Val Tyr Tyr Cys Gln
Gln Tyr Gly Asp Ser Pro 85 90 95 Ser Pro Gly Leu Thr Phe Gly Gly
Gly Thr Lys Leu Glu Phe Lys 100 105 110 3012PRTHomo sapiens 30Arg
Ala Ser Gln Ile Leu His Ser Tyr Asn Leu Ala 1 5 10 317PRTHomo
sapiens 31Gly Ala Tyr Asn Arg Ala Ser 1 5 3212PRTHomo sapiens 32Gln
Gln Tyr Gly Asp Ser Pro Ser Pro Gly Leu Thr 1 5 10 331425DNAHomo
sapiens 33atgaaacacc tgtggttctt cctcctactg atggcggctc ccagatgggt
cctgtcccag 60ctgcaactgc ttgagtcggg cccaagactg gtgaaggctt cggagaccct
gtcactcacc 120tgcagtgtcc ctatgggctc catcctccaa aatgattatc
attgggcctg ggtccgccag 180cccccaggga ggggcctgga gtggattggg
agtgttcact atagacaaaa atcctactac 240agcccgtccc tcaagagccg
agtcttcatg tccgtagaca cgtccagaga ccagttctcc 300ctaaaactct
tctctctggc cgccgcggac acggccgtat attattgtgc gagacataat
360cgggaagatt attatgacag taatgcctac tttgacgagt ggggcctggg
agctcggatc 420accgtctcga gcgcctccac caagggccca tcggtcttcc
ccctggcacc ctcctccaag 480agcacctctg ggggcacagc ggccctgggc
tgcctggtca aggactactt ccccgaaccg 540gtgacggtgt cgtggaactc
aggcgccctg accagcggcg tgcacacctt cccggctgtc 600ctacagtcct
caggactcta ctccctcagc agcgtggtga ccgtgccctc cagcagcttg
660ggcacccaga cctacatctg caacgtgaat cacaagccca gcaacaccaa
ggtggacaag 720agagttgagc ccaaatcttg tgacaaaact cacacatgcc
caccgtgccc agcacctgaa 780ctcctggggg gaccgtcagt cttcctcttc
cccccaaaac ccaaggacac cctcatgatc 840tcccggaccc ctgaggtcac
atgcgtggtg gtggacgtga gccacgaaga ccctgaggtc 900aagttcaact
ggtacgtgga cggcgtggag gtgcataatg ccaagacaaa gccgcgggag
960gagcagtaca acagcacgta ccgtgtggtc agcgtcctca ccgtcctgca
ccaggactgg 1020ctgaatggca aggagtacaa gtgcaaggtc tccaacaaag
ccctcccagc ccccatcgag 1080aaaaccatct ccaaagccaa agggcagccc
cgagaaccac aggtgtacac cctgccccca 1140tcccgggagg agatgaccaa
gaaccaggtc agcctgacct gcctggtcaa aggcttctat 1200cccagcgaca
tcgccgtgga gtgggagagc aatgggcagc cggagaacaa ctacaagacc
1260acgcctcccg tgctggactc cgacggctcc ttcttcctct atagcaagct
caccgtggac 1320aagagcaggt ggcagcaggg gaacgtcttc tcatgctccg
tgatgcatga ggctctgcac 1380aaccactaca cgcagaagag cctctccctg
tctccgggta aatga 142534375DNAHomo sapiens 34cagctgcaac tgcttgagtc
gggcccaaga ctggtgaagg cttcggagac cctgtcactc 60acctgcagtg tccctatggg
ctccatcctc caaaatgatt atcattgggc ctgggtccgc 120cagcccccag
ggaggggcct ggagtggatt gggagtgttc actatagaca aaaatcctac
180tacagcccgt ccctcaagag ccgagtcttc atgtccgtag acacgtccag
agaccagttc 240tccctaaaac tcttctctct ggccgccgcg gacacggccg
tatattattg tgcgagacat 300aatcgggaag attattatga cagtaatgcc
tactttgacg agtggggcct gggagctcgg 360atcaccgtct cgagc
37535474PRTHomo sapiens 35Met Lys His Leu Trp Phe Phe Leu Leu Leu
Met Ala Ala Pro Arg Trp 1 5 10 15 Val Leu Ser Gln Leu Gln Leu Leu
Glu Ser Gly Pro Arg Leu Val Lys 20 25 30 Ala Ser Glu Thr Leu Ser
Leu Thr Cys Ser Val Pro Met Gly Ser Ile 35 40 45 Leu Gln Asn Asp
Tyr His Trp Ala Trp Val Arg Gln Pro Pro Gly Arg 50 55 60 Gly Leu
Glu Trp Ile Gly Ser Val His Tyr Arg Gln Lys Ser Tyr Tyr 65 70 75 80
Ser Pro Ser Leu Lys Ser Arg Val Phe Met Ser Val Asp Thr Ser Arg 85
90 95 Asp Gln Phe Ser Leu Lys Leu Phe Ser Leu Ala Ala Ala Asp Thr
Ala 100 105 110 Val Tyr Tyr Cys Ala Arg His Asn Arg Glu Asp Tyr Tyr
Asp Ser Asn 115 120 125 Ala Tyr Phe Asp Glu Trp Gly Leu Gly Ala Arg
Ile Thr Val Ser Ser 130 135 140 Ala Ser Thr Lys Gly Pro Ser Val Phe
Pro Leu Ala Pro Ser Ser Lys 145 150 155 160 Ser Thr Ser Gly Gly Thr
Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr 165 170 175 Phe Pro Glu Pro
Val Thr Val Ser Trp Asn Ser Gly Ala Leu Thr Ser 180 185 190 Gly Val
His Thr Phe Pro Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser 195 200 205
Leu Ser Ser Val Val Thr Val Pro Ser Ser Ser Leu Gly Thr Gln Thr 210
215 220 Tyr Ile Cys Asn Val Asn His Lys Pro Ser Asn Thr Lys Val Asp
Lys 225 230 235 240 Arg Val Glu Pro Lys Ser Cys Asp Lys Thr His Thr
Cys Pro Pro Cys 245 250 255 Pro Ala Pro Glu Leu Leu Gly Gly Pro Ser
Val Phe Leu Phe Pro Pro 260 265 270 Lys Pro Lys Asp Thr Leu Met Ile
Ser Arg Thr Pro Glu Val Thr Cys 275 280 285 Val Val Val Asp Val Ser
His Glu Asp Pro Glu Val Lys Phe Asn Trp 290 295 300 Tyr Val Asp Gly
Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu 305 310 315 320 Glu
Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu 325 330
335 His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn
340 345 350 Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala
Lys Gly 355 360 365 Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro
Ser Arg Glu Glu 370 375 380 Met Thr Lys Asn Gln Val Ser Leu Thr Cys
Leu Val Lys Gly Phe Tyr 385 390 395 400 Pro Ser Asp Ile Ala Val Glu
Trp Glu Ser Asn Gly Gln Pro Glu Asn 405 410 415 Asn Tyr Lys Thr Thr
Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe 420 425 430 Leu Tyr Ser
Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn 435 440 445 Val
Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn His Tyr Thr 450 455
460 Gln Lys Ser Leu Ser Leu Ser Pro Gly Lys 465 470 36125PRTHomo
sapiens 36Gln Leu Gln Leu Leu Glu Ser Gly Pro Arg Leu Val Lys Ala
Ser Glu 1 5 10 15 Thr Leu Ser Leu Thr Cys Ser Val Pro Met Gly Ser
Ile Leu Gln Asn 20 25 30 Asp Tyr His Trp Ala Trp Val Arg Gln Pro
Pro Gly Arg Gly Leu Glu 35 40 45 Trp Ile Gly Ser Val His Tyr Arg
Gln Lys Ser Tyr Tyr Ser Pro Ser 50 55 60 Leu Lys Ser Arg Val Phe
Met Ser Val Asp Thr Ser Arg Asp Gln Phe 65 70 75 80 Ser Leu Lys Leu
Phe Ser Leu Ala Ala Ala Asp Thr Ala Val Tyr Tyr 85 90 95 Cys Ala
Arg His Asn Arg Glu Asp Tyr Tyr Asp Ser Asn Ala Tyr Phe 100 105 110
Asp Glu Trp Gly Leu Gly Ala Arg Ile Thr Val Ser Ser 115 120 125
377PRTHomo sapiens 37Gln Asn Asp Tyr His Trp Ala 1 5 3816PRTHomo
sapiens 38Ser Val His Tyr Arg Gln Lys Ser Tyr Tyr Ser Pro Ser Leu
Lys Ser 1 5 10 15 3915PRTHomo sapiens 39His Asn Arg Glu Asp Tyr Tyr
Asp Ser Asn Ala Tyr Phe Asp Glu 1 5 10 15 408PRTHomo sapiens 40Met
Gly Ser Ile Leu Gln Asn Asp 1 5 419PRTHomo sapiens 41Ser Val His
Tyr Arg Gln Lys Ser Tyr 1 5 42702DNAHomo sapiens 42atggccagct
tccctctcct cctcggcgtc cttgcttact gcacagggtc gggggcctcc 60tatgagttgt
ctcagccacc ctcagtgtcc gtgttcccgg gacagacagc aagcatcacc
120tgttctggag atgacttgga aaacaccctt gtttgttggt atcaacaaaa
gtcagggcag 180tcccctgtgt tggtcgtcta tcaagattcc aagcggccct
cagggatccc tgagcgattc 240tctggctcca gagttaaaga cacagccact
ctgaccatca gcgggacgca ggctttcgat 300gaggctgact attattgtca
gacgtggcac aggtccaccg cccagtatgt cttcggacct 360gggaccaagg
tcaccgttct aggtcagccc aaggctgccc cctcggtcac tctgttcccg
420ccctcctctg aggagcttca agccaacaag gccacactgg tgtgtctcat
aagtgacttc 480tacccgggag ccgtgacagt ggcctggaag gcagatagca
gccccgtcaa ggcgggagtg 540gagaccacca caccctccaa acaaagcaac
aacaagtacg cggccagcag ctacctgagc 600ctgacgcctg agcagtggaa
gtcccacaaa agctacagct gccaggtcac gcatgaaggg 660agcaccgtgg
agaagacagt ggcccctaca gaatgttcat ag 70243324DNAHomo sapiens
43tcctatgagt tgtctcagcc accctcagtg tccgtgttcc cgggacagac agcaagcatc
60acctgttctg gagatgactt ggaaaacacc cttgtttgtt ggtatcaaca aaagtcaggg
120cagtcccctg tgttggtcgt ctatcaagat tccaagcggc cctcagggat
ccctgagcga 180ttctctggct ccagagttaa agacacagcc actctgacca
tcagcgggac gcaggctttc 240gatgaggctg actattattg tcagacgtgg
cacaggtcca ccgcccagta tgtcttcgga 300cctgggacca aggtcaccgt tcta
32444233PRTHomo sapiens 44Met Ala Ser Phe Pro Leu Leu Leu Gly Val
Leu Ala Tyr Cys Thr Gly 1 5 10 15 Ser Gly Ala Ser Tyr Glu Leu Ser
Gln Pro Pro Ser Val Ser Val Phe 20 25 30 Pro Gly Gln Thr Ala Ser
Ile Thr Cys Ser Gly Asp Asp Leu Glu Asn 35 40 45 Thr Leu Val Cys
Trp Tyr Gln Gln Lys Ser Gly Gln Ser Pro Val Leu 50 55 60 Val Val
Tyr Gln Asp Ser Lys Arg Pro Ser Gly Ile Pro Glu Arg Phe 65 70 75 80
Ser Gly Ser Arg Val Lys Asp Thr Ala Thr Leu Thr Ile Ser Gly Thr 85
90 95 Gln Ala Phe Asp Glu Ala Asp Tyr Tyr Cys Gln Thr Trp His Arg
Ser 100 105 110 Thr Ala Gln Tyr Val Phe Gly Pro Gly Thr Lys Val Thr
Val Leu Gly 115 120 125 Gln Pro Lys Ala Ala Pro Ser Val Thr Leu Phe
Pro Pro Ser Ser Glu 130 135 140 Glu Leu Gln Ala Asn Lys Ala Thr Leu
Val Cys Leu Ile Ser Asp Phe 145 150 155 160 Tyr Pro Gly Ala Val Thr
Val Ala Trp Lys Ala Asp Ser Ser Pro Val 165 170 175 Lys Ala Gly Val
Glu Thr Thr Thr Pro Ser Lys Gln Ser Asn Asn Lys 180 185 190 Tyr Ala
Ala Ser Ser Tyr Leu Ser Leu Thr Pro Glu Gln Trp Lys Ser 195 200 205
His Lys Ser Tyr Ser Cys Gln Val Thr His Glu Gly Ser Thr Val Glu 210
215 220 Lys Thr Val Ala Pro Thr Glu Cys Ser 225 230 45108PRTHomo
sapiens 45Ser Tyr Glu Leu Ser Gln Pro Pro Ser Val Ser Val Phe Pro
Gly Gln 1 5 10 15 Thr Ala Ser Ile Thr Cys Ser Gly Asp Asp Leu Glu
Asn Thr Leu Val 20 25 30 Cys Trp Tyr Gln Gln Lys Ser Gly Gln Ser
Pro Val Leu Val Val Tyr 35 40 45 Gln Asp Ser Lys Arg Pro Ser Gly
Ile Pro Glu Arg Phe Ser Gly Ser 50 55 60 Arg Val Lys Asp Thr Ala
Thr Leu Thr Ile Ser Gly Thr Gln Ala Phe 65 70 75 80 Asp Glu Ala Asp
Tyr Tyr Cys Gln Thr Trp His Arg Ser Thr Ala Gln 85 90 95 Tyr Val
Phe Gly Pro Gly Thr Lys Val Thr Val Leu 100 105 4611PRTHomo sapiens
46Ser Gly Asp Asp Leu Glu Asn Thr Leu Val Cys 1 5 10 477PRTHomo
sapiens 47Gln Asp Ser Lys Arg Pro Ser 1 5 4811PRTHomo sapiens 48Gln
Thr Trp His Arg Ser Thr Ala Gln Tyr Val 1 5 10 491425DNAHomo
sapiens 49atgaaacacc tgtggttctt cctcctgctg gtggcggctc ccagatgggt
cctgtcccag 60ttgcagctgc
ttgagtcggg cccaggactg gtgaagcctt cggagaccct ttcactcacc
120tgcagtgtct ctggggactc cctcctcagt aatgatcaat actgggcctg
ggtccgccag 180cccccaggga ggggcctgga gtggattggg agtgttcact
atagacgacg aaactactac 240agcccgtccc tggagagccg gatcttcatg
tcagtagaca cgtccagaaa cgagttctcc 300ttaaaagttt tctctgtgac
ggccgcggac acggccgtgt attattgtgc gagacacaat 360tgggaagatt
attatgagag taatgcctac tttgactact ggggcctggg aacccggatc
420accgtctcga gcgcctccac caagggccca tcggtcttcc ccctggcacc
ctcctccaag 480agcacctctg ggggcacagc ggccctgggc tgcctggtca
aggactactt ccccgaaccg 540gtgacggtgt cgtggaactc aggcgccctg
accagcggcg tgcacacctt cccggctgtc 600ctacagtcct caggactcta
ctccctcagc agcgtggtga ccgtgccctc cagcagcttg 660ggcacccaga
cctacatctg caacgtgaat cacaagccca gcaacaccaa ggtggacaag
720agagttgagc ccaaatcttg tgacaaaact cacacatgcc caccgtgccc
agcacctgaa 780ctcctggggg gaccgtcagt cttcctcttc cccccaaaac
ccaaggacac cctcatgatc 840tcccggaccc ctgaggtcac atgcgtggtg
gtggacgtga gccacgaaga ccctgaggtc 900aagttcaact ggtacgtgga
cggcgtggag gtgcataatg ccaagacaaa gccgcgggag 960gagcagtaca
acagcacgta ccgtgtggtc agcgtcctca ccgtcctgca ccaggactgg
1020ctgaatggca aggagtacaa gtgcaaggtc tccaacaaag ccctcccagc
ccccatcgag 1080aaaaccatct ccaaagccaa agggcagccc cgagaaccac
aggtgtacac cctgccccca 1140tcccgggagg agatgaccaa gaaccaggtc
agcctgacct gcctggtcaa aggcttctat 1200cccagcgaca tcgccgtgga
gtgggagagc aatgggcagc cggagaacaa ctacaagacc 1260acgcctcccg
tgctggactc cgacggctcc ttcttcctct atagcaagct caccgtggac
1320aagagcaggt ggcagcaggg gaacgtcttc tcatgctccg tgatgcatga
ggctctgcac 1380aaccactaca cgcagaagag cctctccctg tctccgggta aatga
142550375DNAHomo sapiens 50cagttgcagc tgcttgagtc gggcccagga
ctggtgaagc cttcggagac cctttcactc 60acctgcagtg tctctgggga ctccctcctc
agtaatgatc aatactgggc ctgggtccgc 120cagcccccag ggaggggcct
ggagtggatt gggagtgttc actatagacg acgaaactac 180tacagcccgt
ccctggagag ccggatcttc atgtcagtag acacgtccag aaacgagttc
240tccttaaaag ttttctctgt gacggccgcg gacacggccg tgtattattg
tgcgagacac 300aattgggaag attattatga gagtaatgcc tactttgact
actggggcct gggaacccgg 360atcaccgtct cgagc 37551474PRTHomo sapiens
51Met Lys His Leu Trp Phe Phe Leu Leu Leu Val Ala Ala Pro Arg Trp 1
5 10 15 Val Leu Ser Gln Leu Gln Leu Leu Glu Ser Gly Pro Gly Leu Val
Lys 20 25 30 Pro Ser Glu Thr Leu Ser Leu Thr Cys Ser Val Ser Gly
Asp Ser Leu 35 40 45 Leu Ser Asn Asp Gln Tyr Trp Ala Trp Val Arg
Gln Pro Pro Gly Arg 50 55 60 Gly Leu Glu Trp Ile Gly Ser Val His
Tyr Arg Arg Arg Asn Tyr Tyr 65 70 75 80 Ser Pro Ser Leu Glu Ser Arg
Ile Phe Met Ser Val Asp Thr Ser Arg 85 90 95 Asn Glu Phe Ser Leu
Lys Val Phe Ser Val Thr Ala Ala Asp Thr Ala 100 105 110 Val Tyr Tyr
Cys Ala Arg His Asn Trp Glu Asp Tyr Tyr Glu Ser Asn 115 120 125 Ala
Tyr Phe Asp Tyr Trp Gly Leu Gly Thr Arg Ile Thr Val Ser Ser 130 135
140 Ala Ser Thr Lys Gly Pro Ser Val Phe Pro Leu Ala Pro Ser Ser Lys
145 150 155 160 Ser Thr Ser Gly Gly Thr Ala Ala Leu Gly Cys Leu Val
Lys Asp Tyr 165 170 175 Phe Pro Glu Pro Val Thr Val Ser Trp Asn Ser
Gly Ala Leu Thr Ser 180 185 190 Gly Val His Thr Phe Pro Ala Val Leu
Gln Ser Ser Gly Leu Tyr Ser 195 200 205 Leu Ser Ser Val Val Thr Val
Pro Ser Ser Ser Leu Gly Thr Gln Thr 210 215 220 Tyr Ile Cys Asn Val
Asn His Lys Pro Ser Asn Thr Lys Val Asp Lys 225 230 235 240 Arg Val
Glu Pro Lys Ser Cys Asp Lys Thr His Thr Cys Pro Pro Cys 245 250 255
Pro Ala Pro Glu Leu Leu Gly Gly Pro Ser Val Phe Leu Phe Pro Pro 260
265 270 Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr
Cys 275 280 285 Val Val Val Asp Val Ser His Glu Asp Pro Glu Val Lys
Phe Asn Trp 290 295 300 Tyr Val Asp Gly Val Glu Val His Asn Ala Lys
Thr Lys Pro Arg Glu 305 310 315 320 Glu Gln Tyr Asn Ser Thr Tyr Arg
Val Val Ser Val Leu Thr Val Leu 325 330 335 His Gln Asp Trp Leu Asn
Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn 340 345 350 Lys Ala Leu Pro
Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly 355 360 365 Gln Pro
Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg Glu Glu 370 375 380
Met Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr 385
390 395 400 Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro
Glu Asn 405 410 415 Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp
Gly Ser Phe Phe 420 425 430 Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser
Arg Trp Gln Gln Gly Asn 435 440 445 Val Phe Ser Cys Ser Val Met His
Glu Ala Leu His Asn His Tyr Thr 450 455 460 Gln Lys Ser Leu Ser Leu
Ser Pro Gly Lys 465 470 52125PRTHomo sapiens 52Gln Leu Gln Leu Leu
Glu Ser Gly Pro Gly Leu Val Lys Pro Ser Glu 1 5 10 15 Thr Leu Ser
Leu Thr Cys Ser Val Ser Gly Asp Ser Leu Leu Ser Asn 20 25 30 Asp
Gln Tyr Trp Ala Trp Val Arg Gln Pro Pro Gly Arg Gly Leu Glu 35 40
45 Trp Ile Gly Ser Val His Tyr Arg Arg Arg Asn Tyr Tyr Ser Pro Ser
50 55 60 Leu Glu Ser Arg Ile Phe Met Ser Val Asp Thr Ser Arg Asn
Glu Phe 65 70 75 80 Ser Leu Lys Val Phe Ser Val Thr Ala Ala Asp Thr
Ala Val Tyr Tyr 85 90 95 Cys Ala Arg His Asn Trp Glu Asp Tyr Tyr
Glu Ser Asn Ala Tyr Phe 100 105 110 Asp Tyr Trp Gly Leu Gly Thr Arg
Ile Thr Val Ser Ser 115 120 125 537PRTHomo sapiens 53Ser Asn Asp
Gln Tyr Trp Ala 1 5 5416PRTHomo sapiens 54Ser Val His Tyr Arg Arg
Arg Asn Tyr Tyr Ser Pro Ser Leu Glu Ser 1 5 10 15 5515PRTHomo
sapiens 55His Asn Trp Glu Asp Tyr Tyr Glu Ser Asn Ala Tyr Phe Asp
Tyr 1 5 10 15 568PRTHomo sapiens 56Gly Asp Ser Leu Leu Ser Asn Asp
1 5 579PRTHomo sapiens 57Ser Val His Tyr Arg Arg Arg Asn Tyr 1 5
58702DNAHomo sapiens 58atggccagct tccctctctt cctcggcgtc cttgcttact
gcacaggatc gggggcctcc 60tttgacttga ctcagccacc ctcagtgtcc gtgtccccag
gacagaccgc aaccatcacc 120tgttctggag atcaattgga aaataccttt
gtttgctggt atcaacagag gtcaggccag 180gcccctgtgt tggtcatcta
tcaaggttcc aagcggccct cagggatccc tgagcgattc 240tctggctcca
ggtctgggaa cacagccact ctgaccatca gcaggaccca ggctttggat
300gaggctgact attactgtca ggcgtgggac aggtccaccg cccactatgt
cttcggacct 360gggaccaagg tcaccgttct aggtcagccc aaggctgccc
cctcggtcac tctgttcccg 420ccctcctctg aggagcttca agccaacaag
gccacactgg tgtgtctcat aagtgacttc 480tacccgggag ccgtgacagt
ggcctggaag gcagatagca gccccgtcaa ggcgggagtg 540gagaccacca
caccctccaa acaaagcaac aacaagtacg cggccagcag ctacctgagc
600ctgacgcctg agcagtggaa gtcccacaaa agctacagct gccaggtcac
gcatgaaggg 660agcaccgtgg agaagacagt ggcccctaca gaatgttcat ag
70259324DNAHomo sapiens 59tcctttgact tgactcagcc accctcagtg
tccgtgtccc caggacagac cgcaaccatc 60acctgttctg gagatcaatt ggaaaatacc
tttgtttgct ggtatcaaca gaggtcaggc 120caggcccctg tgttggtcat
ctatcaaggt tccaagcggc cctcagggat ccctgagcga 180ttctctggct
ccaggtctgg gaacacagcc actctgacca tcagcaggac ccaggctttg
240gatgaggctg actattactg tcaggcgtgg gacaggtcca ccgcccacta
tgtcttcgga 300cctgggacca aggtcaccgt tcta 32460233PRTHomo sapiens
60Met Ala Ser Phe Pro Leu Phe Leu Gly Val Leu Ala Tyr Cys Thr Gly 1
5 10 15 Ser Gly Ala Ser Phe Asp Leu Thr Gln Pro Pro Ser Val Ser Val
Ser 20 25 30 Pro Gly Gln Thr Ala Thr Ile Thr Cys Ser Gly Asp Gln
Leu Glu Asn 35 40 45 Thr Phe Val Cys Trp Tyr Gln Gln Arg Ser Gly
Gln Ala Pro Val Leu 50 55 60 Val Ile Tyr Gln Gly Ser Lys Arg Pro
Ser Gly Ile Pro Glu Arg Phe 65 70 75 80 Ser Gly Ser Arg Ser Gly Asn
Thr Ala Thr Leu Thr Ile Ser Arg Thr 85 90 95 Gln Ala Leu Asp Glu
Ala Asp Tyr Tyr Cys Gln Ala Trp Asp Arg Ser 100 105 110 Thr Ala His
Tyr Val Phe Gly Pro Gly Thr Lys Val Thr Val Leu Gly 115 120 125 Gln
Pro Lys Ala Ala Pro Ser Val Thr Leu Phe Pro Pro Ser Ser Glu 130 135
140 Glu Leu Gln Ala Asn Lys Ala Thr Leu Val Cys Leu Ile Ser Asp Phe
145 150 155 160 Tyr Pro Gly Ala Val Thr Val Ala Trp Lys Ala Asp Ser
Ser Pro Val 165 170 175 Lys Ala Gly Val Glu Thr Thr Thr Pro Ser Lys
Gln Ser Asn Asn Lys 180 185 190 Tyr Ala Ala Ser Ser Tyr Leu Ser Leu
Thr Pro Glu Gln Trp Lys Ser 195 200 205 His Lys Ser Tyr Ser Cys Gln
Val Thr His Glu Gly Ser Thr Val Glu 210 215 220 Lys Thr Val Ala Pro
Thr Glu Cys Ser 225 230 61108PRTHomo sapiens 61Ser Phe Asp Leu Thr
Gln Pro Pro Ser Val Ser Val Ser Pro Gly Gln 1 5 10 15 Thr Ala Thr
Ile Thr Cys Ser Gly Asp Gln Leu Glu Asn Thr Phe Val 20 25 30 Cys
Trp Tyr Gln Gln Arg Ser Gly Gln Ala Pro Val Leu Val Ile Tyr 35 40
45 Gln Gly Ser Lys Arg Pro Ser Gly Ile Pro Glu Arg Phe Ser Gly Ser
50 55 60 Arg Ser Gly Asn Thr Ala Thr Leu Thr Ile Ser Arg Thr Gln
Ala Leu 65 70 75 80 Asp Glu Ala Asp Tyr Tyr Cys Gln Ala Trp Asp Arg
Ser Thr Ala His 85 90 95 Tyr Val Phe Gly Pro Gly Thr Lys Val Thr
Val Leu 100 105 6211PRTHomo sapiens 62Ser Gly Asp Gln Leu Glu Asn
Thr Phe Val Cys 1 5 10 637PRTHomo sapiens 63Gln Gly Ser Lys Arg Pro
Ser 1 5 6411PRTHomo sapiens 64Gln Ala Trp Asp Arg Ser Thr Ala His
Tyr Val 1 5 10
* * * * *
References