U.S. patent application number 15/846259 was filed with the patent office on 2018-07-19 for in vivo imaging using anti-il-6 antibodies.
The applicant listed for this patent is ALDERBIO HOLDINGS LLC. Invention is credited to Katie ANDERSON, Anne Elisabeth CARVALHO JENSEN, Benjamin H. DUTZAR, Leon F. GARCIA-MARTINEZ, Brian R. KOVACEVICH, John A. LATHAM, Ethan W. OJALA, Jeffrey T.L. SMITH.
Application Number | 20180201668 15/846259 |
Document ID | / |
Family ID | 42196491 |
Filed Date | 2018-07-19 |
United States Patent
Application |
20180201668 |
Kind Code |
A1 |
GARCIA-MARTINEZ; Leon F. ;
et al. |
July 19, 2018 |
In vivo Imaging using Anti-IL-6 Antibodies
Abstract
The present invention is directed to therapeutic methods using
IL-6 antagonists such as an Ab 1 antibody or antibody fragment
having binding specificity for IL-6 to prevent or treat disease or
to improve survivability or quality of life of a patient in need
thereof. In preferred embodiments these patients will comprise
those exhibiting (or at risk of developing) an elevated serum
C-reactive protein level, reduced serum albumin level, elevated
D-dimer or other cogulation cascade related protein(s), cachexia,
fever, weakness and/or fatigue prior to treatment. The subject
therapies also may include the administration of other actives such
as chemotherapeutics, anti-coagulants, statins, and others.
Inventors: |
GARCIA-MARTINEZ; Leon F.;
(Woodinville, WA) ; CARVALHO JENSEN; Anne Elisabeth;
(Wenatchee, WA) ; ANDERSON; Katie; (Kirkland,
WA) ; DUTZAR; Benjamin H.; (Seattle, WA) ;
OJALA; Ethan W.; (Snohomish, WA) ; KOVACEVICH; Brian
R.; (Snohomish, WA) ; LATHAM; John A.;
(Seattle, WA) ; SMITH; Jeffrey T.L.; (Bellevue,
WA) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
ALDERBIO HOLDINGS LLC |
Las Vegas |
NV |
US |
|
|
Family ID: |
42196491 |
Appl. No.: |
15/846259 |
Filed: |
December 19, 2017 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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14725435 |
May 29, 2015 |
9879074 |
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15846259 |
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13682214 |
Nov 20, 2012 |
9085615 |
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14725435 |
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12502581 |
Jul 14, 2009 |
8323649 |
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13682214 |
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61117839 |
Nov 25, 2008 |
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61117811 |
Nov 25, 2008 |
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61117861 |
Nov 25, 2008 |
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Current U.S.
Class: |
1/1 |
Current CPC
Class: |
C07K 16/248 20130101;
C07K 2317/56 20130101; C07K 2317/569 20130101; C07K 2317/92
20130101; C07K 2317/41 20130101; C07K 2317/34 20130101; C07K
2317/94 20130101; A61K 2039/505 20130101; C07K 2317/622 20130101;
A61K 2039/545 20130101; A61P 37/02 20180101; A61K 45/06 20130101;
C07K 2317/55 20130101; C07K 2317/76 20130101; Y02A 50/30 20180101;
A61K 39/39533 20130101; C07K 2317/24 20130101; C07K 16/18 20130101;
A61K 2039/54 20130101; C07K 2317/21 20130101; Y02A 50/412 20180101;
C07K 2317/54 20130101 |
International
Class: |
C07K 16/18 20060101
C07K016/18; A61K 45/06 20060101 A61K045/06; A61K 39/395 20060101
A61K039/395; C07K 16/24 20060101 C07K016/24 |
Claims
1-203. (canceled)
204. A method of in vivo imaging which detects the presence of
cells which express IL-6 comprising administering an effective
amount of at least one anti-IL-6 antibody or antibody fragment,
wherein said wherein the antibody or antibody fragment comprises: a
variable light chain polypeptide comprising: a polypeptide having
at least 90% identity to SEQ ID NO: 709, and a variable heavy chain
polypeptide comprising: a polypeptide having at least 90% identity
to SEQ ID NO: 657, further wherein said anti-IL-6 antibody or
antibody fragment comprises the variable light chain
complementarity determining region (CDR) polypeptides of SEQ ID
NO:4, 5 and 6 and comprises the variable heavy chain
complementarity determining region (CDR) polypeptides of SEQ ID
NO:7, 8 or 120, and 9, and wherein said administration further
includes the administration of a radionuclide or fluorophore that
facilitates detection of the antibody at IL-6 expressing disease
sites.
205. The method of claim 204, wherein said in vivo imaging is used
to detect IL-6 expressing tumors or metastases in a subject with
cancer or metastasis.
206. The method of claim 204, wherein said in vivo imaging is used
to detect IL-6 expressing cells at sites of pathology in a subject
with a chronic autoimmune disorder associated with IL-6 expressing
cells.
207. The method of claim 204, wherein said in vivo imaging is used
to detect IL-6 expressing cells at sites of pathology in a subject
with a chronic inflammatory disorder associated with IL-6
expressing cells.
208. The method of claim 204, wherein said in vivo imaging is used
to facilitate design of an appropriate therapeutic regimen.
209. The method of claim 208, wherein said therapeutic regimen
comprises radiotherapy, chemotherapy or a combination thereof.
210. The method of claim 204, wherein the light chain possesses at
least 95% sequence identity to the light chain polypeptide
comprised in SEQ ID NO: 709.
211. The method of claim 204, wherein the heavy chain possesses at
least 95% sequence identity to the heavy chain polypeptide
comprised in SEQ ID NO: 657.
212. The method of claim 204, wherein the light chain possesses at
least 97% sequence identity to the light chain polypeptide
comprised in SEQ ID NO: 709.
213. The method of claim 204, wherein the heavy chain possesses at
least 97% sequence identity to the heavy chain polypeptide
comprised in SEQ ID NO: 657.
214. The method of claim 204, wherein the light chain possesses at
least 99% sequence identity to the light chain polypeptide
comprised in SEQ ID NO: 709.
215. The method of claim 204, wherein the heavy chain possesses at
least 99% sequence identity to the heavy chain polypeptide
comprised in SEQ ID NO: 657.
216. The method of claim 204, wherein the light chain possesses
100% sequence identity to the light chain polypeptide comprised in
SEQ ID NO: 709.
217. The method of claim 204, wherein the heavy chain possesses
100% sequence identity to the heavy chain polypeptide comprised in
SEQ ID NO: 657.
218. The method of claim 204, wherein said antibody comprises light
chain CDR1, CDR2 and CDR3 polypeptides and heavy chain CDR1, CDR2
and CDR3 identical to those on SEQ ID NO:4, 5 and 6 and SEQ ID
NO:7, 120 and 9 respectively.
219. The method of claim 204, wherein said antibody comprises light
chain CDR1, CDR2 and CDR3 polypeptides and heavy chain CDR1, CDR2
and CDR3 polypeptides identical to those on SEQ ID NO:4, 5 and 6
and SEQ ID NO:7, 8 or 120 and 9 respectively.
220. The method of claim 204 wherein the antibody is selected from
a human, humanized, single chain, or chimeric antibody and the
antibody fragment is selected from a Fab, Fab', F(ab').sub.2, Fv,
or scFv.
Description
RELATED APPLICATION DISCLOSURE
[0001] This application claims the benefit of U.S. Provisional
Applications Ser. Nos. 61/117,861, 61/117,839, and 61/117,811, each
filed Nov. 25, 2008, the disclosure of each of which is hereby
incorporated by reference in its entirety.
[0002] This application is also related to U.S. Ser. No. 12/391,717
filed Feb. 24, 2009, U.S. Ser. No. 12/366,567 filed Feb. 5, 2009,
U.S. Ser. No. 12/323,147 filed Nov. 25, 2008, U.S. Ser. No.
12/323,194 filed Nov. 25, 2008, U.S. Ser. No. 12/323,066 filed Nov.
25, 2008, U.S. Ser. No. 12/153,612 filed May 21, 2008, U.S. Ser.
No. 60/924,550 filed May 21, 2007, U.S. Ser. No. 12/153,611 filed
May 21, 2008, U.S. Ser. No. 60/924,551 filed May 21, 2007, and U.S.
Ser. No. 12/124,723 filed May 21, 2008, the disclosure of each of
which is hereby incorporated by reference in its entirety.
[0003] The sequence listing in the file named "67858o700601.txt"
having a size of 331,787 bytes that was created Jul. 7, 2009 is
hereby incorporated by reference in its entirety.
BACKGROUND
1. Field of the Art
[0004] This invention is an extension of Applicants' prior
invention disclosed in the above-referenced patent applications
relating to novel anti-IL-6 antibodies, novel therapies and
therapeutic protocols utilizing anti-IL-6 antibodies, and
pharmaceutical formulations containing anti-IL-6 antibodies. In
preferred embodiments, an anti-IL-6 antibody is Ab1, which includes
rabbit or humanized forms thereof, as well as heavy chains, light
chains, fragments, variants, and CDRs thereof, or an antibody or
antibody fragment that specifically binds to the same linear or
conformational epitope(s) on an intact human IL-6 polypeptide
fragment thereof as Ab1. Preferably, the anti-IL-6 antibody is an
IL-6 antagonist. In preferred embodiments, the anti-IL-6 antibody
has an in vivo half-life of at least about 25 days, an in vivo
effect of raising albumin, has an in vivo effect of lowering
C-reactive protein, has an in vivo effect of restoring a normal
coagulation profile, possesses a binding affinity (Kd) for IL-6 of
less than about 50 picomolar, and/or has a rate of dissociation
(K.sub.off) from IL-6 of less than or equal to 10.sup.-4
S.sup.-1.
[0005] The invention also pertains to methods of screening for
diseases and disorders associated with IL-6, and methods of
preventing or treating diseases or disorders associated with IL-6
by administering said antibody or a fragment or a variant
thereof.
[0006] In one aspect, this invention pertains to methods of
improving survivability or quality of life of a patient in need
thereof, comprising administering to the patient an anti-IL-6
antibody, such as Ab1 or a fragment or variant thereof, whereby the
patient's C-reactive protein ("CRP") level is lowered, and/or the
patient's albumin level is raised, and optionally monitoring the
patient to determine the patient's CRP and/or albumin level.
[0007] In another aspect, this invention relates to methods of
lowering the C-reactive protein level in a patient in need thereof,
comprising administering to the patient an IL-6 antagonist such as
Ab1, whereby the patient's CRP level is lowered, and monitoring the
patient to assess the CRP level. In another aspect, this invention
relates to methods of raising the albumin level in a patient in
need thereof, comprising administering to the patient an IL-6
antagonist such as Ab1, whereby the patient's serum albumin level
is raised, and monitoring the patient to assess the albumin
level.
[0008] In another aspect, this invention pertains to methods of
preventing or treating cachexia, weakness, fatigue, and/or fever in
a patient in need thereof, e.g., a patient showing elevated CRP
levels, comprising administering to the patient an anti-IL-6
antibody or antibody fragment or variant thereof, whereby the
patient's cachexia, weakness, fatigue, and/or fever is improved or
restored to a normal condition, and optionally monitoring the
patient to assess cachexia, weakness, fatigue, and/or fever.
[0009] In another embodiment, this invention pertains to methods of
preventing or treating thrombosis in a patient in a state of
hypercoagulation, comprising administering to the patient an
anti-IL-6 antibody, such as Ab1 or a fragment or variant thereof,
whereby the patient's coagulation profile is improved or restored
to a normal condition, and optionally monitoring the patient to
assess coagulation profile.
[0010] In another aspect the invention provides novel
pharmaceutical compositions and their use in novel combination
therapies and comprising administration of an anti-IL-6 antibody,
such as Ab1 or a fragment or variant thereof, and at least one
other therapeutic compound such as a statin, anti-coagulant,
anti-emetic, anti-nausea agent, anti-cachexia agent, chemotherapy
agent, anti-cytokine agent, etc.
2. Description of Related Art
[0011] Weight loss, fatigue, and muscular weakness are very common
symptoms of patients with advanced forms of cancer, and these
symptoms can worsen as the cancer continues to progress. Fatigue,
weight loss and muscular weakness can have significant negative
effects on the recovery of patients with advanced forms of cancer,
for example by disrupting lifestyles and relationships and
affecting the willingness or ability of patients to continue cancer
treatments. Known methods of addressing fatigue, weight loss and
muscular weakness include regular routines of fitness and exercise,
methods of conserving the patient's energy, and treatments that
address anemia-induced fatigue and muscular weakness. Nevertheless,
there remains a need in the art for methods and/or treatments that
improve fatigue, weight loss and muscular weakness in cancer
patients.
[0012] Thrombosis is a significant cause of mortality in cancer
patients. Bick, N Engl J Med 349:109-111 (2003). For example,
serious, life-threatening thrombotic events occur in approximately
6% of lung cancer patients. Alguire et al., J Clin Oncol 2004 Vol
22 (July 15th Supplement) No. 14S: 8082. Cancer patients often
exhibit hypercoagulation, in which the coagulation system has an
increased clotting tendency. Rickles and Edwards, Blood 62:14-31
(1983). Markers of hypercoagulation correlate with poor patient
outcome for at least some cancers. Bick, Semin Thromb Hemostat
18:353-372 (1992); Buccheri et al., Cancer 97:3044-3052 (2003);
Wojtukiewicz, Blood Coagul Fibrinolysis 3:429-437 (1992). Causes of
hypercoagulation include the cancer itself and the cancer
treatments (e.g., chemotherapy). Hypercoagulation results in an
increased risk of thrombitic events, which can be further
exacerbated when patients become bed-ridden. When not
contraindicated, anticoagulant therapy has conferred survival
benefit in some cancers. Lebeau et al., Cancer 74:38-45 (1994);
Chahinian et al., J Clin Oncol 7:993-1002 (1989). However,
therapeutic options are often limited because many cancer patients
are at an elevated risk of major bleeding, precluding
administration of anticoagulants that could otherwise be given
prophylactically to reduce the risk of thrombosis. In summary, the
available methods for prevention of thrombosis in cancer patients
are unsatisfactory, and thus there is a need for new therapies.
Such therapies would enhance cancer patient survival and promote
better quality of life.
[0013] Thrombosis can also be a significant cause of adverse events
and mortality in other patient groups, including those with chronic
illness or chronic inflammation, surgical patients, bed-ridden
individuals, and orthopedic patients. When they are not otherwise
contraindicated, preventative methods include calf compression and
anticoagulants (e.g. low molecular weight heparin). These
preventative methods can reduce--but not eliminate--the risk of
thrombosis. Because these preventative methods are not always
effective and are contraindicated for some patients, and because
anticoagulants can cause potentially lethal side-effects such as
major bleeding, there is a need for alternative methods to prevent
thrombosis in these patients. Such methods should improve patient
outcomes.
[0014] Interleukin-6 (hereinafter "IL-6") (also known as
interferon-.beta..sub.2; B-cell differentiation factor; B-cell
stimulatory factor-2; hepatocyte stimulatory factor; hybridoma
growth factor; and plasmacytoma growth factor) is a multifunctional
cytokine involved in numerous biological processes such as the
regulation of the acute inflammatory response, the modulation of
specific immune responses including B- and T-cell differentiation,
bone metabolism, thrombopoiesis, epidermal proliferation, menses,
neuronal cell differentiation, neuroprotection, aging, cancer, and
the inflammatory reaction occurring in Alzheimer's disease. See A.
Papassotiropoulos et al, Neurobiology of Aging, 22:863-871
(2001).
[0015] IL-6 is a member of a family of cytokines that promote
cellular responses through a receptor complex consisting of at
least one subunit of the signal-transducing glycoprotein gp130 and
the IL-6 receptor ("IL-6R") (also known as gp80). The IL-6R may
also be present in a soluble form ("sIL-6R"). IL-6 binds to IL-6R,
which then dimerizes the signal-transducing receptor gp130. See
Jones, S A, J. Immunology, 175:3463-3468 (2005).
[0016] In humans, the gene encoding IL-6 is organized in five exons
and four introns, and maps to the short arm of chromosome 7 at
7p21. Translation of IL-6 RNA and post-translational processing
result in the formation of a 21 to 28 kDa protein with 184 amino
acids in its mature form. See A. Papassotiropoulos, et al,
Neurobiology of Aging, 22:863-871 (2001).
[0017] As set forth in greater detail herein IL-6 is believed to
play a role in the development of a multitude of diseases and
disorders, including but not limited to fatigue, cachexia,
autoimmune diseases, diseases of the skeletal system, cancer, heart
disease, obesity, diabetes, asthma, alzheimer's disease and
multiple sclerosis. Due to the perceived involvement of IL-6 in a
wide range of diseases and disorders, there remains a need in the
art for compositions and methods useful for preventing or treating
diseases associated with IL-6, as well as methods of screening to
identify patients having diseases or disorders associated with
IL-6. Particularly preferred anti-IL-6 compositions are those
having minimal or minimizing adverse reactions when administered to
the patient. Compositions or methods that reduce or inhibit
diseases or disorders associated with IL-6 are beneficial to the
patient in need thereof.
[0018] The function of IL-6 is not restricted to the immune
response as it acts in hematopoiesis, thrombopoiesis, osteoclast
formation, elicitation of hepatic acute phase response resulting in
the elevation of C-reactive protein (CRP) and serum amyloid A (SAA)
protein. It is known to be a growth factor for epidermal
keratinocytes, renal mesangial cells, myeloma and plasmacytoma
cells (Grossman et al., 1989 Prot Natl Acad Sci., 86, (16)
6367-6371; Horii et al., 1989, J Immunol, 143, 12, 3949-3955;
Kawano et al., 1988, Nature 332, 6159, 83-85). IL-6 is produced by
a wide range of cell types including monocytes/macrophages,
fibroblasts, epidermal keratinocytes, vascular endothelial cells,
renal messangial cells, glial cells, condrocytes, T and B-cells and
some tumour cells (Akira et al, 1990, FASEB J., 4, 11, 2860-2867).
Except for tumour cells that constitutively produce IL-6, normal
cells do not express IL-6 unless appropriately stimulated.
[0019] Elevated IL-6 levels have been observed in many types of
cancer, including breast cancer, leukemia, ovarian cancer, prostate
cancer, pancreatic cancer, lymphoma, lung cancer, renal cell
carcinoma, colorectal cancer, and multiple myeloma (e.g., Chopra et
al., 2004, MJAFI 60:45-49; Songur et al., 2004, Tumori 90:196-200;
Blay et al., 1992, Cancer Research 52:3317-3322; Nikiteas et al.,
2005, World J. Gasterenterol. 11:1639-1643; reviewed in Heikkila et
al., 2008, Eur J Cancer, 44:937-945). As noted above, IL-6 is known
or suspected to play a role in promoting proliferation or survival
of at least some types of cancer. Moreover, some of these studies
have demonstrated correlation between IL-6 levels and patient
oucome. Together, these results suggest the possibility that
inhibition of IL-6 can be therapeutically beneficial. Indeed,
clinical studies (reviewed in Trikha et al., 2003, Clinical Cancer
Research 9:4653-4665) have shown some improvement in patient
outcomes due to administration of various anti-IL-6 antibodies,
particularly in those cancers in which IL-6 plays a direct role
promoting cancer cell proliferation or survival.
[0020] As noted above, IL-6 stimulates the hepatic acute phase
response, resulting in increased production of CRP and elevated
serum CRP levels. For this reason, C-reactive protein (CRP) has
been reported to comprise a surrogate marker of IL-6 activity.
Thus, elevated IL-6 activity can be detected through measurement of
serum CRP. Conversely, effective suppression of IL-6 activity,
e.g., through administration of a neutralizing anti-IL-6 antibody,
can be detected by the resulting decrease in serum CRP levels.
[0021] A recent clinical trial demonstrated that administration of
rosuvastatin to apparently healthy individuals having elevated CRP
(greater than 2.0 mg/1) reduced their CRP levels by 37% and greatly
decreased the incidence of myocardial infarction, stroke, arterial
revascularization, hospitalization for unstable angina, or death
from cardiovascular causes. Ridker et al., N Engl J Med. 2008 Nov.
9 [Epub ahead of print].
[0022] In addition to its direct role in pathogenesis of some
cancers and other diseases, chronically elevated IL-6 levels appear
to adversely affect patient well-being and quality of life. For
example, elevated IL-6 levels have been reported to be associated
with cachexia and fever, and reduced serum albumin. Gauldie et al.,
1987, PNAS 84:7251-7253; Heinric et al., 1990, 265:621-636; Zamir
et al., 1993, Metabolism 42:204-208; Zamir et al., 1992, Arch Surg,
127:170-174. Inhibition of IL-6 by a neutralizing antibody has been
reported to ameliorate fever and cachexia in cancer patients,
though improvement in these patients' serum albumin level has not
been reported (Emille et al., 1994, Blood, 84:2472-2479; Blay et
al., 1992, Cancer Research 52:3317-3322; Bataille et al., 1995,
Blood, 86: 685-691).
[0023] Numerous studies have suggested that CRP is a valuable
prognostic factor in cancer patients, with elevated CRP levels
predicting poor outcome. See, e.g., Hefler et al, Clin Cancer Res,
2008 Feb. 1; 14(3):710-4; Nagaoka et al, Liver Int, 2007 October;
27(8):1091-7; Heikkila et al, J Epidemiol Community Health, 2007
September; 61(9):824-33, Review; Hara et al, Anticancer Res, 2007
July-August; 27(4C):3001-4; Polterauer et al, Gynecol Oncol, 2007
October; 107(1):114-7, Epub 2007 Jul. 6; Tingstedt et al, Scand J
Gastroenterol, 2007 June; 42(6):754-9; Suh et al, Support Care
Cancer, 2007 June; 15(6):613-20, Epub 2007 Jan. 18; Gerhardt et al,
World J Gastroenterol, 2006 Sep. 14; 12(34):5495-500; McArdle et
al, Urol Int, 2006; 77(2):127-9; Guillem et al, Dis Esophagus,
2005; 18(3):146-50; Brown et al, Cancer, 2005 Jan. 15;
103(2):377-82. Decreased serum albumin (hypoalbuminemia) is also
associated with increased morbidity and mortality in many critical
illnesses, including cancers (e.g., Vigano et al., Arch Intern Med,
2000 Mar. 27; 160(6):861-8; Hauser et al., Support Care Cancer,
2006 October; 14(10):999-1011; Seve et al., Cancer, 2006 Dec. 1;
107(11):2698-705). The apparent link between hypoalbuminemia and
poor patient oucome might suggest that restoring albumin levels
through direct albumin infusion could promote patient survival,
however, albumin infusion alone has not improved survival of
patients with advanced cancer (Demirkazik et al., Proc Am Soc Clin
Oncol 21: 2002 (abstr 2892)) or other critically ill patients
groups (reviewed in Wilkes et al., Ann Intern Med, 2001 Aug. 7;
135(3):149-64).
[0024] The Glasgow Prognostic Score (GPS) is an inflammation-based
prognostic score that combines levels of albumin (<35 mg/L=1
point) and CRP (>10 mg/L=1 point) (Forrest et al., Br J Cancer,
2004 May 4; 90(9):1704-6). Since its introduction in 2004, the
Glasgow Prognostic Score has already been shown to have prognostic
value as a predictor of mortality in numerous cancers, including
gastro-oesophageal cancer, non-small-cell lung cancer, colorectal
cancer, breast cancer, ovarian cancer, bronchogenic cancer, and
metastatic renal cancer (Forrest et al., Br J Cancer, 2004 May 4;
90(9):1704-6; Sharma et al., Clin Colorectal Cancer, 2008
September; 7(5):331-7; Sharma et al., Eur J Cancer, 2008 January;
44(2):251-6; McMillan et al., Nutr Cancer, 2001; 41(1-2):64-9;
McMillan, Proc Nutr Soc, 2008 August; 67(3):257-62; Ramsey et al.,
Cancer, 2007 Jan. 15; 109(2):205-12). Because the combination of
elevated CRP and reduced albumin predicts cancer patient mortality,
a treatment that both lowers CRP and raises albumin would suggest a
strong possiblity of also promoting patient survival.
[0025] U.S. patent application publication no. 20080081041
(relating to treatment of cancer using an anti-IL-6 antibody)
discloses that since IL-6 is associated with disease activity and
since CRP is a surrogate marker of IL-6 activity, sustained
suppression of CRP by neutralization of IL-6 by their anti-IL-6
antibody (CNTO 328, Zaki et al., Int J Cancer, 2004 Sep. 10;
111(4):592-5) may be assumed necessary to achieve biological
activity. The same patent application indicates that the
relationship between IL-6 and CRP in patients with benign and
malignant prostate disease was previously examined by McArdle
(McArdle et al. 2004 Br J Cancer 91(10):1755-1757). McArdle
reportedly found no significant differences between the
concentrations of IL-6 and CRP in the patients with benign disease
compared with prostate cancer patients, in the cancer patients
there was a significant increase in both IL-6 and CRP concentration
with increasing tumor grade. The median serum CRP value for the 86
subjects with prostate cancer was 1.8 mg/L. Based thereoon the
inventors in this patent application postulate a proposed dose and
schedule wherein 6 mg/kg of an anti-IL-6 antibody (CNTO 328) is
administered every 2 weeks and allege that this is likely to
achieve sustained suppression of CRP in subjects with metastatic
HRPC.
[0026] IL-6 signaling is mediated by the Jak-Tyk family of
cytoplasmic tyrosine kinases, including JAK1, JAK2, and JAK3
(reviewed in Murray J Immunol. 2007 Mar. 1; 178(5):2623-9). Sivash
et al. report abrogation of IL-6-mediated JAK signalling by the
cyclopentenone prostaglandin 15d-PGD.sub.2 in oral squamous
carcinoma cells. British Journal of Cancer (2004) 91, 1074-1080.
These results suggest that inhibitors of JAK1, JAK2, or JAK3 could
be employed as antagonists of IL-6.
[0027] Ulanova et al. report that inhibition of the nonreceptor
protein tyrosine kinase Syk (using siRNA) decreased production of
IL-6 by epithelial cells. Am J Physiol Lung Cell Mol Physiol. 2005
March; 288(3):L497-507. These results suggest that an inhibitor of
Syk could be employed as an antagonist of IL-6.
[0028] Kedar et al. report that treatment with thalidomide
significantly reduced serum levels of CRP and IL-6 to normal or
near normal levels in a substantial fraction of renal cell
carcinoma patients. Int J Cancer. 2004 Jun. 10; 110(2):260-5. These
results suggest that thalidomide, and possibly derivatives thereof,
such as lenalidomide, may be useful antagonists of IL-6.
[0029] In addition, another published patent application, US
20070292420 teaches a Phase I dose escalating study using an
anti-IL-6 (cCLB-8) antibody for treating refractory patients with
advanced stage multiple myeloma (N=12) and indicate that this study
demonstrated that some patients had disease stabilization. The
application also reports that after discontinuation of treatment
there was acceleration in the increase of M protein levels,
suggesting disease re-bound after the withdrawal of therapy.
Anti-IL-6 cCLB-8 antibody inhibited free circulating IL-6.
[0030] The application also indicates that this antibody trial
resulted in no toxicity (except transient thrombocytopenia in two
heavily pretreated patients) or allergic reactions were observed
and that C-reactive protein (CRP) decreased below detection level
in all patients. Their antibody (cCLB-8 antibody) reportedly
possessed a circulating half-life of 17.8 days, and that there was
no human anti-chimeric antibody (HACA) immune response observed
(van Zaanen et al. 1998). They allege that the administration of
CNTO 328 did not cause changes in blood pressure, pulse rate,
temperature, hemoglobin, liver functions and renal functions.
Except for transient thrombocytopenia in two heavily pretreated
patients, no toxicity or allergic reactions allegedly were
observed, and there was no human anti-chimeric antibody (HACA)
immune response observed. Three patients in their study reportedly
developed infection-related complications during therapy, however,
a possible relation with anti-IL-6 cCLB-8 antibody was concluded by
the inventors to be unlikely because infectious complications are
reportedly common in end stage multiple myeloma and are a major
cause of death. They conclude based on their results that this
anti-IL-6 cCLB-8 antibody was safe in multiple myeloma
patients.
[0031] Certain of the anti-IL-6 antibodies disclosed herein have
also been disclosed in the following published and unpublished
patent applications, which are co-owned by the assignee of the
present application: U.S. 2009/0028784, WO 2008/144763, U.S. Ser.
No. 12/391,717 filed Feb. 24, 2009 (Atty. Docket No. 67858.702201),
and U.S. Ser. No. 12/366,567 filed Feb. 5, 2009 (Atty. Docket No.
67858.702101).
[0032] Other anti-IL-6 antibodies have been disclosed in the
following U.S. patents and published patent applications: U.S. Pat.
Nos. 7,482,436; 7,291,721; 6,121,423; 2008/0075726; 2007/0178098;
2007/0154481; 2006/0257407; and 2006/0188502.
[0033] As noted above, elevated IL-6 has been implicated in
pathogenesis of cachexia, weakness, fatigue, and fever. Diseases
and disorders associated with fatigue include, but are not limited
to, general fatigue, exercise-induced fatigue, cancer-related
fatigue, inflammatory disease-related fatigue and chronic fatigue
syndrome. See, for example, Esper D H, et al, The cancer cachexia
syndrome: a review of metabolic and clinical manifestations, Nutr
Clin Pract., 2005 August; 20 (4):369-76; Vgontzas A N, et al, IL-6
and its circadian secretion in humans, Neuroimmunomodulation, 2005;
12(3):131-40; Robson-Ansley, P J, et al, Acute interleukin-6
administration impairs athletic performance in healthy, trained
male runners, Can J Appl Physiol., 2004 August; 29(4):411-8;
Shephard R J., Cytokine responses to physical activity, with
particular reference to IL-6: sources, actions, and clinical
implications, Crit Rev Immunol., 2002; 22(3):165-82; Arnold, M C,
et al, Using an interleukin-6 challenge to evaluate
neuropsychological performance in chronic fatigue syndrome, Psychol
Med., 2002 August; 32(6):1075-89; Kurzrock R., The role of
cytokines in cancer-related fatigue, Cancer, 2001 Sep. 15; 92(6
Suppl):1684-8; Nishimoto N, et al, Improvement in Castleman's
disease by humanized anti-interleukin-6 receptor antibody therapy,
Blood, 2000 Jan. 1; 95 (1):56-61; Vgontzas A N, et al, Circadian
interleukin-6 secretion and quantity and depth of sleep, J Clin
Endocrinol Metab., 1999 August; 84(8):2603-7; and Spath-Schwalbe E,
et al, Acute effects of recombinant human interleukin 6 on
endocrine and central nervous sleep functions in healthy men, J
Clin Endocrinol Metab., 1998 May; 83(5):1573-9; the disclosures of
each of which are herein incorporated by reference in their
entireties.
[0034] Diseases and disorders associated with cachexia include, but
are not limited to, cancer-related cachexia, cardiac-related
cachexia, respiratory-related cachexia, renal-related cachexia and
age-related cachexia. See, for example, Barton, B E., Interleukin-6
and new strategies for the treatment of cancer, hyperproliferative
diseases and paraneoplastic syndromes, Expert Opin Ther Targets,
2005 August; 9(4):737-52; Zaki M H, et al, CNTO 328, a monoclonal
antibody to IL-6, inhibits human tumor-induced cachexia in nude
mice, Int J Cancer, 2004 Sep. 10; 111(4):592-5; Trikha M, et al,
Targeted anti-interleukin-6 monoclonal antibody therapy for cancer:
a review of the rationale and clinical evidence, Clin Cancer Res.,
2003 Oct. 15; 9(13):4653-65; Lelli G, et al, Treatment of the
cancer anorexia-cachexia syndrome: a critical reappraisal, J
Chemother., 2003 June; 15(3):220-5; Argiles J M, et al, Cytokines
in the pathogenesis of cancer cachexia, Curr Opin Clin Nutr Metab
Care, 2003 July; 6(4):401-6; Barton B E., IL-6-like cytokines and
cancer cachexia: consequences of chronic inflammation, Immunol
Res., 2001; 23(1):41-58; Yamashita J I, et al, Medroxyprogesterone
acetate and cancer cachexia: interleukin-6 involvement, Breast
Cancer, 2000; 7(2):130-5; Yeh S S, et al, Geriatric cachexia: the
role of cytokines, Am J Clin Nutr., 1999 August; 70(2):183-97;
Strassmann G, et al, Inhibition of experimental cancer cachexia by
anti-cytokine and anti-cytokine-receptor therapy, Cytokines Mol
Ther., 1995 June; 1(2):107-13; Fujita J, et al, Anti-interleukin-6
receptor antibody prevents muscle atrophy in colon-26
adenocarcinoma-bearing mice with modulation of lysosomal and
ATP-ubiquitin-dependent proteolytic pathways, Int J Cancer, 1996
Nov. 27; 68(5):637-43; Tsujinaka T, et al, Interleukin 6 receptor
antibody inhibits muscle atrophy and modulates proteolytic systems
in interleukin 6 transgenic mice, J Clin Invest., 1996 Jan. 1;
97(1):244-9; Emilie D, et al, Administration of an
anti-interleukin-6 monoclonal antibody to patients with acquired
immunodeficiency syndrome and lymphoma: effect on lymphoma growth
and on B clinical Symptoms, Blood, 1994 Oct. 15; 84 (8):2472-9; and
Strassmann G, et al, Evidence for the involvement of interleukin 6
in experimental cancer cachexia, J Clin Invest., 1992 May;
89(5):1681-4; the disclosures of each of which are herein
incorporated by reference in their entireties.
[0035] Another cachexia-related disease is failure to thrive, also
known as faltering growth, in which a child exhibits a rate of
weight gain less than expected. Failure to thrive is typically
defined as weight below the third percentile or a decrease in the
percentile rank of 2 major growth parameters in a short period.
Failure to thrive results from heterogeneous medical and
psychosocial causes, and the cause sometimes eludes diagnosis. One
recent study (totaling 34 patients) reported a statistically
significant elevation in IL-6 levels in patients diagnosed with
failure to thrive. Shaoul et al. J Pediatr Gastroenterol Nutr.,
2003 October; 37(4):487-91.
SUMMARY
[0036] The present invention is an extension of Applicants'
previous invention which is directed to specific antibodies and
fragments and variants thereof having binding specificity for IL-6,
in particular antibodies having specific epitopic specificity
and/or functional properties and novel therapies using these and
other anti-IL-6 antibodies. One embodiment of the invention
encompasses specific humanized antibodies and fragments and
variants thereof capable of binding to IL-6 and/or the IL-6/IL-6R
complex. These antibodies may bind soluble IL-6 or cell surface
expressed IL-6. Also, these antibodies may inhibit the formation or
the biological effects of one or more of IL-6, IL-6/IL-6R
complexes, IL-6/IL-6R/gp130 complexes and/or multimers of
IL-6/IL-6R/gp130. The present invention relates to novel therapies
and therapeutic protocols using anti-IL-6 antibodies, preferably
those described herein. In particular, the present invention
pertains to methods of preventing or treating thrombosis in a
patient in need thereof, e.g., a patient showing elevated D-dimer
and/or CRP levels prior to treatment, comprising administering to
the patient an IL-6 antagonist, such as those identified infra,
e.g., an anti-IL-6 antibody (such as Ab1) or antibody fragment or
variant thereof, whereby the patient's coagulation profile is
improved or restored to a normal condition. In some embodiments
these methods may further include the administration of other
actives such as statins that may further help (synergize) with the
IL-6 antagonist such as Ab1 and thereby more effectively treat or
prevent thrombosis.
[0037] The present invention also pertains to methods of improving
survivability or quality of life of a patient in need thereof,
e.g., a patient showing elevated CRP levels and/or lowered albumin
levels, comprising administering to the patient an IL-6 antagonist,
such as those identified infra, e.g., an anti-IL-6 antibody (e.g.,
Ab1) or antibody fragment or variant thereof, whereby the patient's
C-reactive protein ("CRP") level is lowered, and/or the patient's
albumin level is raised. In some embodiments these methods may
further include the administration of other actives such as statins
that may further help (synergize) with the IL-6 antagonist such as
Ab1 and thereby more effectively treat the patient.
[0038] Another embodiment of the invention relates to Ab1,
including rabbit and humanized forms thereof, as well as heavy
chains, light chains, fragments, variants, and CDRs thereof.
[0039] In a preferred embodiment this is effected by the
administration of the antibodies described herein, comprising the
sequences of the V.sub.H, V.sub.L and CDR polypeptides described
herein, and the polynucleotides encoding them. In more specific
embodiments of the invention these antibodies will block gp130
activation and/or possess binding affinities (Kds) less than 50
picomolar and/or K.sub.off values less than or equal to 10.sup.-4
S.sup.-1.
[0040] In another embodiment of the invention these antibodies and
humanized versions will be derived from rabbit immune cells (B
lymphocytes) and may be selected based on their homology (sequence
identity) to human germ line sequences. These antibodies may
require minimal or no sequence modifications, thereby facilitating
retention of functional properties after humanization.
[0041] In another embodiment of the invention the subject
antibodies may be selected based on their activity in functional
assays such as IL-6 driven T1165 proliferation assays, IL-6
simulated HepG2 haptoglobin production assays, and the like. A
further embodiment of the invention is directed to fragments from
anti-IL-6 antibodies encompassing V.sub.H, V.sub.L and CDR
polypeptides, e.g., derived from rabbit immune cells and the
polynucleotides encoding the same, as well as the use of these
antibody fragments and the polynucleotides encoding them in the
creation of novel antibodies and polypeptide compositions capable
of recognizing IL-6 and/or IL-6/IL-6R complexes or IL-6/IL-6R/gp130
complexes and/or multimers thereof.
[0042] The invention also contemplates the administration of
conjugates of anti-IL-6 antibodies and binding fragments and
variants thereof conjugated to one or more functional or detectable
moieties. The invention also contemplates methods of making said
humanized anti-IL-6 or anti-IL-6/IL-6R complex antibodies and
binding fragments and variants thereof. In one embodiment, binding
fragments include, but are not limited to, Fab, Fab', F(ab').sub.2,
Fv and scFv fragments.
[0043] Embodiments of the invention pertain to the use of anti-IL-6
antibodies for the diagnosis, assessment and treatment of diseases
and disorders associated with IL-6 or aberrant expression thereof.
The invention also contemplates the use of fragments or variants of
anti-IL-6 antibodies for the diagnosis, assessment and treatment of
diseases and disorders associated with IL-6 or aberrant expression
thereof. Preferred usages of the subject antibodies are the
treatment and prevention of cancer associated fatigue, and/or
cachexia and rheumatoid arthritis.
[0044] Other embodiments of the invention relate to the production
of anti-IL-6 antibodies in recombinant host cells, preferably
diploid yeast such as diploid Pichia and other yeast strains.
[0045] Another embodiment of the invention relates to methods of
improving survivability or quality of life of a patient diagnosed
with cancer, comprising administering to the patient an anti-IL-6
antibody or antibody fragment or variant thereof, whereby the
patient's serum C-reactive protein ("CRP") level is reduced, and
monitoring the patient to assess the reduction in the patient's
serum CRP level, wherein the anti-IL-6 antibody or antibody
fragment or variant thereof may specifically bind to the same
linear or conformational epitope(s) and/or compete for binding to
the same linear or conformational epitope(s) on an intact human
IL-6 polypeptide or antibody fragment or variant thereof thereof as
an anti-IL-6 antibody selected from the group consisting of Ab1 and
fragments and variants thereof.
[0046] Another embodiment of the invention relates to methods of
improving muscular strength in a patient diagnosed with cancer,
comprising administering to the patient an anti-IL-6 antibody or
antibody fragment or variant thereof, whereby the patient's
muscular strength is improved, and monitoring the patient to assess
muscular strength, wherein the anti-IL-6 antibody or antibody
fragment or variant thereof may specifically bind to the same
linear or conformational epitope(s) and/or compete for binding to
the same linear or conformational epitope(s) on an intact human
IL-6 polypeptide or fragment thereof as an anti-IL-6 antibody
selected from the group consisting of AbI and fragments and
variants thereof. In such methods preferably the patient's muscular
strength is improved by at least about 15% within approximately 4
weeks of administering the anti-IL-6 antibody or antibody fragment
or variant thereof, as measured by the Hand Grip Strength test and
more preferably the patient's muscular strength is improved by at
least about 20% within approximately 4 weeks of administering the
anti-IL-6 antibody or antibody fragment or variant thereof, as
measured by the Hand Grip Strength test.
[0047] Another embodiment of the invention relates to methods of
increasing serum albumin in a patient in need thereof, comprising
administering to the patient an anti-IL-6 antibody or antibody
fragment or variant thereof, whereby the patient's serum albumin
level is improved, and monitoring the patient to assess serum
albumin level, wherein the anti-IL-6 antibody or antibody fragment
or variant thereof may specifically bind to the same linear or
conformational epitope(s) and/or compete for binding to the same
linear or conformational epitope(s) on an intact human IL-6
polypeptide or antibody fragment or variant thereof thereof as an
anti-IL-6 antibody selected from the group consisting of AbI and
fragments and variants thereof. Preferably, these methods are
effected under conditions whereby the patient's survivability is
improved, and/or under conditions wherein the serum albumin level
is increased by about 5 g/L within approximately 6 weeks of
administering the anti-IL-6 antibody or antibody fragment or
variant thereof. These patients will include, without limitation
thereto, those diagnosed with rheumatoid arthritis, cancer,
advanced cancer, liver disease, renal disease, inflammatory bowel
disease, celiac's disease, trauma, burns, other diseases associated
with reduced serum albumin, or any combination thereof.
[0048] In an embodiment of the invention, the patient may have been
diagnosed with rheumatoid arthritis, juvenile rheumatoid arthritis,
psoriasis, psoriatic arthropathy, ankylosing spondylitis, systemic
lupus erythematosis, Crohn's disease, ulcerative colitis,
pemphigus, dermatomyositis, polymyositis, polymyalgia rheumatica,
giant cell arteritis, vasculitis, polyarteritis nodosa, Wegener's
granulomatosis, Kawasaki disease, isolated CNS vasculitis,
Churg-Strauss arteritis, microscopic polyarteritis, microscopic
polyangiitis, Henoch-Schonlein purpura, essential cryoglobulinemic
vasculitis, rheumatoid vasculitis, cryoglobulinemia, relapsing
polychondritis, Behcet's disease, Takayasu's arteritis, ischemic
heart disease, stroke, multiple sclerosis, sepsis, vasculitis
secondary to viral infection (e.g., hepatitis B, hepatitis C, HIV,
cytomegalovirus, Epstein-Barr virus, Parvo B19 virus, etc.),
Buerger's Disease, cancer, advanced cancer, Osteoarthritis,
systemic sclerosis, CREST syndrome, Reiter's disease, Paget's
disease of bone, Sjogran's syndrome, diabetes type 1, diabetes type
2, familial Mediterrean fever, autoimmune thrombocytopenia,
autoimmune hemolytic anemia, autoimmune thyroid diseases,
pernicious anemia, vitiligo, alopecia areata, primary biliary
cirrhosis, autoimmune chronic active hepatitis, alcoholic
cirrhosis, viral hepatitis including hepatitis B and C, other organ
specific autoimmune diseases, burns, idiopathic pulmonary fibrosis,
chronic obsructive pulmonary disease, allergic asthma, other
allergic conditions or any combination thereof.
[0049] In an embodiment of the invention, the patient may have an
elevated C-reactive protein (CRP) level prior to treatment
[0050] Another embodiment of the invention relates to methods of
improving survivability or quality of life of a patient in need
thereof, comprising administering to the patient an IL-6 antagonist
such as Ab1, whereby the patient's serum C-reactive protein ("CRP")
level is reduced, and monitoring the patient to assess the
reduction in the patient's serum CRP level.
[0051] Another embodiment of the invention relates to methods of
improving survivability or quality of life of a patient in need
thereof, comprising administering to the patient an IL-6 antagonist
such as Ab1, whereby the patient's serum albumin level is
increased, and monitoring the patient to assess the increase in the
patient's serum albumin level.
[0052] Another embodiment of the invention relates to methods of
improving survivability or quality of life of a patient in need
thereof, comprising administering to the patient an IL-6 antagonist
such as Ab1, whereby the patient's serum CRP level is reduced and
the patient's serum albumin level is increased, and monitoring the
patient to assess the reduction in the patient's serum CRP level
and the increase in the patient's serum albumin level.
[0053] Another embodiment of the invention relates to methods of
preventing or treating thrombosis in a patient in a state of
hypercoagulation, comprising administering to the patient an IL-6
antagonist, e.g. an anti-IL-6 antibody (e.g., Ab1) or antibody
fragment or variant thereof, whereby the patient's coagulation
profile is improved or restored to a normal condition, and
monitoring the patient to assess coagulation profile. In such
methods if the IL-6 antagonist is an anti-IL-6 antibody or antibody
fragment or variant thereof preferably this antibody may
specifically bind to the same linear or conformational epitope(s)
and/or compete for binding to the same linear or conformational
epitope(s) on an intact human IL-6 polypeptide or fragment thereof
as an anti-IL-6 antibody selected from the group consisting of Ab1
and fragments and variants thereof. In the inventive methods of
preventing or treating thrombosis, the patient's coagulation
profile is assessed by measurement of the patient's serum level of
one or more of D-dimer, Factor II, Factor V, Factor VIII, Factor
IX, Factor XI, Factor XII, F/fibrin degradation products,
thrombin-antithrombin III complex, fibrinogen, plasminogen,
prothrombin, and von Willebrand factor and preferably by a method
including measuring the patient's serum D-dimer level prior to
administration of the anti-IL-6 antibody, and administering the
anti-IL-6 antibody or antibody fragment or variant thereof if the
patient's serum D-dimer level is elevated. In addition, the levels
of C reactive protein may also be asessed in the patient prior to
treatment and if elevated this may be used as a further indicator
as to an increased risk of thrombosis in the patient.
[0054] An embodiment of the invention relates to methods of
treating a patient having a disease or condition associated with
hypercoagulation, which may comprise administering to the patient
an IL-6 antagonist such as Ab1, whereby the patient's coagulation
profile is improved or restored to normal, and monitoring the
patient to assess coagulation profile.
[0055] In an embodiment of the invention, the patient may have
elevated serum D-dimer levels prior to treatment.
[0056] In an embodiment of the invention, the patient may have a
reduced serum albumin level prior to treatment.
[0057] In an embodiment of the invention, the patient's Glasgow
Prognostic Score (GPS) may be improved following the treatment.
[0058] In an embodiment of the invention, the patient may have an
elevated serum CRP level prior to treatment.
[0059] In an embodiment of the invention, the method may further
comprise the administration of at least one statin.
[0060] In an embodiment of the invention, the IL-6 antagonist may
target IL-6, IL-6 receptor alpha, gp130, p38 MAP kinase, JAK1,
JAK2, JAK3, SYK, or any combination thereof.
[0061] In an embodiment of the invention, the IL-6 antagonist may
comprise an antibody, an antibody fragment, a peptide, a
glycoalkoid, an antisense nucleic acid, a ribozyme, a retinoid, an
avemir, a small molecule, or any combination thereof.
[0062] In an embodiment of the invention, the IL-6 antagonist may
comprise an anti-IL-6R, anti-gp130, anti-p38 MAP kinase, anti-JAK1,
anti-JAK2, anti-JAK3, or anti-SYK antibody or antibody
fragment.
[0063] In one embodiment of the invention, the IL-6 antagonist may
comprise a small molecule comprising thalidomide, lenalidomide, or
any combination thereof.
[0064] In an embodiment of the invention, the antagonist may
comprise an anti-IL-6 antibody (e.g., Ab1) or antibody fragment or
variant thereof.
[0065] In an embodiment of the invention, the anti-IL-6 antibody or
antibody fragment or variant thereof may specifically bind to the
same linear or conformational epitope(s) and/or compete for binding
to the same linear or conformational epitope(s) on an intact human
IL-6 polypeptide or fragment thereof as an anti-IL-6 antibody
selected from the group consisting of Ab1 and fragments and
variants thereof.
[0066] In an embodiment of the invention, the anti-IL-6 antibody
may bind to the same linear or conformational epitope(s) and/or
compete for binding to the same linear or conformational epitope(s)
on an intact human IL-6 polypeptide or a fragment thereof as
Ab1.
[0067] In an embodiment of the invention, the anti-IL-6 antibody or
antibody fragment or variant thereof may specifically bind to the
same linear or conformational epitope(s) on an intact human IL-6
polypeptide or fragment thereof as an anti-IL-6 antibody selected
from the group consisting of AbI and fragments and variants
thereof.
[0068] In an embodiment of the invention, the anti-IL-6 antibody or
antibody fragment or variant thereof may specifically bind to the
same linear or conformational epitope(s) on an intact human IL-6
polypeptide or a fragment thereof as AbI.
[0069] In an embodiment of the invention, the anti-IL-6 antibody or
antibody fragment or variant thereof may specifically bind to the
same linear or conformational epitopes on an intact IL-6
polypeptide or fragment thereof that is (are) specifically bound by
AbI and wherein said epitope(s) when ascertained by epitopic
mapping using overlapping linear peptide fragments which span the
full length of the native human IL-6 polypeptide includes one or
more residues comprised in IL-6 fragments selected from those
respectively encompassing amino acid residues 37-5I, amino acid
residues 70-84, amino acid residues I69-I83, amino acid residues
3I-45 and/or amino acid residues 58-72.
[0070] In an embodiment of the invention, the anti-IL-6 antibody or
antibody fragment or variant thereof may comprise at least 2
complementarity determining regions (CDRs) in each the variable
light and the variable heavy regions which are identical to those
contained in an anti-IL-6 antibody selected from the group
consisting of AbI and fragments and variants thereof. In certain
embodiments, antibodies containing these CDRs may be constructed
using appropriate human frameworks based on the humanization
methods disclosed herein.
[0071] In an embodiment of the invention, the anti-IL-6 antibody or
antibody fragment or variant thereof may comprise at least 2
complementarity determining regions (CDRs) in each the variable
light and the variable heavy regions which are identical to those
contained in AbI.
[0072] In an embodiment of the invention, all of the CDRs in the
anti-IL-6 antibody or antibody fragment or variant thereof may be
identical to the CDRs contained in an anti-IL-6 antibody selected
from the group consisting of AbI and fragments and variants
thereof.
[0073] In an embodiment of the invention, all of the CDRs in the
anti-IL-6 antibody or antibody fragment or variant thereof may be
identical to the CDRs contained in AbI.
[0074] In an embodiment of the invention, the anti-IL-6 antibody or
antibody fragment or variant thereof may be aglycosylated.
[0075] In an embodiment of the invention, the anti-IL-6 antibody or
antibody fragment or variant thereof may contain an Fc region that
has been modified to alter effector function, half-life,
proteolysis, and/or glycosylation.
[0076] In an embodiment of the invention, the anti-IL-6 antibody or
antibody fragment or variant thereof may be a human, humanized,
single chain or chimeric antibody.
[0077] In an embodiment of the invention, the anti-IL-6 antibody or
antibody fragment or variant thereof may be a humanized antibody
derived from a rabbit (parent) anti-IL-6 antibody.
[0078] In an embodiment of the invention, the framework regions
(FRs) in the variable light region and the variable heavy regions
of said anti-IL-6 antibody or antibody fragment or variant thereof
respectively may be human FRs which are unmodified or which have
been modified by the substitution of at most 2 or 3 human FR
residues in the variable light or heavy chain region with the
corresponding FR residues of the parent rabbit antibody, and the
human FRs may have been derived from human variable heavy and light
chain antibody sequences which have been selected from a library of
human germline antibody sequences based on their high level of
homology to the corresponding rabbit variable heavy or light chain
regions relative to other human germline antibody sequences
contained in the library.
[0079] In an embodiment of the invention, the anti-IL-6 antibody or
antibody fragment or variant thereof may be administered to the
patient with a frequency at most once per period of approximately
four weeks, approximately eight weeks, approximately twelve weeks,
approximately sixteen weeks, approximately twenty weeks, or
approximately twenty-four weeks.
[0080] In an embodiment of the invention, the patient's coagulation
profile may remain improved for an entire period intervening two
consecutive anti-IL-6 antibody administrations.
[0081] In an embodiment of the invention, the patient's serum CRP
level may remain decreased and/or serum albumin level may remain
raised for an entire period intervening two consecutive anti-IL-6
antibody administrations.
[0082] In an embodiment of the invention, the patient's cachexia,
weakness, fatigue, and/or fever may remain improved for an entire
period intervening two consecutive anti-IL-6 antibody
administrations.
[0083] In an embodiment of the invention, the patient may have been
diagnosed with cancer selected from Acanthoma, Acinic cell
carcinoma, Acoustic neuroma, Acral lentiginous melanoma,
Acrospiroma, Acute eosinophilic leukemia, Acute lymphoblastic
leukemia, Acute megakaryoblastic leukemia, Acute monocytic
leukemia, Acute myeloblastic leukemia with maturation, Acute
myeloid dendritic cell leukemia, Acute myeloid leukemia, Acute
promyelocytic leukemia, Adamantinoma, Adenocarcinoma, Adenoid
cystic carcinoma, Adenoma, Adenomatoid odontogenic tumor,
Adrenocortical carcinoma, Adult T-cell leukemia, Aggressive NK-cell
leukemia, AIDS-Related Cancers, AIDS-related lymphoma, Alveolar
soft part sarcoma, Ameloblastic fibroma, Anal cancer, Anaplastic
large cell lymphoma, Anaplastic thyroid cancer, Angioimmunoblastic
T-cell lymphoma, Angiomyolipoma, Angiosarcoma, Appendix cancer,
Astrocytoma, Atypical teratoid rhabdoid tumor, Basal cell
carcinoma, Basal-like carcinoma, B-cell leukemia, B-cell lymphoma,
Bellini duct carcinoma, Biliary tract cancer, Bladder cancer,
Blastoma, Bone Cancer, Bone tumor, Brain Stem Glioma, Brain Tumor,
Breast Cancer, Brenner tumor, Bronchial Tumor, Bronchioloalveolar
carcinoma, Brown tumor, Burkitt's lymphoma, Cancer of Unknown
Primary Site, Carcinoid Tumor, Carcinoma, Carcinoma in situ,
Carcinoma of the penis, Carcinoma of Unknown Primary Site,
Carcinosarcoma, Castleman's Disease, Central Nervous System
Embryonal Tumor, Cerebellar Astrocytoma, Cerebral Astrocytoma,
Cervical Cancer, Cholangiocarcinoma, Chondroma, Chondrosarcoma,
Chordoma, Choriocarcinoma, Choroid plexus papilloma, Chronic
Lymphocytic Leukemia, Chronic monocytic leukemia, Chronic
myelogenous leukemia, Chronic Myeloproliferative Disorder, Chronic
neutrophilic leukemia, Clear-cell tumor, Colon Cancer, Colorectal
cancer, Craniopharyngioma, Cutaneous T-cell lymphoma, Degos
disease, Dermatofibrosarcoma protuberans, Dermoid cyst,
Desmoplastic small round cell tumor, Diffuse large B cell lymphoma,
Dysembryoplastic neuroepithelial tumor, Embryonal carcinoma,
Endodermal sinus tumor, Endometrial cancer, Endometrial Uterine
Cancer, Endometrioid tumor, Enteropathy-associated T-cell lymphoma,
Ependymoblastoma, Ependymoma, Epithelioid sarcoma, Erythroleukemia,
Esophageal cancer, Esthesioneuroblastoma, Ewing Family of Tumor,
Ewing Family Sarcoma, Ewing's sarcoma, Extracranial Germ Cell
Tumor, Extragonadal Germ Cell Tumor, Extrahepatic Bile Duct Cancer,
Extramammary Paget's disease, Fallopian tube cancer, Fetus in fetu,
Fibroma, Fibrosarcoma, Follicular lymphoma, Follicular thyroid
cancer, Gallbladder Cancer, Gallbladder cancer, Ganglioglioma,
Ganglioneuroma, Gastric Cancer, Gastric lymphoma, Gastrointestinal
cancer, Gastrointestinal Carcinoid Tumor, Gastrointestinal Stromal
Tumor, Gastrointestinal stromal tumor, Germ cell tumor, Germinoma,
Gestational choriocarcinoma, Gestational Trophoblastic Tumor, Giant
cell tumor of bone, Glioblastoma multiforme, Glioma, Gliomatosis
cerebri, Glomus tumor, Glucagonoma, Gonadoblastoma, Granulosa cell
tumor, Hairy Cell Leukemia, Hairy cell leukemia, Head and Neck
Cancer, Head and neck cancer, Heart cancer, Hemangioblastoma,
Hemangiopericytoma, Hemangiosarcoma, Hematological malignancy,
Hepatocellular carcinoma, Hepatosplenic T-cell lymphoma, Hereditary
breast-ovarian cancer syndrome, Hodgkin Lymphoma, Hodgkin's
lymphoma, Hypopharyngeal Cancer, Hypothalamic Glioma, Inflammatory
breast cancer, Intraocular Melanoma, Islet cell carcinoma, Islet
Cell Tumor, Juvenile myelomonocytic leukemia, Kaposi Sarcoma,
Kaposi's sarcoma, Kidney Cancer, Klatskin tumor, Krukenberg tumor,
Laryngeal Cancer, Laryngeal cancer, Lentigo maligna melanoma,
Leukemia, Leukemia, Lip and Oral Cavity Cancer, Liposarcoma, Lung
cancer, Luteoma, Lymphangioma, Lymphangiosarcoma,
Lymphoepithelioma, Lymphoid leukemia, Lymphoma, Macroglobulinemia,
Malignant Fibrous Histiocytoma, Malignant fibrous histiocytoma,
Malignant Fibrous Histiocytoma of Bone, Malignant Glioma, Malignant
Mesothelioma, Malignant peripheral nerve sheath tumor, Malignant
rhabdoid tumor, Malignant triton tumor, MALT lymphoma, Mantle cell
lymphoma, Mast cell leukemia, Mediastinal germ cell tumor,
Mediastinal tumor, Medullary thyroid cancer, Medulloblastoma,
Medulloblastoma, Medulloepithelioma, Melanoma, Melanoma,
Meningioma, Merkel Cell Carcinoma, Mesothelioma, Mesothelioma,
Metastatic Squamous Neck Cancer with Occult Primary, Metastatic
urothelial carcinoma, Mixed Mullerian tumor, Monocytic leukemia,
Mouth Cancer, Mucinous tumor, Multiple Endocrine Neoplasia
Syndrome, Multiple Myeloma, Multiple myeloma, Mycosis Fungoides,
Mycosis fungoides, Myelodysplastic Disease, Myelodysplastic
Syndromes, Myeloid leukemia, Myeloid sarcoma, Myeloproliferative
Disease, Myxoma, Nasal Cavity Cancer, Nasopharyngeal Cancer,
Nasopharyngeal carcinoma, Neoplasm, Neurinoma, Neuroblastoma,
Neuroblastoma, Neurofibroma, Neuroma, Nodular melanoma, Non-Hodgkin
Lymphoma, Non-Hodgkin lymphoma, Nonmelanoma Skin Cancer, Non-Small
Cell Lung Cancer, Ocular oncology, Oligoastrocytoma,
Oligodendroglioma, Oncocytoma, Optic nerve sheath meningioma, Oral
Cancer, Oral cancer, Oropharyngeal Cancer, Osteosarcoma,
Osteosarcoma, Ovarian Cancer, Ovarian cancer, Ovarian Epithelial
Cancer, Ovarian Germ Cell Tumor, Ovarian Low Malignant Potential
Tumor, Paget's disease of the breast, Pancoast tumor, Pancreatic
Cancer, Pancreatic cancer, Papillary thyroid cancer,
Papillomatosis, Paraganglioma, Paranasal Sinus Cancer, Parathyroid
Cancer, Penile Cancer, Perivascular epithelioid cell tumor,
Pharyngeal Cancer, Pheochromocytoma, Pineal Parenchymal Tumor of
Intermediate Differentiation, Pineoblastoma, Pituicytoma, Pituitary
adenoma, Pituitary tumor, Plasma Cell Neoplasm, Pleuropulmonary
blastoma, Polyembryoma, Precursor T-lymphoblastic lymphoma, Primary
central nervous system lymphoma, Primary effusion lymphoma, Primary
Hepatocellular Cancer, Primary Liver Cancer, Primary peritoneal
cancer, Primitive neuroectodermal tumor, Prostate cancer,
Pseudomyxoma peritonei, Rectal Cancer, Renal cell carcinoma,
Respiratory Tract Carcinoma Involving the NUT Gene on Chromosome
15, Retinoblastoma, Rhabdomyoma, Rhabdomyosarcoma, Richter's
transformation, Sacrococcygeal teratoma, Salivary Gland Cancer,
Sarcoma, Schwannomatosis, Sebaceous gland carcinoma, Secondary
neoplasm, Seminoma, Serous tumor, Sertoli-Leydig cell tumor, Sex
cord-stromal tumor, Sezary Syndrome, Signet ring cell carcinoma,
Skin Cancer, Small blue round cell tumor, Small cell carcinoma,
Small Cell Lung Cancer, Small cell lymphoma, Small intestine
cancer, Soft tissue sarcoma, Somatostatinoma, Soot wart, Spinal
Cord Tumor, Spinal tumor, Splenic marginal zone lymphoma, Squamous
cell carcinoma, Stomach cancer, Superficial spreading melanoma,
Supratentorial Primitive Neuroectodermal Tumor, Surface
epithelial-stromal tumor, Synovial sarcoma, T-cell acute
lymphoblastic leukemia, T-cell large granular lymphocyte leukemia,
T-cell leukemia, T-cell lymphoma, T-cell prolymphocytic leukemia,
Teratoma, Terminal lymphatic cancer, Testicular cancer, Thecoma,
Throat Cancer, Thymic Carcinoma, Thymoma, Thyroid cancer,
Transitional Cell Cancer of Renal Pelvis and Ureter, Transitional
cell carcinoma, Urachal cancer, Urethral cancer, Urogenital
neoplasm, Uterine sarcoma, Uveal melanoma, Vaginal Cancer, Verner
Morrison syndrome, Verrucous carcinoma, Visual Pathway Glioma,
Vulvar Cancer, Waldenstrom's macroglobulinemia, Warthin's tumor,
Wilms' tumor, or any combination thereof.
[0084] In an embodiment of the invention, the patient may have been
diagnosed with a cancer selected from Colorectal Cancer, Non-Small
Cell Lung Cancer, Cholangiocarcinoma, Mesothelioma, Castleman's
disease, Renal Cell Carcinoma, or any combination thereof.
[0085] In an embodiment of the invention, the anti-IL-6 antibody or
antibody fragment or variant thereof may comprise a heavy chain
polypeptide sequence selected from the group consisting of: SEQ ID
NO: 3, 18, 19, 652, 656, 657, 658, 66I, 664, 665, 704, and 708; and
may further comprise a VL polypeptide sequence selected from the
group consisting of: SEQ ID NO: 2, 20, 647, 651, 660, 666, 699,
702, 706, and 709 or a variant thereof wherein one or more of the
framework residues (FR residues) in said VH or VL polypeptide may
have been substituted with another amino acid residue resulting in
an anti-IL-6 antibody or antibody fragment or variant thereof that
specifically binds human IL-6.
[0086] In an embodiment of the invention, one or more of said FR
residues may be substituted with an amino acid present at the
corresponding site in a parent rabbit anti-IL-6 antibody from which
the complementarity determining regions (CDRs) contained in said VH
or VL polypeptides have been derived or by a conservative amino
acid substitution.
[0087] In an embodiment of the invention, said anti-IL-6 antibody
or antibody fragment or variant thereof may be humanized.
[0088] In an embodiment of the invention, said anti-IL-6 antibody
or antibody fragment or variant thereof may be chimeric.
[0089] In an embodiment of the invention, said anti-IL-6 antibody
or antibody fragment or variant thereof further may comprise a
human Fc.
[0090] In an embodiment of the invention, said human Fc may be
derived from IgG1, IgG2, IgG3, IgG4, IgG5, IgG6, IgG7, IgG8, IgG9,
IgG10, IgG11, IgG12, IgG13, IgG14, IgG15, IgG16, IgG17, IgG18 or
IgG19.
[0091] In an embodiment of the invention, the anti-IL-6 antibody or
antibody fragment or variant thereof may comprise a polypeptide
having at least 90% sequence homology to one or more of the
polypeptide sequences of SEQ ID NO: 3, 18, 19, 652, 656, 657, 658,
66I, 664, 665, 704, 708, 2, 20, 647, 651, 660, 666, 699, 702, 706,
and 709.
[0092] In an embodiment of the invention, the anti-IL-6 antibody or
antibody fragment or variant thereof may have an elimination
half-life of at least about 22 days, at least about 25 days, or at
least about 30 days.
[0093] In an embodiment of the invention, the IL-6 antagonist such
as AbI may be co-administered with a chemotherapy agent. In an
embodiment of the invention, the chemotherapy agent include without
limitation thereto: VEGF antagonists, EGFR antagonists, platins,
taxols, irinotecan, 5-fluorouracil, gemcytabine, leucovorine,
steroids, cyclophosphamide, melphalan, vinca alkaloids (e.g.,
vinblastine, vincristine, vindesine and vinorelbine), mustines,
tyrosine kinase inhibitors, radiotherapy, sex hormone antagonists,
selective androgen receptor modulators, selective estrogen receptor
modulators, PDGF antagonists, TNF antagonists, IL-1 antagonists,
interleukins (e.g. IL-12 or IL-2), IL-12R antagonists, Toxin
conjugated monoclonal antibodies, tumor antigen specific monoclonal
antibodies, Erbitux, Avastin, Pertuzumab, anti-CD20 antibodies,
Rituxan, ocrelizumab, ofatumumab, DXL625, herceptin, or any
combination thereof.
[0094] In an embodiment of the invention, the another therapeutic
compound may be a statin.
[0095] In an embodiment of the invention, the anti-IL-6 antibody or
antibody fragment or variant thereof may be directly or indirectly
attached to a detectable label or therapeutic agent.
[0096] In an embodiment of the invention, the anti-IL-6 antibody or
antibody fragment may be Ab1 or a fragment or variant thereof
thereof.
[0097] In an embodiment of the invention, the disease or condition
may be selected from acute venous thrombosis, pulmonary embolism,
thrombosis during pregnancy, hemorrhagic skin necrosis, acute or
chronic disseminated intravascular coagulation (DIC), clot
formation from surgery, long bed rest, long periods of
immobilization, venous thrombosis, fulminant meningococcemia, acute
thrombotic stroke, acute coronary occlusion, acute peripheral
arterial occlusion, massive pulmonary embolism, axillary vein
thrombosis, massive iliofemoral vein thrombosis, occluded arterial
cannulae, occluded venous cannulae, cardiomyopathy, venoocclusive
disease of the liver, hypotension, decreased cardiac output,
decreased vascular resistance, pulmonary hypertension, diminished
lung compliance, leukopenia, thrombocytopenia, heparin-induced
thrombocytopenia (HIT), heparin-induced thrombocytopenia and
thrombosis (HITT), atrial fibrillation, implantation of a
prosthetic heart valve, genetic susceptibility to thrombosis,
factor V Leiden, prothrombin gene mutation,
methylenetetrahydrofolate reductase (MTHFR) polymorphism,
platelet-receptor polymorphism, trauma, fractures, burns, or any
combination thereof.
[0098] In an embodiment of the invention, the disease or condition
may be selected from cancer, rheumatoid arthritis, AIDS, heart
disease, dehydration, malnutrition, lead exposure, malaria,
respiratory disease, old age, hypothyroidism, tuberculosis,
hypopituitarism, neurasthenia, hypernatremia, hyponatremia, renal
disease, splenica, ankylosing spondylitis, failure to thrive
(faltering growth), or any combination thereof.
[0099] In an embodiment of the invention, the method may include
administration of an antagonist of a cachexia-associated factor,
weakness-associated factor, fatigue-associated factor, and/or
fever-associated factor. The cachexia-associated factor,
weakness-associated factor, fatigue-associated factor, and/or
fever-associated factor may be selected from tumor necrosis
factor-alpha, Interferon gamma, Interleukin 1 alpha, Interleukin 1
beta, Interleukin 6, proteolysis inducing factor,
leukemia-inhibitory factor, or any combination thereof.
[0100] In an embodiment of the invention, the method may include
administration of an anti-cachexia agent selected from cannabis,
dronabinol (Marinol), nabilone (Cesamet), cannabidiol,
cannabichromene, tetrahydrocannabinol, Sativex, megestrol acetate,
or any combination thereof.
[0101] In an embodiment of the invention, the method may include
administration of an anti-nausea or antiemetic agent selected from
5-HT3 receptor antagonists, ajwain, alizapride, anticholinergics,
antihistamines, aprepitant, benzodiazepines, cannabichromene,
cannabidiol, cannabinoids, cannabis, casopitant, chlorpromazine,
cyclizine, dexamethasone, dexamethasone, dimenhydrinate (Gravol),
diphenhydramine, dolasetron, domperidone, dopamine antagonists,
doxylamine, dronabinol (Marinol), droperidol, emetrol, ginger,
granisetron, haloperidol, hydroxyzine, hyoscine, lorazepam,
meclizine, metoclopramide, midazolam, muscimol, nabilone (Cesamet),
nkl receptor antagonists, ondansetron, palonosetron, peppermint,
Phenergan, prochlorperazine, Promacot, promethazine, Pentazine,
propofol, sativex, tetrahydrocannabinol, trimethobenzamide,
tropisetron, nandrolone, stilbestrol, thalidomide, lenalidomide,
ghrelin agonists, myostatin antagonists, anti-myostatin antibodies,
selective androgen receptor modulators, selective estrogen receptor
modulators, angiotensin AII antagonists, beta two adenergic
receptor agonists, beta three adenergic receptor agonists, or any
combination thereof.
[0102] In an embodiment of the invention, the patient's fever may
be assessed by measurement of patient's body temperature.
[0103] In an embodiment of the invention, the method may include
measuring the patient's body temperature prior to administration of
the anti-IL-6 antibody, and administering the anti-IL-6 antibody or
antibody fragment or variant thereof if the patient's body
temperature is higher than about 38.degree. C.
[0104] In an embodiment of the invention, the method may include
measuring the patient's body temperature within 24 hours prior to
administration of the anti-IL-6 antibody, and administering the
anti-IL-6 antibody or antibody fragment or variant thereof if the
patient's body temperature measurement indicates that a fever was
present.
[0105] In an embodiment of the invention, the method may further
include measuring the patient's body weight prior to administration
of the anti-IL-6 antibody, and administering the anti-IL-6 antibody
or antibody fragment or variant thereof if the patient's weight has
declined by greater than approximately 5% within approximately 30
days, or if the patient's lean body mass index is less than about
17 kg/m2 (male patient) or less than about 14 kg/m2 (female
patient).
[0106] In an embodiment of the invention, the method may include
measuring the patient's muscular strength prior to administration
of the anti-IL-6 antibody, and administering the anti-IL-6 antibody
or antibody fragment or variant thereof if the patient's muscular
strength has declined by greater than approximately 20% within
approximately 30 days.
[0107] In an embodiment of the invention, the method may result in
a prolonged improvement in cachexia, weakness, fatigue, and/or
fever in the patient.
[0108] In an embodiment of the invention, the patient's body mass
may be raised by approximately 1 kilogram within approximately 4
weeks of administration of the anti-IL-6 antibody or antibody
fragment or variant thereof.
[0109] In an embodiment of the invention, the patient's cachexia
may be measurably improved within about 4 weeks of anti-IL-6
antibody administration.
[0110] In an embodiment of the invention, the patient's cachexia
may be assessed by measurement of the patient's total body mass,
lean body mass, lean body mass index, and/or appendicular lean body
mass.
[0111] In an embodiment of the invention, the measurement of the
patient's body mass may discount (subtract) the estimated weight of
the patient's tumor(s) and/or extravascular fluid
collection(s).
[0112] In an embodiment of the invention, the patient's cachexia
may remain measurably improved approximately 8 weeks after
anti-IL-6 antibody administration.
[0113] In an embodiment of the invention, the patient's weakness
may be measurably improved within about 4 weeks of anti-IL-6
antibody administration.
[0114] In an embodiment of the invention, the patient's weakness
may be measured by the hand grip strength test.
[0115] In an embodiment of the invention, the patient's hand grip
strength may be improved by at least about 15%, or at least about
20%.
[0116] In an embodiment of the invention, the patient's weakness
may remain measurably improved approximately 8 weeks after
anti-IL-6 antibody administration.
[0117] In an embodiment of the invention, the patient's fatigue may
be measurably improved within about 1 week of anti-IL-6 antibody
administration.
[0118] In an embodiment of the invention, the patient's fatigue may
be measured by the FACIT-F FS test.
[0119] In an embodiment of the invention, the patient's FACIT-F FS
score may be improved by at least about 10 points.
[0120] In an embodiment of the invention, the patient's fatigue may
remain measurably improved approximately 8 weeks after anti-IL-6
antibody administration.
[0121] In an embodiment of the invention, the patient's fever may
be measurably improved within about 1 week of anti-IL-6 antibody
administration.
[0122] In an embodiment of the invention, the patient's fever may
remain measurably improved approximately 8 weeks after anti-IL-6
antibody administration.
[0123] In an embodiment of the invention, the patient's quality of
life may be improved.
[0124] In an embodiment of the invention, may include
administration of one or more anti-coagulants or statins.
[0125] In an embodiment of the invention, the one or more
anti-coagulants may be selected from abciximab (ReoPro),
acenocoumarol, antithrombin III, argatroban, aspirin, bivalirudin
(Angiomax), clopidogrel, dabigatran, dabigatran etexilate
(Pradaxa/Pradax), desirudin (Revasc/Iprivask), dipyridamole,
eptifibatide (Integrilin), fondaparinux, heparin, hirudin,
idraparinux, lepirudin (Refludan), low molecular weight heparin,
melagatran, phenindione, phenprocoumon, ticlopidine, tirofiban
(Aggrastat), warfarin, ximelagatran, ximelagatran (Exanta/Exarta),
or any combination thereof.
[0126] In an embodiment of the invention, the one or more statins
may be selected from atorvastatin, cerivastatin, fluvastatin,
lovastatin, mevastatin, pitavastatin, pravastatin, rosuvastatin,
simvastatin, or any combination thereof.
[0127] In an embodiment of the invention, the patient's coagulation
profile may be assessed by measurement of the patient's serum level
of one or more of D-dimer, Factor II, Factor V, Factor VIII, Factor
IX, Factor XI, Factor XII, F/fibrin degradation products,
thrombin-antithrombin III complex, fibrinogen, plasminogen,
prothrombin, and von Willebrand factor.
[0128] In an embodiment of the invention, the patient's coagulation
profile may be assessed by a functional measurement of clotting
ability.
[0129] In an embodiment of the invention, the functional
measurement of clotting ability may be selected from prothrombin
time (PT), prothrombin ratio (PR), international normalized ratio
(INR), or any combination thereof.
[0130] In an embodiment of the invention, the method may include
measuring the patient's international normalized ratio (INR) prior
to administration of the IL-6 antagonist, and administering to the
patient an IL-6 antagonist such as Ab1 if the patient's INR is less
than about 0.9.
[0131] In an embodiment of the invention, the invention may include
measuring the patient's international normalized ratio (INR) prior
to administration of the IL-6 antagonist, and administering to the
patient an IL-6 antagonist such as Ab1 if the patient's INR is less
than about 0.5.
54.) In an embodiment of the invention, the patient's INR may be
raised to more than approximately 0.9 within 4 weeks of
administering to the patient an IL-6 antagonist.
[0132] In an embodiment of the invention, the method may include
measuring the patient's serum D-dimer level prior to administration
of the IL-6 antagonist, and administering the IL-6 antagonist such
as Ab1 if the patient's serum serum D-dimer level is above the
normal reference range.
[0133] In an embodiment of the invention, the patient's serum
D-dimer level may be lowered to less than the upper limit of the
normal reference range within 4 weeks of administering to the
patient an IL-6 antagonist.
[0134] In an embodiment of the invention, the method may result in
a prolonged improvement in the patient's coagulation profile.
[0135] In an embodiment of the invention, the patient's coagulation
profile may be measurably improved within about 2 weeks of
administration of the IL-6 antagonist.
[0136] In an embodiment of the invention, the patient's coagulation
profile may remain measurably improved approximately 12 weeks after
administering to the patient an IL-6 antagonist.
[0137] In an embodiment of the invention, the patient's
survivability may be improved.
[0138] In an embodiment of the invention, the IL-6 antagonist may
be an antisense nucleic acid.
[0139] In an embodiment of the invention, the IL-6 antagonist may
be an antisense nucleic acid, for example comprising at least
approximately 10 nucleotides of a sequence encoding IL-6, IL-6
receptor alpha, gp130, p38 MAP kinase, JAK1, JAK2, JAK3, or
SYK.
[0140] In an embodiment of the invention, the antisense nucleic
acid may comprise DNA, RNA, peptide nucleic acid, locked nucleic
acid, morpholino (phosphorodiamidate morpholino oligo), glycerol
nucleic acid, threose nucleic acid, or any combination thereof.
[0141] In an embodiment of the invention, the IL-6 antagonist may
comprise Actemra (Tocilizumab), Remicade, Zenapax, or any
combination thereof.
[0142] In an embodiment of the invention, the IL-6 antagonist may
comprise a polypeptide having a sequence comprising a fragment of
IL-6, IL-6 receptor alpha, gp130, p38 MAP kinase, JAK1, JAK2, JAK3,
SYK, or any combination thereof, such as a fragment or full-length
polypeptide that is at least 40 amino acids in length.
[0143] In an embodiment of the invention, the IL-6 antagonist may
comprise a soluble IL-6, IL-6 receptor alpha, gp130, p38 MAP
kinase, JAK1, JAK2, JAK3, SYK, or any combination thereof.
[0144] In an embodiment of the invention, the IL-6 antagonist may
be coupled to a half-life increasing moiety.
[0145] In an embodiment of the invention, the method may include
measuring the patient's serum CRP level prior to administration of
the anti-IL-6 antibody, and administering the anti-IL-6 antibody or
antibody fragment or variant thereof if the patient's serum CRP
level is at least approximately 5 mg/L.
[0146] In an embodiment of the invention, the patient's serum CRP
level may be reduced to less than approximately 5 mg/L within 1
week of administration of the IL-6 antagonist.
[0147] In an embodiment of the invention, the patient's serum CRP
level may be reduced to below 1 mg/L within 1 week of
administration of the IL-6 antagonist.
[0148] In an embodiment of the invention, treatment may result in a
prolonged reduction in serum CRP level of the patient.
[0149] In an embodiment of the invention, the patient's serum CRP
level may be reduced to below 10 mg/L within about 1 week of IL-6
antagonist administration.
[0150] In an embodiment of the invention, 14 days after IL-6
antagonist administration the patient's serum CRP level may remain
below 10 mg/L.
[0151] In an embodiment of the invention, 21 days after IL-6
antagonist administration the patient's serum CRP level may remain
below 10 mg/L.
[0152] In an embodiment of the invention, 28 days after IL-6
antagonist administration the patient's serum CRP level may remain
below 10 mg/L.
[0153] In an embodiment of the invention, 35 days after IL-6
antagonist administration the patient's serum CRP level may remain
below 10 mg/L.
[0154] In an embodiment of the invention, 42 days after IL-6
antagonist administration the patient's serum CRP level may remain
below 10 mg/L.
[0155] In an embodiment of the invention, 49 days after IL-6
antagonist administration the patient's serum CRP level may remain
below 10 mg/L.
[0156] In an embodiment of the invention, 56 days after IL-6
antagonist administration the patient's serum CRP level may remain
below 10 mg/L.
[0157] In an embodiment of the invention, the patient's
survivability is improved.
[0158] In an embodiment of the invention, the method may include
measuring the patient's serum albumin level prior to administration
of the IL-6 antagonist, and administering the IL-6 antagonist such
as Ab1 if the patient's serum albumin level is less than
approximately 35 g/L.
[0159] In an embodiment of the invention, the patient's serum
albumin level may be increased to more than approximately 35 g/L
within about 5 weeks of administration of the IL-6 antagonist.
[0160] In an embodiment of the invention, treatment may result in a
prolonged increase in serum albumin level of the patient.
[0161] In an embodiment of the invention, 42 days after IL-6
antagonist administration the patient's serum albumin level may
remain above 35 g/L.
[0162] In an embodiment of the invention, 49 days after IL-6
antagonist administration the patient's serum albumin level may
remain above 35 g/L.
[0163] In an embodiment of the invention, 56 days after IL-6
antagonist administration the patient's serum albumin level may
remain above 35 g/L.
[0164] In an embodiment of the invention, the patient's serum
albumin level may be increased by about 5 g/L within approximately
5 weeks of administering the IL-6 antagonist.
[0165] In an embodiment of the invention, the patient may have been
diagnosed with rheumatoid arthritis, cancer, advanced cancer, liver
disease, renal disease, inflammatory bowel disease, celiac's
disease, trauma, burns, other diseases associated with reduced
serum albumin, or any combination thereof.
[0166] In an embodiment of the invention, the method may further
comprise administration of one or more statins to the patient,
including without limitation thereto atorvastatin, cerivastatin,
fluvastatin, lovastatin, mevastatin, pitavastatin, pravastatin,
rosuvastatin, simvastatin, or any combination thereof.
[0167] Another embodiment of the invention relates to a composition
comprising an IL-6 antagonist such as Ab1, and an anti-coagulant.
In an embodiment of the invention, the one or more anti-coagulants
may be selected from abciximab (ReoPro), acenocoumarol,
antithrombin III, argatroban, aspirin, bivalirudin (Angiomax),
clopidogrel, dabigatran, dabigatran etexilate (Pradaxa/Pradax),
desirudin (Revasc/Iprivask), dipyridamole, eptifibatide
(Integrilin), fondaparinux, heparin, hirudin, idraparinux,
lepirudin (Refludan), low molecular weight heparin, melagatran,
phenindione, phenprocoumon, ticlopidine, tirofiban (Aggrastat),
warfarin, ximelagatran, ximelagatran (Exanta/Exarta), or any
combination thereof.
[0168] Another embodiment of the invention relates to a composition
comprising an IL-6 antagonist such as Ab1, and a chemotherapy
agent. In an embodiment of the invention, the chemotherapy agent
may be selected from VEGF antagonists, EGFR antagonists, platins,
taxols, irinotecan, 5-fluorouracil, gemcytabine, leucovorine,
steroids, cyclophosphamide, melphalan, vinca alkaloids (e.g.,
vinblastine, vincristine, vindesine and vinorelbine), mustines,
tyrosine kinase inhibitors, radiotherapy, sex hormone antagonists,
selective androgen receptor modulators, selective estrogen receptor
modulators, PDGF antagonists, TNF antagonists, IL-1 antagonists,
interleukins (e.g. IL-12 or IL-2), IL-12R antagonists, Toxin
conjugated monoclonal antibodies, tumor antigen specific monoclonal
antibodies, Erbitux, Avastin, Pertuzumab, anti-CD20 antibodies,
Rituxan, ocrelizumab, ofatumumab, DXL625, herceptin, or any
combination thereof.
BRIEF DESCRIPTION OF THE DRAWINGS
[0169] FIG. 1 shows that a variety of unique epitopes were
recognized by the collection of anti-IL-6 antibodies prepared by
the antibody selection protocol. Epitope variability was confirmed
by antibody-IL-6 binding competition studies (ForteBio Octet).
[0170] FIG. 2 shows alignments of variable light and variable heavy
sequences between a rabbit antibody variable light and variable
heavy sequences and homologous human sequences and the final
humanized sequences. Framework regions are identified FR1-FR4.
Complementarity determining regions are identified as CDR1-CDR3.
Amino acid residues are numbered as shown. The initial rabbit
sequences are called RbtVL and RbtVH for the variable light and
variable heavy sequences respectively. Three of the most similar
human germline antibody sequences, spanning from Framework 1
through to the end of Framework 3, are aligned below the rabbit
sequences. The human sequence that is considered the most similar
to the rabbit sequence is shown first. In this example those most
similar sequences are L12A for the light chain and 3-64-04 for the
heavy chain. Human CDR3 sequences are not shown. The closest human
Framework 4 sequence is aligned below the rabbit Framework 4
sequence. The vertical dashes indicate a residue where the rabbit
residue is identical with one or more of the human residues at the
same position. The bold residues indicate that the human residue at
that position is identical to the rabbit residue at the same
position. The final humanized sequences are called VLh and VHh for
the variable light and variable heavy sequences respectively. The
underlined residues indicate that the residue is the same as the
rabbit residue at that position but different than the human
residues at that position in the three aligned human sequences.
[0171] FIG. 3 demonstrates the high correlation between the IgG
produced and antigen specificity for an exemplary IL-6 protocol. 9
of 11 wells showed specific IgG correlation with antigen
recognition.
[0172] FIG. 4 provides the .alpha.-2-macroglobulin (A2M) dose
response curve for antibody Ab1 administered intravenously at
different doses one hour after a 100 .mu.g/kg s.c. dose of human
IL-6.
[0173] FIG. 5 provides survival data for the antibody Ab1
progression groups versus control groups.
[0174] FIG. 6 provides additional survival data for the antibody
Ab1 regression groups versus control groups.
[0175] FIG. 7 provides survival data for polyclonal human IgG at 10
mg/kg i.v. every three days (270-320 mg tumor size) versus antibody
Ab1 at 10 mg/kg i.v. every three days (270-320 mg tumor size).
[0176] FIG. 8 provides survival data for polyclonal human IgG at 10
mg/kg i.v. every three days (400-527 mg tumor size) versus antibody
Ab1 at 10 mg/kg i.v. every three days (400-527 mg tumor size).
[0177] FIG. 9 provides a pharamcokinetic profile of antibody Ab1 in
cynomolgus monkey. Plasma levels of antibody Ab1 were quantitated
through antigen capture ELISA. This protein displays a half life of
between 12 and 17 days consistent with other full length humanized
antibodies.
[0178] FIG. 10 (A-D) provides binding data for antibodies Ab4, Ab3,
Ab8 and Ab2, respectively. FIG. 10 E provides binding data for
antibodies Ab1, Ab6 and Ab7.
[0179] FIG. 11 summarizes the binding data of FIG. 10 (A-E) in
tabular form.
[0180] FIG. 12 presents the sequences of the 15 amino acid peptides
used in the peptide mapping experiment of Example 14.
[0181] FIG. 13 presents the results of the blots prepared in
Example 14.
[0182] FIG. 14 presents the results of the blots prepared in
Example 14.
[0183] FIG. 15A shows affinity and binding kinetics of Ab1 for IL-6
of various species.
[0184] FIG. 15B demonstrates inhibition of IL-6 by Ab1 in the T1165
cell proliferation assay.
[0185] FIG. 16. shows the mean plasma concentration of Ab1
resulting from a single administration of Ab1 to healthy male
subjects in several dosage groups.
[0186] FIG. 17 shows mean area under the plasma Ab1 concentration
time curve (AUC) for the dosage groups shown in FIG. 16.
[0187] FIG. 18 shows mean peak plasma Ab1 concentration (C.sub.max)
for the dosage groups shown in FIG. 16.
[0188] FIG. 19 summarizes Ab1 pharmacokinetic measurements of the
dosage groups shown in FIG. 16.
[0189] FIG. 20 shows the mean plasma concentration of Ab1 resulting
from a single administration of Ab1 to patients with advanced
cancer.
[0190] FIG. 21 illustrates the unprecedented elimination half-life
of Ab1 compared with other anti-IL-6 antibodies.
[0191] FIG. 22 shows increased hemoglobin concentration following
administration of Ab1 to patients with advanced cancer.
[0192] FIG. 23 shows mean plasma lipid concentrations following
administration of Ab1 to patients with advanced cancer.
[0193] FIG. 24 shows mean neutrophil counts following
administration of Ab1 to patients with advanced cancer.
[0194] FIG. 25 demonstrates suppression of serum CRP levels in
healthy individuals.
[0195] FIG. 26 (A-B) demonstrates suppression of serum CRP levels
in advanced cancer patients.
[0196] FIG. 27 shows prevention of weight loss by Ab1 in a mouse
cancer cachexia model.
[0197] FIG. 28 shows the physical appearance of representative
Ab1-treated and control mice in a cancer cachexia model.
[0198] FIG. 29 demonstrates that Ab1 promotes weight gain in
advanced cancer patients.
[0199] FIG. 30 demonstrates that Ab1 reduces fatigue in advanced
cancer patients.
[0200] FIG. 31 demonstrates that Ab1 promotes hand grip strength in
advanced cancer patients.
[0201] FIG. 32 demonstrates that Ab1 suppresses an acute phase
protein (Serum Amyloid A) in mice.
[0202] FIG. 33 demonstrates that Ab1 increase plasma albumin
concentration in advanced cancer patients.
[0203] FIGS. 34 and 35 shows alignments between a rabbit antibody
light and variable heavy sequences and homologous human sequences
and the final humanized sequences. Framework regions are identified
FR1-FR4. Complementarity determining regions are identified as
CDR1-CDR3.
[0204] FIGS. 36 and 37 shows alignments between light and variable
heavy sequences, respectively, of different forms of Ab1. Framework
regions are identified FR1-FR4. Complementarity determining regions
are identified as CDR1-CDR3. Sequence differences within the CDR
regions highlighted.
DETAILED DESCRIPTION
Definitions
[0205] It is to be understood that this invention is not limited to
the particular methodology, protocols, cell lines, animal species
or genera, and reagents described, as such may vary. It is also to
be understood that the terminology used herein is for the purpose
of describing particular embodiments only, and is not intended to
limit the scope of the present invention which will be limited only
by the appended claims.
[0206] The term "variants" (as applied to antibodies including Ab1)
includes single-chain antibodies, dimers, multimers, sequene
variants, domain substitution variants, etc. Single-chain
antibodies such as SMIPs, shark antibodies, nanobodies (e.g.,
Camelidiae antibodies). Sequence variants can be specified by
percentage identity (or similarity) e.g., 99%, 95%, 90%, 85%, 80%,
70%, 60%, etc. or by numbers of permitted conservative or
non-conservative substitutions. Domain substitution variants
include replacement of a domain of one protein with a similar
domain of a related protein. A similar domain may be identified by
similarity of sequence, structure (actual or predicted), or
function. For example, domain substitution variants include the
substitution of one or more CDRs and/or framework regions.
[0207] As used herein the singular forms "a", "and", and "the"
include plural referents unless the context clearly dictates
otherwise. Thus, for example, reference to "a cell" includes a
plurality of such cells and reference to "the protein" includes
reference to one or more proteins and equivalents thereof known to
those skilled in the art, and so forth. All technical and
scientific terms used herein have the same meaning as commonly
understood to one of ordinary skill in the art to which this
invention belongs unless clearly indicated otherwise.
[0208] Interleukin-6 (IL-6): As used herein, interleukin-6 (IL-6)
encompasses not only the following 212 amino acid sequence
available as GenBank Protein Accession No. NP_000591:
MNSFSTSAFGPVAFSLGLLLVLPAAFPAPVPPGEDSKDVAAPHRQPLTSSERIDKQIRYIL
DGISALRKETCNKSNMCESSKEALAENNLNLPKMAEKDGCFQSGFNEETCLVKIITGLLE
FEVYLEYLQNRFESSEEQARAVQMSTKVLIQFLQKKAKNLDAITTPDPTTNASLLTKLQA
QNQWLQDMTTHLILRSFKEFLQSSLRALRQM (SEQ ID NO: 1), but also any
pre-pro, pro- and mature forms of this IL-6 amino acid sequence, as
well as mutants and variants including allelic variants of this
sequence.
[0209] IL-6 antagonist: As used herein, the terms "IL-6
antagonist," and grammatical variants thereof include any
composition that prevents, inhibits, or lessens the effect(s) of
IL-6 signaling. Generally, such antagonists may reduce the levels
or activity of IL-6, IL-6 receptor alpha, gp130, or a molecule
involved in IL-6 signal transduction, or may reduce the levels or
activity complexes between the foregoing (e.g., reducing the
activity of an IL-6/IL-6 receptor complex). Antagonists include
antisense nucleic acids, including DNA, RNA, or a nucleic acid
analogue such as a peptide nucleic acid, locked nucleic acid,
morpholino (phosphorodiamidate morpholino oligo), glycerol nucleic
acid, or threose nucleic acid. See Heasman, Dev Biol. 2002 Mar. 15;
243(2):209-14; Hannon and Rossi, Nature. 2004 Sep. 16;
431(7006):371-8; Paul et al., Nat Biotechnol. 2002 May;
20(5):505-8; Zhang et al., J Am Chem Soc. 2005 Mar. 30;
127(12):4174-5; Wahlestedt et al., Proc Natl Acad Sci USA. 2000 May
9; 97(10):5633-8; Hanvey et al., 1992 Nov. 27; 258(5087):1481-5;
Braasch et al., Biochemistry. 2002 Apr. 9; 41(14):4503-10; Schoning
et al., Science. 2000 Nov. 17; 290(5495):1347-51. In addition IL-6
antagonists specifically include peptides that block IL-6 signaling
such as those described in any of U.S. Pat. Nos. 6,599,875;
6,172,042; 6,838,433; 6,841,533; 5,210,075 et al. Also, IL-6
antagonists according to the invention may include p38 MAP kinase
inhibitors such as those reported in US20070010529 et al. given
this kinase's role in cytokine production and more particularly
IL-6 production. Further, IL-6 antagonosts according to the
invention include the glycoalkaloid compounds reported in
US20050090453 as well as other IL-6 antagonist compounds isolatable
using the IL-6 antagononist screening assays reported therein.
Other IL-6 antagonists include antibodies, such as anti-IL-6
antibodies, anti-IL-6 receptor alpha antibodies, anti-gp130
antibodies, and anti-p38 MAP kinase antibodies including (but not
limited to) the anti-IL-6 antibodies disclosed herein, Actemra
(Tocilizumab), Remicade, Zenapax, or any combination thereof. Other
IL-6 antagonists include portions or fragments of molecules
involved in IL-6 signaling, such as IL-6, IL-6 receptor alpha, and
gp130, which may be native, mutant, or variant sequence, and may
optionally be coupled to other moieties (such as
half-life-increasing moieties, e.g. an Fc domain). For example, an
IL-6 antagonist may be a soluble IL-6 receptor or fragment, a
soluble IL-6 receptor:Fc fusion protein, a small molecule inhibitor
of IL-6, an anti-IL-6 receptor antibody or antibody fragment or
variant thereof, antisense nucleic acid, etc. Other IL-6
antagonists include avemirs, such as C326 (Silverman et al., Nat
Biotechnol. 2005 December; 23(12):1556-61) and small molecules,
such as synthetic retinoid AM80 (tamibarotene) (Takeda et al.,
Arterioscler Thromb Vasc Biol. 2006 May; 26(5):1177-83). Such IL-6
antagonists may be administered by any means known in the art,
including contacting a subject with nucleic acids which encode or
cause to be expressed any of the foregoing polypeptides or
antisense sequences.
[0210] Thrombosis: As used herein, thrombosis refers to a thrombus
(blood clot) inside a blood vessel. The term encompasses, without
limitation, arterial and venous thrombosis, including deep vein
thrombosis, portal vein thrombosis, jugular vein thrombosis, renal
vein thrombosis, stroke, myocardial infarction, Budd-Chiari
syndrome, Paget-Schroetter disease, and cerebral venous sinus
thrombosis. Diseases and conditions associated with thrombosis
include, without limitation, acute venous thrombosis, pulmonary
embolism, thrombosis during pregnancy, hemorrhagic skin necrosis,
acute or chronic disseminated intravascular coagulation (DIC), clot
formation from surgery, long bed rest, long periods of
immobilization, venous thrombosis, fulminant meningococcemia, acute
thrombotic stroke, acute coronary occlusion, acute peripheral
arterial occlusion, massive pulmonary embolism, axillary vein
thrombosis, massive iliofemoral vein thrombosis, occluded arterial
cannulae, occluded venous cannulae, cardiomyopathy, venoocclusive
disease of the liver, hypotension, decreased cardiac output,
decreased vascular resistance, pulmonary hypertension, diminished
lung compliance, leukopenia, and thrombocytopenia.
[0211] D-Dimer: As used herein, D-dimer refers to a fibrin
degradation product produced during the break down of blood clots
by the enzyme plasmin. Monoclonal antibodies specifically reactive
against D-dimer are readily available, e.g. DD-3B6/22 (Elms et al.,
1986, Am J Clin Pathol. 85:360-4). Clinical measurements of D-dimer
levels are routinely performed, e.g., using a red blood cell
agglutination test, ELISA, etc. (reviewed in Dempfle, Semin Vasc
Med, 2005 November; 5(4):315-20). Measurements of D-dimer may vary
depending on the measurement method and testing lab; nonetheless, a
normal "reference range" may be readily established for any
particular method and testing lab, e.g. by taking measurements from
healthy individuals. Accordingly, an elevated D-dimer level is
understood by persons skilled in the art to refer to a D-dimer
level that is above the reference range for the partcular method
and testing lab.
[0212] Coagulation profile: As used herein, coagulation profile
refers generally to the functioning of the coagulation system. Both
the tissue factor (extrinsic) and contact activation (intrinsic)
pathways of clotting are components of the coagulation profile. A
normal coagulation profile refers to coagulation functioning as in
a normal, healthy individual, i.e., maintaining balance between
ability to control bleeding and tendency towards excessive clotting
(thrombotic tendency). An abnormal coagulation profile may be a
decrease or an increase in coagulation tendency. One particularly
abnormal coagulation profile is hypercoagulation, which refers to a
greatly increased risk of excessive clot formation, resulting in
high risk of thrombosis. Coagulation profile may be assessed by
various tests and assays known in the art, such as: the activated
partial thromboplastin time (aPTT) test; prothrombin time (PT) test
(typical reference range of 12 to 15 second); measurements derived
from the PT test, such as prothrombin ratio (PR) and international
normalized ratio (INR) (typical reference range 0.8 to 1.2);
fibrinogen testing (e.g. the Clauss method (Clauss A, "Rapid
Physiological Coagulation Method for the Determination of
Fibrinogen [German]," Acta Haematol, 1957, 17:237-46) or the Ellis
method (Ellis B C and Stransky A, "A Quick and Accurate Method for
the Determination of Fibrinogen in Plasma," J Lab Clin Med, 1961,
58:477-88); assays for activated protein C resistance, protein C,
protein S, and antithrombin; assays for antiphospholipid antibodies
(lupus anticoagulant and anticardiolipin antibodies); elevated
homocysteine; assays for plasminogen, dysfibrinogenemia, heparin
cofactor II, or platelet hyperaggregability. Other assays useful to
assess coagulation profile include measurement of clotting factors
and/or indicators of clotting, such as serum levels of D-dimer,
Factor II, Factor V, Factor VIII, Factor IX, Factor XI, Factor XII,
F/fibrin degradation products, thrombin-antithrombin III complex,
thrombocytosis, fibrinogen, plasminogen, prothrombin, and von
Willebrand factor. Worsening in coagulation profile refers to a
measureable change in an indicator of coagulation, e.g., any of the
aforementioned assays, that reflects a deterioration of the normal
coagulation tendency, such that the measured value becomes abnormal
or deviates farther from the normal range than previously.
Improvement in coagulation profile refers to a measureable change
in an indicator of coagulation, e.g., any of the aforementioned
assays, that reflects a partial or full restoration of the normal
coagulation tendency, i.e., after a therapeutic intervention, such
as administration of an anti-IL-6 antibody, the measured value is
in the normal range or closer to the normal range than prior to the
therapeutic intervention.
[0213] Disease or condition: As used herein, "disease or condition"
refers to a disease or condition that a patient has been diagnosed
with or is suspected of having, particularly a disease or condition
associated with elevated IL-6. A disease or condition encompasses,
without limitation thereto, the side-effects of medications or
treatments (such as radiation therapy), as well as idiopathic
conditions characterized by symptoms that include elevated
IL-6.
[0214] Cachexia: As used herein, cachexia, also known as wasting
disease, refers to any disease marked especially by progressive
emaciation, weakness, general ill health, malnutrition, loss of
body mass, loss of muscle mass, or an accelerated loss of skeletal
muscle in the context of a chronic inflammatory response (reviewed
in Kotler, Ann Intern Med. 2000 Oct. 17; 133(8):622-34). Diseases
and conditions in which cachexia is frequently observed include
cancer, rheumatoid arthritis, AIDS, heart disease, dehydration,
malnutrition, lead exposure, malaria, respiratory disease, old age,
hypothyroidism, tuberculosis, hypopituitarism, neurasthenia,
hypernatremia, hyponatremia, renal disease, splenica, ankylosing
spondylitis, failure to thrive (faltering growth) and other
diseases, particularly chronic diseases. Cachexia may also be
idiopathic (arising from an uncertain cause). Weight assessment in
a patient is understood to exclude growths or fluid accumulations,
e.g. tumor weight, extravascular fluid accumulation, etc. Cachexia
may be assessed by measureent of a patient's total body mass
(exclusive of growths or fluid accumulations), total lean
(fat-free) body mass, lean mass of the arms and legs (appendicular
lean mass, e.g. measured using dual-energy x-ray absorptiometry or
bioelectric impedance spectroscopy), and/or lean body mass index
(lean body mass divided by the square of the patient's height). See
Kotler, Ann Intern Med. 2000 Oct. 17; 133(8):622-34; Marcora et
al., Rheumatology (Oxford). 2006 November; 45(11):1385-8.
[0215] Weakness: As used herein, weakness refers physical fatigue,
which typically manifests as a loss of muscle strength and/or
endurance. Weakness may be central (affecting most or all of the
muscles in the body) or peripheral (affecting a subset of muscles).
Weakness includes "true weakness," in which a patient's muscles
have a decrease in some measure of peak and/or sustained force
output, and "perceived weakness," in which a patient perceives that
a greater effort is required for performance of a task even though
objectively measured strength remains nearly the same, and may be
objectively measured or self-reported by the patient. For example,
weakness may be objectively measured using the hand grip strength
test (a medically recognized test for evaluating muscle strength),
typically employing a handgrip dynamometer.
[0216] Fatigue: As used herein, fatigue refers to mental fatigue
(for physical fatigue see "weakness"). Fatigue includes drowsiness
(somnolence) and/or decreased attention. Fatigue may be measured
using a variety of tests known in the art, such as the FACIT-F
(Functional Assessment of Chronic Illness Therapy-Fatigue) test.
See, e.g., Cella, D., Lai, J. S., Chang, C. H., Peterman, A., &
Slavin, M. (2002). Fatigue in cancer patients compared with fatigue
in the general population. Cancer, 94(2), 528-538; Cella, D., Eton,
D. T., Lai, F J-S., Peterman, A. H & Merkel, D. E. (2002).
Combining anchor and distribution based methods to derive minimal
clinically important differences on the Functional Assessment of
Cancer Therapy anemia and fatigue scales. Journal of Pain &
Symptom Management, 24 (6) 547-561.
[0217] Fever: As used herein, "fever" refers to a body temperature
set-point that is elevated by at least 1 to 2 degrees Celsius.
Fever is often associated with a subjective feeling of hypothermia
exhibited as a cold sensation, shivering, increased heart rate and
respiration rate by which the individual's body reaches the
increased set-point. As is well understood in the medical arts,
normal body temperature typically varies with activity level and
time of day, with highest temperatures observed in the afternoon
and early evening hours, and lowest temperatures observed during
the second half of the sleep cycle, and temperature measurements
may be influenced by external factors such as mouth breathing,
consumption of food or beverage, smoking, or ambient temperature
(depending on the type of measurement). Moreover, the normal
temperature set point for individuals may vary by up to about 0.5
degrees Celsius, thus a medical professional may interpret an
individual's temperature in view of these factors to diagnose
whether a fever is present. Generally speaking, a fever is
typically diagnosed by a core body temperature above 38.0 degrees
Celsius, an oral temperature above 37.5 degrees Celsius, or an
axillary temperature above 37.2 degrees Celsius.
[0218] Improved: As used herein, "improved," "improvement," and
other grammatical variants, includes any beneficial change
resulting from a treatment. A beneficial change is any way in which
a patient's condition is better than it would have been in the
absence of the treatment. "Improved" includes prevention of an
undesired condition, slowing the rate at which a condition worsens,
delaying the development of an undesired condition, and restoration
to an essentially normal condition. For example, improvement in
cachexia encompasses any increase in patient's mass, such as total
body mass (excluding weight normally excluded during assessment of
cachexia, e.g. tumor weight, extravascular fluid accumulation,
etc.), lean body mass, and/or appendicular lean mass, as well as
any delay or slowing in the rate of loss of mass, or prevention or
slowing of loss of mass associated with a disease or condition with
which the patient has been diagnosed. For another example,
improvement in weakness encompasses any increase in patient's
strength, as well as any delay or slowing in the rate of loss of
strength, or prevention or slowing of loss of strength associated
with a disease or condition with which the patient has been
diagnosed. For yet another example, improvement in fatigue
encompasses any decrease in patient's fatigue, as well as any delay
or slowing in the rate of increase of fatigue, or prevention or
slowing of increase in fatigue associated with a disease or
condition with which the patient has been diagnosed. For still
another example, improvement in fever encompasses any decrease in
patient's fever, as well as any delay or slowing in the rate of
increase in fever, or prevention or slowing of increase in fever
associated with a disease or condition with which the patient has
been diagnosed.
[0219] C-Reactive Protein (CRP): As used herein, C-Reactive Protein
(CRP) encompasses not only the following 224 amino acid sequence
available as GenBank Protein Accession No. NP_000558:
[0220] MEKLLCFLVLTSLSHAFGQTDMSRKAFVFPKESDTSYVSLKAPLTKPLKA
FTVCLHFYTELSSTRGYSIFSYATKRQDNEILIFWSKDIGYSFTVGGSEILFEVPEVTVAPV
HICTSWESASGIVEFWVDGKPRVRKSLKKGYTVGAEASIILGQEQDSFGGNFEGSQSLVG
DIGNVNMWDFVLSPDEINTIYLGGPFSPNVLNWRALKYEVQGEVFTKPQLWP (SEQ ID NO:
726), but also any pre-pro, pro- and mature forms of this CRP amino
acid sequence, as well as mutants and variants including allelic
variants of this sequence. CRP levels, e.g. in the serum, liver,
tumor, or elsewhere in the body, can be readily measured using
routine methods and commercially available reagents, e.g. ELISA,
antibody test strip, immunoturbidimetry, rapid immunodiffusion,
visual agglutination, Western blot, Northern blot, etc. As
mentioned above CRP levels may in addition be measured in patients
having or at risk of developing thrombosis according to the
invention.
[0221] Interleukin-6 receptor (IL-6R); also called IL-6 receptor
alpha (IL-6RA): As used herein, "interleukin-6 receptor" ("IL-6R";
also "IL-6 receptor alpha" or "IL-6RA") encompasses not only the
following 468 amino acid sequence available as Swiss-Prot Protein
Accession No. P08887:
MLAVGCALLAALLAAPGAALAPRRCPAQEVARGVLTSLPGDSVTLTCPGVEPEDNATV
HWVLRKPAAGSHPSRWAGMGRRLLLRSVQLEIDSGNYSCYRAGRPAGTVHLLVDVPPE
EPQLSCFRKSPLSNVVCEWGPRSTPSLTTKAVLLVRKFQNSPAEDFQEPCQYSQESQKFS
CQLAVPEGDSSFYIVSMCVASSVGSKFSKTQTFQGCGILQPDPPANITVTAVARNPRWLS
VTWQDPHSWNSSFYRLRFELRYRAERSKTFTTWMVKDLQHHCVIHDAWSGLRHVVQL
RAQEEFGQGEWSEWSPEAMGTPWTESRSPPAENEVSTPMQALTTNKDDDNILFRDSAN
ATSLPVQDSSSVPLPTFLVAGGSLAFGTLLCIAIVLRFKKTWKLRALKEGKTSMHPPYSL
GQLVPERPRPTPVLVPLISPPVSPSSLGSDNTSSHNRPDARDPRSPYDISNTDYFFPR (SEQ ID
NO: 727), but also any pre-pro, pro- and mature forms of this amino
acid sequence, as well as mutants and variants including allelic
variants of this sequence.
[0222] gp130: As used herein, gp130 (also called Interleukin-6
receptor subunit beta) encompasses not only the following 918
precursor amino acid sequence available as Swiss-Prot Protein
Accession No. P40189:
MLTLQTWVVQALFIFLTTESTGELLDPCGYISPESPVVQLHSNFTAVCVLKEKCMDYFH
VNANYIVWKTNHFTIPKEQYTIINRTASSVTFTDIASLNIQLTCNILTFGQLEQNVYGITIIS
GLPPEKPKNLSCIVNEGKKMRCEWDGGRETHLETNFTLKSEWATHKFADCKAKRDTPT
SCTVDYSTVYFVNIEVWVEAENALGKVTSDHINFDPVYKVKPNPPHNLSVINSEELSSIL
KLTWTNPSIKSVIILKYNIQYRTKDASTWSQIPPEDTASTRSSFTVQDLKPFTEYVFRIRC
MKEDGKGYWSDWSEEASGITYEDRPSKAPSFWYKIDPSHTQGYRTVQLVWKTLPPFEA
NGKILDYEVTLTRWKSHLQNYTVNATKLTVNLTNDRYLATLTVRNLVGKSDAAVLTIP
ACDFQATHPVMDLKAFPKDNMLWVEWTTPRESVKKYILEWCVLSDKAPCITDWQQED
GTVHRTYLRGNLAESKCYLITVTPVYADGPGSPESIKAYLKQAPPSKGPTVRTKKVGKN
EAVLEWDQLPVDVQNGFIRNYTIFYRTIIGNETAVNVDSSHTEYTLSSLTSDTLYMVRM
AAYTDEGGKDGPEFTFTTPKFAQGEIEAIVVPVCLAFLLTTLLGVLFCFNKRDLIKKHIWP
NVPDPSKSHIAQWSPHTPPRHNFNSKDQMYSDGNFTDVSVVEIEANDKKPFPEDLKSLD
LFKKEKINTEGHSSGIGGSSCMSSSRPSISSSDENESSQNTSSTVQYSTVVHSGYRHQVPS
VQVFSRSESTQPLLDSEERPEDLQLVDHVDGGDGILPRQQYFKQNCSQUESSPDISHFER
SKQVSSVNEEDFVRLKQQISDHISQSCGSGQMKMFQEVSAADAFGPGTEGQVERFETVG
MEAATDEGMPKSYLPQTVRQGGYMPQ (SEQ ID NO: 728), but also any pre-pro,
pro- and mature forms of this amino acid sequence, such as the
mature form encoded by amino acids 23 through 918 of the sequence
shown, as well as mutants and variants including allelic variants
of this sequence.
[0223] Glasgow Prognostic Score (GPS): As used herein, Glasgow
Prognostic Score (GPS) refers to an inflammation-based prognostic
score that awards one point for a serum albumin level less than
<35 mg/L and one point for a CRP level above 10 mg/L. Thus, a
GPS of 0 indicates normal albumin and CRP, a GPS of 1 indicates
reduced albumin or elevated CRP, and a GPS of 2 indicates both
reduced albumin and elevated CRP.
[0224] Effective amount: As used herein, "effective amount,"
"amount effective to," "amount of X effective to" and the like,
refer to an amount of an active ingredient that is effective to
relieve or reduce to some extent one or more of the symptoms of the
disease in need of treatment, or to retard initiation of clinical
markers or symptoms of a disease in need of prevention, when the
compound is administered. Thus, an effective amount refers to an
amount of the active ingredient which exhibit effects such as (i)
reversing the rate of progress of a disease; (ii) inhibiting to
some extent further progress of the disease; and/or, (iii)
relieving to some extent (or, preferably, eliminating) one or more
symptoms associated with the disease. The effective amount may be
empirically determined by experimenting with the compounds
concerned in known in vivo and in vitro model systems for a disease
in need of treatment. The context in which the phrase "effective
amount" is used may indicate a particular desired effect. For
example, "an amount of an anti-IL-6 antibody effective to prevent
or treat a hypercoagulable state" and similar phrases refer to an
amount of anti-IL-6 antibody that, when administered to a subject,
will cause a measurable improvement in the subject's coagulation
profile, or prevent, slow, delay, or arrest, a worsening of the
coagulation profile for which the subject is at risk. Similarly,
"an amount of an anti-IL-6 antibody effective to reduce serum CRP
levels" and similar phrases refer to an amount of anti-IL-6
antibody that, when administered to a subject, will cause a
measurable decrease in serum CRP levels, or prevent, slow, delay,
or arrest, an increase in serum CRP levels for which the subject is
at risk. Similarly, "an amount of an anti-IL-6 antibody effective
to increase serum albumin levels" and similar phrases refer to an
amount of anti-IL-6 antibody that, when administered to a subject,
will cause a measurable increase in serum albumin levels, or
prevent, slow, delay, or arrest, a decrease in serum albumin levels
for which the subject is at risk. Similarly, "an amount of an
anti-IL-6 antibody effective to reduce weakness" and similar
phrases refer to an amount of anti-IL-6 antibody that, when
administered to a subject, will cause a measurable decrease in
weakness as determined by the hand grip strength test. Similarly,
"an amount of an anti-IL-6 antibody effective to increase weight"
and similar phrases refer to an amount of anti-IL-6 antibody that,
when administered to a subject, will cause a measurable increase in
a patient's weight. An effective amount will vary according to the
weight, sex, age and medical history of the individual, as well as
the severity of the patient's condition(s), the type of disease(s),
mode of administration, and the like. An effective amount may be
readily determined using routine experimentation, e.g., by
titration (administration of increasing dosages until an effective
dosage is found) and/or by reference to amounts that were effective
for prior patients. Generally, the anti-IL-6 antibodies of the
present invention will be administered in dosages ranging between
about 0.1 mg/kg and about 20 mg/kg of the patient's
body-weight.
[0225] Prolonged improvement in coagulation profile: As used
herein, "prolonged improvement in coagulation profile" and similar
phrases refer to a measurable improvement in the subject's
coagulation profile relative to the initial coagulation profile
(i.e. the coagulation profile at a time before treatment begins)
that is detectable within about a week from when treatment begins
(e.g. administration of an IL-6 antagonist such as AbI) and remains
improved for a prolonged duration, e.g., at least about 14 days, at
least about 21 days, at least about 28 days, at least about 35
days, at least about 40 days, at least about 50 days, at least
about 60 days, at least about 70 days, at least about 11 weeks, or
at least about 12 weeks from when the treatment begins.
[0226] Prolonged reduction in serum CRP: As used herein, "prolonged
reduction in serum CRP" and similar phrases refer to a measurable
decrease in serum CRP level relative to the initial serum CRP level
(i.e. the serum CRP level at a time before treatment begins) that
is detectable within about a week from when a treatment begins
(e.g. administration of an anti-IL-6 antibody) and remains below
the initial serum CRP level for an prolonged duration, e.g. at
least about 14 days, at least about 21 days, at least about 28
days, at least about 35 days, at least about 40 days, at least
about 50 days, at least about 60 days, at least about 70 days, at
least about 11 weeks, or at least about 12 weeks from when the
treatment begins.
[0227] Prolonged increase in serum albumin: As used herein,
"prolonged increase in serum albumin" and similar phrases refer to
a measurable decrease in serum albumin level relative to the
initial serum albumin level (i.e. the serum albumin level at a time
before treatment begins) that is detectable within about a week
from when a treatment begins (e.g. administration of an anti-IL-6
antibody) and remains above the initial serum albumin level for an
prolonged duration, e.g. at least about 14 days, at least about 21
days, at least about 28 days, at least about 35 days, at least
about 40 days, at least about 50 days, at least about 60 days, at
least about 70 days, at least about 11 weeks, or at least about 12
weeks from when the treatment begins.
[0228] Prolonged improvement in cachexia: As used herein,
"prolonged improvement in cachexia" refers to a measureable
improvement patient's body mass, lean body mass, apendicular lean
body mass, and/or lean body mass index, relative to the initial
level (i.e. the level at a time before treatment begins) that is
detectable within about 4 weeks and remains improved for a
prolonged duration, e.g. at least about 35 days, at least about 40
days, at least about 50 days, at least about 60 days, at least
about 70 days, at least about 11 weeks, or at least about 12 weeks
from when the treatment begins.
[0229] Prolonged improvement in weakness: As used herein,
"prolonged improvement in weakness" refers to a measureable
improvement in muscular strength, relative to the initial level
(i.e. the level at a time before treatment begins) that is
detectable within about 2 weeks and remains improved for a
prolonged duration, e.g. at least about 21 days, at least about 28
days, at least about 35 days, at least about 40 days, at least
about 50 days, at least about 60 days, at least about 70 days, at
least about 11 weeks, or at least about 12 weeks from when the
treatment begins.
[0230] Prolonged improvement in fatigue: As used herein, "prolonged
improvement in fatigue" refers to a measureable improvement in
fatigue, relative to the initial level (i.e. the level at a time
before treatment begins) that is detectable within about 1 week and
remains improved for a prolonged duration, e.g. at least about 14
days, at least about 21 days, at least about 28 days, at least
about 35 days, at least about 40 days, at least about 50 days, at
least about 60 days, at least about 70 days, at least about 11
weeks, or at least about 12 weeks from when the treatment
begins.
[0231] Prolonged improvement in fever: As used herein, "prolonged
improvement in fever" refers to a measureable decrease in fever
(e.g. peak temperature or amount of time that temperature is
elevated), relative to the initial level (i.e. the level at a time
before treatment begins) that is detectable within about 1 week and
remains improved for a prolonged duration, e.g. at least about 14
days, at least about 21 days, at least about 28 days, at least
about 35 days, at least about 40 days, at least about 50 days, at
least about 60 days, at least about 70 days, at least about 11
weeks, or at least about 12 weeks from when the treatment
begins.
[0232] Mating competent yeast species: In the present invention
this is intended to broadly encompass any diploid or tetraploid
yeast which can be grown in culture. Such species of yeast may
exist in a haploid, diploid, or tetraploid form. The cells of a
given ploidy may, under appropriate conditions, proliferate for
indefinite number of generations in that form. Diploid cells can
also sporulate to form haploid cells. Sequential mating can result
in tetraploid strains through further mating or fusion of diploid
strains. In the present invention the diploid or polyploidal yeast
cells are preferably produced by mating or spheroplast fusion.
[0233] In one embodiment of the invention, the mating competent
yeast is a member of the Saccharomycetaceae family, which includes
the genera Arxiozyma; Ascobotryozyma; Citeromyces; Debaryomyces;
Dekkera; Eremothecium; Issatchenkia; Kazachstania; Kluyveromyces;
Kodamaea; Lodderomyces; Pachysolen; Pichia; Saccharomyces;
Saturnispora; Tetrapisispora; Torulaspora; Williopsis; and
Zygosaccharomyces. Other types of yeast potentially useful in the
invention include Yarrowia, Rhodosporidium, Candida, Hansenula,
Filobasium, Filobasidellla, Sporidiobolus, Bullera, Leucosporidium
and Filobasidella.
[0234] In a preferred embodiment of the invention, the mating
competent yeast is a member of the genus Pichia. In a further
preferred embodiment of the invention, the mating competent yeast
of the genus Pichia is one of the following species: Pichia
pastoris, Pichia methanolica, and Hansenula polymorpha (Pichia
angusta). In a particularly preferred embodiment of the invention,
the mating competent yeast of the genus Pichia is the species
Pichia pastoris.
[0235] Haploid Yeast Cell: A cell having a single copy of each gene
of its normal genomic (chromosomal) complement.
[0236] Polyploid Yeast Cell: A cell having more than one copy of
its normal genomic (chromosomal) complement.
[0237] Diploid Yeast Cell: A cell having two copies (alleles) of
essentially every gene of its normal genomic complement, typically
formed by the process of fusion (mating) of two haploid cells.
[0238] Tetraploid Yeast Cell: A cell having four copies (alleles)
of essentially every gene of its normal genomic complement,
typically formed by the process of fusion (mating) of two haploid
cells. Tetraploids may carry two, three, four, or more different
expression cassettes. Such tetraploids might be obtained in S.
cerevisiae by selective mating homozygotic heterothallic a/a and
alpha/alpha diploids and in Pichia by sequential mating of haploids
to obtain auxotrophic diploids. For example, a [met his] haploid
can be mated with [ade his] haploid to obtain diploid [his]; and a
[met arg] haploid can be mated with [ade arg] haploid to obtain
diploid [arg]; then the diploid [his].times.diploid [arg] to obtain
a tetraploid prototroph. It will be understood by those of skill in
the art that reference to the benefits and uses of diploid cells
may also apply to tetraploid cells.
[0239] Yeast Mating: The process by which two haploid yeast cells
naturally fuse to form one diploid yeast cell.
[0240] Meiosis: The process by which a diploid yeast cell undergoes
reductive division to form four haploid spore products. Each spore
may then germinate and form a haploid vegetatively growing cell
line.
[0241] Selectable Marker: A selectable marker is a gene or gene
fragment that confers a growth phenotype (physical growth
characteristic) on a cell receiving that gene as, for example
through a transformation event. The selectable marker allows that
cell to survive and grow in a selective growth medium under
conditions in which cells that do not receive that selectable
marker gene cannot grow. Selectable marker genes generally fall
into several types, including positive selectable marker genes such
as a gene that confers on a cell resistance to an antibiotic or
other drug, temperature when two ts mutants are crossed or a ts
mutant is transformed; negative selectable marker genes such as a
biosynthetic gene that confers on a cell the ability to grow in a
medium without a specific nutrient needed by all cells that do not
have that biosynthetic gene, or a mutagenized biosynthetic gene
that confers on a cell inability to grow by cells that do not have
the wild type gene; and the like. Suitable markers include but are
not limited to: ZEO; G418; LYS3; MET1; MET3a; ADE1; ADE3; URA3; and
the like.
[0242] Expression Vector: These DNA vectors contain elements that
facilitate manipulation for the expression of a foreign protein
within the target host cell. Conveniently, manipulation of
sequences and production of DNA for transformation is first
performed in a bacterial host, e.g. E. coli, and usually vectors
will include sequences to facilitate such manipulations, including
a bacterial origin of replication and appropriate bacterial
selection marker. Selection markers encode proteins necessary for
the survival or growth of transformed host cells grown in a
selective culture medium. Host cells not transformed with the
vector containing the selection gene will not survive in the
culture medium. Typical selection genes encode proteins that (a)
confer resistance to antibiotics or other toxins, (b) complement
auxotrophic deficiencies, or (c) supply critical nutrients not
available from complex media. Exemplary vectors and methods for
transformation of yeast are described, for example, in Burke, D.,
Dawson, D., & Stearns, T. (2000). Methods in yeast genetics: a
Cold Spring Harbor Laboratory course manual. Plainview, N.Y.: Cold
Spring Harbor Laboratory Press.
[0243] Expression vectors for use in the methods of the invention
will further include yeast specific sequences, including a
selectable auxotrophic or drug marker for identifying transformed
yeast strains. A drug marker may further be used to amplify copy
number of the vector in a yeast host cell.
[0244] The polypeptide coding sequence of interest is operably
linked to transcriptional and translational regulatory sequences
that provide for expression of the polypeptide in yeast cells.
These vector components may include, but are not limited to, one or
more of the following: an enhancer element, a promoter, and a
transcription termination sequence. Sequences for the secretion of
the polypeptide may also be included, e.g. a signal sequence, and
the like. A yeast origin of replication is optional, as expression
vectors are often integrated into the yeast genome.
[0245] In one embodiment of the invention, the polypeptide of
interest is operably linked, or fused, to sequences providing for
optimized secretion of the polypeptide from yeast diploid
cells.
[0246] Nucleic acids are "operably linked" when placed into a
functional relationship with another nucleic acid sequence. For
example, DNA for a signal sequence is operably linked to DNA for a
polypeptide if it is expressed as a preprotein that participates in
the secretion of the polypeptide; a promoter or enhancer is
operably linked to a coding sequence if it affects the
transcription of the sequence. Generally, "operably linked" means
that the DNA sequences being linked are contiguous, and, in the
case of a secretory leader, contiguous and in reading frame.
However, enhancers do not have to be contiguous. Linking is
accomplished by ligation at convenient restriction sites or
alternatively via a PCR/recombination method familiar to those
skilled in the art (Gateway.RTM. Technology; Invitrogen, Carlsbad
Calif.). If such sites do not exist, the synthetic oligonucleotide
adapters or linkers are used in accordance with conventional
practice.
[0247] Promoters are untranslated sequences located upstream (5')
to the start codon of a structural gene (generally within about 100
to 1000 bp) that control the transcription and translation of
particular nucleic acid sequences to which they are operably
linked. Such promoters fall into several classes: inducible,
constitutive, and repressible promoters (that increase levels of
transcription in response to absence of a repressor). Inducible
promoters may initiate increased levels of transcription from DNA
under their control in response to some change in culture
conditions, e.g., the presence or absence of a nutrient or a change
in temperature.
[0248] The yeast promoter fragment may also serve as the site for
homologous recombination and integration of the expression vector
into the same site in the yeast genome; alternatively a selectable
marker is used as the site for homologous recombination. Pichia
transformation is described in Cregg et al. (1985) Mol. Cell. Biol.
5:3376-3385.
[0249] Examples of suitable promoters from Pichia include the AOX1
and promoter (Cregg et al. (1989) Mol. Cell. Biol. 9:1316-1323);
ICL1 promoter (Menendez et al. (2003) Yeast 20(13):1097-108);
glyceraldehyde-3-phosphate dehydrogenase promoter (GAP) (Waterham
et al. (1997) Gene 186(1):37-44); and FLD1 promoter (Shen et al.
(1998) Gene 216(1):93-102). The GAP promoter is a strong
constitutive promoter and the AOX and FLD1 promoters are
inducible.
[0250] Other yeast promoters include ADH1, alcohol dehydrogenase
II, GAL4, PHO3, PHOS, Pyk, and chimeric promoters derived
therefrom. Additionally, non-yeast promoters may be used in the
invention such as mammalian, insect, plant, reptile, amphibian,
viral, and avian promoters. Most typically the promoter will
comprise a mammalian promoter (potentially endogenous to the
expressed genes) or will comprise a yeast or viral promoter that
provides for efficient transcription in yeast systems.
[0251] The polypeptides of interest may be produced recombinantly
not only directly, but also as a fusion polypeptide with a
heterologous polypeptide, e.g. a signal sequence or other
polypeptide having a specific cleavage site at the N-terminus of
the mature protein or polypeptide. In general, the signal sequence
may be a component of the vector, or it may be a part of the
polypeptide coding sequence that is inserted into the vector. The
heterologous signal sequence selected preferably is one that is
recognized and processed through one of the standard pathways
available within the host cell. The S. cerevisiae alpha factor
pre-pro signal has proven effective in the secretion of a variety
of recombinant proteins from P. pastoris. Other yeast signal
sequences include the alpha mating factor signal sequence, the
invertase signal sequence, and signal sequences derived from other
secreted yeast polypeptides. Additionally, these signal peptide
sequences may be engineered to provide for enhanced secretion in
diploid yeast expression systems. Other secretion signals of
interest also include mammalian signal sequences, which may be
heterologous to the protein being secreted, or may be a native
sequence for the protein being secreted. Signal sequences include
pre-peptide sequences, and in some instances may include propeptide
sequences. Many such signal sequences are known in the art,
including the signal sequences found on immunoglobulin chains,
e.g., K28 preprotoxin sequence, PHA-E, FACE, human MCP-1, human
serum albumin signal sequences, human Ig heavy chain, human Ig
light chain, and the like. For example, see Hashimoto et. al.
Protein Eng 11(2) 75 (1998); and Kobayashi et. al. Therapeutic
Apheresis 2(4) 257 (1998).
[0252] Transcription may be increased by inserting a
transcriptional activator sequence into the vector. These
activators are cis-acting elements of DNA, usually about from 10 to
300 bp, which act on a promoter to increase its transcription.
Transcriptional enhancers are relatively orientation and position
independent, having been found 5' and 3' to the transcription unit,
within an intron, as well as within the coding sequence itself. The
enhancer may be spliced into the expression vector at a position 5'
or 3' to the coding sequence, but is preferably located at a site
5' from the promoter.
[0253] Expression vectors used in eukaryotic host cells may also
contain sequences necessary for the termination of transcription
and for stabilizing the mRNA. Such sequences are commonly available
from 3' to the translation termination codon, in 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.
[0254] Construction of suitable vectors containing one or more of
the above-listed components employs standard ligation techniques or
PCR/recombination methods. Isolated plasmids or DNA fragments are
cleaved, tailored, and re-ligated in the form desired to generate
the plasmids required or via recombination methods. For analysis to
confirm correct sequences in plasmids constructed, the ligation
mixtures are used to transform host cells, and successful
transformants selected by antibiotic resistance (e.g. ampicillin or
Zeocin) where appropriate. Plasmids from the transformants are
prepared, analyzed by restriction endonuclease digestion and/or
sequenced.
[0255] As an alternative to restriction and ligation of fragments,
recombination methods based on att sites and recombination enzymes
may be used to insert DNA sequences into a vector. Such methods are
described, for example, by Landy (1989) Ann. Rev. Biochem.
58:913-949; and are known to those of skill in the art. Such
methods utilize intermolecular DNA recombination that is mediated
by a mixture of lambda and E. coli--encoded recombination proteins.
Recombination occurs between specific attachment (att) sites on the
interacting DNA molecules. For a description of att sites see
Weisberg and Landy (1983) Site-Specific Recombination in Phage
Lambda, in Lambda I I, Weisberg, ed. (Cold Spring Harbor, N.Y.:
Cold Spring Harbor Press), pp. 211-250. The DNA segments flanking
the recombination sites are switched, such that after
recombination, the att sites are hybrid sequences comprised of
sequences donated by each parental vector. The recombination can
occur between DNAs of any topology.
[0256] Att sites may be introduced into a sequence of interest by
ligating the sequence of interest into an appropriate vector;
generating a PCR product containing att B sites through the use of
specific primers; generating a cDNA library cloned into an
appropriate vector containing att sites; and the like.
[0257] Folding, as used herein, refers to the three-dimensional
structure of polypeptides and proteins, where interactions between
amino acid residues act to stabilize the structure. While
non-covalent interactions are important in determining structure,
usually the proteins of interest will have intra- and/or
intermolecular covalent disulfide bonds formed by two cysteine
residues. For naturally occurring proteins and polypeptides or
derivatives and variants thereof, the proper folding is typically
the arrangement that results in optimal biological activity, and
can conveniently be monitored by assays for activity, e.g. ligand
binding, enzymatic activity, etc.
[0258] In some instances, for example where the desired product is
of synthetic origin, assays based on biological activity will be
less meaningful. The proper folding of such molecules may be
determined on the basis of physical properties, energetic
considerations, modeling studies, and the like.
[0259] The expression host may be further modified by the
introduction of sequences encoding one or more enzymes that enhance
folding and disulfide bond formation, i.e. foldases, chaperonins,
etc. Such sequences may be constitutively or inducibly expressed in
the yeast host cell, using vectors, markers, etc. as known in the
art. Preferably the sequences, including transcriptional regulatory
elements sufficient for the desired pattern of expression, are
stably integrated in the yeast genome through a targeted
methodology.
[0260] For example, the eukaryotic PDI is not only an efficient
catalyst of protein cysteine oxidation and disulfide bond
isomerization, but also exhibits chaperone activity. Co-expression
of PDI can facilitate the production of active proteins having
multiple disulfide bonds. Also of interest is the expression of BIP
(immunoglobulin heavy chain binding protein); cyclophilin; and the
like. In one embodiment of the invention, each of the haploid
parental strains expresses a distinct folding enzyme, e.g. one
strain may express BIP, and the other strain may express PDI or
combinations thereof.
[0261] The terms "desired protein" or "target protein" are used
interchangeably and refer generally to a humanized antibody or a
binding portion thereof described herein. The term "antibody" is
intended to include any polypeptide chain-containing molecular
structure with a specific shape that fits to and recognizes an
epitope, where one or more non-covalent binding interactions
stabilize the complex between the molecular structure and the
epitope. The archetypal antibody molecule is the immunoglobulin,
and all types of immunoglobulins, IgG, IgM, IgA, IgE, IgD, etc.,
from all sources, e.g. human, rodent, rabbit, cow, sheep, pig, dog,
other mammals, chicken, other avians, etc., are considered to be
"antibodies." A preferred source for producing antibodies useful as
starting material according to the invention is rabbits. Numerous
antibody coding sequences have been described; and others may be
raised by methods well-known in the art. Examples thereof include
chimeric antibodies, human antibodies and other non-human mammalian
antibodies, humanized antibodies, single chain antibodies such as
scFvs, camelbodies, nanobodies, IgNAR (single-chain antibodies
derived from sharks), small-modular immunopharmaceuticals (SMIPs),
and antibody fragments such as Fabs, Fab', F(ab).sub.2 and the
like. See Streltsov V A, et al., Structure of a shark IgNAR
antibody variable domain and modeling of an early-developmental
isotype, Protein Sci. 2005 November; 14(11):2901-9. Epub 2005 Sep.
30; Greenberg A S, et al., A new antigen receptor gene family that
undergoes rearrangement and extensive somatic diversification in
sharks, Nature. 1995 Mar. 9; 374(6518):168-73; Nuttall S D, et al.,
Isolation of the new antigen receptor from wobbegong sharks, and
use as a scaffold for the display of protein loop libraries, Mol
Immunol. 2001 August; 38(4):313-26; Hamers-Casterman C, et al.,
Naturally occurring antibodies devoid of light chains, Nature. 1993
Jun. 3; 363(6428):446-8; Gill D S, et al., Biopharmaceutical drug
discovery using novel protein scaffolds, Curr Opin Biotechnol. 2006
December; 17(6):653-8. Epub 2006 Oct. 19.
[0262] For example, antibodies or antigen binding fragments or
variants thereofs may be produced by genetic engineering. In this
technique, as with other methods, antibody-producing cells are
sensitized to the desired antigen or immunogen. The messenger RNA
isolated from antibody producing cells is used as a template to
make cDNA using PCR amplification. A library of vectors, each
containing one heavy chain gene and one light chain gene retaining
the initial antigen specificity, is produced by insertion of
appropriate sections of the amplified immunoglobulin cDNA into the
expression vectors. A combinatorial library is constructed by
combining the heavy chain gene library with the light chain gene
library. This results in a library of clones which co-express a
heavy and light chain (resembling the Fab fragment or antigen
binding fragment of an antibody molecule). The vectors that carry
these genes are co-transfected into a host cell. When antibody gene
synthesis is induced in the transfected host, the heavy and light
chain proteins self-assemble to produce active antibodies that can
be detected by screening with the antigen or immunogen.
[0263] Antibody coding sequences of interest include those encoded
by native sequences, as well as nucleic acids that, by virtue of
the degeneracy of the genetic code, are not identical in sequence
to the disclosed nucleic acids, and variants thereof. Variant
polypeptides can include amino acid (aa) substitutions, additions
or deletions. The amino acid substitutions can be conservative
amino acid substitutions or substitutions to eliminate
non-essential amino acids, such as to alter a glycosylation site,
or to minimize misfolding by substitution or deletion of one or
more cysteine residues that are not necessary for function.
Variants can be designed so as to retain or have enhanced
biological activity of a particular region of the protein (e.g., a
functional domain, catalytic amino acid residues, etc). Variants
also include fragments of the polypeptides disclosed herein,
particularly biologically active fragments and/or fragments
corresponding to functional domains. Techniques for in vitro
mutagenesis of cloned genes are known. Also included in the subject
invention are polypeptides that have been modified using ordinary
molecular biological techniques so as to improve their resistance
to proteolytic degradation or to optimize solubility properties or
to render them more suitable as a therapeutic agent.
[0264] Chimeric antibodies may be made by recombinant means by
combining the variable light and heavy chain regions (V.sub.L and
V.sub.H), obtained from antibody producing cells of one species
with the constant light and heavy chain regions from another.
Typically chimeric antibodies utilize rodent or rabbit variable
regions and human constant regions, in order to produce an antibody
with predominantly human domains. The production of such chimeric
antibodies is well known in the art, and may be achieved by
standard means (as described, e.g., in U.S. Pat. No. 5,624,659,
incorporated herein by reference in its entirety). It is further
contemplated that the human constant regions of chimeric antibodies
of the invention may be selected from IgG1, IgG2, IgG3, IgG4, IgG5,
IgG6, IgG7, IgG8, IgG9, IgG10, IgG11, IgG12, IgG13, IgG14, IgG15,
IgG16, IgG17, IgG18 or IgG19 constant regions.
[0265] Humanized antibodies are engineered to contain even more
human-like immunoglobulin domains, and incorporate only the
complementarity-determining regions of the animal-derived antibody.
This is accomplished by carefully examining the sequence of the
hyper-variable loops of the variable regions of the monoclonal
antibody, and fitting them to the structure of the human antibody
chains. Although facially complex, the process is straightforward
in practice. See, e.g., U.S. Pat. No. 6,187,287, incorporated fully
herein by reference.
[0266] In addition to entire immunoglobulins (or their recombinant
counterparts), immunoglobulin fragments comprising the epitope
binding site (e.g., Fab', F(ab').sub.2, or other fragments) may be
synthesized. "Fragment," or minimal immunoglobulins may be designed
utilizing recombinant immunoglobulin techniques. For instance "Fv"
immunoglobulins for use in the present invention may be produced by
synthesizing a fused variable light chain region and a variable
heavy chain region. Combinations of antibodies are also of
interest, e.g. diabodies, which comprise two distinct Fv
specificities. In another embodiment of the invention, SMIPs (small
molecule immunopharmaceuticals), camelbodies, nanobodies, and IgNAR
are encompassed by immunoglobulin fragments.
[0267] Immunoglobulins and fragments thereof may be modified
post-translationally, e.g. to add effector moieties such as
chemical linkers, detectable moieties, such as fluorescent dyes,
enzymes, toxins, substrates, bioluminescent materials, radioactive
materials, chemiluminescent moieties and the like, or specific
binding moieties, such as streptavidin, avidin, or biotin, and the
like may be utilized in the methods and compositions of the present
invention. Examples of additional effector molecules are provided
infra.
[0268] The term "polyploid yeast that stably expresses or expresses
a desired secreted heterologous polypeptide for prolonged time"
refers to a yeast culture that secretes said polypeptide for at
least several days to a week, more preferably at least a month,
still more preferably at least 1-6 months, and even more preferably
for more than a year at threshold expression levels, typically at
least 10-25 mg/liter and preferably substantially greater.
[0269] The term "polyploidal yeast culture that secretes desired
amounts of recombinant polypeptide" refers to cultures that stably
or for prolonged periods secrete at least 10-25 mg/liter of
heterologous polypeptide, more preferably at least 50-500 mg/liter,
and most preferably 500-1000 mg/liter or more.
[0270] A polynucleotide sequence "corresponds" to a polypeptide
sequence if translation of the polynucleotide sequence in
accordance with the genetic code yields the polypeptide sequence
(i.e., the polynucleotide sequence "encodes" the polypeptide
sequence), one polynucleotide sequence "corresponds" to another
polynucleotide sequence if the two sequences encode the same
polypeptide sequence.
[0271] A "heterologous" region or domain of a DNA construct is an
identifiable segment of DNA within a larger DNA molecule that is
not found in association with the larger molecule in nature. Thus,
when the heterologous region encodes a mammalian gene, the gene
will usually be flanked by DNA that does not flank the mammalian
genomic DNA in the genome of the source organism. Another example
of a heterologous region is a construct where the coding sequence
itself is not found in nature (e.g., a cDNA where the genomic
coding sequence contains introns, or synthetic sequences having
codons different than the native gene). Allelic variations or
naturally-occurring mutational events do not give rise to a
heterologous region of DNA as defined herein.
[0272] A "coding sequence" is an in-frame sequence of codons that
(in view of the genetic code) correspond to or encode a protein or
peptide sequence. Two coding sequences correspond to each other if
the sequences or their complementary sequences encode the same
amino acid sequences. A coding sequence in association with
appropriate regulatory sequences may be transcribed and translated
into a polypeptide. A polyadenylation signal and transcription
termination sequence will usually be located 3' to the coding
sequence. A "promoter sequence" is a DNA regulatory region capable
of binding RNA polymerase in a cell and initiating transcription of
a downstream (3' direction) coding sequence. Promoter sequences
typically contain additional sites for binding of regulatory
molecules (e.g., transcription factors) which affect the
transcription of the coding sequence. A coding sequence is "under
the control" of the promoter sequence or "operatively linked" to
the promoter when RNA polymerase binds the promoter sequence in a
cell and transcribes the coding sequence into mRNA, which is then
in turn translated into the protein encoded by the coding
sequence.
[0273] Vectors are used to introduce a foreign substance, such as
DNA, RNA or protein, into an organism or host cell. Typical vectors
include recombinant viruses (for polynucleotides) and liposomes or
other lipid aggregates (for polypeptides and/or polynucleotides). A
"DNA vector" is a replicon, such as plasmid, phage or cosmid, to
which another polynucleotide segment may be attached so as to bring
about the replication of the attached segment. An "expression
vector" is a DNA vector which contains regulatory sequences which
will direct polypeptide synthesis by an appropriate host cell. This
usually means a promoter to bind RNA polymerase and initiate
transcription of mRNA, as well as ribosome binding sites and
initiation signals to direct translation of the mRNA into a
polypeptide(s). Incorporation of a polynucleotide sequence into an
expression vector at the proper site and in correct reading frame,
followed by transformation of an appropriate host cell by the
vector, enables the production of a polypepide encoded by said
polynucleotide sequence. Exemplary expression vectors and
techniques for their use are described in the following
publications: Old et al., Principles of Gene Manipulation: An
Introduction to Genetic Engineering, Blackwell Scientific
Publications, 4th edition, 1989; Sambrook et al., Molecular
Cloning: A Laboratory Manual, 2nd Edition, Cold Spring Harbor
Laboratory Press, 1989; Sambrook et al., Molecular Cloning: A
Laboratory Manual, 3rd Edition, Cold Spring Harbor Laboratory
Press, 2001; Gorman, "High Efficiency Gene Transfer into Mammalian
Cells," in DNA Cloning, Volume II, Glover, D. M., Ed., IRL Press,
Washington, D.C., pp. 143 190 (1985).
[0274] For example, a liposomes or other lipid aggregate may
comprise a lipid such as phosphatidylcholines (lecithins) (PC),
phosphatidylethanolamines (PE), lysolecithins,
lysophosphatidylethanolamines, phosphatidylserines (PS),
phosphatidylglycerols (PG), phosphatidylinositol (PI),
sphingomyelins, cardiolipin, phosphatidic acids (PA), fatty acids,
gangliosides, glucolipids, glycolipids, mono-, di or triglycerides,
ceramides, cerebrosides and combinations thereof; a cationic lipid
(or other cationic amphiphile) such as
1,2-dioleyloxy-3-(trimethylamino) propane (DOTAP);
N-cholesteryloxycarbaryl-3,7,12-triazapentadecane-1,15-diamine
(CTAP); N-[1-(2,3,
-ditetradecyloxy)propyl]-N,N-dimethyl-N-hydroxyethyl ammonium
bromide (DMRIE);
N-[1-(2,3,-dioleyloxy)propyl]-N,N-dimethyl-N-hydroxy ethylammonium
bromide (DOME); N-[1-(2,3-dioleyloxy)
propyl]-N,N,N-trimethylammonium chloride (DOTMA); 3 beta
[N--(N',N'-dimethylaminoethane)carbamoly] cholesterol (DC-Choi);
and dimethyldioctadecylammonium (DDAB); dioleoylphosphatidyl
ethanolamine (DOPE), cholesterol-containing DOPC; and combinations
thereof; and/or a hydrophilic polymer such as polyvinylpyrrolidone,
polyvinylmethylether, polymethyloxazoline, polyethyloxazoline,
polyhydroxypropyloxazoline, polyhydroxypropylmethacrylamide,
polymethacrylamide, polydimethylacrylamide,
polyhydroxypropylmethacrylate, polyhydroxyethylacrylate,
hydroxymethylcellulose, hydroxyethylcellulose, polyethyleneglycol,
polyaspartamide and combinations thereof. Other suitable cationic
lipids are described in Miller, Angew. Chem. Int. Ed. 37:1768 1785
(1998), and Cooper et al., Chem. Eur. J. 4(1): 137 151 (1998).
Liposomes can be crosslinked, partially crosslinked, or free from
crosslinking. Crosslinked liposomes can include crosslinked as well
as non-crosslinked components. Suitable cationic liposomes or
cytofectins are commercially available and can also be prepared as
described in Sipkins et al., Nature Medicine, 1998, 4(5):(1998),
623 626 or as described in Miller, supra. Exemplary liposomes
includes a polymerizable zwitterionic or neutral lipid, a
polymerizable integrin targeting lipid and a polymerizable cationic
lipid suitable for binding a nucleic acid. Liposomes can optionally
include peptides that provide increased efficiency, for example as
described in U.S. Pat. No. 7,297,759. Additional exemplary
liposomes and other lipid aggregates are described in U.S. Pat. No.
7,166,298.
[0275] "Amplification" of polynucleotide sequences is the in vitro
production of multiple copies of a particular nucleic acid
sequence. The amplified sequence is usually in the form of DNA. A
variety of techniques for carrying out such amplification are
described in a review article by Van Brunt (1990, Bio/Technol.,
8(4):291-294). Polymerase chain reaction or PCR is a prototype of
nucleic acid amplification, and use of PCR herein should be
considered exemplary of other suitable amplification
techniques.
[0276] The general structure of antibodies in vertebrates now is
well understood (Edelman, G. M., Ann. N.Y. Acad. Sci., 190: 5
(1971)). Antibodies consist of two identical light polypeptide
chains of molecular weight approximately 23,000 daltons (the "light
chain"), and two identical heavy chains of molecular weight
53,000-70,000 (the "heavy chain"). The four chains are joined by
disulfide bonds in a "Y" configuration wherein the light chains
bracket the heavy chains starting at the mouth of the "Y"
configuration. The "branch" portion of the "Y" configuration is
designated the F.sub.ab region; the stem portion of the "Y"
configuration is designated the F.sub.C region. The amino acid
sequence orientation runs from the N-terminal end at the top of the
"Y" configuration to the C-terminal end at the bottom of each
chain. The N-terminal end possesses the variable region having
specificity for the antigen that elicited it, and is approximately
100 amino acids in length, there being slight variations between
light and heavy chain and from antibody to antibody.
[0277] The variable region is linked in each chain to a constant
region that extends the remaining length of the chain and that
within a particular class of antibody does not vary with the
specificity of the antibody (i.e., the antigen eliciting it). There
are five known major classes of constant regions that determine the
class of the immunoglobulin molecule (IgG, IgM, IgA, IgD, and IgE
corresponding to .gamma., .mu., .alpha., .delta., and .epsilon.
(gamma, mu, alpha, delta, or epsilon) heavy chain constant
regions). The constant region or class determines subsequent
effector function of the antibody, including activation of
complement (Kabat, E. A., Structural Concepts in Immunology and
Immunochemistry, 2nd Ed., p. 413-436, Holt, Rinehart, Winston
(1976)), and other cellular responses (Andrews, D. W., et al.,
Clinical Immunobiology, pp 1-18, W. B. Sanders (1980); Kohl, S., et
al., Immunology, 48: 187 (1983)); while the variable region
determines the antigen with which it will react. Light chains are
classified as either .kappa. (kappa) or .lamda. (lambda). Each
heavy chain class can be paired with either kappa or lambda light
chain. The light and heavy chains are covalently bonded to each
other, and the "tail" portions of the two heavy chains are bonded
to each other by covalent disulfide linkages when the
immunoglobulins are generated either by hybridomas or by B
cells.
[0278] The expression "variable region" or "VR" refers to the
domains within each pair of light and heavy chains in an antibody
that are involved directly in binding the antibody to the antigen.
Each heavy chain has at one end a variable domain (V.sub.H)
followed by a number of constant domains. Each light chain has a
variable domain (V.sub.L) at one end and a constant domain at its
other end; the constant domain of the light chain is aligned with
the first constant domain of the heavy chain, and the light chain
variable domain is aligned with the variable domain of the heavy
chain.
[0279] The expressions "complementarity determining region,"
"hypervariable region," or "CDR" refer to one or more of the
hyper-variable or complementarity determining regions (CDRs) found
in the variable regions of light or heavy chains of an antibody
(See Kabat, E. A. et al., Sequences of Proteins of Immunological
Interest, National Institutes of Health, Bethesda, Md., (1987)).
These expressions include the hypervariable regions as defined by
Kabat et al. ("Sequences of Proteins of Immunological Interest,"
Kabat E., et al., US Dept. of Health and Human Services, 1983) or
the hypervariable loops in 3-dimensional structures of antibodies
(Chothia and Lesk, J Mol. Biol. 196 901-917 (1987)). The CDRs in
each chain are held in close proximity by framework regions and,
with the CDRs from the other chain, contribute to the formation of
the antigen binding site. Within the CDRs there are select amino
acids that have been described as the selectivity determining
regions (SDRs) which represent the critical contact residues used
by the CDR in the antibody-antigen interaction (Kashmiri, S.,
Methods, 36:25-34 (2005)).
[0280] The expressions "framework region" or "FR" refer to one or
more of the framework regions within the variable regions of the
light and heavy chains of an antibody (See Kabat, E. A. et al.,
Sequences of Proteins of Immunological Interest, National
Institutes of Health, Bethesda, Md., (1987)). These expressions
include those amino acid sequence regions interposed between the
CDRs within the variable regions of the light and heavy chains of
an antibody.
[0281] Ab1 Anti-IL-6 Antibodies and Binding Fragments Thereof
[0282] The invention includes antibodies having binding specificity
to IL-6 and possessing a variable light chain sequence comprising
the sequence set forth below:
MDTRAPTQLLGLLLLWLPGARCAYDMTQTPASVSAAVGGTVTIKCQASQSINNELSWY
QQKPGQRPKLLIYRASTLASGVSSRFKGSGSGTEFTLTISDLECADAATYYCQQGYSLRN
IDNAFGGGTEVVVKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNN (SEQ ID NO: 2) and
humanized versions and variants thereof including those set forth
in FIGS. 2 and 34-37, and those identified in Table 1.
[0283] The invention also includes antibodies having binding
specificity to IL-6 and possessing a variable heavy chain sequence
comprising the sequence set forth below:
METGLRWLLLVAVLKGVQCQSLEESGGRLVTPGTPLTLTCTASGFSLSNYYVTWVRQA
PGKGLEWIGIIYGSDETAYATWAIGRFTISKTSTTVDLKMTSLTAADTATYFCARDDSSD
WDAKFNLWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVK (SEQ ID NO: 3) and
humanized versions and variants thereof including those set forth
in FIGS. 2 and 34-37, and those identified in Table 1.
[0284] The invention further includes antibodies having binding
specificity to IL-6 and possessing a variable heavy chain sequence
which is a modified version of SEQ ID NO:3 wherein the tryptophan
residue in CDR2 is changed to a serine as set forth below:
METGLRWLLLVAVLKGVQCQSLEESGGRLVTPGTPLTLTCTASGFSLSNYYVTWVRQA
PGKGLEWIGIIYGSDETAYATSAIGRFTISKTSTTVDLKMTSLTAADTATYFCARDDSSD
WDAKFNLWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVK (SEQ ID NO: 658)
and humanized versions and variants thereof including those set
forth in FIGS. 2 and 34-37, and those identified in Table 1.
[0285] The invention further contemplates antibodies comprising one
or more of the polypeptide sequences of SEQ ID NO: 4; SEQ ID NO: 5;
and SEQ ID NO: 6 which correspond to the
complementarity-determining regions (CDRs, or hypervariable
regions) of the variable light chain sequence of SEQ ID NO: 2,
and/or one or more of the polypeptide sequences of SEQ ID NO: 7;
SEQ ID NO: 8 or 120; and SEQ ID NO: 9 which correspond to the
complementarity-determining regions (CDRs, or hypervariable
regions) of the variable heavy chain sequence of SEQ ID NO: 3 or
19, or combinations of these polypeptide sequences. In another
embodiment of the invention, the antibodies of the invention
include combinations of the CDRs and the variable heavy and light
chain sequences set forth above.
[0286] In another embodiment, the invention contemplates other
antibodies, such as for example chimeric antibodies, comprising one
or more of the polypeptide sequences of SEQ ID NO: 4; SEQ ID NO: 5;
and SEQ ID NO: 6 which correspond to the
complementarity-determining regions (CDRs, or hypervariable
regions) of the variable light chain sequence of SEQ ID NO: 2,
and/or one or more of the polypeptide sequences of SEQ ID NO: 7;
SEQ ID NO: 8 or 120; and SEQ ID NO: 9 which correspond to the
complementarity-determining regions (CDRs, or hypervariable
regions) of the variable heavy chain sequence of SEQ ID NO: 3 or
19, or combinations of these polypeptide sequences. In another
embodiment of the invention, the antibodies of the invention
include combinations of the CDRs and the variable heavy and light
chain sequences set forth above.
[0287] The invention also contemplates fragments of the antibody
having binding specificity to IL-6. In one embodiment of the
invention, antibody fragments of the invention comprise, or
alternatively consist of, the polypeptide sequence of SEQ ID NO: 2,
20, 647, 651, 660, 666, 699, 702, 706, or 709. In another
embodiment of the invention, antibody fragments of the invention
comprise, or alternatively consist of, the polypeptide sequence of
SEQ ID NO: 3, 18, 19, 652, 656, 657, 658, 66I, 664, 665, 704, or
708.
[0288] In a further embodiment of the invention, fragments of the
antibody having binding specificity to IL-6 comprise, or
alternatively consist of, one or more of the polypeptide sequences
of SEQ ID NO: 4; SEQ ID NO: 5; and SEQ ID NO: 6 which correspond to
the complementarity-determining regions (CDRs, or hypervariable
regions) of the variable light chain sequence of SEQ ID NO: 2.
[0289] In a further embodiment of the invention, fragments of the
antibody having binding specificity to IL-6 comprise, or
alternatively consist of, one or more of the polypeptide sequences
of SEQ ID NO: 7; SEQ ID NO: 8 or SEQ ID NO: 120; and SEQ ID NO: 9
which correspond to the complementarity-determining regions (CDRs,
or hypervariable regions) of the variable heavy chain sequence of
SEQ ID NO: 3 or 19.
[0290] The invention also contemplates antibody fragments which
include one or more of the antibody fragments described herein. In
one embodiment of the invention, fragments of the antibodies having
binding specificity to IL-6 comprise, or alternatively consist of,
one, two, three or more, including all of the following antibody
fragments: the variable light chain region of SEQ ID NO: 2; the
variable heavy chain region of SEQ ID NO: 3; the
complementarity-determining regions (SEQ ID NO: 4; SEQ ID NO: 5;
and SEQ ID NO: 6) of the variable light chain region of SEQ ID NO:
2; and the complementarity-determining regions (SEQ ID NO: 7; SEQ
ID NO: 8 or SEQ ID NO: 120; and SEQ ID NO: 9) of the variable heavy
chain region of SEQ ID NO: 3 or 19.
[0291] The invention also contemplates variants wherein either of
the heavy chain polypeptide sequences of SEQ ID NO: 18 or SEQ ID
NO: 19 is substituted for the heavy chain polypeptide sequence of
SEQ ID NO: 3; the light chain polypeptide sequence of SEQ ID NO: 20
is substituted for the light chain polypeptide sequence of SEQ ID
NO: 2; and the heavy chain CDR sequence of SEQ ID NO: 120 is
substituted for the heavy chain CDR sequence of SEQ ID NO: 8.
[0292] In a preferred embodiment of the invention, the anti-IL-6
antibody is Ab1, comprising SEQ ID NO: 2 and SEQ ID NO: 3, or the
alternative SEQ ID NOs set forth in the preceding paragraph, and
having at least one of the biological activities set forth
herein.
[0293] Sequences of anti-IL-6 antibodies of the present invention
are shown in Table 1. Exemplary sequence variants other alternative
forms of the heavy and light chains of Ab1 through Ab7 are shown.
The antibodies of the present invention encompass additional
sequence variants, including conservative substitutions,
substitution of one or more CDR sequences and/or FR sequences,
etc.
[0294] Exemplary Ab1 embodiments include an antibody comprising a
variant of the light chain and/or heavy chain. Exemplary variants
of the light chain of Ab1 include the sequence of any of the Ab1
light chains shown (i.e., any of SEQ ID NO: 2, 20, 647, 651, 660,
666, 699, 702, 706, or 709) wherein the entire CDR1 sequence is
replaced or wherein one or more residues in the CDR1 sequence is
substituted by the residue in the corresponding position of any of
the other light chain CDR1 sequences set forth (i.e., any of SEQ ID
NO: 23, 39, 55, 71, 87, 103, 124, 140, 156, 172, 188, 204, 220,
236, 252, 268, 284, 300, 316, 332, 348, 364, 380, 396, 412, 428,
444, 460, 476, 492, 508, 524, 540, 556, or 572); and/or wherein the
entire CDR2 sequence is replaced or wherein one or more residues in
the CDR2 sequence is substituted by the residue in the
corresponding position of any of the other light chain CDR2
sequences set forth (i.e., any of SEQ ID NO: 24, 40, 56, 72, 88,
104, 125, 141, 157, 173, 189, 205, 221, 237, 253, 269, 285, 301,
317, 333, 349, 365, 381, 397, 413, 429, 445, 461, 477, 493, 509,
525, 541, 557, or 573); and/or wherein the entire CDR3 sequence is
replaced or wherein one or more residues in the CDR3 sequence is
substituted by the residue in the corresponding position of any of
the other light chain CDR3 sequences set forth (i.e., any of SEQ ID
NO: 25, 41, 57, 73, 89, 105, 126, 142, 158, 174, 190, 206, 222,
238, 254, 270, 286, 302, 318, 334, 350, 366, 382, 398, 414, 430,
446, 462, 478, 494, 510, 526, 542, 558, or 574).
[0295] Exemplary variants of the heavy chain of Ab1 include the
sequence of any of the Ab1 heavy chains shown (i.e., any of SEQ ID
NO: 3, 18, 19, 652, 656, 657, 658, 661, 664, 665, 704, or 708)
wherein the entire CDR1 sequence is replaced or wherein one or more
residues in the CDR1 sequence is substituted by the residue in the
corresponding position of any of the other heavy chain CDR1
sequences set forth (i.e., any of SEQ ID NO: 26, 42, 58, 74, 90,
106, 127, 143, 159, 175, 191, 207, 223, 239, 255, 271, 287, 303,
319, 335, 351, 367, 383, 399, 415, 431, 447, 463, 479, 495, 511,
527, 543, 559, or 575); and/or wherein the entire CDR2 sequence is
replaced or wherein one or more residues in the CDR2 sequence is
substituted by the residue in the corresponding position of an Ab1
heavy chain CDR2, such as those set forth in Table 1 (i.e., any of
SEQ ID NO: 8, or 120) or any of the other heavy chain CDR2
sequences set forth (i.e., any of SEQ ID NO: 27, 43, 59, 75, 91,
107, 121, 128, 144, 160, 176, 192, 208, 224, 240, 256, 272, 288,
304, 320, 336, 352, 368, 384, 400, 416, 432, 448, 464, 480, 496,
512, 528, 544, 560, or 576); and/or wherein the entire CDR3
sequence is replaced or wherein one or more residues in the CDR3
sequence is substituted by the residue in the corresponding
position of any of the other heavy chain CDR3 sequences set forth
(i.e., any of SEQ ID NO: 28, 44, 60, 76, 92, 108, 129, 145, 161,
177, 193, 209, 225, 241, 257, 273, 289, 305, 321, 337, 353, 369,
385, 401, 417, 433, 449, 465, 481, 497, 513, 529, 545, 561, or
577).
[0296] In another embodiment, the invention contemplates other
antibodies, such as for example chimeric or humanized antibodies,
comprising one or more of the polypeptide sequences of SEQ ID NO:
4; SEQ ID NO: 5; and SEQ ID NO: 6 which correspond to the
complementarity-determining regions (CDRs, or hypervariable
regions) of the variable light chain sequence of SEQ ID NO: 2,
and/or one or more of the polypeptide sequences of SEQ ID NO: 7
(CDR1); SEQ ID NO:8 (CDR2); SEQ ID NO:120 (CDR2); and SEQ ID NO: 9
(CDR3) which correspond to the complementarity-determining regions
(CDRs, or hypervariable regions) of the variable heavy chain
sequence of SEQ ID NO:3 or SEQ ID NO:19, or combinations of these
polypeptide sequences. In another embodiment of the invention, the
antibodies of the invention include combinations of the CDRs and
the variable heavy and light chain sequences set forth above
including those set forth in FIGS. 2 and 34-37, and those
identified in Table 1.
[0297] In another embodiment the anti-IL-6 antibody of the
invention is one comprising at least one of the following: a CDR1
light chain encoded by the sequence in SEQ ID NO:12 or SEQ ID
NO:694; a light chain CDR2 encoded by the sequence in SEQ ID NO:13;
a light chain CDR3 encoded by the sequence in SEQ ID NO:14 or SEQ
ID NO:695; a heavy chain CDR1 encoded by the sequence in SEQ ID
NO:15, a heavy chain CDR2 encoded by SEQ ID NO:16 or SEQ ID NO:696
and a heavy chain CDR3 encoded by SEQ ID NO:17 or SEQ ID NO:697. In
addition the invention embraces such nucleic acid sequences and
variants thereof.
[0298] In another embodiment the invention is directed to amino
acid sequences corresponding to the CDRs of said anti-IL-6 antibody
which are selected from SEQ ID NO:4 (CDR1), SEQ ID NO:5 (CDR2), SEQ
ID NO:6 (CDR3), SEQ ID NO:7, SEQ ID NO:120 and SEQ ID NO:9.
[0299] In another embodiment the anti-IL-6 antibody of the
invention comprises a light chain nucleic acid sequence of SEQ ID
NO: 10, 662, 698, 701, 705, 720, 721, 722, or 723; and/or a heavy
chain nucleic acid sequence of SEQ ID NO: 11, 663, 700, 703, 707,
724, or 725. In addition the invention is directed to the
corresponding polypeptides encoded by any of the foregoing nucleic
acid sequences and combinations thereof.
[0300] In a specific embodiment of the invention the anti-IL-6
antibodies or a portion thereof will be encoded by a nucleic acid
sequence selected from those comprised in SEQ ID NO: 10, 12, 13,
14, 662, 694, 695, 698, 701, 705, 720, 721, 722, 723, 11, 15, 16,
17, 663, 696, 697, 700, 703, 707, 724, and 725. For example the
CDR1 in the light chain may be encoded by SEQ ID NO:12 or 694, the
CDR2 in the light chain may be encoded by SEQ ID NO:13, the CDR3 in
the light chain may be encoded by SEQ ID NO:14 or 695; the CDR1 in
the heavy chain may be encoded by SEQ ID NO:15, the CDR2 in the
heavy chain may be encoded by SEQ ID NO:16 or 696, the CDR3 in the
heavy chain may be encoded by SEQ ID NO: 17 or 697. As discussed
infra antibodies containing these CDRs may be constructed using
appropriate human frameworks based on the humanization methods
disclosed herein.
[0301] In another specific embodiment of the invention the variable
light chain will be encoded by SEQ ID NO: I0, 662, 698, 70I, 705,
720, 72I, 722, or 723 and the variable heavy chain of the anti-IL-6
antibodies will be encoded by SEQ ID NO: II, 663, 700, 703, 707,
724, or 725.
[0302] In a more specific embodiment variable light and heavy
chains of the anti-IL-6 antibody respectively will be encoded by
SEQ ID NO: I0 and II, or SEQ ID NO:698 and SEQ ID NO:700, or SEQ ID
NO:70I and SEQ ID NO:703 or SEQ ID NO:705 and SEQ ID NO:707.
[0303] In another specific embodiment the invention covers nucleic
acid constructs containing any of the foregoing nucleic acid
sequences and combinations thereof as well as recombinant cells
containing these nucleic acid sequences and constructs containing
wherein these nucleic acid sequences or constructs may be
extrachromosomal or integrated into the host cell genome
[0304] In another specific embodiment the invention covers
polypeptides containing any of the CDRs or combinations thereof
recited in SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7, SEQ
ID NO:8, SEQ ID NO:I20, SEQ ID NO:9 or polypeptides comprising any
of the variable light polypeptides comprised in SEQ ID NO: 2, 20,
647, 65I, 660, 666, 699, 702, 706, or 709 and/or the variable heavy
polypeptides comprised in SEQ ID NO: 3 I8, I9, 652, 656, 657, 658,
66I, 664, 665, 704, or 708. These polypeptides optionally may be
attached directly or indirectly to other immunoglobulin
polypeptides or effector moieties such as therapeutic or detectable
entities.
[0305] In another embodiment the anti-IL-6 antibody is one
comprising at least one of the following: a variable light chain
encoded by the sequence in SEQ ID NO: TO or SEQ ID NO:698 or SEQ ID
NO:70I or SEQ ID NO:705 and a variable chain encoded by the
sequence in SEQ ID NO:II or SEQ ID NO:700 or SEQ ID NO:703 or SEQ
ID NO:707.
[0306] In another embodiment the anti-IL-6 antibody is a variant of
the foregoing sequences that includes one or more substitution in
the framework and/or CDR sequences and which has one or more of the
properties of AbI in vitro and/or upon in vivo administration.
[0307] These in vitro and in vivo properties are described in more
detail in the examples below and include: competing with AbI for
binding to IL-6 and/or peptides thereof; having a binding affinity
(Kd) for IL-6 of less than about 50 picomolar, and/or a rate of
dissociation (K.sub.off) from IL-6 of less than or equal to
10.sup.-4 S.sup.-1; having an in-vivo half-life of at least about
22 days in a healthy human subject; ability to prevent or treat
hypoalbumemia; ability to prevent or treat elevated CRP; ability to
prevent or treat abnormal coagulation; and/or ability to decrease
the risk of thrombosis in an individual having a disease or
condition associated with increased risk of thrombosis. Additional
non-limiting examples of anti-IL-6 activity are set forth herein,
for example, under the heading "Anti-IL-6 Activity."
[0308] In another embodiment the anti-IL-6 antibody includes one or
more of the Ab1 light-chain and/or heavy chain CDR sequences (see
Table 1) or variant(s) thereof which has one or more of the
properties of Ab1 in vitro and/or upon in vivo administration
(examples of such properties are discussed in the preceding
paragraph). One of skill in the art would understand how to combine
these CDR sequences to form an antigen-binding surface, e.g. by
linkage to one or more scaffold which may comprise human or other
mammalian framework sequences, or their functional orthologs
derived from a SMIP, camelbody, nanobody, IgNAR or other
immunoglobulin or other engineered antibody. For example,
embodiments may specifically bind to human IL-6 and include one,
two, three, four, five, six, or more of the following CDR sequences
or variants thereof:
[0309] a polypeptide having at least 72.7% (i.e., 8 out of 11 amino
acids) identity to the light chain CDR1 of SEQ ID NO: 4;
[0310] a polypeptide having at least 81.8% (i.e., 9 out of 11 amino
acids) identity to the light chain CDR1 of SEQ ID NO: 4;
[0311] a polypeptide having at least 90.9% (i.e., 10 out of 11
amino acids) identity to the light chain CDR1 of SEQ ID NO: 4;
[0312] a polypeptide having 100% (i.e., 11 out of 11 amino acids)
identity to the light chain CDR1 of SEQ ID NO: 4;
[0313] a polypeptide having at least 85.7% (i.e., 6 out of 7 amino
acids) identity to the light chain CDR2 of SEQ ID NO: 5;
[0314] a polypeptide having 100% (i.e., 7 out of 7 amino acids)
identity to the light chain CDR2 of SEQ ID NO: 5;
[0315] a polypeptide having at least 50% (i.e., 6 out of 12 amino
acids) identity to the light chain CDR3 of SEQ ID NO: 6;
[0316] a polypeptide having at least 58.3% (i.e., 7 out of 12 amino
acids) identity to the light chain CDR3 of SEQ ID NO: 6;
[0317] a polypeptide having at least 66.6% (i.e., 8 out of 12 amino
acids) identity to the light chain CDR3 of SEQ ID NO: 6;
[0318] a polypeptide having at least 75% (i.e., 9 out of 12 amino
acids) identity to the light chain CDR3 of SEQ ID NO: 6;
[0319] a polypeptide having at least 83.3% (i.e., 10 out of 12
amino acids) identity to the light chain CDR3 of SEQ ID NO: 6;
[0320] a polypeptide having at least 91.6% (i.e., 11 out of 12
amino acids) identity to the light chain CDR3 of SEQ ID NO: 6;
[0321] a polypeptide having 100% (i.e., 12 out of 12 amino acids)
identity to the light chain CDR3 of SEQ ID NO: 6;
[0322] a polypeptide having at least 80% (i.e., 4 out of 5 amino
acids) identity to the heavy chain CDR1 of SEQ ID NO: 7;
[0323] a polypeptide having 100% (i.e., 5 out of 5 amino acids)
identity to the heavy chain CDR1 of SEQ ID NO: 7;
[0324] a polypeptide having at least 50% (i.e., 8 out of 16 amino
acids) identity to the heavy chain CDR2 of SEQ ID NO: 120;
[0325] a polypeptide having at least 56.2% (i.e., 9 out of 16 amino
acids) identity to the heavy chain CDR2 of SEQ ID NO: 120;
[0326] a polypeptide having at least 62.5% (i.e., 10 out of 16
amino acids) identity to the heavy chain CDR2 of SEQ ID NO:
120;
[0327] a polypeptide having at least 68.7% (i.e., 11 out of 16
amino acids) identity to the heavy chain CDR2 of SEQ ID NO:
120;
[0328] a polypeptide having at least 75% (i.e., 12 out of 16 amino
acids) identity to the heavy chain CDR2 of SEQ ID NO: 120;
[0329] a polypeptide having at least 81.2% (i.e., 13 out of 16
amino acids) identity to the heavy chain CDR2 of SEQ ID NO:
120;
[0330] a polypeptide having at least 87.5% (i.e., 14 out of 16
amino acids) identity to the heavy chain CDR2 of SEQ ID NO:
120;
[0331] a polypeptide having at least 93.7% (i.e., 15 out of 16
amino acids) identity to the heavy chain CDR2 of SEQ ID NO:
120;
[0332] a polypeptide having 100% (i.e., 16 out of 16 amino acids)
identity to the heavy chain CDR2 of SEQ ID NO: 120;
[0333] a polypeptide having at least 33.3% (i.e., 4 out of 12 amino
acids) identity to the heavy chain CDR3 of SEQ ID NO: 9;
[0334] a polypeptide having at least 41.6% (i.e., 5 out of 12 amino
acids) identity to the heavy chain CDR3 of SEQ ID NO: 9;
[0335] a polypeptide having at least 50% (i.e., 6 out of 12 amino
acids) identity to the heavy chain CDR3 of SEQ ID NO: 9;
[0336] a polypeptide having at least 58.3% (i.e., 7 out of 12 amino
acids) identity to the heavy chain CDR3 of SEQ ID NO: 9;
[0337] a polypeptide having at least 66.6% (i.e., 8 out of 12 amino
acids) identity to the heavy chain CDR3 of SEQ ID NO: 9;
[0338] a polypeptide having at least 75% (i.e., 9 out of 12 amino
acids) identity to the heavy chain CDR3 of SEQ ID NO: 9;
[0339] a polypeptide having at least 83.3% (i.e., 10 out of 12
amino acids) identity to the heavy chain CDR3 of SEQ ID NO: 9;
[0340] a polypeptide having at least 91.6% (i.e., 11 out of 12
amino acids) identity to the heavy chain CDR3 of SEQ ID NO: 9;
[0341] a polypeptide having 100% (i.e., 12 out of 12 amino acids)
identity to the heavy chain CDR3 of SEQ ID NO: 9;
[0342] a polypeptide having at least 90.9% (i.e., 10 out of 11
amino acids) similarity to the light chain CDR1 of SEQ ID NO:
4;
[0343] a polypeptide having 100% (i.e., 11 out of 11 amino acids)
similarity to the light chain CDR1 of SEQ ID NO: 4;
[0344] a polypeptide having at least 85.7% (i.e., 6 out of 7 amino
acids) similarity to the light chain CDR2 of SEQ ID NO: 5;
[0345] a polypeptide having 100% (i.e., 7 out of 7 amino acids)
similarity to the light chain CDR2 of SEQ ID NO: 5;
[0346] a polypeptide having at least 66.6% (i.e., 8 out of 12 amino
acids) similarity to the light chain CDR3 of SEQ ID NO: 6;
[0347] a polypeptide having at least 75% (i.e., 9 out of 12 amino
acids) similarity to the light chain CDR3 of SEQ ID NO: 6;
[0348] a polypeptide having at least 83.3% (i.e., 10 out of 12
amino acids) similarity to the light chain CDR3 of SEQ ID NO:
6;
[0349] a polypeptide having at least 91.6% (i.e., 11 out of 12
amino acids) similarity to the light chain CDR3 of SEQ ID NO:
6;
[0350] a polypeptide having 100% (i.e., 12 out of 12 amino acids)
similarity to the light chain CDR3 of SEQ ID NO: 6;
[0351] a polypeptide having at least 80% (i.e., 4 out of 5 amino
acids) similarity to the heavy chain CDR1 of SEQ ID NO: 7;
[0352] a polypeptide having 100% (i.e., 5 out of 5 amino acids)
similarity to the heavy chain CDR1 of SEQ ID NO: 7;
[0353] a polypeptide having at least 56.2% (i.e., 9 out of 16 amino
acids) similarity to the heavy chain CDR2 of SEQ ID NO: 120;
[0354] a polypeptide having at least 62.5% (i.e., 10 out of 16
amino acids) similarity to the heavy chain CDR2 of SEQ ID NO:
120;
[0355] a polypeptide having at least 68.7% (i.e., 11 out of 16
amino acids) similarity to the heavy chain CDR2 of SEQ ID NO:
120;
[0356] a polypeptide having at least 75% (i.e., 12 out of 16 amino
acids) similarity to the heavy chain CDR2 of SEQ ID NO: 120;
[0357] a polypeptide having at least 81.2% (i.e., 13 out of 16
amino acids) similarity to the heavy chain CDR2 of SEQ ID NO:
120;
[0358] a polypeptide having at least 87.5% (i.e., 14 out of 16
amino acids) similarity to the heavy chain CDR2 of SEQ ID NO:
120;
[0359] a polypeptide having at least 93.7% (i.e., 15 out of 16
amino acids) similarity to the heavy chain CDR2 of SEQ ID NO:
120;
[0360] a polypeptide having 100% (i.e., 16 out of 16 amino acids)
similarity to the heavy chain CDR2 of SEQ ID NO: 120;
[0361] a polypeptide having at least 50% (i.e., 6 out of 12 amino
acids) similarity to the heavy chain CDR3 of SEQ ID NO: 9;
[0362] a polypeptide having at least 58.3% (i.e., 7 out of 12 amino
acids) similarity to the heavy chain CDR3 of SEQ ID NO: 9;
[0363] a polypeptide having at least 66.6% (i.e., 8 out of 12 amino
acids) similarity to the heavy chain CDR3 of SEQ ID NO: 9;
[0364] a polypeptide having at least 75% (i.e., 9 out of 12 amino
acids) similarity to the heavy chain CDR3 of SEQ ID NO: 9;
[0365] a polypeptide having at least 83.3% (i.e., 10 out of 12
amino acids) similarity to the heavy chain CDR3 of SEQ ID NO:
9;
[0366] a polypeptide having at least 91.6% (i.e., 11 out of 12
amino acids) similarity to the heavy chain CDR3 of SEQ ID NO:
9;
[0367] a polypeptide having 100% (i.e., 12 out of 12 amino acids)
similarity to the heavy chain CDR3 of SEQ ID NO: 9.
[0368] Other exemplary embodiments include one or more
polynucleotides encoding any of the foregoing, e.g., a
polynucleotide encoding a polypeptide that specifically binds to
human IL-6 and includes one, two, three, four, five, six, or more
of the following CDRs or variants thereof:
[0369] a polynucleotide encoding a polypeptide having at least
72.7% (i.e., 8 out of 11 amino acids) identity to the light chain
CDR1 of SEQ ID NO: 4;
[0370] a polynucleotide encoding a polypeptide having at least
81.8% (i.e., 9 out of 11 amino acids) identity to the light chain
CDR1 of SEQ ID NO: 4;
[0371] a polynucleotide encoding a polypeptide having at least
90.9% (i.e., 10 out of 11 amino acids) identity to the light chain
CDR1 of SEQ ID NO: 4;
[0372] a polynucleotide encoding a polypeptide having 100% (i.e.,
11 out of 11 amino acids) identity to the light chain CDR1 of SEQ
ID NO: 4;
[0373] a polynucleotide encoding a polypeptide having at least
85.7% (i.e., 6 out of 7 amino acids) identity to the light chain
CDR2 of SEQ ID NO: 5;
[0374] a polynucleotide encoding a polypeptide having 100% (i.e., 7
out of 7 amino acids) identity to the light chain CDR2 of SEQ ID
NO: 5;
[0375] a polynucleotide encoding a polypeptide having at least 50%
(i.e., 6 out of 12 amino acids) identity to the light chain CDR3 of
SEQ ID NO: 6;
[0376] a polynucleotide encoding a polypeptide having at least
58.3% (i.e., 7 out of 12 amino acids) identity to the light chain
CDR3 of SEQ ID NO: 6;
[0377] a polynucleotide encoding a polypeptide having at least
66.6% (i.e., 8 out of 12 amino acids) identity to the light chain
CDR3 of SEQ ID NO: 6;
[0378] a polynucleotide encoding a polypeptide having at least 75%
(i.e., 9 out of 12 amino acids) identity to the light chain CDR3 of
SEQ ID NO: 6;
[0379] a polynucleotide encoding a polypeptide having at least
83.3% (i.e., 10 out of 12 amino acids) identity to the light chain
CDR3 of SEQ ID NO: 6;
[0380] a polynucleotide encoding a polypeptide having at least
91.6% (i.e., 11 out of 12 amino acids) identity to the light chain
CDR3 of SEQ ID NO: 6;
[0381] a polynucleotide encoding a polypeptide having 100% (i.e.,
12 out of 12 amino acids) identity to the light chain CDR3 of SEQ
ID NO: 6;
[0382] a polynucleotide encoding a polypeptide having at least 80%
(i.e., 4 out of 5 amino acids) identity to the heavy chain CDR1 of
SEQ ID NO: 7;
[0383] a polynucleotide encoding a polypeptide having 100% (i.e., 5
out of 5 amino acids) identity to the heavy chain CDR1 of SEQ ID
NO: 7;
[0384] a polynucleotide encoding a polypeptide having at least 50%
(i.e., 8 out of 16 amino acids) identity to the heavy chain CDR2 of
SEQ ID NO: 120;
[0385] a polynucleotide encoding a polypeptide having at least
56.2% (i.e., 9 out of 16 amino acids) identity to the heavy chain
CDR2 of SEQ ID NO: 120;
[0386] a polynucleotide encoding a polypeptide having at least
62.5% (i.e., 10 out of 16 amino acids) identity to the heavy chain
CDR2 of SEQ ID NO: 120;
[0387] a polynucleotide encoding a polypeptide having at least
68.7% (i.e., 11 out of 16 amino acids) identity to the heavy chain
CDR2 of SEQ ID NO: 120;
[0388] a polynucleotide encoding a polypeptide having at least 75%
(i.e., 12 out of 16 amino acids) identity to the heavy chain CDR2
of SEQ ID NO: 120;
[0389] a polynucleotide encoding a polypeptide having at least
81.2% (i.e., 13 out of 16 amino acids) identity to the heavy chain
CDR2 of SEQ ID NO: 120;
[0390] a polynucleotide encoding a polypeptide having at least
87.5% (i.e., 14 out of 16 amino acids) identity to the heavy chain
CDR2 of SEQ ID NO: 120;
[0391] a polynucleotide encoding a polypeptide having at least
93.7% (i.e., 15 out of 16 amino acids) identity to the heavy chain
CDR2 of SEQ ID NO: 120;
[0392] a polynucleotide encoding a polypeptide having 100% (i.e.,
16 out of 16 amino acids) identity to the heavy chain CDR2 of SEQ
ID NO: 120;
[0393] a polynucleotide encoding a polypeptide having at least
33.3% (i.e., 4 out of 12 amino acids) identity to the heavy chain
CDR3 of SEQ ID NO: 9;
[0394] a polynucleotide encoding a polypeptide having at least
41.6% (i.e., 5 out of 12 amino acids) identity to the heavy chain
CDR3 of SEQ ID NO: 9;
[0395] a polynucleotide encoding a polypeptide having at least 50%
(i.e., 6 out of 12 amino acids) identity to the heavy chain CDR3 of
SEQ ID NO: 9;
[0396] a polynucleotide encoding a polypeptide having at least
58.3% (i.e., 7 out of 12 amino acids) identity to the heavy chain
CDR3 of SEQ ID NO: 9;
[0397] a polynucleotide encoding a polypeptide having at least
66.6% (i.e., 8 out of 12 amino acids) identity to the heavy chain
CDR3 of SEQ ID NO: 9;
[0398] a polynucleotide encoding a polypeptide having at least 75%
(i.e., 9 out of 12 amino acids) identity to the heavy chain CDR3 of
SEQ ID NO: 9;
[0399] a polynucleotide encoding a polypeptide having at least
83.3% (i.e., 10 out of 12 amino acids) identity to the heavy chain
CDR3 of SEQ ID NO: 9;
[0400] a polynucleotide encoding a polypeptide having at least
91.6% (i.e., 11 out of 12 amino acids) identity to the heavy chain
CDR3 of SEQ ID NO: 9;
[0401] a polynucleotide encoding a polypeptide having 100% (i.e.,
12 out of 12 amino acids) identity to the heavy chain CDR3 of SEQ
ID NO: 9;
[0402] a polynucleotide encoding a polypeptide having at least
90.9% (i.e., 10 out of 11 amino acids) similarity to the light
chain CDR1 of SEQ ID NO: 4;
[0403] a polynucleotide encoding a polypeptide having 100% (i.e.,
11 out of 11 amino acids) similarity to the light chain CDR1 of SEQ
ID NO: 4;
[0404] a polynucleotide encoding a polypeptide having at least
85.7% (i.e., 6 out of 7 amino acids) similarity to the light chain
CDR2 of SEQ ID NO: 5;
[0405] a polynucleotide encoding a polypeptide having 100% (i.e., 7
out of 7 amino acids) similarity to the light chain CDR2 of SEQ ID
NO: 5;
[0406] a polynucleotide encoding a polypeptide having at least
66.6% (i.e., 8 out of 12 amino acids) similarity to the light chain
CDR3 of SEQ ID NO: 6;
[0407] a polynucleotide encoding a polypeptide having at least 75%
(i.e., 9 out of 12 amino acids) similarity to the light chain CDR3
of SEQ ID NO: 6;
[0408] a polynucleotide encoding a polypeptide having at least
83.3% (i.e., 10 out of 12 amino acids) similarity to the light
chain CDR3 of SEQ ID NO: 6;
[0409] a polynucleotide encoding a polypeptide having at least
91.6% (i.e., 11 out of 12 amino acids) similarity to the light
chain CDR3 of SEQ ID NO: 6;
[0410] a polynucleotide encoding a polypeptide having 100% (i.e.,
12 out of 12 amino acids) similarity to the light chain CDR3 of SEQ
ID NO: 6;
[0411] a polynucleotide encoding a polypeptide having at least 80%
(i.e., 4 out of 5 amino acids) similarity to the heavy chain CDR1
of SEQ ID NO: 7;
[0412] a polynucleotide encoding a polypeptide having 100% (i.e., 5
out of 5 amino acids) similarity to the heavy chain CDR1 of SEQ ID
NO: 7;
[0413] a polynucleotide encoding a polypeptide having at least
56.2% (i.e., 9 out of 16 amino acids) similarity to the heavy chain
CDR2 of SEQ ID NO: 120;
[0414] a polynucleotide encoding a polypeptide having at least
62.5% (i.e., 10 out of 16 amino acids) similarity to the heavy
chain CDR2 of SEQ ID NO: 120;
[0415] a polynucleotide encoding a polypeptide having at least
68.7% (i.e., 11 out of 16 amino acids) similarity to the heavy
chain CDR2 of SEQ ID NO: 120;
[0416] a polynucleotide encoding a polypeptide having at least 75%
(i.e., 12 out of 16 amino acids) similarity to the heavy chain CDR2
of SEQ ID NO: 120;
[0417] a polynucleotide encoding a polypeptide having at least
81.2% (i.e., 13 out of 16 amino acids) similarity to the heavy
chain CDR2 of SEQ ID NO: 120;
[0418] a polynucleotide encoding a polypeptide having at least
87.5% (i.e., 14 out of 16 amino acids) similarity to the heavy
chain CDR2 of SEQ ID NO: 120;
[0419] a polynucleotide encoding a polypeptide having at least
93.7% (i.e., 15 out of 16 amino acids) similarity to the heavy
chain CDR2 of SEQ ID NO: 120;
[0420] a polynucleotide encoding a polypeptide having 100% (i.e.,
16 out of 16 amino acids) similarity to the heavy chain CDR2 of SEQ
ID NO: 120;
[0421] a polynucleotide encoding a polypeptide having at least 50%
(i.e., 6 out of 12 amino acids) similarity to the heavy chain CDR3
of SEQ ID NO: 9;
[0422] a polynucleotide encoding a polypeptide having at least
58.3% (i.e., 7 out of 12 amino acids) similarity to the heavy chain
CDR3 of SEQ ID NO: 9;
[0423] a polynucleotide encoding a polypeptide having at least
66.6% (i.e., 8 out of 12 amino acids) similarity to the heavy chain
CDR3 of SEQ ID NO: 9;
[0424] a polynucleotide encoding a polypeptide having at least 75%
(i.e., 9 out of 12 amino acids) similarity to the heavy chain CDR3
of SEQ ID NO: 9;
[0425] a polynucleotide encoding a polypeptide having at least
83.3% (i.e., 10 out of 12 amino acids) similarity to the heavy
chain CDR3 of SEQ ID NO: 9;
[0426] a polynucleotide encoding a polypeptide having at least
91.6% (i.e., 11 out of 12 amino acids) similarity to the heavy
chain CDR3 of SEQ ID NO: 9;
[0427] a polynucleotide encoding a polypeptide having 100% (i.e.,
12 out of 12 amino acids) similarity to the heavy chain CDR3 of SEQ
ID NO: 9.
TABLE-US-00001 TABLE 1 Sequences of exemplary anti-IL-6 antibodies.
Antibody chains CDR1 CDR2 CDR3 Antibody PRT. Nuc. PRT. Nuc. PRT.
Nuc. PRT. Nuc. Ab1 light chains * 2 10 4 12 5 13 6 14 20 720 4 12 5
13 6 14 647 721 4 12 5 13 6 14 651 4 12 5 13 6 14 660 662 4 12 5 13
6 14 666 722 4 12 5 13 6 14 699 698 4 694 5 13 6 695 702 701 4 694
5 13 6 695 706 705 4 694 5 13 6 695 709 723 4 12 5 13 6 14 Human
light 648 710 713 chains used in 649 711 714 Ab1 humanization 650
712 715 Ab1 heavy chains 3 11 7 15 8 16 9 17 18 7 15 8 16 9 17 19
724 7 15 120 696 9 17 652 725 7 15 8 16 9 17 656 7 15 8 16 9 17 657
700 7 15 659 696 9 697 658 7 15 120 696 9 17 661 663 7 15 8 16 9 17
664 7 15 8 16 9 17 665 7 15 120 696 9 17 704 703 7 15 120 696 9 697
708 707 7 15 120 696 9 697 Human heavy 653 716 717 chains used in
654 716 717 Ab1 humanization 655 74 82 718 Ab2 light chains 21 29
23 31 24 32 25 33 667 669 23 31 24 32 25 33 Ab2 heavy chains 22 30
26 34 27 35 28 36 668 670 26 34 27 35 28 36 Ab3 light chains 37 45
39 47 40 48 41 49 671 673 39 47 40 48 41 49 Ab3 heavy chains 38 46
42 50 43 51 44 52 672 674 42 50 43 51 44 52 Ab4 light chains 53 61
55 63 56 64 57 65 675 677 55 63 56 64 57 65 Ab4 heavy chains 54 62
58 66 59 67 60 68 676 678 58 66 59 67 60 68 Ab5 light chains 69 77
71 79 72 80 73 81 679 681 71 79 72 80 73 81 Ab5 heavy chains 70 78
74 82 75 83 76 84 680 682 74 82 75 83 76 84 Ab6 light chains 85 93
87 95 88 96 89 97 683 685 87 95 88 96 89 97 Ab6 heavy chains 86 94
90 98 91 99 92 100 684 686 90 98 91 99 92 100 Ab7 light chains 101
109 103 111 104 112 105 113 119 103 111 104 112 105 113 687 689 103
111 104 112 105 113 693 103 111 104 112 105 113 Ab7 heavy chains
102 110 106 114 107 115 108 116 117 106 114 107 115 108 116 118 106
114 121 108 116 688 690 106 114 107 115 108 116 691 106 114 107 115
108 116 692 106 114 121 108 116 Ab8 light chain 122 130 124 132 125
133 126 134 Ab8 heavy chain 123 131 127 135 128 136 129 137 Ab9
light chain 138 146 140 148 141 149 142 150 Ab9 heavy chain 139 147
143 151 144 152 145 153 Ab10 light chain 154 162 156 164 157 165
158 166 Ab10 heavy chain 155 163 159 167 160 168 161 169 Ab11 light
chain 170 178 172 180 173 181 174 182 Ab11 heavy chain 171 179 175
183 176 184 177 185 Ab12 light chain 186 194 188 196 189 197 190
198 Ab12 heavy chain 187 195 191 199 192 200 193 201 Ab13 light
chain 202 210 204 212 205 213 206 214 Ab13 heavy chain 203 211 207
215 208 216 209 217 Ab14 light chain 218 226 220 228 221 229 222
230 Ab14 heavy chain 219 227 223 231 224 232 225 233 Ab15 light
chain 234 242 236 244 237 245 238 246 Ab15 heavy chain 235 243 239
247 240 248 241 249 Ab16 light chain 250 258 252 260 253 261 254
262 Ab16 heavy chain 251 259 255 263 256 264 257 265 Ab17 light
chain 266 274 268 276 269 277 270 278 Ab17 heavy chain 267 275 271
279 272 280 273 281 Ab18 light chain 282 290 284 292 285 293 286
294 Ab18 heavy chain 283 291 287 295 288 296 289 297 Ab19 light
chain 298 306 300 308 301 309 302 310 Ab19 heavy chain 299 307 303
311 304 312 305 313 Ab20 light chain 314 322 316 324 317 325 318
326 Ab20 heavy chain 315 323 319 327 320 328 321 329 Ab21 light
chain 330 338 332 340 333 341 334 342 Ab21 heavy chain 331 339 335
343 336 344 337 345 Ab22 light chain 346 354 348 356 349 357 350
358 Ab22 heavy chain 347 355 351 359 352 360 353 361 Ab23 light
chain 362 370 364 372 365 373 366 374 Ab23 heavy chain 363 371 367
375 368 376 369 377 Ab24 light chain 378 386 380 388 381 389 382
390 Ab24 heavy chain 379 387 383 391 384 392 385 393 Ab25 light
chain 394 402 396 404 397 405 398 406 Ab25 heavy chain 395 403 399
407 400 408 401 409 Ab26 light chain 410 418 412 420 413 421 414
422 Ab26 heavy chain 411 419 415 423 416 424 417 425 Ab27 light
chain 426 434 428 436 429 437 430 438 Ab27 heavy chain 427 435 431
439 432 440 433 441 Ab28 light chain 442 450 444 452 445 453 446
454 Ab28 heavy chain 443 451 447 455 448 456 449 457 Ab29 light
chain 458 466 460 468 461 469 462 470 Ab29 heavy chain 459 467 463
471 464 472 465 473 Ab30 light chain 474 482 476 484 477 485 478
486 Ab30 heavy chain 475 483 479 487 480 488 481 489 Ab31 light
chain 490 498 492 500 493 501 494 502 Ab31 heavy chain 491 499 495
503 496 504 497 505 Ab32 light chain 506 514 508 516 509 517 510
518 Ab32 heavy chain 507 515 511 519 512 520 513 521 Ab33 light
chain 522 530 524 532 525 533 526 534 Ab33 heavy chain 523 531 527
535 528 536 529 537 Ab34 light chain 538 546 540 548 541 549 542
550 Ab34 heavy chain 539 547 543 551 544 552 545 553 Ab35 light
chain 554 562 556 564 557 565 558 566 Ab35 heavy chain 555 563 559
567 560 568 561 569 Ab36 light chain 570 578 572 580 573 581 574
582 Ab36 heavy chain 571 579 575 583 576 584 577 585 * Exemplary
sequence variant forms of heavy and light chains are shown on
separate lines. PRT.: Polypeptide sequence. Nuc.: Exemplary coding
sequence.
[0428] For reference, sequence identifiers other than those
included in Table 1 are summarized in Table 2.
TABLE-US-00002 TABLE 2 Summary of sequence identifiers in this
application. SEQ ID Description 1 Human IL-6 586 kappa constant
light chain polypeptide sequence 587 kappa constant light chain
polynucleotide sequence 588 gamma-1 constant heavy chain
polypeptide sequence 589 gamma-1 constant heavy chain
polynucleotide sequence 590-646 Human IL-6 peptides (see FIG. 12
and Example 14) 719 gamma-1 constant heavy chain polypeptide
sequence (differs from SEQ ID NO: 518 at two positions) 726
C-reactive protein polypeptide sequence 727 IL-6 receptor alpha 728
IL-6 receptor beta/gp130
[0429] Such antibody fragments or variants thereof may be present
in one or more of the following non-limiting forms: Fab, Fab',
F(ab').sub.2, Fv and single chain Fv antibody forms. In a preferred
embodiment, the anti-IL-6 antibodies described herein further
comprises the kappa constant light chain sequence comprising the
sequence set forth below:
TABLE-US-00003 (SEQ ID NO: 586)
VAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNS
QESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSF NRGEC.
[0430] In another preferred embodiment, the anti-IL-6 antibodies
described herein further comprises and the gamma-1 constant heavy
chain polypeptide sequence comprising one of the sequences set
forth below:
TABLE-US-00004 (SEQ ID NO: 588)
ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGV
HTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVEP
KSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVS
HEDPEVKFNWYVDGVEVHNAKTKPREEQYASTYRVVSVLTVLHQDWLNGK
EYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTC
LVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRW
QQGNVFSCSVMHEALHNHYTQKSLSLSPGK
[0431] and
TABLE-US-00005 (SEQ ID NO: 719)
ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGV
HTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVEP
KSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVS
HEDPEVKFNWYVDGVEVHNAKTKPREEQYASTYRVVSVLTVLHQDWLNGK
EYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTC
LVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRW
QQGNVFSCSVMHEALHNHYTQKSLSLSPGK.
[0432] Embodiments of antibodies described herein may include a
leader sequence, such as a rabbit Ig leader, albumin pre-peptide, a
yeast mating factor pre pro secretion leader sequence (such as P.
pastoris or Saccharomyces cerevisiae a or alpha factor), or human
HAS leader. Exemplary leader sequences are shown offset from FR1 at
the N-terminus of polypeptides shown in FIGS. 36A and 37A as
follows: rabbit Ig leader sequences in SEQ ID NOs: 2 and 660 (MD .
. . ) and SEQ ID NOs: 3 and 661 (ME . . . ); and an albumin
prepeptide in SEQ ID NOs: 706 and 708, which facilitates secretion.
Other leader sequences known in the art to confer desired
properties, such as secretion, improved stability or half-life,
etc. may also be used, either alone or in combinations with one
another, on the heavy and/or light chains, which may optionally be
cleaved prior to administration to a subject. For example, a
polypeptide may be expressed in a cell or cell-free expression
system that also expresses or includes (or is modified to express
or include) a protease, e.g., a membrane-bound signal peptidase,
that cleaves a leader sequence.
[0433] In another embodiment, the invention contemplates an
isolated anti-IL-6 antibody comprising a V.sub.H polypeptide
sequence selected from the group consisting of: SEQ ID NO: 3, 18,
19, 22, 38, 54, 70, 86, 102, 117, 118, 123, 139, 155, 171, 187,
203, 219, 235, 251, 267, 283, 299, 315, 331, 347, 363, 379, 395,
411, 427, 443, 459, 475, 491, 507, 523, 539, 555 and SEQ ID NO:
571; and further comprising a V.sub.L polypeptide sequence selected
from the group consisting of: SEQ ID NO: 2, 20, 21, 37, 53, 69, 85,
101, 119, 122, 138, 154, 170, 186, 202, 218, 234, 250, 266, 282,
298, 314, 330, 346, 362, 378, 394, 410, 426, 442, 458, 474, 490,
506, 522, 538, 554 and SEQ ID NO: 570 or a variant thereof wherein
one or more of the framework residues (FR residues) or CDR residues
in said V.sub.H or V.sub.L polypeptide has been substituted with
another amino acid residue resulting in an anti-IL-6 antibody that
specifically binds IL-6. The invention contemplates humanized and
chimeric forms of these antibodies. The chimeric antibodies may
include an Fc derived from IgG1, IgG2, IgG3, IgG4, IgG5, IgG6,
IgG7, IgG8, IgG9, IgG10, IgG11, IgG12, IgG13, IgG14, IgG15, IgG16,
IgG17, IgG18 or IgG19 constant regions.
[0434] In one embodiment of the invention, the antibodies or
V.sub.H or V.sub.L polypeptides originate or are selected from one
or more rabbit B cell populations prior to initiation of the
humanization process referenced herein.
[0435] In another embodiment of the invention, the anti-IL-6
antibodies and fragments and variants thereof have binding
specificity for primate homologs of the human IL-6 protein.
Non-limiting examples of primate homologs of the human IL-6 protein
are IL-6 obtained from Macaca fascicularis (also known as the
cynomolgus monkey) and the Rhesus monkey. In another embodiment of
the invention, the anti-IL-6 antibodies and fragments and variants
thereof inhibits the association of IL-6 with IL-6R, and/or the
production of IL-6/IL-6R/gp130 complexes and/or the production of
IL-6/IL-6R/gp130 multimers and/or antagonizes the biological
effects of one or more of the foregoing.
[0436] As stated above, antibodies and fragments and variants
thereof may be modified post-translationally to add effector
moieties such as chemical linkers, detectable moieties such as for
example fluorescent dyes, enzymes, substrates, bioluminescent
materials, radioactive materials, and chemiluminescent moieties, or
functional moieties such as for example streptavidin, avidin,
biotin, a cytotoxin, a cytotoxic agent, and radioactive
materials.
[0437] Regarding detectable moieties, further exemplary enzymes
include, but are not limited to, horseradish peroxidase,
acetylcholinesterase, alkaline phosphatase, beta-galactosidase and
luciferase. Further exemplary fluorescent materials include, but
are not limited to, rhodamine, fluorescein, fluorescein
isothiocyanate, umbelliferone, dichlorotriazinylamine,
phycoerythrin and dansyl chloride. Further exemplary
chemiluminescent moieties include, but are not limited to, luminol.
Further exemplary bioluminescent materials include, but are not
limited to, luciferin and aequorin. Further exemplary radioactive
materials include, but are not limited to, Iodine 125 (.sup.125I),
Carbon 14 (.sup.14C), Sulfur 35 (.sup.35S), Tritium (.sup.3H) and
Phosphorus 32 (32P).
[0438] Regarding functional moieties, exemplary cytotoxic agents
include, but are not limited to, methotrexate, aminopterin,
6-mercaptopurine, 6-thioguanine, cytarabine, 5-fluorouracil
decarbazine; alkylating agents such as mechlorethamine, thioepa
chlorambucil, melphalan, carmustine (BSNU), mitomycin C, lomustine
(CCNU), 1-methylnitrosourea, cyclothosphamide, mechlorethamine,
busulfan, dibromomannitol, streptozotocin, mitomycin C,
cis-dichlorodiamine platinum (II) (DDP) cisplatin and carboplatin
(paraplatin); anthracyclines include daunorubicin (formerly
daunomycin), doxorubicin (adriamycin), detorubicin, carminomycin,
idarubicin, epirubicin, mitoxantrone and bisantrene; antibiotics
include dactinomycin (actinomycin D), bleomycin, calicheamicin,
mithramycin, and anthramycin (AMC); and antimytotic agents such as
the vinca alkaloids, vincristine and vinblastine. Other cytotoxic
agents include paclitaxel (taxol), ricin, pseudomonas exotoxin,
gemcitabine, cytochalasin B, gramicidin D, ethidium bromide,
emetine, etoposide, tenoposide, colchicin, dihydroxy anthracin
dione, 1-dehydrotestosterone, glucocorticoids, procaine,
tetracaine, lidocaine, propranolol, puromycin, procarbazine,
hydroxyurea, asparaginase, corticosteroids, mytotane (O,P'-(DDD)),
interferons, and mixtures of these cytotoxic agents.
[0439] Further cytotoxic agents include, but are not limited to,
chemotherapeutic agents such as carboplatin, cisplatin, paclitaxel,
gemcitabine, calicheamicin, doxorubicin, 5-fluorouracil, mitomycin
C, actinomycin D, cyclophosphamide, vincristine, bleomycin, VEGF
antagonists, EGFR antagonists, platins, taxols, irinotecan,
5-fluorouracil, gemcytabine, leucovorine, steroids,
cyclophosphamide, melphalan, vinca alkaloids (e.g., vinblastine,
vincristine, vindesine and vinorelbine), mustines, tyrosine kinase
inhibitors, radiotherapy, sex hormone antagonists, selective
androgen receptor modulators, selective estrogen receptor
modulators, PDGF antagonists, TNF antagonists, IL-1 antagonists,
interleukins (e.g. IL-12 or IL-2), IL-12R antagonists, Toxin
conjugated monoclonal antibodies, tumor antigen specific monoclonal
antibodies, Erbitux, Avastin, Pertuzumab, anti-CD20 antibodies,
Rituxan, ocrelizumab, ofatumumab, DXL625, herceptin, or any
combination thereof. Toxic enzymes from plants and bacteria such as
ricin, diphtheria toxin and Pseudomonas toxin may be conjugated to
the humanized antibodies, or binding fragments thereof, to generate
cell-type-specific-killing reagents (Youle, et al., Proc. Nat'l
Acad. Sci. USA 77:5483 (1980); Gilliland, et al., Proc. Nat'l Acad.
Sci. USA 77:4539 (1980); Krolick, et al., Proc. Nat'l Acad. Sci.
USA 77:5419 (1980)).
[0440] Other cytotoxic agents include cytotoxic ribonucleases as
described by Goldenberg in U.S. Pat. No. 6,653,104. Embodiments of
the invention also relate to radioimmunoconjugates where a
radionuclide that emits alpha or beta particles is stably coupled
to the antibody, or binding fragments thereof, with or without the
use of a complex-forming agent. Such radionuclides include
beta-emitters such as Phosphorus-32 (.sup.32P), Scandium-47
(.sup.475 c), Copper-67 (.sup.67Cu), Gallium-67 (.sup.67Ga),
Yttrium-88 (.sup.88Y), Yttrium-90 (.sup.90Y), Iodine-125
(.sup.125I) Iodine-131 (.sup.131I), Samarium-153 (.sup.1535 m),
Lutetium-177 (.sup.177Lu), Rhenium-186 (.sup.186Re) or Rhenium-188
(.sup.188Re), and alpha-emitters such as Astatine-211 (211At),
Lead-212 (212Pb), Bismuth-212 (.sup.212Bi) or -213 (.sup.213Bi) or
Actinium-225 (.sup.225Ac).
[0441] Methods are known in the art for conjugating an antibody or
binding fragment thereof to a detectable moiety and the like, such
as for example those methods described by Hunter et al, Nature
144:945 (1962); David et al, Biochemistry 13:1014 (1974); Pain et
al, J. Immunol. Meth. 40:219 (1981); and Nygren, J., Histochem. and
Cytochem. 30:407 (1982).
[0442] Embodiments described herein further include variants and
equivalents that are substantially homologous to the antibodies,
antibody fragments, diabodies, SMIPs, camelbodies, nanobodies,
IgNAR, polypeptides, variable regions and CDRs set forth herein.
These may contain, e.g., conservative substitution mutations,
(i.e., the substitution of one or more amino acids by similar amino
acids). For example, conservative substitution refers to the
substitution of an amino acid with another within the same general
class, e.g., one acidic amino acid with another acidic amino acid,
one basic amino acid with another basic amino acid, or one neutral
amino acid by another neutral amino acid. What is intended by a
conservative amino acid substitution is well known in the art.
[0443] In another embodiment, the invention contemplates
polypeptide sequences having at least 90% or greater sequence
homology to any one or more of the polypeptide sequences of
antibody fragments, variable regions and CDRs set forth herein.
More preferably, the invention contemplates polypeptide sequences
having at least 95% or greater sequence homology, even more
preferably at least 98% or greater sequence homology, and still
more preferably at least 99% or greater sequence homology to any
one or more of the polypeptide sequences of antibody fragments,
variable regions and CDRs set forth herein. Methods for determining
homology between nucleic acid and amino acid sequences are well
known to those of ordinary skill in the art.
[0444] In another embodiment, the invention further contemplates
the above-recited polypeptide homologs of the antibody fragments,
variable regions and CDRs set forth herein further having anti-IL-6
activity. Non-limiting examples of anti-IL-6 activity are set forth
herein, for example, under the heading "Anti-IL-6 Activity,"
infra.
[0445] In another embodiment, the invention further contemplates
the generation and use of anti-idiotypic antibodies that bind any
of the foregoing sequences. In an exemplary embodiment, such an
anti-idiotypic antibody could be administered to a subject who has
received an anti-IL-6 antibody to modulate, reduce, or neutralize,
the effect of the anti-IL-6 antibody. Such anti-idiotypic
antibodies could also be useful for treatment of an autoimmune
disease characterized by the presence of anti-IL-6 antibodies. A
further exemplary use of such anti-idiotypic antibodies is for
detection of the anti-IL-6 antibodies of the present invention, for
example to monitor the levels of the anti-IL-6 antibodies present
in a subject's blood or other bodily fluids.
[0446] The present invention also contemplates anti-IL-6 antibodies
comprising any of the polypeptide or polynucleotide sequences
described herein substituted for any of the other polynucleotide
sequences described herein. For example, without limitation
thereto, the present invention contemplates antibodies comprising
the combination of any of the variable light chain and variable
heavy chain sequences described herein, and further contemplates
antibodies resulting from substitution of any of the CDR sequences
described herein for any of the other CDR sequences described
herein. As noted preferred anti-IL-6 antibodies or fragments or
variants thereof may contain a variable heavy and/or light seqence
as shown in FIG. 34 or 35, such as SEQ ID NO: 651, 657, 709 or
variants thereof wherein one or more CDR or FR residues are
modified without advesely affecting antibody binding to IL-6 or
other desired functional activity.
[0447] Polynucleotides Encoding Anti-IL-6 Antibody Polypeptides
[0448] The invention is further directed to polynucleotides
encoding polypeptides of the antibodies having binding specificity
to IL-6. In one embodiment of the invention, polynucleotides of the
invention comprise, or alternatively consist of, the following
polynucleotide sequence encoding the variable light chain
polypeptide sequence of SEQ ID NO: 2:
[0449] ATGGACACGAGGGCCCCCACTCAGCTGCTGGGGCTCCTGCTGCTCTGG
CTCCCAGGTGCCAGATGTGCCTATGATATGACCCAGACTCCAGCCTCGGTGTCTGCA
GCTGTGGGAGGCACAGTCACCATCAAGTGCCAGGCCAGTCAGAGCATTAACAATGA
ATTATCCTGGTATCAGCAGAAACCAGGGCAGCGTCCCAAGCTCCTGATCTATAGGGC
ATCCACTCTGGCATCTGGGGTCTCATCGCGGTTCAAAGGCAGTGGATCTGGGACAGA
GTTCACTCTCACCATCAGCGACCTGGAGTGTGCCGATGCTGCCACTTACTACTGTCA
ACAGGGTTATAGTCTGAGGAATATTGATAATGCTTTCGGCGGAGGGACCGAGGTGG
TGGTCAAACGTACGGTAGCGGCCCCATCTGTCTTCATCTTCCCGCCATCTGATGAGC
AGTTGAAATCTGGAACTGCCTCTGTTGTGTGCCTGCTGAATAACTT (SEQ ID NO: 10) or
the polynucleotide sequence of SEQ ID NO:662, 698, 701, or 705.
[0450] In another embodiment of the invention, polynucleotides of
the invention comprise, or alternatively consist of, the following
polynucleotide sequence encoding the variable heavy chain
polypeptide sequence of SEQ ID NO: 3:
[0451] ATGGAGACTGGGCTGCGCTGGCTTCTCCTGGTCGCTGTGCTCAAAGGT
GTCCAGTGTCAGTCGCTGGAGGAGTCCGGGGGTCGCCTGGTCACGCCTGGGACACC
CCTGACACTCACCTGCACAGCCTCTGGATTCTCCCTCAGTAACTACTACGTGACCTG
GGTCCGCCAGGCTCCAGGGAAGGGGCTGGAATGGATCGGAATCATTTATGGTAGTG
ATGAAACGGCCTACGCGACCTGGGCGATAGGCCGATTCACCATCTCCAAAACCTCG
ACCACGGTGGATCTGAAAATGACCAGTCTGACAGCCGCGGACACGGCCACCTATTT
CTGTGCCAGAGATGATAGTAGTGACTGGGATGCAAAATTTAACTTGTGGGGCCAAG
GCACCCTGGTCACCGTCTCGAGCGCCTCCACCAAGGGCCCATCGGTCTTCCCCCTGG
CACCCTCCTCCAAGAGCACCTCTGGGGGCACAGCGGCCCTGGGCTGCCTGGTCAAG G (SEQ ID
NO: 11) or the polynucleotide sequence of SEQ ID NO:663, 700, 703,
or 707.
[0452] In a further embodiment of the invention, polynucleotides
encoding fragments or variants of the antibody having binding
specificity to IL-6 comprise, or alternatively consist of, one or
more of the polynucleotide sequences of SEQ ID NO: 12 or 694; SEQ
ID NO: 13; and SEQ ID NO: 14 or 695 which correspond to
polynucleotides encoding the complementarity-determining regions
(CDRs, or hypervariable regions) of the light chain variable
sequence of SEQ ID NO: 2.
[0453] In a further embodiment of the invention, polynucleotides
encoding fragments or variants of the antibody having binding
specificity to IL-6 comprise, or alternatively consist of, one or
more of the polynucleotide sequences of SEQ ID NO: 15; SEQ ID NO:
16 or 696; and SEQ ID NO: 17 or 697 which correspond to
polynucleotides encoding the complementarity-determining regions
(CDRs, or hypervariable regions) of the heavy chain variable
sequence of SEQ ID NO:3 or SEQ ID NO:661 or SEQ ID NO:657 or others
depicted in FIG. 34 or 35.
[0454] The invention also contemplates polynucleotide sequences
including one or more of the polynucleotide sequences encoding
antibody fragments or variants described herein. In one embodiment
of the invention, polynucleotides encoding fragments or variants of
the antibody having binding specificity to IL-6 comprise, or
alternatively consist of, one, two, three or more, including all of
the following polynucleotides encoding antibody fragments: the
polynucleotide SEQ ID NO: 10 encoding the light chain variable
region of SEQ ID NO: 2; the polynucleotide SEQ ID NO: 11 encoding
the heavy chain variable region of SEQ ID NO: 3;
[0455] the polynucleotide SEQ ID NO: 720 encoding the light chain
polypeptide of SEQ ID NO: 20; the polynucleotide SEQ ID NO: 721
encoding the light chain polypeptide of SEQ ID NO: 647; the
polynucleotide SEQ ID NO: 662 encoding the light chain polypeptide
of SEQ ID NO: 660; the polynucleotide SEQ ID NO: 722 encoding the
light chain polypeptide of SEQ ID NO: 666; the polynucleotide SEQ
ID NO: 698 encoding the light chain polypeptide of SEQ ID NO: 699;
the polynucleotide SEQ ID NO: 701 encoding the light chain
polypeptide of SEQ ID NO: 702; the polynucleotide SEQ ID NO: 705
encoding the light chain polypeptide of SEQ ID NO: 706; the
polynucleotide SEQ ID NO: 723 encoding the light chain polypeptide
of SEQ ID NO: 709; the polynucleotide SEQ ID NO: 724 encoding the
heavy chain polypeptide of SEQ ID NO: 19; the polynucleotide SEQ ID
NO: 725 encoding the heavy chain polypeptide of SEQ ID NO: 652; the
polynucleotide SEQ ID NO: 700 encoding the heavy chain polypeptide
of SEQ ID NO: 657; the polynucleotide SEQ ID NO: 663 encoding the
heavy chain polypeptide of SEQ ID NO: 661; the polynucleotide SEQ
ID NO: 703 encoding the heavy chain polypeptide of SEQ ID NO: 704;
the polynucleotide SEQ ID NO: 707 encoding the heavy chain
polypeptide of SEQ ID NO: 708; the polynucleotides of SEQ ID NO:
12, 13, 14, 694 and 695 encoding the complementarity-determining
regions of the aforementioned light chain polypeptides; and the
polynucleotides of SEQ ID NO: 15, 16, 17, 696 and 697 encoding the
complementarity-determining regions of the aforementioned heavy
chain polypeptides.
[0456] Exemplary nucleotide sequences encoding anti-IL-6 antibodies
of the present invention are identified in Table 1, above. The
polynucleotide sequences shown are to be understood to be
illustrative, rather than limiting. One of skill in the art can
readily determine the polynucleotide sequences that would encode a
given polypeptide and can readily generate coding sequences
suitable for expression in a given expression system, such as by
adapting the polynucleotide sequences provided and/or by generating
them de novo, and can readily produce codon-optimized expression
sequences, for example as described in published U.S. Patent
Application no. 2008/0120732 or using other methods known in the
art.
[0457] In another embodiment of the invention, polynucleotides of
the invention further comprise, the following polynucleotide
sequence encoding the kappa constant light chain sequence of SEQ ID
NO: 586:
TABLE-US-00006 (SEQ ID NO: 587)
GTGGCTGCACCATCTGTCTTCATCTTCCCGCCATCTGATGAGCAGTTGAA
ATCTGGAACTGCCTCTGTTGTGTGCCTGCTGAATAACTTCTATCCCAGAG
AGGCCAAAGTACAGTGGAAGGTGGATAACGCCCTCCAATCGGGTAACTCC
CAGGAGAGTGTCACAGAGCAGGACAGCAAGGACAGCACCTACAGCCTCAG
CAGCACCCTGACGCTGAGCAAAGCAGACTACGAGAAACACAAAGTCTACG
CCTGCGAAGTCACCCATCAGGGCCTGAGCTCGCCCGTCACAAAGAGCTTC
AACAGGGGAGAGTGT.
[0458] In another embodiment of the invention, polynucleotides of
the invention further comprise, the following polynucleotide
sequence encoding the gamma-1 constant heavy chain polypeptide
sequence of SEQ ID NO: 588:
TABLE-US-00007 (SEQ ID NO: 589)
GCCTCCACCAAGGGCCCATCGGTCTTCCCCCTGGCACCCTCCTCCAAGAG
CACCTCTGGGGGCACAGCGGCCCTGGGCTGCCTGGTCAAGGACTACTTCC
CCGAACCGGTGACGGTGTCGTGGAACTCAGGCGCCCTGACCAGCGGCGTG
CACACCTTCCCGGCTGTCCTACAGTCCTCAGGACTCTACTCCCTCAGCAG
CGTGGTGACCGTGCCCTCCAGCAGCTTGGGCACCCAGACCTACATCTGCA
ACGTGAATCACAAGCCCAGCAACACCAAGGTGGACAAGAGAGTTGAGCCC
AAATCTTGTGACAAAACTCACACATGCCCACCGTGCCCAGCACCTGAACT
CCTGGGGGGACCGTCAGTCTTCCTCTTCCCCCCAAAACCCAAGGACACCC
TCATGATCTCCCGGACCCCTGAGGTCACATGCGTGGTGGTGGACGTGAGC
CACGAAGACCCTGAGGTCAAGTTCAACTGGTACGTGGACGGCGTGGAGGT
GCATAATGCCAAGACAAAGCCGCGGGAGGAGCAGTACGCCAGCACGTACC
GTGTGGTCAGCGTCCTCACCGTCCTGCACCAGGACTGGCTGAATGGCAAG
GAGTACAAGTGCAAGGTCTCCAACAAAGCCCTCCCAGCCCCCATCGAGAA
AACCATCTCCAAAGCCAAAGGGCAGCCCCGAGAACCACAGGTGTACACCC
TGCCCCCATCCCGGGAGGAGATGACCAAGAACCAGGTCAGCCTGACCTGC
CTGGTCAAAGGCTTCTATCCCAGCGACATCGCCGTGGAGTGGGAGAGCAA
TGGGCAGCCGGAGAACAACTACAAGACCACGCCTCCCGTGCTGGACTCCG
ACGGCTCCTTCTTCCTCTACAGCAAGCTCACCGTGGACAAGAGCAGGTGG
CAGCAGGGGAACGTCTTCTCATGCTCCGTGATGCATGAGGCTCTGCACAA
CCACTACACGCAGAAGAGCCTCTCCCTGTCTCCGGGTAAA.
[0459] In one embodiment, the invention is directed to an isolated
polynucleotide comprising a polynucleotide encoding an anti-IL-6
V.sub.H antibody amino acid sequence selected from SEQ ID NO: 3,
18, 19, 652, 656, 657, 658, 661, 664, 665, 704, and 708 or encoding
a variant thereof wherein at least one framework residue (FR
residue) has been substituted with an amino acid present at the
corresponding position in a rabbit anti-IL-6 antibody V.sub.H
polypeptide or a conservative amino acid substitution. In addition,
the invention specifically encompasses humanized anti-Il-6
antibodies or humanized antibody binding fragments or variants
thereof and nucleic acid sequences encoding the foregoing
comprising the humanized variable heavy chain and/or light chain
polypeptides depicted in the sequences contained in FIG. 2 or
34-37, or those identified in Table 1, or variants thereof wherein
one or more framework or CDR residues may be modified. Preferably,
if any modifications are introduced they will not affect adversely
the binding affinity of the resulting anti-IL-6 antibody or
fragment or variant thereof.
[0460] In another embodiment, the invention is directed to an
isolated polynucleotide comprising the polynucleotide sequence
encoding an anti-IL-6 V.sub.L antibody amino acid sequence selected
from SEQ ID NO: 2, 20, 647, 651, 660, 666, 699, 702, 706, and 709
or encoding a variant thereof wherein at least one framework
residue (FR residue) has been substituted with an amino acid
present at the corresponding position in a rabbit anti-IL-6
antibody V.sub.L polypeptide or a conservative amino acid
substitution.
[0461] In yet another embodiment, the invention is directed to one
or more heterologous polynucleotides comprising a sequence encoding
the polypeptides contained in SEQ ID NO:2 and SEQ ID NO:3; SEQ ID
NO:2 and SEQ ID NO:18; SEQ ID NO:2 and SEQ ID NO:19; SEQ ID NO:20
and SEQ ID NO:3; SEQ ID NO:20 and SEQ ID NO:18; or SEQ ID NO:20 and
SEQ ID NO:19.
[0462] In another embodiment, the invention is directed to an
isolated isolated polynucleotide that expresses a polypeptide
containing at least one CDR polypeptide derived from an anti-IL-6
antibody wherein said expressed polypeptide alone specifically
binds IL-6 or specifically binds IL-6 when expressed in association
with another polynucleotide sequence that expresses a polypeptide
containing at least one CDR polypeptide derived from an anti-IL-6
antibody wherein said at least one CDR is selected from those
contained in the V.sub.L or V.sub.H polypeptides contained in SEQ
ID NO: 3, 18, 19, 652, 656, 657, 658, 661, 664, 665, 704, 708, 2,
20, 647, 651, 660, 666, 699, 702, 706, or 709.
[0463] Host cells and vectors comprising said polynucleotides are
also contemplated.
[0464] In another specific embodiment the invention covers nucleic
acid constructs containing any of the foregoing nucleic acid
sequences and combinations thereof as well as recombinant cells
containing these nucleic acid sequences and constructs containing
wherein these nucleic acid sequences or constructs may be
extrachromosomal or integrated into the host cell genome.
[0465] The invention further contemplates vectors comprising the
polynucleotide sequences encoding the variable heavy and light
chain polypeptide sequences, as well as the individual
complementarity determining regions (CDRs, or hypervariable
regions) set forth herein, as well as host cells comprising said
sequences. In one embodiment of the invention, the host cell is a
yeast cell. In another embodiment of the invention, the yeast host
cell belongs to the genus Pichia.
[0466] In some instances, more than one exemplary polynucleotide
encoding a given polypeptide sequence is provided, as summarized in
Table 3.
TABLE-US-00008 TABLE 3 Multiple exemplary polynucleotides encoding
particular polypeptides. Polypeptide Exemplary coding SEQ ID NO SEQ
ID NOs 4 12, 111, 694 5 13, 112, 389, 501 6 14, 113, 695 9 17, 116,
697 39 47, 260 40 48, 261 60 68, 265 72 80, 325, 565, 581 89 97,
134, 166 103 12, 111, 694 104 13, 112, 389, 501 105 14, 113, 695
108 17, 116, 697 126 97, 134, 166 158 97, 134, 166 190 198, 214 191
199, 215 205 213, 469, 485 206 198, 214 207 199, 215 252 47, 260
253 48, 261 257 68, 265 317 80, 325, 565, 581 333 341, 533 381 13,
112, 389, 501 415 423, 439 431 423, 439 461 213, 469, 485 475 483,
499 476 484, 500 477 213, 469, 485 478 486, 502 479 487, 503 480
488, 504 481 489, 505 491 483, 499 492 484, 500 493 13, 112, 389,
501 494 486, 502 495 487, 503 496 488, 504 497 489, 505 525 341,
533 545 553, 585 554 562, 578 556 564, 580 557 80, 325, 565, 581
558 566, 582 570 562, 578 572 564, 580 573 80, 325, 565, 581 574
566, 582 577 553, 585
[0467] In some instances, multiple sequence identifiers refer to
the same polypeptide or polynucleotide sequence, as summarized in
Table 4. References to these sequence identifers are understood to
be interchangeable, except where context indicates otherwise.
TABLE-US-00009 TABLE 4 Repeated sequences. Each cell lists a group
of repeated sequences included in the sequence listing. SEQ ID NOs
of repeated sequences 4, 103 5, 104, 381, 493 6, 105 9, 108 12, 111
13, 112 14, 113 17, 116 39, 252 40, 253 48, 261 60, 257 68, 265 72,
317, 557, 573 80, 325, 565, 581 89, 126, 158 97, 134, 166 120, 659
190, 206 191, 207 198, 214 199, 215 205, 461, 477 213, 469 333, 525
415, 431 423, 439 475, 491 476, 492 478, 494 479, 495 480, 496 481,
497 483, 499 484, 500 486, 502 487, 503 488, 504 489, 505 545, 577
554, 570 556, 572 558, 574 562, 578 564, 580 566, 582
[0468] Certain exemplary embodiments include polynucleotides that
hybridize under moderately or highly stringent hybridization
conditions to a polynucleotide having one of the exemplary coding
sequences recited in Table 1, and also include polynucleotides that
hybridize under moderately or highly stringent hybridization
conditions to a polynucleotide encoding the same polypeptide as a
polynucleotide having one of the exemplary coding sequences recited
in Table 1, or polypeptide encoded by any of the foregoing
polynucleotides.
[0469] The phrase "high stringency hybridization conditions" refers
to conditions under which a probe will hybridize to its target
subsequence, typically in a complex mixture of nucleic acid, but to
no other sequences. High stringency conditions are sequence
dependent and will be different in different circumstances. Longer
sequences hybridize specifically at higher temperatures. An
extensive guide to the hybridization of nucleic acids is found in
Tijssen, Techniques in Biochemistry and Molecular
Biology--Hybridization with Nucleic Probes, "Overview of principles
of hybridization and the strategy of nucleic acid assays" (1993).
Generally, high stringency conditions are selected to be about
5-10.degree. C. lower than the thermal melting point (Tm) for the
specific sequence at a defined ionic strength pH. The Tm is the
temperature (under defined ionic strength, pH, and nucleic
concentration) at which 50% of the probes complementary to the
target hybridize to the target sequence at equilibrium (as the
target sequences are present in excess, at Tm, 50% of the probes
are occupied at equilibrium). High stringency conditions will be
those in which the salt concentration is less than about 1.0 M
sodium ion, typically about 0.01 to 1.0 M sodium ion concentration
(or other salts) at pH 7.0 to 8.3 and the temperature is at least
about 30.degree. C. for short probes (e.g., 10 to 50 nucleotides)
and at least about 60.degree. C. for long probes (e.g., greater
than 50 nucleotides). High stringency conditions may also be
achieved with the addition of destabilizing agents such as
formamide. For selective or specific hybridization, a positive
signal is at least two times background, optionally 10 times
background hybridization. Exemplary high stringency hybridization
conditions can be as following: 50% formamide, 5.times.SSC, and 1%
SDS, incubating at 42.degree. C., or, 5.times.SSC, 1% SDS,
incubating at 65.degree. C., with wash in 0.2.times.SSC, and 0.1%
SDS at 65.degree. C. Such hybridizations and wash steps can be
carried out for, e.g., 1, 2, 5, 10, 15, 30, 60; or more
minutes.
[0470] Nucleic acids that do not hybridize to each other under high
stringency conditions are still substantially related if the
polypeptides that they encode are substantially related. This
occurs, for example, when a copy of a nucleic acid is created using
the maximum codon degeneracy permitted by the genetic code. In such
cases, the nucleic acids typically hybridize under moderate
stringency hybridization conditions. Exemplary "moderate stringency
hybridization conditions" include a hybridization in a buffer of
40% formamide, I M NaCl, I% SDS at 37.degree. C., and a wash in
I.times.SSC at 45.degree. C. Such hybridizations and wash steps can
be carried out for, e.g., I, 2, 5, I0, I5, 30, 60, or more minutes.
A positive hybridization is at least twice background. Those of
ordinary skill will readily recognize that alternative
hybridization and wash conditions can be utilized to provide
conditions of similar stringency.
Additional Exemplary Embodiments of the Invention
[0471] In another embodiment, the invention contemplates one or
more anti-IL-6 antibodies or antibody fragments or variants thereof
which may specifically bind to the same linear or conformational
epitope(s) and/or compete for binding to the same linear or
conformational epitope(s) on an intact human IL-6 polypeptide or
fragment thereof as an anti-IL-6 antibody selected from the group
consisting of AbI and fragments and variants thereof. In a
preferred embodiment, the anti-IL-6 antibody or fragment or variant
thereof may specifically bind to the same linear or conformational
epitope(s) and/or compete for binding to the same linear or
conformational epitope(s) on an intact human IL-6 polypeptide or a
fragment thereof as AbI.
[0472] In another embodiment of the invention, the anti-IL-6
antibody which may specifically bind to the same linear or
conformational epitopes on an intact IL-6 polypeptide or fragment
thereof that is (are) specifically bound by AbI may bind to an IL-6
epitope(s) ascertained by epitopic mapping using overlapping linear
peptide fragments which span the full length of the native human
IL-6 polypeptide. In one embodiment of the invention, the IL-6
epitope comprises, or alternatively consists of, one or more
residues comprised in IL-6 fragments selected from those
respectively encompassing amino acid residues 37-5I, amino acid
residues 70-84, amino acid residues I69-I83, amino acid residues
3I-45 and/or amino acid residues 58-72.
[0473] The invention is also directed to an anti-IL-6 antibody that
binds with the same IL-6 epitope and/or competes with an anti-IL-6
antibody for binding to IL-6 as an antibody or antibody fragment
disclosed herein, including but not limited to an anti-IL-6
antibody selected from AbI and fragments and variants thereof.
[0474] In another embodiment, the invention is also directed to an
isolated anti-IL-6 antibody or antibody fragment or variant thereof
comprising one or more of the CDRs contained in the V.sub.H
polypeptide sequences selected from the group consisting of: SEQ ID
NO: 3, 18, 19, 22, 38, 54, 70, 86, 102, 117, 118, 123, 139, 155,
171, 187, 203, 219, 235, 251, 267, 283, 299, 315, 331, 347, 363,
379, 395, 411, 427, 443, 459, 475, 491, 507, 523, 539, 555 and SEQ
ID NO: 571 and/or one or more of the CDRs contained in the V.sub.L
polypeptide sequence consisting of: 2, 20, 21, 37, 53, 69, 85, 101,
119, 122, 138, 154, 170, 186, 202, 218, 234, 250, 266, 282, 298,
314, 330, 346, 362, 378, 394, 410, 426, 442, 458, 474, 490, 506,
522, 538, 554 and SEQ ID NO: 570.
[0475] In one embodiment of the invention, the anti-IL-6 antibody
discussed in the two prior paragraphs comprises at least 2
complementarity determining regions (CDRs) in each the variable
light and the variable heavy regions which are identical to those
contained in an anti-IL-6 antibody selected from the group
consisting of Ab1 and fragments and variants thereof.
[0476] In a preferred embodiment, the anti-IL-6 antibody discussed
above comprises at least 2 complementarity determining regions
(CDRs) in each the variable light and the variable heavy regions
which are identical to those contained in Ab1. In another
embodiment, all of the CDRs of the anti-IL-6 antibody discussed
above are identical to the CDRs contained in an anti-IL-6 antibody
selected from the group consisting of Ab1 and fragments and
variants thereof. In a preferred embodiment of the invention, all
of the CDRs of the anti-IL-6 antibody discussed above are identical
to the CDRs contained in Ab1.
[0477] The invention further contemplates that the one or more
anti-IL-6 antibodies discussed above are aglycosylated; that
contain an Fc region that has been modified to alter effector
function, half-life, proteolysis, and/or glycosylation; are human,
humanized, single chain or chimeric; and are a humanized antibody
derived from a rabbit (parent) anti-IL-6 antibody.
[0478] The invention further contemplates one or more anti-IL-6
antibodies wherein the framework regions (FRs) in the variable
light region and the variable heavy regions of said antibody
respectively are human FRs which are unmodified or which have been
modified by the substitution of at most 2 or 3 human FR residues in
the variable light or heavy chain region with the corresponding FR
residues of the parent rabbit antibody, and wherein said human FRs
have been derived from human variable heavy and light chain
antibody sequences which have been selected from a library of human
germline antibody sequences based on their high level of homology
to the corresponding rabbit variable heavy or light chain regions
relative to other human germline antibody sequences contained in
the library.
[0479] In one embodiment of the invention, the anti-IL-6 antibody
or fragment or variant thereof may specifically bind to IL-6
expressing human cells and/or to circulating soluble IL-6 molecules
in vivo, including IL-6 expressed on or by human cells in a patient
with a disease associated with cells that express IL-6.
[0480] In another embodiment, the disease is selected from general
fatigue, exercise-induced fatigue, cancer-related fatigue,
inflammatory disease-related fatigue, chronic fatigue syndrome,
fibromyalgia, cancer-related cachexia, cardiac-related cachexia,
respiratory-related cachexia, renal-related cachexia, age-related
cachexia, rheumatoid arthritis, systemic lupus erythematosis (SLE),
systemic juvenile idiopathic arthritis, psoriasis, psoriatic
arthropathy, ankylosing spondylitis, inflammatory bowel disease
(IBD), polymyalgia rheumatica, giant cell arteritis, autoimmune
vasculitis, graft versus host disease (GVHD), Sjogren's syndrome,
adult onset Still's disease, rheumatoid arthritis, systemic
juvenile idiopathic arthritis, osteoarthritis, osteoporosis,
Paget's disease of bone, osteoarthritis, multiple myeloma,
Hodgkin's lymphoma, non-Hodgkin's lymphoma, prostate cancer,
leukemia, renal cell cancer, multicentric Castleman's disease,
ovarian cancer, drug resistance in cancer chemotherapy, cancer
chemotherapy toxicity, ischemic heart disease, atherosclerosis,
obesity, diabetes, asthma, multiple sclerosis, Alzheimer's disease,
cerebrovascular disease, fever, acute phase response, allergies,
anemia, anemia of inflammation (anemia of chronic disease),
hypertension, depression, depression associated with a chronic
illness, thrombosis, thrombocytosis, acute heart failure, metabolic
syndrome, miscarriage, obesity, chronic prostatitis,
glomerulonephritis, pelvic inflammatory disease, reperfusion
injury, transplant rejection, graft versus host disease (GVHD),
avian influenza, smallpox, pandemic influenza, adult respiratory
distress syndrome (ARDS), severe acute respiratory syndrome (SARS),
sepsis, and systemic inflammatory response syndrome (SIRS). In a
preferred embodiment, the disease is selected from a cancer,
inflammatory disorder, viral disorder, or autoimmune disorder. In a
particularly preferred embodiment, the disease is arthritis,
cachexia, and wasting syndrome
[0481] The invention further contemplates anti-IL-6 antibodies or
fragments or variants thereof directly or indirectly attached to a
detectable label or therapeutic agent.
[0482] The invention also contemplates one or more nucleic acid
sequences which result in the expression of an anti-IL-6 antibody
or antibody fragment or variant thereof as set forth above,
including those comprising, or alternatively consisting of, yeast
or human preferred codons. The invention also contemplates vectors
(including plasmids or recombinant viral vectors) comprising said
nucleic acid sequence(s). The invention also contemplates host
cells or recombinant host cells expressing at least one of the
antibodies set forth above, including a mammalian, yeast,
bacterial, and insect cells. In a preferred embodiment, the host
cell is a yeast cell. In a further preferred embodiment, the yeast
cell is a diploidal yeast cell. In a more preferred embodiment, the
yeast cell is a Pichia yeast.
[0483] The invention also contemplates a method of treatment
comprising administering to a patient with a disease or condition
associated with IL-6 expressing cells a therapeutically effective
amount of at least one anti-IL-6 antibody or fragment or variant
thereof. The diseases that may be treated are presented in the
non-limiting list set forth above. In a preferred embodiment, the
disease is selected from a cancer, autoimmune disease, or
inflammatory condition. In a particularly preferred embodiment, the
disease is cancer or viral infection. In another embodiment the
treatment further includes the administration of another
therapeutic agent or regimen selected from chemotherapy,
radiotherapy, cytokine administration or gene therapy.
[0484] The invention further contemplates a method of in vivo
imaging which detects the presence of cells which express IL-6
comprising administering a diagnostically effective amount of at
least one anti-IL-6 antibody. In one embodiment, said
administration further includes the administration of a
radionuclide or fluorophore that facilitates detection of the
antibody at IL-6 expressing disease sites. In another embodiment of
the invention, the method of in vivo imaging is used to detect IL-6
expressing tumors or metastases or is used to detect the presence
of sites of autoimmune disorders associated with IL-6 expressing
cells. In a further embodiment, the results of said in vivo imaging
method are used to facilitate design of an appropriate therapeutic
regimen, including therapeutic regimens including radiotherapy,
chemotherapy or a combination thereof.
[0485] Anti-IL-6 Activity
[0486] As stated previously, IL-6 is a member of a family of
cytokines that promote cellular responses through a receptor
complex consisting of at least one subunit of the
signal-transducing glycoprotein gpI30 and the IL-6 receptor
(IL-6R). The IL-6R may also be present in a soluble form (sIL-6R).
IL-6 binds to IL-6R, which then dimerizes the signal-transducing
receptor gpI30.
[0487] It is believed that the anti-IL-6 antibodies of the
invention, or IL-6 binding fragments or variants thereof, are
useful by exhibiting anti-IL-6 activity. In one non-limiting
embodiment of the invention, the anti-IL-6 antibodies of the
invention, or IL-6 binding fragments or variants thereof, exhibit
anti-IL-6 activity by binding to IL-6 which may be soluble IL-6 or
cell surface expressed IL-6 and/or may prevent or inhibit the
binding of IL-6 to IL-6R and/or activation (dimerization) of the
gpI30 signal-transducing glycoprotein and the formation of
IL-6/IL-6R/gpI30 multimers and the biological effects of any of the
foregoing. The subject anti-IL-6 antibodies may possess different
antagonistic activities based on where (i.e., epitope) the
particular antibody binds IL-6 and/or how it affects the formation
of the foregoing IL-6 complexes and/or multimers and the biological
effects thereof. Consequently, different anti-IL-6 antibodies
according to the invention e.g., may be better suited for
preventing or treating conditions involving the formation and
accumulation of substantial soluble IL-6 such as rheumatoid
arthritis whereas other antibodies may be favored in treatments
wherein the prevention of IL-6/IL-6R/gpI30 or IL-6/IL-6R/gpI30
multimers is a desired therapeutic outcome. This can be determined
in binding and other assays.
[0488] The anti-IL-6 activity of the anti-IL-6 antibody of the
present invention, and fragments and variants thereof having
binding specificity to IL-6, may also be described by their
strength of binding or their affinity for IL-6. This also may
affect their therapeutic properties. In one embodiment of the
invention, the anti-IL-6 antibodies of the present invention, and
fragments thereof having binding specificity to IL-6, bind to IL-6
with a dissociation constant (KD) of less than or equal to
5.times.I0.sup.-7, I0.sup.-7, 5.times.I0.sup.-8, I0.sup.-8,
5.times.I0.sup.-9, I0.sup.-9, 5.times.I0.sup.-10, I0.sup.-10,
5.times.I0.sup.-11, I0.sup.-11, 5.times.I0.sup.-12, I0.sup.-12,
5.times.I0.sup.-13, I0.sup.-13, 5.times.I0.sup.-14, I0.sup.-14,
5.times.I0.sup.-15 or I0.sup.-15. Preferably, the anti-IL-6
antibodies and fragments and variants thereof bind IL-6 with a
dissociation constant of less than or equal to
5.times.I0.sup.-10.
[0489] In another embodiment of the invention, the anti-IL-6
activity of the anti-IL-6 antibodies of the present invention, and
fragments and variants thereof having binding specificity to IL-6,
bind to IL-6 with an off-rate of less than or equal to 10.sup.-4
S.sup.-1, 5.times.10.sup.-5 S.sup.-1, 10.sup.-5 S.sup.-1,
5.times.10.sup.-6 S.sup.-1, 10.sup.-6 S.sup.-1, 5.times.10.sup.-7
S.sup.-1, or 10.sup.-7 S.sup.-1. In one embodiment of the
invention, the anti-IL-6 antibodies of the invention, and fragments
and variants thereof having binding specificity to IL-6, bind to a
linear or conformational IL-6 epitope.
[0490] In a further embodiment of the invention, the anti-IL-6
activity of the anti-IL-6 antibodies of the present invention, and
fragments and variants thereof having binding specificity to IL-6,
exhibit anti-IL-6 activity by ameliorating or reducing the symptoms
of, or alternatively treating, or preventing, diseases and
disorders associated with IL-6. Non-limiting examples of diseases
and disorders associated with IL-6 are set forth infra. As noted
cancer-related fatigue, cachexia and rheumatoid arthritis are
preferred indications for the subject anti-IL-6 antibodies.
[0491] In another embodiment of the invention, the anti-IL-6
antibodies described herein, or IL-6 binding fragments and variants
thereof, do not have binding specificity for IL-6R or the gp-130
signal-transducing glycoprotein.
[0492] B-cell Screening and Isolation
[0493] In one embodiment, the present invention provides methods of
isolating a clonal population of antigen-specific B cells that may
be used for isolating at least one antigen-specific cell. As
described and exemplified infra, these methods contain a series of
culture and selection steps that can be used separately, in
combination, sequentially, repetitively, or periodically.
Preferably, these methods are used for isolating at least one
antigen-specific cell, which can be used to produce a monoclonal
antibody, which is specific to a desired antigen, or a nucleic acid
sequence corresponding to such an antibody.
[0494] In one embodiment, the present invention provides a method
comprising the steps of:
[0495] a. preparing a cell population comprising at least one
antigen-specific B cell;
[0496] b. enriching the cell population, e.g., by chromatography,
to form an enriched cell population comprising at least one
antigen-specific B cell;
[0497] c. isolating a single B cell from the enriched B cell
population; and
[0498] d. determining whether the single B cell produces an
antibody specific to the antigen.
[0499] In another embodiment, the present invention provides an
improvement to a method of isolating a single, antibody-producing B
cell, the improvement comprising enriching a B cell population
obtained from a host that has been immunized or naturally exposed
to an antigen, wherein the enriching step precedes any selection
steps, comprises at least one culturing step, and results in a
clonal population of B cells that produces a single monoclonal
antibody specific to said antigen.
[0500] Throughout this application, a "clonal population of B
cells" refers to a population of B cells that only secrete a single
antibody specific to a desired antigen. That is to say that these
cells produce only one type of monoclonal antibody specific to the
desired antigen.
[0501] In the present application, "enriching" a cell population
cells means increasing the frequency of desired cells, typically
antigen-specific cells, contained in a mixed cell population, e.g.,
a B cell-containing isolate derived from a host that is immunized
against a desired antigen. Thus, an enriched cell population
encompasses a cell population having a higher frequency of
antigen-specific cells as a result of an enrichment step, but this
population of cells may contain and produce different
antibodies.
[0502] The general term "cell population" encompasses pre- and a
post-enrichment cell populations, keeping in mind that when
multiple enrichment steps are performed, a cell population can be
both pre- and post-enrichment. For example, in one embodiment, the
present invention provides a method:
[0503] a. harvesting a cell population from an immunized host to
obtain a harvested cell population;
[0504] b. creating at least one single cell suspension from the
harvested cell population;
[0505] c. enriching at least one single cell suspension to form a
first enriched cell population;
[0506] d. enriching the first enriched cell population to form a
second enriched cell population;
[0507] e. enriching the second enriched cell population to form a
third enriched cell population; and
[0508] f. selecting an antibody produced by an antigen-specific
cell of the third enriched cell population.
[0509] Each cell population may be used directly in the next step,
or it can be partially or wholly frozen for long- or short-term
storage or for later steps. Also, cells from a cell population can
be individually suspended to yield single cell suspensions. The
single cell suspension can be enriched, such that a single cell
suspension serves as the pre-enrichment cell population. Then, one
or more antigen-specific single cell suspensions together form the
enriched cell population; the antigen-specific single cell
suspensions can be grouped together, e.g., re-plated for further
analysis and/or antibody production.
[0510] In one embodiment, the present invention provides a method
of enriching a cell population to yield an enriched cell population
having an antigen-specific cell frequency that is about 50% to
about 100%, or increments therein. Preferably, the enriched cell
population has an antigen-specific cell frequency greater than or
equal to about 50%, 60%, 70%, 75%, 80%, 90%, 95%, 99%, or 100%.
[0511] In another embodiment, the present invention provides a
method of enriching a cell population whereby the frequency of
antigen-specific cells is increased by at least about 2-fold,
5-fold, 10-fold, 20-fold, 50-fold, 100-fold, or increments
therein.
[0512] Throughout this application, the term "increment" is used to
define a numerical value in varying degrees of precision, e.g., to
the nearest 10, 1, 0.1, 0.01, etc. The increment can be rounded to
any measurable degree of precision, and the increment need not be
rounded to the same degree of precision on both sides of a range.
For example, the range 1 to 100 or increments therein includes
ranges such as 20 to 80, 5 to 50, and 0.4 to 98. When a range is
open-ended, e.g., a range of less than 100, increments therein
means increments between 100 and the measurable limit. For example,
less than 100 or increments therein means 0 to 100 or increments
therein unless the feature, e.g., temperature, is not limited by
0.
[0513] Antigen-specificity can be measured with respect to any
antigen. The antigen can be any substance to which an antibody can
bind including, but not limited to, peptides, proteins or fragments
thereof; carbohydrates; organic and inorganic molecules; receptors
produced by animal cells, bacterial cells, and viruses; enzymes;
agonists and antagonists of biological pathways; hormones; and
cytokines. Exemplary antigens include, but are not limited to,
IL-2, IL-4, IL-6, IL-10, IL-I2, IL-I3, IL-I8, IFN-.alpha.,
IFN-.gamma., BAFF, CXCLI3, IP-10, VEGF, EPO, EGF, HRG, Hepatocyte
Growth Factor (HGF) and Hepcidin. Preferred antigens include IL-6,
IL-I3, TNF-.alpha., VEGF-.alpha., Hepatocyte Growth Factor (HGF)
and Hepcidin. In a method utilizing more than one enrichment step,
the antigen used in each enrichment step can be the same as or
different from one another. Multiple enrichment steps with the same
antigen may yield a large and/or diverse population of
antigen-specific cells; multiple enrichment steps with different
antigens may yield an enriched cell population with
cross-specificity to the different antigens.
[0514] Enriching a cell population can be performed by any
cell-selection means known in the art for isolating
antigen-specific cells. For example, a cell population can be
enriched by chromatographic techniques, e.g., Miltenyi bead or
magnetic bead technology. The beads can be directly or indirectly
attached to the antigen of interest. In a preferred embodiment, the
method of enriching a cell population includes at least one
chromatographic enrichment step.
[0515] A cell population can also be enriched by performed by any
antigen-specificity assay technique known in the art, e.g., an
ELISA assay or a halo assay. ELISA assays include, but are not
limited to, selective antigen immobilization (e.g., biotinylated
antigen capture by streptavidin, avidin, or neutravidin coated
plate), non-specific antigen plate coating, and through an antigen
build-up strategy (e.g., selective antigen capture followed by
binding partner addition to generate a heteromeric protein-antigen
complex). The antigen can be directly or indirectly attached to a
solid matrix or support, e.g., a column. A halo assay comprises
contacting the cells with antigen-loaded beads and labeled
anti-host antibody specific to the host used to harvest the B
cells. The label can be, e.g., a fluorophore. In one embodiment, at
least one assay enrichment step is performed on at least one single
cell suspension. In another embodiment, the method of enriching a
cell population includes at least one chromatographic enrichment
step and at least one assay enrichment step.
[0516] Methods of "enriching" a cell population by size or density
are known in the art. See, e.g., U.S. Pat. No. 5,627,052. These
steps can be used in the present method in addition to enriching
the cell population by antigen-specificity.
[0517] The cell populations of the present invention contain at
least one cell capable of recognizing an antigen.
Antigen-recognizing cells include, but are not limited to, B cells,
plasma cells, and progeny thereof. In one embodiment, the present
invention provides a clonal cell population containing a single
type of antigen-specific B-cell, i.e., the cell population produces
a single monoclonal antibody specific to a desired antigen.
[0518] In such embodiment, it is believed that the clonal
antigen-specific population of B cells consists predominantly of
antigen-specific, antibody-secreting cells, which are obtained by
the novel culture and selection protocol provided herein.
Accordingly, the present invention also provides methods for
obtaining an enriched cell population containing at least one
antigen-specific, antibody-secreting cell. In one embodiment, the
present invention provides an enriched cell population containing
about 50% to about 100%, or increments therein, or greater than or
equal to about 60%, 70%, 80%, 90%, or 100% of antigen-specific,
antibody-secreting cells.
[0519] In one embodiment, the present invention provides a method
of isolating a single B cell by enriching a cell population
obtained from a host before any selection steps, e.g., selecting a
particular B cell from a cell population and/or selecting an
antibody produced by a particular cell. The enrichment step can be
performed as one, two, three, or more steps. In one embodiment, a
single B cell is isolated from an enriched cell population before
confirming whether the single B cell secretes an antibody with
antigen-specificity and/or a desired property.
[0520] In one embodiment, a method of enriching a cell population
is used in a method for antibody production and/or selection. Thus,
the present invention provides a method comprising enriching a cell
population before selecting an antibody. The method can include the
steps of: preparing a cell population comprising at least one
antigen-specific cell, enriching the cell population by isolating
at least one antigen-specific cell to form an enriched cell
population, and inducing antibody production from at least one
antigen-specific cell. In a preferred embodiment, the enriched cell
population contains more than one antigen-specific cell. In one
embodiment, each antigen-specific cell of the enriched population
is cultured under conditions that yield a clonal antigen-specific B
cell population before isolating an antibody producing cell
therefrom and/or producing an antibody using said B cell, or a
nucleic acid sequence corresponding to such an antibody. In
contrast to prior techniques where antibodies are produced from a
cell population with a low frequency of antigen-specific cells, the
present invention allows antibody selection from among a high
frequency of antigen-specific cells. Because an enrichment step is
used prior to antibody selection, the majority of the cells,
preferably virtually all of the cells, used for antibody production
are antigen-specific. By producing antibodies from a population of
cells with an increased frequency of antigen specificity, the
quantity and variety of antibodies are increased.
[0521] In the antibody selection methods of the present invention,
an antibody is preferably selected after an enrichment step and a
culture step that results in a clonal population of
antigen-specific B cells. The methods can further comprise a step
of sequencing a selected antibody or portions thereof from one or
more isolated, antigen-specific cells. Any method known in the art
for sequencing can be employed and can include sequencing the heavy
chain, light chain, variable region(s), and/or complementarity
determining region(s) (CDR).
[0522] In addition to the enrichment step, the method for antibody
selection can also include one or more steps of screening a cell
population for antigen recognition and/or antibody functionality.
For example, the desired antibodies may have specific structural
features, such as binding to a particular epitope or mimicry of a
particular structure; antagonist or agonist activity; or
neutralizing activity, e.g., inhibiting binding between the antigen
and a ligand. In one embodiment, the antibody functionality screen
is ligand-dependent. Screening for antibody functionality includes,
but is not limited to, an in vitro protein-protein interaction
assay that recreates the natural interaction of the antigen ligand
with recombinant receptor protein; and a cell-based response that
is ligand dependent and easily monitored (e.g., proliferation
response). In one embodiment, the method for antibody selection
includes a step of screening the cell population for antibody
functionality by measuring the inhibitory concentration (IC50). In
one embodiment, at least one of the isolated, antigen-specific
cells produces an antibody having an IC50 of less than about 100,
50, 30, 25, 10 .mu.g/mL, or increments therein.
[0523] In addition to the enrichment step, the method for antibody
selection can also include one or more steps of screening a cell
population for antibody binding strength. Antibody binding strength
can be measured by any method known in the art (e.g., Biacore). In
one embodiment, at least one of the isolated, antigen-specific
cells produces an antibody having a high antigen affinity, e.g., a
dissociation constant (Kd) of less than about 5.times.10.sup.-10
M-1, preferably about 1.times.10.sup.-13 to 5.times.10.sup.-10,
1.times.10.sup.-12 to 1.times.10.sup.-10, 1.times.10.sup.-12 to
7.5.times.10.sup.-11, 1.times.10.sup.-11 to 2.times.10.sup.-11,
about 1.5.times.10.sup.-11 or less, or increments therein. In this
embodiment, the antibodies are said to be affinity mature. In a
preferred embodiment, the affinity of the antibodies is comparable
to or higher than the affinity of any one of Panorex.RTM.
(edrecolomab), Rituxan.RTM. (rituximab), Herceptin.RTM.
(traztuzumab), Mylotarg.RTM. (gentuzumab), Campath.RTM.
(alemtuzumab), Zevalin.TM. (ibritumomab), Erbitux.TM. (cetuximab),
Avastin.TM. (bevicizumab), Raptiva.TM. (efalizumab), Remicade.RTM.
(infliximab), Humira.TM. (adalimumab), and Xolair.TM. (omalizumab).
Preferably, the affinity of the antibodies is comparable to or
higher than the affinity of Humira.TM.. The affinity of an antibody
can also be increased by known affinity maturation techniques. In
one embodiment, at least one cell population is screened for at
least one of, preferably both, antibody functionality and antibody
binding strength.
[0524] In addition to the enrichment step, the method for antibody
selection can also include one or more steps of screening a cell
population for antibody sequence homology, especially human
homology. In one embodiment, at least one of the isolated,
antigen-specific cells produces an antibody that has a homology to
a human antibody of about 50% to about 100%, or increments therein,
or greater than about 60%, 70%, 80%, 85%, 90%, or 95% homologous.
The antibodies can be humanized to increase the homology to a human
sequence by techniques known in the art such as CDR grafting or
selectivity determining residue grafting (SDR).
[0525] In another embodiment, the present invention also provides
the antibodies themselves according to any of the embodiments
described above in terms of IC50, Kd, and/or homology.
[0526] The B cell selection protocol disclosed herein has a number
of intrinsic advantages versus other methods for obtaining
antibody-secreting B cells and monoclonal antibodies specific to
desired target antigens. These advantages include, but are not
restricted to, the following:
[0527] First, it has been found that when these selection
procedures are utilized with a desired antigen such as IL-6 or
TNF-.alpha., the methods reproducibly result in antigen-specific B
cells capable of generating what appears to be a substantially
comprehensive complement of antibodies, i.e., antibodies that bind
to the various different epitopes of the antigen. Without being
bound by theory, it is hypothesized that the comprehensive
complement is attributable to the antigen enrichment step that is
performed prior to initial B cell recovery. Moreover, this
advantage allows for the isolation and selection of antibodies with
different properties as these properties may vary depending on the
epitopic specificity of the particular antibody.
[0528] Second, it has been found that the B cell selection protocol
reproducibly yields a clonal B cell culture containing a single B
cell, or its progeny, secreting a single monoclonal antibody that
generally binds to the desired antigen with a relatively high
binding affinity, i.e. picomolar or better antigen binding
affinities. By contrast, prior antibody selection methods tend to
yield relatively few high affinity antibodies and therefore require
extensive screening procedures to isolate an antibody with
therapeutic potential. Without being bound by theory, it is
hypothesized that the protocol results in both in vivo B cell
immunization of the host (primary immunization) followed by a
second in vitro B cell stimulation (secondary antigen priming step)
that may enhance the ability and propensity of the recovered clonal
B cells to secrete a single high affinity monoclonal antibody
specific to the antigen target.
[0529] Third, it has been observed (as shown herein with IL-6
specific B cells) that the B cell selection protocol reproducibly
yields enriched B cells producing IgG's that are, on average,
highly selective (antigen specific) to the desired target.
Antigen-enriched B cells recovered by these methods are believed to
contain B cells capable of yielding the desired full complement of
epitopic specificities as discussed above.
[0530] Fourth, it has been observed that the B cell selection
protocols, even when used with small antigens, i.e., peptides of
100 amino acids or less, e.g., 5-50 amino acids long, reproducibly
give rise to a clonal B cell culture that secretes a single high
affinity antibody to the small antigen, e.g., a peptide. This is
highly surprising as it is generally quite difficult, labor
intensive, and sometimes not even feasible to produce high affinity
antibodies to small peptides. Accordingly, the invention can be
used to produce therapeutic antibodies to desired peptide targets,
e.g., viral, bacterial or autoantigen peptides, thereby allowing
for the production of monoclonal antibodies with very discrete
binding properties or even the production of a cocktail of
monoclonal antibodies to different peptide targets, e.g., different
viral strains. This advantage may especially be useful in the
context of the production of a therapeutic or prophylactic vaccine
having a desired valency, such as an HPV vaccine that induces
protective immunity to different HPV strains.
[0531] Fifth, the B cell selection protocol, particularly when used
with B cells derived from rabbits, tends to reproducibly yield
antigen-specific antibody sequences that are very similar to
endogenous human immunoglobulins (around 90% similar at the amino
acid level) and that contain CDRs that possess a length very
analogous to human immunoglobulins and therefore require little or
no sequence modification (typically at most only a few CDR residues
may be modified in the parent antibody sequence and no framework
exogenous residues introduced) in order to eliminate potential
immunogenicity concerns. In particular, preferably the recombinant
antibody will contain only the host (rabbit) CDR1 and CDR2 residues
required for antigen recognition and the entire CDR3. Thereby, the
high antigen binding affinity of the recovered antibody sequences
produced according to the B cell and antibody selection protocol
remains intact or substantially intact even with humanization.
[0532] In sum, these method can be used to produce antibodies
exhibiting higher binding affinities to more distinct epitopes by
the use of a more efficient protocol than was previously known.
[0533] In a specific embodiment, the present invention provides a
method for identifying a single B cell that secretes an antibody
specific to a desired antigen and that optionally possesses at
least one desired functional property such as affinity, avidity,
cytolytic activity, and the like by a process including the
following steps:
[0534] a. immunizing a host against an antigen;
[0535] b. harvesting B cells from the host;
[0536] c. enriching the harvested B cells to increase the frequency
of antigen-specific cells;
[0537] d. creating at least one single cell suspension;
[0538] e. culturing a sub-population from the single cell
suspension under conditions that favor the survival of a single
antigen-specific B cell per culture well;
[0539] f isolating B cells from the sub-population; and
[0540] g. determining whether the single B cell produces an
antibody specific to the antigen.
[0541] Typically, these methods will further comprise an additional
step of isolating and sequencing, in whole or in part, the
polypeptide and nucleic acid sequences encoding the desired
antibody. These sequences or modified versions or portions thereof
can be expressed in desired host cells in order to produce
recombinant antibodies to a desired antigen.
[0542] As noted previously, it is believed that the clonal
population of B cells predominantly comprises antibody-secreting B
cells producing antibody against the desired antigen. It is also
believed based on experimental results obtained with several
antigens and with different B cell populations that the clonally
produced B cells and the isolated antigen-specific B cells derived
therefrom produced according to the invention secrete a monoclonal
antibody that is typically of relatively high affinity and moreover
is capable of efficiently and reproducibly producing a selection of
monoclonal antibodies of greater epitopic variability as compared
to other methods of deriving monoclonal antibodies from cultured
antigen-specific B cells. In an exemplary embodiment the population
of immune cells used in such B cell selection methods will be
derived from a rabbit. However, other hosts that produce
antibodies, including non-human and human hosts, can alternatively
be used as a source of immune B cells. It is believed that the use
of rabbits as a source of B cells may enhance the diversity of
monoclonal antibodies that may be derived by the methods. Also, the
antibody sequences derived from rabbits according to the invention
typically possess sequences having a high degree of sequence
identity to human antibody sequences making them favored for use in
humans since they should possess little antigenicity. In the course
of humanization, the final humanized antibody contains a much lower
foreign/host residue content, usually restricted to a subset of the
host CDR residues that differ dramatically due to their nature
versus the human target sequence used in the grafting. This
enhances the probability of complete activity recovery in the
humanized antibody protein.
[0543] The methods of antibody selection using an enrichment step
disclosed herein include a step of obtaining a immune
cell-containing cell population from an immunized host. Methods of
obtaining an immune cell-containing cell population from an
immunized host are known in the art and generally include inducing
an immune response in a host and harvesting cells from the host to
obtain one or more cell populations. The response can be elicited
by immunizing the host against a desired antigen. Alternatively,
the host used as a source of such immune cells can be naturally
exposed to the desired antigen such as an individual who has been
infected with a particular pathogen such as a bacterium or virus or
alternatively has mounted a specific antibody response to a cancer
that the individual is afflicted with.
[0544] Host animals are well-known in the art and include, but are
not limited to, guinea pig, rabbit, mouse, rat, non-human primate,
human, as well as other mammals and rodents, chicken, cow, pig,
goat, and sheep. Preferably the host is a mammal, more preferably,
rabbit, mouse, rat, or human. When exposed to an antigen, the host
produces antibodies as part of the native immune response to the
antigen. As mentioned, the immune response can occur naturally, as
a result of disease, or it can be induced by immunization with the
antigen. Immunization can be performed by any method known in the
art, such as, by one or more injections of the antigen with or
without an agent to enhance immune response, such as complete or
incomplete Freund's adjuvant. In another embodiment, the invention
also contemplates intrasplenic immunization. As an alternative to
immunizing a host animal in vivo, the method can comprise
immunizing a host cell culture in vitro.
[0545] After allowing time for the immune response (e.g., as
measured by serum antibody detection), host animal cells are
harvested to obtain one or more cell populations. In a preferred
embodiment, a harvested cell population is screened for antibody
binding strength and/or antibody functionality. A harvested cell
population is preferably from at least one of the spleen, lymph
nodes, bone marrow, and/or peripheral blood mononuclear cells
(PBMCs). The cells can be harvested from more than one source and
pooled. Certain sources may be preferred for certain antigens. For
example, the spleen, lymph nodes, and PBMCs are preferred for IL-6;
and the lymph nodes are preferred for TNF. The cell population is
harvested about 20 to about 90 days or increments therein after
immunization, preferably about 50 to about 60 days. A harvested
cell population and/or a single cell suspension therefrom can be
enriched, screened, and/or cultured for antibody selection. The
frequency of antigen-specific cells within a harvested cell
population is usually about 1% to about 5%, or increments
therein.
[0546] In one embodiment, a single cell suspension from a harvested
cell population is enriched, preferably by using Miltenyi beads.
From the harvested cell population having a frequency of
antigen-specific cells of about 1% to about 5%, an enriched cell
population is thus derived having a frequency of antigen-specific
cells approaching 100%.
[0547] The method of antibody selection using an enrichment step
includes a step of producing antibodies from at least one
antigen-specific cell from an enriched cell population. Methods of
producing antibodies in vitro are well known in the art, and any
suitable method can be employed. In one embodiment, an enriched
cell population, such as an antigen-specific single cell suspension
from a harvested cell population, is plated at various cell
densities, such as 50, 100, 250, 500, or other increments between 1
and 1000 cells per well. Preferably, the sub-population comprises
no more than about 10,000 antigen-specific, antibody-secreting
cells, more preferably about 50-10,000, about 50-5,000, about
50-1,000, about 50-500, about 50-250 antigen-specific,
antibody-secreting cells, or increments therein. Then, these
sub-populations are cultured with suitable medium (e.g., an
activated T cell conditioned medium, particularly 1-5% activated
rabbit T cell conditioned medium) on a feeder layer, preferably
under conditions that favor the survival of a single proliferating
antibody-secreting cell per culture well. The feeder layer,
generally comprised of irradiated cell matter, e.g., EL4B cells,
does not constitute part of the cell population. The cells are
cultured in a suitable media for a time sufficient for antibody
production, for example about 1 day to about 2 weeks, about 1 day
to about 10 days, at least about 3 days, about 3 to about 5 days,
about 5 days to about 7 days, at least about 7 days, or other
increments therein. In one embodiment, more than one sub-population
is cultured simultaneously. Preferably, a single antibody-producing
cell and progeny thereof survives in each well, thereby providing a
clonal population of antigen-specific B cells in each well. At this
stage, the immunoglobulin G (IgG) produced by the clonal population
is highly correlative with antigen specificity. In a preferred
embodiment, the IgGs exhibit a correlation with antigen specificity
that is greater than about 50%, more preferably greater than 70%,
85%, 90%, 95%, 99%, or increments therein. See FIG. 3, which
demonstrates an exemplary correlation for IL-6. The correlations
were demonstrated by setting up B cell cultures under limiting
conditions to establish single antigen-specific antibody products
per well. Antigen-specific versus general IgG synthesis was
compared. Three populations were observed: IgG that recognized a
single format of antigen (biotinylated and direct coating),
detectable IgG and antigen recognition irrespective of
immobilization, and IgG production alone. IgG production was highly
correlated with antigen-specificity.
[0548] A supernatant containing the antibodies is optionally
collected, which can be can be enriched, screened, and/or cultured
for antibody selection according to the steps described above. In
one embodiment, the supernatant is enriched (preferably by an
antigen-specificity assay, especially an ELISA assay) and/or
screened for antibody functionality.
[0549] In another embodiment, the enriched, preferably clonal,
antigen-specific B cell population from which a supernatant
described above is optionally screened in order to detect the
presence of the desired secreted monoclonal antibody is used for
the isolation of a few B cells, preferably a single B cell, which
is then tested in an appropriate assay in order to confirm the
presence of a single antibody-producing B cell in the clonal B cell
population. In one embodiment about 1 to about 20 cells are
isolated from the clonal B cell population, preferably less than
about 15, 12, 10, 5, or 3 cells, or increments therein, most
preferably a single cell. The screen is preferably effected by an
antigen-specificity assay, especially a halo assay. The halo assay
can be performed with the full length protein, or a fragment
thereof. The antibody-containing supernatant can also be screened
for at least one of: antigen binding affinity; agonism or
antagonism of antigen-ligand binding, induction or inhibition of
the proliferation of a specific target cell type; induction or
inhibition of lysis of a target cell, and induction or inhibition
of a biological pathway involving the antigen.
[0550] The identified antigen-specific cell can be used to derive
the corresponding nucleic acid sequences encoding the desired
monoclonal antibody. (An AluI digest can confirm that only a single
monoclonal antibody type is produced per well.) As mentioned above,
these sequences can be mutated, such as by humanization, in order
to render them suitable for use in human medicaments.
[0551] As mentioned, the enriched B cell population used in the
process can also be further enriched, screened, and/or cultured for
antibody selection according to the steps described above which can
be repeated or performed in a different order. In a preferred
embodiment, at least one cell of an enriched, preferably clonal,
antigen-specific cell population is isolated, cultured, and used
for antibody selection.
[0552] Thus, in one embodiment, the present invention provides a
method comprising:
[0553] a. harvesting a cell population from an immunized host to
obtain a harvested cell population;
[0554] b. creating at least one single cell suspension from a
harvested cell population;
[0555] c. enriching at least one single cell suspension, preferably
by chromatography, to form a first enriched cell population;
[0556] d. enriching the first enriched cell population, preferably
by ELISA assay, to form a second enriched cell population which
preferably is clonal, i.e., it contains only a single type of
antigen-specific B cell;
[0557] e. enriching the second enriched cell population, preferably
by halo assay, to form a third enriched cell population containing
a single or a few number of B cells that produce an antibody
specific to a desired antigen; and
[0558] f. selecting an antibody produced by an antigen-specific
cell isolated from the third enriched cell population.
[0559] The method can further include one or more steps of
screening the harvested cell population for antibody binding
strength (affinity, avidity) and/or antibody functionality.
Suitable screening steps include, but are not limited to, assay
methods that detect: whether the antibody produced by the
identified antigen-specific B cell produces an antibody possessing
a minimal antigen binding affinity, whether the antibody agonizes
or antagonizes the binding of a desired antigen to a ligand;
whether the antibody induces or inhibits the proliferation of a
specific cell type; whether the antibody induces or elicits a
cytolytic reaction against target cells; whether the antibody binds
to a specific epitope; and whether the antibody modulates (inhibits
or agonizes) a specific biological pathway or pathways involving
the antigen.
[0560] Similarly, the method can include one or more steps of
screening the second enriched cell population for antibody binding
strength and/or antibody functionality.
[0561] The method can further include a step of sequencing the
polypeptide sequence or the corresponding nucleic acid sequence of
the selected antibody. The method can also include a step of
producing a recombinant antibody using the sequence, a fragment
thereof, or a genetically modified version of the selected
antibody. Methods for mutating antibody sequences in order to
retain desired properties are well known to those skilled in the
art and include humanization, chimerisation, production of single
chain antibodies; these mutation methods can yield recombinant
antibodies possessing desired effector function, immunogenicity,
stability, removal or addition of glycosylation, and the like. The
recombinant antibody can be produced by any suitable recombinant
cell, including, but not limited to mammalian cells such as CHO,
COS, BHK, HEK-293, bacterial cells, yeast cells, plant cells,
insect cells, and amphibian cells. In one embodiment, the
antibodies are expressed in polyploidal yeast cells, i.e., diploid
yeast cells, particularly Pichia.
[0562] In one embodiment, the method comprises:
[0563] a. immunizing a host against an antigen to yield host
antibodies;
[0564] b. screening the host antibodies for antigen specificity and
neutralization;
[0565] c. harvesting B cells from the host;
[0566] d. enriching the harvested B cells to create an enriched
cell population having an increased frequency of antigen-specific
cells;
[0567] e. culturing one or more sub-populations from the enriched
cell population under conditions that favor the survival of a
single B cell to produce a clonal population in at least one
culture well;
[0568] f determining whether the clonal population produces an
antibody specific to the antigen;
[0569] g. isolating a single B cell; and
[0570] h. sequencing the nucleic acid sequence of the antibody
produced by the single B cell.
[0571] Methods of Humanizing Antibodies
[0572] In another embodiment of the invention, there is provided a
method for humanizing antibody heavy and light chains. In this
embodiment, the following method is followed for the humanization
of the heavy and light chains:
[0573] Light Chain
[0574] 1. Identify the amino acid that is the first one following
the signal peptide sequence. This is the start of Framework 1. The
signal peptide starts at the first initiation methionine and is
typically, but not necessarily 22 amino acids in length for rabbit
light chain protein sequences. The start of the mature polypeptide
can also be determined experimentally by N-terminal protein
sequencing, or can be predicted using a prediction algorithm. This
is also the start of Framework 1 as classically defined by those in
the field.
[0575] Example: RbtVL Amino acid residue 1 in FIG. 2, starting
`AYDM . . . `
[0576] 2. Identify the end of Framework 3. This is typically 86-90
amino acids following the start of Framework 1 and is typically a
cysteine residue preceded by two tyrosine residues. This is the end
of the Framework 3 as classically defined by those in the
field.
[0577] Example: RbtVL amino acid residue 88 in FIG. 2, ending as
`TYYC`
[0578] 3. Use the rabbit light chain sequence of the polypeptide
starting from the beginning of Framework 1 to the end of Framework
3 as defined above and perform a sequence homology search for the
most similar human antibody protein sequences. This will typically
be a search against human germline sequences prior to antibody
maturation in order to reduce the possibility of immunogenicity,
however any human sequences can be used. Typically a program like
BLAST can be used to search a database of sequences for the most
homologous. Databases of human antibody sequences can be found from
various sources such as NCBI (National Center for Biotechnology
Information).
[0579] Example: RbtVL amino acid sequence from residues numbered 1
through 88 in FIG. 2 is BLASTed against a human antibody germline
database. The top three unique returned sequences are shown in FIG.
2 as L12A, V1 and Vx02.
[0580] 4. Generally the most homologous human germline variable
light chain sequence is then used as the basis for humanization.
However those skilled in the art may decide to use another sequence
that wasn't the highest homology as determined by the homology
algorithm, based on other factors including sequence gaps and
framework similarities.
[0581] Example: In FIG. 2, L12A was the most homologous human
germline variable light chain sequence and is used as the basis for
the humanization of RbtVL.
[0582] 5. Determine the framework and CDR arrangement (FR1, FR2,
FR3, CDR1 & CDR2) for the human homolog being used for the
light chain humanization. This is using the traditional layout as
described in the field. Align the rabbit variable light chain
sequence with the human homolog, while maintaining the layout of
the framework and CDR regions.
[0583] Example: In FIG. 2, the RbtVL sequence is aligned with the
human homologous sequence L12A, and the framework and CDR domains
are indicated.
[0584] 6. Replace the human homologous light chain sequence CDR1
and CDR2 regions with the CDR1 and CDR2 sequences from the rabbit
sequence. If there are differences in length between the rabbit and
human CDR sequences then use the entire rabbit CDR sequences and
their lengths. It is possible that the specificity, affinity and/or
immunogenicity of the resulting humanized antibody may be unaltered
if smaller or larger sequence exchanges are performed, or if
specific residue(s) are altered, however the exchanges as described
have been used successfully, but do not exclude the possibility
that other changes may be permitted.
[0585] Example: In FIG. 2, the CDR1 and CDR2 amino acid residues of
the human homologous variable light chain L12A are replaced with
the CDR1 and CDR2 amino acid sequences from the RbtVL rabbit
antibody light chain sequence. The human L12A frameworks 1, 2 and 3
are unaltered. The resulting humanized sequence is shown below as
VLh from residues numbered 1 through 88. Note that the only
residues that are different from the L12A human sequence are
underlined, and are thus rabbit-derived amino acid residues. In
this example only 8 of the 88 residues are different than the human
sequence.
[0586] 7. After framework 3 of the new hybrid sequence created in
Step 6, attach the entire CDR3 of the rabbit light chain antibody
sequence. The CDR3 sequence can be of various lengths, but is
typically 9 to 15 amino acid residues in length. The CDR3 region
and the beginning of the following framework 4 region are defined
classically and identifiable by those skilled in the art. Typically
the beginning of Framework 4, and thus after the end of CDR3
consists of the sequence `FGGG . . . `, however some variation may
exist in these residues.
[0587] Example: In FIG. 2, the CDR3 of RbtVL (amino acid residues
numbered 89-100) is added after the end of framework 3 in the
humanized sequence indicated as VLh.
[0588] 8. The rabbit light chain framework 4, which is typically
the final 11 amino acid residues of the variable light chain and
begins as indicated in Step 7 above and typically ends with the
amino acid sequence ` . . . VVKR` is replaced with the nearest
human light chain framework 4 homolog, usually from germline
sequence. Frequently this human light chain framework 4 is of the
sequence `FGGGTKVEIKR`. It is possible that other human light chain
framework 4 sequences that are not the most homologous or otherwise
different may be used without affecting the specificity, affinity
and/or immunogenicity of the resulting humanized antibody. This
human light chain framework 4 sequence is added to the end of the
variable light chain humanized sequence immediately following the
CDR3 sequence from Step 7 above. This is now the end of the
variable light chain humanized amino acid sequence.
[0589] Example: In FIG. 2, Framework 4 (FR4) of the RbtVL rabbit
light chain sequence is shown above a homologous human FR4
sequence. The human FR4 sequence is added to the humanized variable
light chain sequence (VLh) right after the end of the CD3 region
added in Step 7 above.
[0590] In addition, FIGS. 34 and 35 depict preferred humanized
anti-IL-6 variable heavy and variable light chain sequences
humanized from the variable heavy and light regions in Ab1
according to the invention. These humanized light and heavy chain
regions are respectively contained in the polypeptides contained in
SEQ ID NO:647, or 651 and in SEQ ID NO:652, 656, 657 or 658. The
CDR2 of the humanized variable heavy region in SEQ ID NO:657
(containing a serine substitution in CDR2) is contained in SEQ ID
NO:658. Alignments illustrating variants of the light and heavy
chains are shown in FIGS. 36 and 37, respectively, with sequence
differences within the CDR regions highlighted. Sequence
identifiers of CDR sequences and of exemplary coding sequences are
summarized in Table 1, above.
[0591] Heavy Chain
[0592] 1. Identify the amino acid that is the first one following
the signal peptide sequence. This is the start of Framework 1. The
signal peptide starts at the first initiation methionine and is
typically 19 amino acids in length for rabbit heavy chain protein
sequences. Typically, but not necessarily always, the final 3 amino
acid residues of a rabbit heavy chain signal peptide are ` . . .
VQC`, followed by the start of Framework 1. The start of the mature
polypeptide can also be determined experimentally by N-terminal
protein sequencing, or can be predicted using a prediction
algorithm. This is also the start of Framework 1 as classically
defined by those in the field.
[0593] Example: RbtVH Amino acid residue 1 in FIG. 2, starting
`QEQL . . . `
[0594] 2. Identify the end of Framework 3. This is typically 95-100
amino acids following the start of Framework 1 and typically has
the final sequence of ` . . . CAR` (although the alanine can also
be a valine). This is the end of the Framework 3 as classically
defined by those in the field.
[0595] Example: RbtVH amino acid residue 98 in FIG. 2, ending
as
[0596] 3. Use the rabbit heavy chain sequence of the polypeptide
starting from the beginning of Framework 1 to the end of Framework
3 as defined above and perform a sequence homology search for the
most similar human antibody protein sequences. This will typically
be against a database of human germline sequences prior to antibody
maturation in order to reduce the possibility of immunogenicity,
however any human sequences can be used. Typically a program like
BLAST can be used to search a database of sequences for the most
homologous. Databases of human antibody sequences can be found from
various sources such as NCBI (National Center for Biotechnology
Information).
[0597] Example: RbtVH amino acid sequence from residues numbered 1
through 98 in FIG. 2 is BLASTed against a human antibody germline
database. The top three unique returned sequences are shown in FIG.
2 as 3-64-04, 3-66-04, and 3-53-02.
[0598] 4. Generally the most homologous human germline variable
heavy chain sequence is then used as the basis for humanization.
However those skilled in the art may decide to use another sequence
that wasn't the most homologous as determined by the homology
algorithm, based on other factors including sequence gaps and
framework similarities.
[0599] Example: 3-64-04 in FIG. 2 was the most homologous human
germline variable heavy chain sequence and is used as the basis for
the humanization of RbtVH.
[0600] 5. Determine the framework and CDR arrangement (FR1, FR2,
FR3, CDR1 & CDR2) for the human homolog being used for the
heavy chain humanization. This is using the traditional layout as
described in the field. Align the rabbit variable heavy chain
sequence with the human homolog, while maintaining the layout of
the framework and CDR regions.
[0601] Example: In FIG. 2, the RbtVH sequence is aligned with the
human homologous sequence 3-64-04, and the framework and CDR
domains are indicated.
[0602] 6. Replace the human homologous heavy chain sequence CDR1
and CDR2 regions with the CDR1 and CDR2 sequences from the rabbit
sequence. If there are differences in length between the rabbit and
human CDR sequences then use the entire rabbit CDR sequences and
their lengths. In addition, it may be necessary to replace the
final three amino acids of the human heavy chain Framework 1 region
with the final three amino acids of the rabbit heavy chain
Framework 1. Typically but not always, in rabbit heavy chain
Framework 1 these three residues follow a Glycine residue preceded
by a Serine residue. In addition, it may be necessary replace the
final amino acid of the human heavy chain Framework 2 region with
the final amino acid of the rabbit heavy chain Framework 2.
Typically, but not necessarily always, this is a Glycine residue
preceded by an Isoleucine residue in the rabbit heavy chain
Framework 2. It is possible that the specificity, affinity and/or
immunogenicity of the resulting humanized antibody may be unaltered
if smaller or larger sequence exchanges are performed, or if
specific residue(s) are altered, however the exchanges as described
have been used successfully, but do not exclude the possibility
that other changes may be permitted. For example, a tryptophan
amino acid residue typically occurs four residues prior to the end
of the rabbit heavy chain CDR2 region, whereas in human heavy chain
CDR2 this residue is typically a Serine residue. Changing this
rabbit tryptophan residue to a the human Serine residue at this
position has been demonstrated to have minimal to no effect on the
humanized antibody's specificity or affinity, and thus further
minimizes the content of rabbit sequence-derived amino acid
residues in the humanized sequence.
[0603] Example: In FIG. 2, The CDR1 and CDR2 amino acid residues of
the human homologous variable heavy chain are replaced with the
CDR1 and CDR2 amino acid sequences from the RbtVH rabbit antibody
light chain sequence, except for the boxed residue, which is
tryptophan in the rabbit sequence (position number 63) and Serine
at the same position in the human sequence, and is kept as the
human Serine residue. In addition to the CDR1 and CDR2 changes, the
final three amino acids of Framework 1 (positions 28-30) as well as
the final residue of Framework 2 (position 49) are retained as
rabbit amino acid residues instead of human. The resulting
humanized sequence is shown below as VHh from residues numbered 1
through 98. Note that the only residues that are different from the
3-64-04 human sequence are underlined, and are thus rabbit-derived
amino acid residues. In this example only 15 of the 98 residues are
different than the human sequence.
[0604] 7. After framework 3 of the new hybrid sequence created in
Step 6, attach the entire CDR3 of the rabbit heavy chain antibody
sequence. The CDR3 sequence can be of various lengths, but is
typically 5 to 19 amino acid residues in length. The CDR3 region
and the beginning of the following framework 4 region are defined
classically and are identifiable by those skilled in the art.
Typically the beginning of framework 4, and thus after the end of
CDR3 consists of the sequence WGXG . . . (where X is usually Q or
P), however some variation may exist in these residues.
[0605] Example: The CDR3 of RbtVH (amino acid residues numbered
99-110) is added after the end of framework 3 in the humanized
sequence indicated as VHh.
[0606] 8. The rabbit heavy chain framework 4, which is typically
the final 11 amino acid residues of the variable heavy chain and
begins as indicated in Step 7 above and typically ends with the
amino acid sequence ` . . . TVSS` is replaced with the nearest
human heavy chain framework 4 homolog, usually from germline
sequence. Frequently this human heavy chain framework 4 is of the
sequence `WGQGTLVTVSS`. It is possible that other human heavy chain
framework 4 sequences that are not the most homologous or otherwise
different may be used without affecting the specificity, affinity
and/or immunogenicity of the resulting humanized antibody. This
human heavy chain framework 4 sequence is added to the end of the
variable heavy chain humanized sequence immediately following the
CDR3 sequence from Step 7 above. This is now the end of the
variable heavy chain humanized amino acid sequence.
[0607] Example: In FIG. 2, framework 4 (FR4) of the RbtVH rabbit
heavy chain sequence is shown above a homologous human heavy FR4
sequence. The human FR4 sequence is added to the humanized variable
heavy chain sequence (VHh) right after the end of the CD3 region
added in Step 7 above.
[0608] Methods of Producing Antibodies and Fragments Thereof
[0609] The invention is also directed to the production of the
antibodies described herein or fragments thereof. Recombinant
polypeptides corresponding to the antibodies described herein or
fragments thereof are secreted from polyploidal, preferably diploid
or tetraploid strains of mating competent yeast. In an exemplary
embodiment, the invention is directed to methods for producing
these recombinant polypeptides in secreted form for prolonged
periods using cultures comprising polyploid yeast, i.e., at least
several days to a week, more preferably at least a month or several
months, and even more preferably at least 6 months to a year or
longer. These polyploid yeast cultures will express at least 10-25
mg/liter of the polypeptide, more preferably at least 50-250
mg/liter, still more preferably at least 500-1000 mg/liter, and
most preferably a gram per liter or more of the recombinant
polypeptide(s).
[0610] In one embodiment of the invention a pair of genetically
marked yeast haploid cells are transformed with expression vectors
comprising subunits of a desired heteromultimeric protein. One
haploid cell comprises a first expression vector, and a second
haploid cell comprises a second expression vector. In another
embodiment diploid yeast cells will be transformed with one or more
expression vectors that provide for the expression and secretion of
one or more of the recombinant polypeptides. In still another
embodiment a single haploid cell may be transformed with one or
more vectors and used to produce a polyploidal yeast by fusion or
mating strategies. In yet another embodiment a diploid yeast
culture may be transformed with one or more vectors providing for
the expression and secretion of a desired polypeptide or
polypeptides. These vectors may comprise vectors e.g., linearized
plasmids or other linear DNA products that integrate into the yeast
cell's genome randomly, through homologous recombination, or using
a recombinase such as Cre/Lox or Flp/Frt. Optionally, additional
expression vectors may be introduced into the haploid or diploid
cells; or the first or second expression vectors may comprise
additional coding sequences; for the synthesis of heterotrimers;
heterotetramers; etc. The expression levels of the non-identical
polypeptides may be individually calibrated, and adjusted through
appropriate selection, vector copy number, promoter strength and/or
induction and the like. The transformed haploid cells are
genetically crossed or fused. The resulting diploid or tetraploid
strains are utilized to produce and secrete fully assembled and
biologically functional proteins, humanized antibodies described
herein or fragments thereof.
[0611] The use of diploid or tetraploid cells for protein
production provides for unexpected benefits. The cells can be grown
for production purposes, i.e. scaled up, and for extended periods
of time, in conditions that can be deleterious to the growth of
haploid cells, which conditions may include high cell density;
growth in minimal media; growth at low temperatures; stable growth
in the absence of selective pressure; and which may provide for
maintenance of heterologous gene sequence integrity and maintenance
of high level expression over time. Without wishing to be bound
thereby, the inventors theorize that these benefits may arise, at
least in part, from the creation of diploid strains from two
distinct parental haploid strains. Such haploid strains can
comprise numerous minor autotrophic mutations, which mutations are
complemented in the diploid or tetraploid, enabling growth and
enhanced production under highly selective conditions.
[0612] Transformed mating competent haploid yeast cells provide a
genetic method that enables subunit pairing of a desired protein.
Haploid yeast strains are transformed with each of two expression
vectors, a first vector to direct the synthesis of one polypeptide
chain and a second vector to direct the synthesis of a second,
non-identical polypeptide chain. The two haploid strains are mated
to provide a diploid host where optimized target protein production
can be obtained.
[0613] Optionally, additional non-identical coding sequence(s) are
provided. Such sequences may be present on additional expression
vectors or in the first or the second expression vectors. As is
known in the art, multiple coding sequences may be independently
expressed from individual promoters; or may be coordinately
expressed through the inclusion of an "internal ribosome entry
site" or "IRES", which is an element that promotes direct internal
ribosome entry to the initiation codon, such as ATG, of a cistron
(a protein encoding region), thereby leading to the cap-independent
translation of the gene. IRES elements functional in yeast are
described by Thompson et al. (2001) P.N.A.S. 98:12866-12868.
[0614] In one embodiment of the invention, antibody sequences are
produced in combination with a secretory J chain, which provides
for enhanced stability of IgA (see U.S. Pat. Nos. 5,959,177; and
5,202,422).
[0615] In a preferred embodiment the two haploid yeast strains are
each auxotrophic, and require supplementation of media for growth
of the haploid cells. The pair of auxotrophs are complementary,
such that the diploid product will grow in the absence of the
supplements required for the haploid cells. Many such genetic
markers are known in yeast, including requirements for amino acids
(e.g. met, lys, his, arg, etc.), nucleosides (e.g. ura3, ade1,
etc.); and the like. Amino acid markers may be preferred for the
methods of the invention. Alternatively diploid cells which contain
the desired vectors can be selected by other means, e.g., by use of
other markers, such as green fluorescent protein, antibiotic
resistance genes, various dominant selectable markers, and the
like.
[0616] Two transformed haploid cells may be genetically crossed and
diploid strains arising from this mating event selected by their
hybrid nutritional requirements and/or antibiotic resistance
spectra. Alternatively, populations of the two transformed haploid
strains are spheroplasted and fused, and diploid progeny
regenerated and selected. By either method, diploid strains can be
identified and selectively grown based on their ability to grow in
different media than their parents. For example, the diploid cells
may be grown in minimal medium that may include antibiotics. The
diploid synthesis strategy has certain advantages. Diploid strains
have the potential to produce enhanced levels of heterologous
protein through broader complementation to underlying mutations,
which may impact the production and/or secretion of recombinant
protein. Furthermore, once stable strains have been obtained, any
antibiotics used to select those strains do not necessarily need to
be continuously present in the growth media.
[0617] As noted above, in some embodiments a haploid yeast may be
transformed with a single or multiple vectors and mated or fused
with a non-transformed cell to produce a diploid cell containing
the vector or vectors. In other embodiments, a diploid yeast cell
may be transformed with one or more vectors that provide for the
expression and secretion of a desired heterologous polypeptide by
the diploid yeast cell.
[0618] In one embodiment of the invention, two haploid strains are
transformed with a library of polypeptides, e.g. a library of
antibody heavy or light chains. Transformed haploid cells that
synthesize the polypeptides are mated with the complementary
haploid cells. The resulting diploid cells are screened for
functional protein. The diploid cells provide a means of rapidly,
conveniently and inexpensively bringing together a large number of
combinations of polypeptides for functional testing. This
technology is especially applicable for the generation of
heterodimeric protein products, where optimized subunit synthesis
levels are critical for functional protein expression and
secretion.
[0619] In another embodiment of the invention, the expression level
ratio of the two subunits is regulated in order to maximize product
generation. Heterodimer subunit protein levels have been shown
previously to impact the final product generation (Simmons L C, J
Immunol Methods. 2002 May 1; 263(1-2):133-47). Regulation can be
achieved prior to the mating step by selection for a marker present
on the expression vector. By stably increasing the copy number of
the vector, the expression level can be increased. In some cases,
it may be desirable to increase the level of one chain relative to
the other, so as to reach a balanced proportion between the
subunits of the polypeptide. Antibiotic resistance markers are
useful for this purpose, e.g. Zeocin resistance marker, G418
resistance, etc. and provide a means of enrichment for strains that
contain multiple integrated copies of an expression vector in a
strain by selecting for transformants that are resistant to higher
levels of Zeocin or G418. The proper ratio, e.g. 1:1; 1:2; etc. of
the subunit genes may be important for efficient protein
production. Even when the same promoter is used to transcribe both
subunits, many other factors contribute to the final level of
protein expressed and therefore, it can be useful to increase the
number of copies of one encoded gene relative to the other.
Alternatively, diploid strains that produce higher levels of a
polypeptide, relative to single copy vector strains, are created by
mating two haploid strains, both of which have multiple copies of
the expression vectors.
[0620] Host cells are transformed with the above-described
expression vectors, mated to form diploid strains, and cultured in
conventional nutrient media modified as appropriate for inducing
promoters, selecting transformants or amplifying the genes encoding
the desired sequences. A number of minimal media suitable for the
growth of yeast are known in the art. Any of these media may be
supplemented as necessary with salts (such as sodium chloride,
calcium, magnesium, and phosphate), buffers (such as phosphate,
HEPES), nucleosides (such as adenosine and thymidine), antibiotics,
trace elements, and glucose or an equivalent energy source. Any
other necessary supplements may also be included at appropriate
concentrations that would be known to those skilled in the art. The
culture conditions, such as temperature, pH and the like, are those
previously used with the host cell selected for expression, and
will be apparent to the ordinarily skilled artisan.
[0621] Secreted proteins are recovered from the culture medium. A
protease inhibitor, such as phenyl methyl sulfonyl fluoride (PMSF)
may be useful to inhibit proteolytic degradation during
purification, and antibiotics may be included to prevent the growth
of adventitious contaminants. The composition may be concentrated,
filtered, dialyzed, etc., using methods known in the art.
[0622] The diploid cells of the invention are grown for production
purposes. Such production purposes desirably include growth in
minimal media, which media lacks pre-formed amino acids and other
complex biomolecules, e.g., media comprising ammonia as a nitrogen
source, and glucose as an energy and carbon source, and salts as a
source of phosphate, calcium and the like. Preferably such
production media lacks selective agents such as antibiotics, amino
acids, purines, pyrimidines, etc. The diploid cells can be grown to
high cell density, for example at least about 50 g/L; more usually
at least about 100 g/L; and may be at least about 300, about 400,
about 500 g/L or more.
[0623] In one embodiment of the invention, the growth of the
subject cells for production purposes is performed at low
temperatures, which temperatures may be lowered during log phase,
during stationary phase, or both. The term "low temperature" refers
to temperatures of at least about 15.degree. C., more usually at
least about 17.degree. C., and may be about 20.degree. C., and is
usually not more than about 25.degree. C., more usually not more
than about 22.degree. C. In another embodiment of the invention,
the low temperature is usually not more than about 28.degree. C.
Growth temperature can impact the production of full-length
secreted proteins in production cultures, and decreasing the
culture growth temperature can strongly enhance the intact product
yield. The decreased temperature appears to assist intracellular
trafficking through the folding and post-translational processing
pathways used by the host to generate the target product, along
with reduction of cellular protease degradation.
[0624] The methods of the invention provide for expression of
secreted, active protein, preferably a mammalian protein. In one
embodiment, secreted, "active antibodies", as used herein, refers
to a correctly folded multimer of at least two properly paired
chains, which accurately binds to its cognate antigen. Expression
levels of active protein are usually at least about 10-50 mg/liter
culture, more usually at least about 100 mg/liter, preferably at
least about 500 mg/liter, and may be 1000 mg/liter or more.
[0625] The methods of the invention can provide for increased
stability of the host and heterologous coding sequences during
production. The stability is evidenced, for example, by maintenance
of high levels of expression of time, where the starting level of
expression is decreased by not more than about 20%, usually not
more than 10%, and may be decreased by not more than about 5% over
about 20 doublings, 50 doublings, 100 doublings, or more.
[0626] The strain stability also provides for maintenance of
heterologous gene sequence integrity over time, where the sequence
of the active coding sequence and requisite transcriptional
regulatory elements are maintained in at least about 99% of the
diploid cells, usually in at least about 99.9% of the diploid
cells, and preferably in at least about 99.99% of the diploid cells
over about 20 doublings, 50 doublings, 100 doublings, or more.
Preferably, substantially all of the diploid cells maintain the
sequence of the active coding sequence and requisite
transcriptional regulatory elements.
[0627] Other methods of producing antibodies are well known to
those of ordinary skill in the art. For example, methods of
producing chimeric antibodies are now well known in the art (See,
for example, U.S. Pat. No. 4,816,567 to Cabilly et al.; Morrison et
al., P.N.A.S. USA, 81:8651-55 (1984); Neuberger, M. S. et al.,
Nature, 314:268-270 (1985); Boulianne, G. L. et al., Nature,
312:643-46 (1984), the disclosures of each of which are herein
incorporated by reference in their entireties).
[0628] Likewise, other methods of producing humanized antibodies
are now well known in the art (See, for example, U.S. Pat. Nos.
5,530,101, 5,585,089, 5,693,762, and 6,180,370 to Queen et al; U.S.
Pat. Nos. 5,225,539 and 6,548,640 to Winter; U.S. Pat. Nos.
6,054,297, 6,407,213 and 6,639,055 to Carter et al; U.S. Pat. No.
6,632,927 to Adair; Jones, P. T. et al, Nature, 321:522-525 (1986);
Reichmann, L., et al, Nature, 332:323-327 (1988); Verhoeyen, M, et
al, Science, 239:1534-36 (1988), the disclosures of each of which
are herein incorporated by reference in their entireties).
[0629] Antibody polypeptides of the invention having IL-6 binding
specificity may also be produced by constructing, using
conventional techniques well known to those of ordinary skill in
the art, an expression vector containing an operon and a DNA
sequence encoding an antibody heavy chain in which the DNA sequence
encoding the CDRs required for antibody specificity is derived from
a non-human cell source, preferably a rabbit B-cell source, while
the DNA sequence encoding the remaining parts of the antibody chain
is derived from a human cell source.
[0630] A second expression vector is produced using the same
conventional means well known to those of ordinary skill in the
art, said expression vector containing an operon and a DNA sequence
encoding an antibody light chain in which the DNA sequence encoding
the CDRs required for antibody specificity is derived from a
non-human cell source, preferably a rabbit B-cell source, while the
DNA sequence encoding the remaining parts of the antibody chain is
derived from a human cell source.
[0631] The expression vectors are transfected into a host cell by
convention techniques well known to those of ordinary skill in the
art to produce a transfected host cell, said transfected host cell
cultured by conventional techniques well known to those of ordinary
skill in the art to produce said antibody polypeptides.
[0632] The host cell may be co-transfected with the two expression
vectors described above, the first expression vector containing DNA
encoding an operon and a light chain-derived polypeptide and the
second vector containing DNA encoding an operon and a heavy
chain-derived polypeptide. The two vectors contain different
selectable markers, but preferably achieve substantially equal
expression of the heavy and light chain polypeptides.
Alternatively, a single vector may be used, the vector including
DNA encoding both the heavy and light chain polypeptides. The
coding sequences for the heavy and light chains may comprise
cDNA.
[0633] The host cells used to express the antibody polypeptides may
be either a bacterial cell such as E. coli, or a eukaryotic cell.
In a particularly preferred embodiment of the invention, a
mammalian cell of a well-defined type for this purpose, such as a
myeloma cell or a Chinese hamster ovary (CHO) cell line may be
used.
[0634] The general methods by which the vectors may be constructed,
transfection methods required to produce the host cell and
culturing methods required to produce the antibody polypeptides
from said host cells all include conventional techniques. Although
preferably the cell line used to produce the antibody is a
mammalian cell line, any other suitable cell line, such as a
bacterial cell line such as an E. coli-derived bacterial strain, or
a yeast cell line, may alternatively be used.
[0635] Similarly, once produced the antibody polypeptides may be
purified according to standard procedures in the art, such as for
example cross-flow filtration, ammonium sulphate precipitation,
affinity column chromatography and the like.
[0636] The antibody polypeptides described herein may also be used
for the design and synthesis of either peptide or non-peptide
mimetics that would be useful for the same therapeutic applications
as the antibody polypeptides of the invention. See, for example,
Saragobi et al, Science, 253:792-795 (1991), the contents of which
is herein incorporated by reference in its entirety.
Exemplary Embodiments of Heavy and Light Chain Polypeptides and
Polynucleotides
[0637] This section recites exemplary embodiments of heavy and
light chain polypeptides, as well as exemplary polynucleotides
encoding such polypeptides. These exemplary polynucleotides are
suitable for expression in the disclosed Pichia expression
system.
[0638] In certain embodiments, the present invention encompasses
polynucleotides having at least 70%, such as at least 75%, at least
80%, at least 85%, at least 90%, at least 91%, at least 92%, at
least 93%, at least 94%, at least 95%, at least 96%, at least 97%,
at least 98%, at least 99%, or 100% identity to the polynucleotides
recited in this application or that encode polypeptides recited in
this application, or that hybridize to said polynucleotides under
conditions of low-stringency, moderate-stringency, or
high-stringency conditions, preferably those that encode
polypeptides (e.g. an immunoglobulin heavy and light chain, a
single-chain antibody, an antibody fragment, etc.) that have at
least one of the biological activities set forth herein, including
without limitation thereto specific binding to an IL-6 polypeptide.
In another aspect, the invention encompasses a composition
comprising such a polynucleotide and/or a polypeptide encoded by
such a polynucleotide. In yet another aspect, the invention
encompasses a method of treatment of a disease or condition
associated with IL-6 or that may be prevented, treated, or
ameliorated with an IL-6 antagonist such as Ab1 (e.g. cachexia,
cancer fatigue, arthritis, etc.) comprising administration of a
composition comprising such a polynucleotide and/or
polypeptide.
[0639] In certain preferred embodiments, a heavy chain polypeptide
will comprise one or more of the CDR sequences of the heavy and/or
light chain polypeptides recited herein (including those contained
in the heavy and light chain polypeptides recited herein) and one
or more of the framework region polypeptides recited herein,
including those depicted in FIGS. 2 and 34-37 or Table 1, and
contained in the heavy and light chain polypeptide sequences
recited herein. In certain preferred embodiments, a heavy chain
polypeptide will comprise one or more Framework 4 region sequences
as depicted in FIGS. 2 and 34-37 or Table 1, or as contained in a
heavy or light chain polypeptide recited herein.
[0640] In certain preferred embodiments, a light chain polypeptide
will comprise one or more of the CDR sequences of the heavy and/or
light chain polypeptides recited herein (including those contained
in the heavy and light chain polypeptides recited herein) and one
or more of the Framework region polypeptides recited herein,
including those depicted in FIGS. 2 and 34-37 or Table 1, and
contained in the heavy and light chain polypeptide sequences
recited herein. In certain preferred embodiments, a light chain
polypeptide will comprise one or more Framework 4 region sequences
as depicted in FIGS. 2 and 34-37 or Table 1, or as contained in a
heavy or light chain polypeptide recited herein.
[0641] In any of the embodiments recited herein, certain of the
sequences recited may be substituted for each other, unless the
context indicates otherwise. The recitation that particular
sequences may be substituted for one another, where such
recitations are made, are understood to be illustrative rather than
limiting, and it is also understood that such substitutions are
encompassed even when no illustrative examples of substitutions are
recited, For example, wherever one or more of the Ab1 light chain
polypeptides is recited, e.g. any of SEQ ID NO: 2, 20, 647, 651,
660, 666, 699, 702, 706, or 709, another Ab1 light chain
polypeptide may be substituted unless the context indicates
otherwise. Similarly, wherever one of the Ab1 heavy chain
polypeptides is recited, e.g. any of SEQ ID NO: 3, 18, 19, 652,
656, 657, 661, 664, 665, 657, 658, 704, or 708, another Ab1 heavy
chain polypeptide may be substituted unless the context indicates
otherwise. Likewise, wherever one of the Ab1 light chain
polynucleotides is recited, e.g. any of SEQ ID NO:10, 662, 698,
701, or 705, another Ab1 light chain polynucleotide may be
substituted unless the context indicates otherwise. Similarly,
wherever one of the Ab1 heavy chain polynucleotides is recited,
e.g. any of SEQ ID NO:11, 663, 700, 703, or 707, another Ab1 heavy
chain polynucleotide may be substituted unless the context
indicates otherwise. Additionally, recitation of any member of any
of the following groups is understood to encompass substitution by
any other member of the group, as follows: Ab2 Light chain
polypeptides (SEQ ID NO:21 and 667); Ab2 Light chain
polynucleotides (SEQ ID NO:29 and 669); Ab2 Heavy chain
polypeptides (SEQ ID NO:22 and 668); Ab2 Heavy chain
polynucleotides (SEQ ID NO:30 and 670); Ab3 Light chain
polypeptides (SEQ ID NO:37 and 671); Ab3 Light chain
polynucleotides (SEQ ID NO:45 and 673); Ab3 Heavy chain
polypeptides (SEQ ID NO:38 and 672); Ab3 Heavy chain
polynucleotides (SEQ ID NO:46 and 674); Ab4 Light chain
polypeptides (SEQ ID NO:53 and 675); Ab4 Light chain
polynucleotides (SEQ ID NO:61 and 677); Ab4 Heavy chain
polypeptides (SEQ ID NO:54 and 676); Ab4 Heavy chain
polynucleotides (SEQ ID NO:62 and 678); Ab5 Light chain
polypeptides (SEQ ID NO:69 and 679); Ab5 Light chain
polynucleotides (SEQ ID NO:77 and 681); Ab5 Heavy chain
polypeptides (SEQ ID NO:70 and 680); Ab5 Heavy chain
polynucleotides (SEQ ID NO:78 and 682); Ab6 Light chain
polypeptides (SEQ ID NO:85 and 683); Ab6 Light chain
polynucleotides (SEQ ID NO:93 and 685); Ab6 Heavy chain
polypeptides (SEQ ID NO:86 and 684); Ab6 Heavy chain
polynucleotides (SEQ ID NO:94 and 686); Ab7 Light chain
polypeptides (SEQ ID NO:101, 119, 687, 693); Ab7 Light chain
polynucleotides (SEQ ID NO:109 and 689); Ab7 Heavy chain
polypeptides (SEQ ID NO:102, 117, 118, 688, 691, and 692); Ab7
Heavy chain polynucleotides (SEQ ID NO:110 and 690); Ab1 Light
Chain CDR1 polynucleotides (SEQ ID NO:12 and 694); Ab1 Light Chain
CDR3 polynucleotides (SEQ ID NO:14 and 695); Ab1 Heavy Chain CDR2
polynucleotides (SEQ ID NO:16 and 696) and Ab1 Heavy Chain CDR3
polynucleotides (SEQ ID NO:17 and 697).
[0642] Exemplary Ab1-encoding polynucleotide sequences are recited
as follows:
TABLE-US-00010 SEQ ID NO: 662:
ATGGACACGAGGGCCCCCACTCAGCTGCTGGGGCTCCTGCTGCTCTGGCT
CCCAGGTGCCAGATGTGCCTATGATATGACCCAGACTCCAGCCTCGGTGT
CTGCAGCTGTGGGAGGCACAGTCACCATCAAGTGCCAGGCCAGTCAGAGC
ATTAACAATGAATTATCCTGGTATCAGCAGAAACCAGGGCAGCGTCCCAA
GCTCCTGATCTATAGGGCATCCACTCTGGCATCTGGGGTCTCATCGCGGT
TCAAAGGCAGTGGATCTGGGACAGAGTTCACTCTCACCATCAGCGACCTG
GAGTGTGCCGATGCTGCCACTTACTACTGTCAACAGGGTTATAGTCTGAG GAATATTGATAATGCT
SEQ ID NO: 663: ATGGAGACTGGGCTGCGCTGGCTTCTCCTGGTCGCTGTGCTCAAAGGTGT
CCAGTGTCAGTCGCTGGAGGAGTCCGGGGGTCGCCTGGTCACGCCTGGGA
CACCCCTGACACTCACCTGCACAGCCTCTGGATTCTCCCTCAGTAACTAC
TACGTGACCTGGGTCCGCCAGGCTCCAGGGAAGGGGCTGGAATGGATCGG
AATCATTTATGGTAGTGATGAAACGGCCTACGCGACCTGGGCGATAGGCC
GATTCACCATCTCCAAAACCTCGACCACGGTGGATCTGAAAATGACCAGT
CTGACAGCCGCGGACACGGCCACCTATTTCTGTGCCAGAGATGATAGTAG
TGACTGGGATGCAAAATTTAACTTG SEQ ID NO: 698:
GCTATCCAGATGACCCAGTCTCCTTCCTCCCTGTCTGCATCTGTAGGAGA
CAGAGTCACCATCACTTGCCAGGCCAGTCAGAGCATTAACAATGAGTTAT
CCTGGTATCAGCAGAAACCAGGGAAAGCCCCTAAGCTCCTGATCTATAGG
GCATCCACTCTGGCATCTGGGGTCCCATCAAGGTTCAGCGGCAGTGGATC
TGGGACAGACTTCACTCTCACCATCAGCAGCCTGCAGCCTGATGATTTTG
CAACTTATTACTGCCAACAGGGTTATAGTCTGAGGAACATTGATAATGCT
TTCGGCGGAGGGACCAAGGTGGAAATCAAACGTACG SEQ ID NO: 700:
GAGGTGCAGCTGGTGGAGTCTGGGGGAGGCTTGGTCCAGCCTGGGGGGTC
CCTGAGACTCTCCTGTGCAGCCTCTGGATTCTCCCTCAGTAACTACTACG
TGACCTGGGTCCGTCAGGCTCCAGGGAAGGGGCTGGAGTGGGTCGGCATC
ATCTATGGTAGTGATGAAACCGCCTACGCTACCTCCGCTATAGGCCGATT
CACCATCTCCAGAGACAATTCCAAGAACACCCTGTATCTTCAAATGAACA
GCCTGAGAGCTGAGGACACTGCTGTGTATTACTGTGCTAGAGATGATAGT
AGTGACTGGGATGCAAAGTTCAACTTGTGGGGCCAAGGGACCCTCGTCAC CGTCTCGAGC SEQ
ID NO: 701: GCTATCCAGATGACCCAGTCTCCTTCCTCCCTGTCTGCATCTGTAGGAGA
CAGAGTCACCATCACTTGCCAGGCCAGTCAGAGCATTAACAATGAGTTAT
CCTGGTATCAGCAGAAACCAGGGAAAGCCCCTAAGCTCCTGATCTATAGG
GCATCCACTCTGGCATCTGGGGTCCCATCAAGGTTCAGCGGCAGTGGATC
TGGGACAGACTTCACTCTCACCATCAGCAGCCTGCAGCCTGATGATTTTG
CAACTTATTACTGCCAACAGGGTTATAGTCTGAGGAACATTGATAATGCT
TTCGGCGGAGGGACCAAGGTGGAAATCAAACGTACGGTGGCTGCACCATC
TGTCTTCATCTTCCCGCCATCTGATGAGCAGTTGAAATCTGGAACTGCCT
CTGTTGTGTGCCTGCTGAATAACTTCTATCCCAGAGAGGCCAAAGTACAG
TGGAAGGTGGATAACGCCCTCCAATCGGGTAACTCCCAGGAGAGTGTCAC
AGAGCAGGACAGCAAGGACAGCACCTACAGCCTCAGCAGCACCCTGACGC
TGAGCAAAGCAGACTACGAGAAACACAAAGTCTACGCCTGCGAAGTCACC
CATCAGGGCCTGAGCTCGCCCGTCACAAAGAGCTTCAACAGGGGAGAGTG T SEQ ID NO:
703: GAGGTGCAGCTGGTGGAGTCTGGGGGAGGCTTGGTCCAGCCTGGGGGGTC
CCTGAGACTCTCCTGTGCAGCCTCTGGATTCTCCCTCAGTAACTACTACG
TGACCTGGGTCCGTCAGGCTCCAGGGAAGGGGCTGGAGTGGGTCGGCATC
ATCTATGGTAGTGATGAAACCGCCTACGCTACCTCCGCTATAGGCCGATT
CACCATCTCCAGAGACAATTCCAAGAACACCCTGTATCTTCAAATGAACA
GCCTGAGAGCTGAGGACACTGCTGTGTATTACTGTGCTAGAGATGATAGT
AGTGACTGGGATGCAAAGTTCAACTTGTGGGGCCAAGGGACCCTCGTCAC
CGTCTCGAGCGCCTCCACCAAGGGCCCATCGGTCTTCCCCCTGGCACCCT
CCTCCAAGAGCACCTCTGGGGGCACAGCGGCCCTGGGCTGCCTGGTCAAG
GACTACTTCCCCGAACCGGTGACGGTGTCGTGGAACTCAGGCGCCCTGAC
CAGCGGCGTGCACACCTTCCCGGCTGTCCTACAGTCCTCAGGACTCTACT
CCCTCAGCAGCGTGGTGACCGTGCCCTCCAGCAGCTTGGGCACCCAGACC
TACATCTGCAACGTGAATCACAAGCCCAGCAACACCAAGGTGGACAAGAG
AGTTGAGCCCAAATCTTGTGACAAAACTCACACATGCCCACCGTGCCCAG
CACCTGAACTCCTGGGGGGACCGTCAGTCTTCCTCTTCCCCCCAAAACCC
AAGGACACCCTCATGATCTCCCGGACCCCTGAGGTCACATGCGTGGTGGT
GGACGTGAGCCACGAAGACCCTGAGGTCAAGTTCAACTGGTACGTGGACG
GCGTGGAGGTGCATAATGCCAAGACAAAGCCGCGGGAGGAGCAGTACGCC
AGCACGTACCGTGTGGTCAGCGTCCTCACCGTCCTGCACCAGGACTGGCT
GAATGGCAAGGAGTACAAGTGCAAGGTCTCCAACAAAGCCCTCCCAGCCC
CCATCGAGAAAACCATCTCCAAAGCCAAAGGGCAGCCCCGAGAACCACAG
GTGTACACCCTGCCCCCATCCCGGGAGGAGATGACCAAGAACCAGGTCAG
CCTGACCTGCCTGGTCAAAGGCTTCTATCCCAGCGACATCGCCGTGGAGT
GGGAGAGCAATGGGCAGCCGGAGAACAACTACAAGACCACGCCTCCCGTG
CTGGACTCCGACGGCTCCTTCTTCCTCTACAGCAAGCTCACCGTGGACAA
GAGCAGGTGGCAGCAGGGGAACGTCTTCTCATGCTCCGTGATGCATGAGG
CTCTGCACAACCACTACACGCAGAAGAGCCTCTCCCTGTCTCCGGGTAAA SEQ ID NO: 705:
ATGAAGTGGGTAACCTTTATTTCCCTTCTGTTTCTCTTTAGCAGCGCTTA
TTCCGCTATCCAGATGACCCAGTCTCCTTCCTCCCTGTCTGCATCTGTAG
GAGACAGAGTCACCATCACTTGCCAGGCCAGTCAGAGCATTAACAATGAG
TTATCCTGGTATCAGCAGAAACCAGGGAAAGCCCCTAAGCTCCTGATCTA
TAGGGCATCCACTCTGGCATCTGGGGTCCCATCAAGGTTCAGCGGCAGTG
GATCTGGGACAGACTTCACTCTCACCATCAGCAGCCTGCAGCCTGATGAT
TTTGCAACTTATTACTGCCAACAGGGTTATAGTCTGAGGAACATTGATAA
TGCTTTCGGCGGAGGGACCAAGGTGGAAATCAAACGTACGGTGGCTGCAC
CATCTGTCTTCATCTTCCCGCCATCTGATGAGCAGTTGAAATCTGGAACT
GCCTCTGTTGTGTGCCTGCTGAATAACTTCTATCCCAGAGAGGCCAAAGT
ACAGTGGAAGGTGGATAACGCCCTCCAATCGGGTAACTCCCAGGAGAGTG
TCACAGAGCAGGACAGCAAGGACAGCACCTACAGCCTCAGCAGCACCCTG
ACGCTGAGCAAAGCAGACTACGAGAAACACAAAGTCTACGCCTGCGAAGT
CACCCATCAGGGCCTGAGCTCGCCCGTCACAAAGAGCTTCAACAGGGGAG AGTGT SEQ ID NO:
707: ATGAAGTGGGTAACCTTTATTTCCCTTCTGTTTCTCTTTAGCAGCGCTTA
TTCCGAGGTGCAGCTGGTGGAGTCTGGGGGAGGCTTGGTCCAGCCTGGGG
GGTCCCTGAGACTCTCCTGTGCAGCCTCTGGATTCTCCCTCAGTAACTAC
TACGTGACCTGGGTCCGTCAGGCTCCAGGGAAGGGGCTGGAGTGGGTCGG
CATCATCTATGGTAGTGATGAAACCGCCTACGCTACCTCCGCTATAGGCC
GATTCACCATCTCCAGAGACAATTCCAAGAACACCCTGTATCTTCAAATG
AACAGCCTGAGAGCTGAGGACACTGCTGTGTATTACTGTGCTAGAGATGA
TAGTAGTGACTGGGATGCAAAGTTCAACTTGTGGGGCCAAGGGACCCTCG
TCACCGTCTCGAGCGCCTCCACCAAGGGCCCATCGGTCTTCCCCCTGGCA
CCCTCCTCCAAGAGCACCTCTGGGGGCACAGCGGCCCTGGGCTGCCTGGT
CAAGGACTACTTCCCCGAACCGGTGACGGTGTCGTGGAACTCAGGCGCCC
TGACCAGCGGCGTGCACACCTTCCCGGCTGTCCTACAGTCCTCAGGACTC
TACTCCCTCAGCAGCGTGGTGACCGTGCCCTCCAGCAGCTTGGGCACCCA
GACCTACATCTGCAACGTGAATCACAAGCCCAGCAACACCAAGGTGGACA
AGAGAGTTGAGCCCAAATCTTGTGACAAAACTCACACATGCCCACCGTGC
CCAGCACCTGAACTCCTGGGGGGACCGTCAGTCTTCCTCTTCCCCCCAAA
ACCCAAGGACACCCTCATGATCTCCCGGACCCCTGAGGTCACATGCGTGG
TGGTGGACGTGAGCCACGAAGACCCTGAGGTCAAGTTCAACTGGTACGTG
GACGGCGTGGAGGTGCATAATGCCAAGACAAAGCCGCGGGAGGAGCAGTA
CGCCAGCACGTACCGTGTGGTCAGCGTCCTCACCGTCCTGCACCAGGACT
GGCTGAATGGCAAGGAGTACAAGTGCAAGGTCTCCAACAAAGCCCTCCCA
GCCCCCATCGAGAAAACCATCTCCAAAGCCAAAGGGCAGCCCCGAGAACC
ACAGGTGTACACCCTGCCCCCATCCCGGGAGGAGATGACCAAGAACCAGG
TCAGCCTGACCTGCCTGGTCAAAGGCTTCTATCCCAGCGACATCGCCGTG
GAGTGGGAGAGCAATGGGCAGCCGGAGAACAACTACAAGACCACGCCTCC
CGTGCTGGACTCCGACGGCTCCTTCTTCCTCTACAGCAAGCTCACCGTGG
ACAAGAGCAGGTGGCAGCAGGGGAACGTCTTCTCATGCTCCGTGATGCAT
GAGGCTCTGCACAACCACTACACGCAGAAGAGCCTCTCCCTGTCTCCGGG TAAA SEQ ID NO:
720: ATCCAGATGACCCAGTCTCCTTCCTCCCTGTCTGCATCTGTAGGAGACAG
AGTCACCATCACTTGCCAGGCCAGTCAGAGCATTAACAATGAGTTATCCT
GGTATCAGCAGAAACCAGGGAAAGCCCCTAAGCTCCTGATCTATAGGGCA
TCCACTCTGGCATCTGGGGTCCCATCAAGGTTCAGCGGCAGTGGATCTGG
GACAGACTTCACTCTCACCATCAGCAGCCTGCAGCCTGATGATTTTGCAA
CTTATTACTGCCAACAGGGTTATAGTCTGAGGAACATTGATAATGCT SEQ ID NO: 721:
GCCTATGATATGACCCAGACTCCAGCCTCGGTGTCTGCAGCTGTGGGAGG
CACAGTCACCATCAAGTGCCAGGCCAGTCAGAGCATTAACAATGAATTAT
CCTGGTATCAGCAGAAACCAGGGCAGCGTCCCAAGCTCCTGATCTATAGG
GCATCCACTCTGGCATCTGGGGTCTCATCGCGGTTCAAAGGCAGTGGATC
TGGGACAGAGTTCACTCTCACCATCAGCGACCTGGAGTGTGCCGATGCTG
CCACTTACTACTGTCAACAGGGTTATAGTCTGAGGAATATTGATAATGCT
TTCGGCGGAGGGACCGAGGTGGTGGTCAAACGT SEQ ID NO: 722:
ATCCAGATGACCCAGTCTCCTTCCTCCCTGTCTGCATCTGTAGGAGACAG
AGTCACCATCACTTGCCAGGCCAGTCAGAGCATTAACAATGAGTTATCCT
GGTATCAGCAGAAACCAGGGAAAGCCCCTAAGCTCCTGATCTATAGGGCA
TCCACTCTGGCATCTGGGGTCCCATCAAGGTTCAGCGGCAGTGGATCTGG
GACAGACTTCACTCTCACCATCAGCAGCCTGCAGCCTGATGATTTTGCAA
CTTATTACTGCCAACAGGGTTATAGTCTGAGGAACATTGATAATGCTTTC
GGCGGAGGGACCAAGGTGGAAATCAAACGTACGGTGGCTGCACCATCTGT
CTTCATCTTCCCGCCATCTGATGAGCAGTTGAAATCTGGAACTGCCTCTG
TTGTGTGCCTGCTGAATAACTTCTATCCCAGAGAGGCCAAAGTACAGTGG
AAGGTGGATAACGCCCTCCAATCGGGTAACTCCCAGGAGAGTGTCACAGA
GCAGGACAGCAAGGACAGCACCTACAGCCTCAGCAGCACCCTGACGCTGA
GCAAAGCAGACTACGAGAAACACAAAGTCTACGCCTGCGAAGTCACCCAT
CAGGGCCTGAGCTCGCCCGTCACAAAGAGCTTCAACAGGGGAGAGTGT SEQ ID NO: 723:
GCTATCCAGATGACCCAGTCTCCTTCCTCCCTGTCTGCATCTGTAGGAGA
CAGAGTCACCATCACTTGCCAGGCCAGTCAGAGCATTAACAATGAGTTAT
CCTGGTATCAGCAGAAACCAGGGAAAGCCCCTAAGCTCCTGATCTATAGG
GCATCCACTCTGGCATCTGGGGTCCCATCAAGGTTCAGCGGCAGTGGATC
TGGGACAGACTTCACTCTCACCATCAGCAGCCTGCAGCCTGATGATTTTG
CAACTTATTACTGCCAACAGGGTTATAGTCTGAGGAACATTGATAATGCT
TTCGGCGGAGGGACCAAGGTGGAAATCAAACGT SEQ ID NO: 724:
GAGGTGCAGCTGGTGGAGTCTGGGGGAGGCTTGGTCCAGCCTGGGGGGTC
CCTGAGACTCTCCTGTGCAGCCTCTGGATTCTCCCTCAGTAACTACTACG
TGACCTGGGTCCGTCAGGCTCCAGGGAAGGGGCTGGAGTGGGTCGGCATC
ATCTATGGTAGTGATGAAACCGCCTACGCTACCTCCGCTATAGGCCGATT
CACCATCTCCAGAGACAATTCCAAGAACACCCTGTATCTTCAAATGAACA
GCCTGAGAGCTGAGGACACTGCTGTGTATTACTGTGCTAGAGATGATAGT
AGTGACTGGGATGCAAAGTTCAACTTG SEQ ID NO: 725:
CAGTCGCTGGAGGAGTCCGGGGGTCGCCTGGTCACGCCTGGGACACCCCT
GACACTCACCTGCACAGCCTCTGGATTCTCCCTCAGTAACTACTACGTGA
CCTGGGTCCGCCAGGCTCCAGGGAAGGGGCTGGAATGGATCGGAATCATT
TATGGTAGTGATGAAACGGCCTACGCGACCTGGGCGATAGGCCGATTCAC
CATCTCCAAAACCTCGACCACGGTGGATCTGAAAATGACCAGTCTGACAG
CCGCGGACACGGCCACCTATTTCTGTGCCAGAGATGATAGTAGTGACTGG
GATGCAAAATTTAACTTGTGGGGCCAAGGCACCCTGGTCACCGTCTCGAG C
[0643] Screening Assays
[0644] The invention also includes screening assays designed to
assist in the identification of diseases and disorders associated
with IL-6 in patients exhibiting symptoms of an IL-6 associated
disease or disorder.
[0645] In one embodiment of the invention, the anti-IL-6 antibodies
of the invention, or IL-6 binding fragments or variants thereof,
are used to detect the presence of IL-6 in a biological sample
obtained from a patient exhibiting symptoms of a disease or
disorder associated with IL-6. The presence of IL-6, or elevated
levels thereof when compared to pre-disease levels of IL-6 in a
comparable biological sample, may be beneficial in diagnosing a
disease or disorder associated with IL-6.
[0646] Another embodiment of the invention provides a diagnostic or
screening assay to assist in diagnosis of diseases or disorders
associated with IL-6 in patients exhibiting symptoms of an IL-6
associated disease or disorder identified herein, comprising
assaying the level of IL-6 expression in a biological sample from
said patient using a post-translationally modified anti-IL-6
antibody or binding fragment or variant thereof. The anti-IL-6
antibody or binding fragment or variant thereof may be
post-translationally modified to include a detectable moiety such
as set forth previously in the disclosure.
[0647] The IL-6 level in the biological sample is determined using
a modified anti-IL-6 antibody or binding fragment or variant
thereof as set forth herein, and comparing the level of IL-6 in the
biological sample against a standard level of IL-6 (e.g., the level
in normal biological samples). The skilled clinician would
understand that some variability may exist between normal
biological samples, and would take that into consideration when
evaluating results.
[0648] The above-recited assay may also be useful in monitoring a
disease or disorder, where the level of IL-6 obtained in a
biological sample from a patient believed to have an IL-6
associated disease or disorder is compared with the level of IL-6
in prior biological samples from the same patient, in order to
ascertain whether the IL-6 level in said patient has changed with,
for example, a treatment regimen.
[0649] The invention is also directed to a method of in vivo
imaging which detects the presence of cells which express IL-6
comprising administering a diagnostically effective amount of a
diagnostic composition. Said in vivo imaging is useful for the
detection and imaging of IL-6 expressing tumors or metastases and
IL-6 expressing inflammatory sites, for example, and can be used as
part of a planning regimen for design of an effective cancer or
arthritis treatment protocol. The treatment protocol may include,
for example, one or more of radiation, chemotherapy, cytokine
therapy, gene therapy, and antibody therapy, as well as an
anti-IL-6 antibody or fragment or variant thereof.
[0650] A skilled clinician would understand that a biological
sample includes, but is not limited to, sera, plasma, urine,
saliva, mucous, pleural fluid, synovial fluid and spinal fluid.
[0651] Methods of Ameliorating or Reducing Symptoms of, or
Treating, or Preventing, Diseases and Disorders Associated with,
IL-6
[0652] In an embodiment of the invention, IL-6 antagonists such as
Ab1 described herein are useful for ameliorating or reducing the
symptoms of, or treating, or preventing, diseases and disorders
associated with IL-6. IL-6 antagonists described herein (e.g., Ab1)
can also be administered in a therapeutically effective amount to
patients in need of treatment of diseases and disorders associated
with IL-6 in the form of a pharmaceutical composition as described
in greater detail below.
[0653] In one embodiment of the invention, IL-6 antagonists
described herein (e.g., Ab1) are useful for ameliorating or
reducing the symptoms of, or treating, or preventing, diseases and
disorders associated with elevated C-reactive protein (CRP). Such
diseases include any disease that exhibits chronic inflammation,
e.g., rheumatoid arthritis, juvenile rheumatoid arthritis,
psoriasis, psoriatic arthropathy, ankylosing spondylitis, systemic
lupus erythematosis, Crohn's disease, ulcerative colitis,
pemphigus, dermatomyositis, polymyositis, polymyalgia rheumatica,
giant cell arteritis, vasculitis, polyarteritis nodosa, Wegener's
granulomatosis, Kawasaki disease, isolated CNS vasculitis,
Churg-Strauss arteritis, microscopic polyarteritis, microscopic
polyangiitis, Henoch-Schonlein purpura, essential cryoglobulinemic
vasculitis, rheumatoid vasculitis, cryoglobulinemia, relapsing
polychondritis, Behcet's disease, Takayasu's arteritis, ischemic
heart disease, stroke, multiple sclerosis, sepsis, vasculitis
secondary to viral infection (e.g., hepatitis B, hepatitis C, HIV,
cytomegalovirus, Epstein-Barr virus, Parvo B19 virus, etc.),
Buerger's Disease, cancer, advanced cancer, Osteoarthritis,
systemic sclerosis, CREST syndrome, Reiter's disease, Paget's
disease of bone, Sjogran's syndrome, diabetes type 1, diabetes type
2, familial Mediterrean fever, autoimmune thrombocytopenia,
autoimmune hemolytic anemia, autoimmune thyroid diseases,
pernicious anemia, vitiligo, alopecia areata, primary biliary
cirrhosis, autoimmune chronic active hepatitis, alcoholic
cirrhosis, viral hepatitis including hepatitis B and C, other organ
specific autoimmune diseases, burns, idiopathic pulmonary fibrosis,
chronic obsructive pulmonary disease, allergic asthma, other
allergic conditions or any combination thereof.
[0654] In one embodiment of the invention, IL-6 antagonists
described herein, such as anti-IL-6 antibodies (e.g., Ab1),
variants thereof, or fragments thereof, are useful for ameliorating
or reducing the symptoms of, or treating, or preventing, diseases
and disorders associated with reduced serum albumin, e.g.
rheumatoid arthritis, cancer, advanced cancer, liver disease, renal
disease, inflammatory bowel disease, celiac's disease, trauma,
burns, other diseases associated with reduced serum albumin, or any
combination thereof.
[0655] In another embodiment of the invention, IL-6 antagonists
described herein are administered to a patient in combination with
another active agent. For example, an IL-6 antagonist such as Ab1
may be co-administered with one or more chemotherapy agents, such
as VEGF antagonists, EGFR antagonists, platins, taxols, irinotecan,
5-fluorouracil, gemcytabine, leucovorine, steroids,
cyclophosphamide, melphalan, vinca alkaloids (e.g., vinblastine,
vincristine, vindesine and vinorelbine), mustines, tyrosine kinase
inhibitors, radiotherapy, sex hormone antagonists, selective
androgen receptor modulators, selective estrogen receptor
modulators, PDGF antagonists, TNF antagonists, IL-1 antagonists,
interleukins (e.g. IL-12 or IL-2), IL-12R antagonists, Toxin
conjugated monoclonal antibodies, tumor antigen specific monoclonal
antibodies, Erbitux, Avastin, Pertuzumab, anti-CD20 antibodies,
Rituxan, ocrelizumab, ofatumumab, DXL625, herceptin, or any
combination thereof.
[0656] In one embodiment of the invention, anti-IL-6 antibodies
described herein, or fragments or variants thereof, are useful for
ameliorating or reducing the symptoms of, or treating, or
preventing, diseases and disorders associated with fatigue.
Diseases and disorders associated with fatigue include, but are not
limited to, general fatigue, exercise-induced fatigue,
cancer-related fatigue, fibromyalgia, inflammatory disease-related
fatigue and chronic fatigue syndrome. See, for example, Esper D H,
et al, The cancer cachexia syndrome: a review of metabolic and
clinical manifestations, Nutr Clin Pract., 2005 August; 20
(4):369-76; Vgontzas A N, et al, IL-6 and its circadian secretion
in humans, Neuroimmunomodulation, 2005; 12(3):131-40;
Robson-Ansley, P J, et al, Acute interleukin-6 administration
impairs athletic performance in healthy, trained male runners, Can
J Appl Physiol., 2004 August; 29(4):411-8; Shephard R J., Cytokine
responses to physical activity, with particular reference to IL-6:
sources, actions, and clinical implications, Crit Rev Immunol.,
2002; 22(3):165-82; Arnold, M C, et al, Using an interleukin-6
challenge to evaluate neuropsychological performance in chronic
fatigue syndrome, Psychol Med., 2002 August; 32(6):1075-89;
Kurzrock R., The role of cytokines in cancer-related fatigue,
Cancer, 2001 Sep. 15; 92(6 Suppl):1684-8; Nishimoto N, et al,
Improvement in Castleman's disease by humanized anti-interleukin-6
receptor antibody therapy, Blood, 2000 Jan. 1; 95 (1):56-61;
Vgontzas A N, et al, Circadian interleukin-6 secretion and quantity
and depth of sleep, J Clin Endocrinol Metab., 1999 August;
84(8):2603-7; and Spath-Schwalbe E, et al, Acute effects of
recombinant human interleukin 6 on endocrine and central nervous
sleep functions in healthy men, J Clin Endocrinol Metab., 1998 May;
83(5):1573-9; the disclosures of each of which are herein
incorporated by reference in their entireties.
[0657] In a preferred embodiment of the invention, anti-IL-6
antibodies described herein, or fragments or variants thereof, are
useful for ameliorating or reducing the symptoms of, or treating,
or preventing, cachexia. Diseases and disorders associated with
cachexia include, but are not limited to, cancer-related cachexia,
cardiac-related cachexia, respiratory-related cachexia,
renal-related cachexia and age-related cachexia. See, for example,
Barton, B E., Interleukin-6 and new strategies for the treatment of
cancer, hyperproliferative diseases and paraneoplastic syndromes,
Expert Opin Ther Targets, 2005 August; 9(4):737-52; Zaki M H, et
al, CNTO 328, a monoclonal antibody to IL-6, inhibits human
tumor-induced cachexia in nude mice, Int J Cancer, 2004 Sep. 10;
111(4):592-5; Trikha M, et al, Targeted anti-interleukin-6
monoclonal antibody therapy for cancer: a review of the rationale
and clinical evidence, Clin Cancer Res., 2003 Oct. 15;
9(13):4653-65; Lelli G, et al, Treatment of the cancer
anorexia-cachexia syndrome: a critical reappraisal, J Chemother.,
2003 June; 15(3):220-5; Argiles J M, et al, Cytokines in the
pathogenesis of cancer cachexia, Curr Opin Clin Nutr Metab Care,
2003 July; 6(4):401-6; Barton B E., IL-6-like cytokines and cancer
cachexia: consequences of chronic inflammation, Immunol Res., 2001;
23(1):41-58; Yamashita J I, et al, Medroxyprogesterone acetate and
cancer cachexia: interleukin-6 involvement, Breast Cancer, 2000;
7(2):130-5; Yeh S S, et al, Geriatric cachexia: the role of
cytokines, Am J Clin Nutr., 1999 August; 70(2):183-97; Strassmann
G, et al, Inhibition of experimental cancer cachexia by
anti-cytokine and anti-cytokine-receptor therapy, Cytokines Mol
Ther., 1995 June; 1(2):107-13; Fujita J, et al, Anti-interleukin-6
receptor antibody prevents muscle atrophy in colon-26
adenocarcinoma-bearing mice with modulation of lysosomal and
ATP-ubiquitin-dependent proteolytic pathways, Int J Cancer, 1996
Nov. 27; 68(5):637-43; Tsujinaka T, et al, Interleukin 6 receptor
antibody inhibits muscle atrophy and modulates proteolytic systems
in interleukin 6 transgenic mice, J Clin Invest., 1996 Jan. 1;
97(1):244-9; Emilie D, et al, Administration of an
anti-interleukin-6 monoclonal antibody to patients with acquired
immunodeficiency syndrome and lymphoma: effect on lymphoma growth
and on B clinical Symptoms, Blood, 1994 Oct. 15; 84 (8):2472-9; and
Strassmann G, et al, Evidence for the involvement of interleukin 6
in experimental cancer cachexia, J Clin Invest., 1992 May;
89(5):1681-4; the disclosures of each of which are herein
incorporated by reference in their entireties.
[0658] In another embodiment of the invention, anti-IL-6 antibodies
described herein, or fragments or variants thereof, are useful for
ameliorating or reducing the symptoms of, or treating, or
preventing, autoimmune diseases and disorders. Diseases and
disorders associated with autoimmunity include, but are not limited
to, rheumatoid arthritis, systemic lupus erythematosis (SLE),
systemic juvenile idiopathic arthritis, psoriasis, psoriatic
arthropathy, ankylosing spondylitis, inflammatory bowel disease
(IBD), polymyalgia rheumatica, giant cell arteritis, autoimmune
vasculitis, graft versus host disease (GVHD), Sjogren's syndrome,
adult onset Still's disease. In a preferred embodiment of the
invention, humanized anti-IL-6 antibodies described herein, or
fragments or variants thereof, are useful for ameliorating or
reducing the symptoms of, or treating, or preventing, rheumatoid
arthritis and systemic juvenile idiopathic arthritis. See, for
example, Nishimoto N., Clinical studies in patients with
Castleman's disease, Crohn's disease, and rheumatoid arthritis in
Japan, Clin Rev Allergy Immunol., 2005 June; 28(3):221-30;
Nishimoto N, et al, Treatment of rheumatoid arthritis with
humanized anti-interleukin-6 receptor antibody: a multicenter,
double-blind, placebo-controlled trial, Arthritis Rheum., 2004
June; 50(6):1761-9; Choy E., Interleukin 6 receptor as a target for
the treatment of rheumatoid arthritis, Ann Rheum Dis., 2003
November; 62 Suppl 2:ii68-9; Nishimoto N, et al, Toxicity,
pharmacokinetics, and dose-finding study of repetitive treatment
with the humanized anti-interleukin 6 receptor antibody MRA in
rheumatoid arthritis. Phase I/II clinical study, J Rheumatol., 2003
July; 30(7):1426-35; Mihara M, et al, Humanized antibody to human
interleukin-6 receptor inhibits the development of collagen
arthritis in cynomolgus monkeys, Clin Immunol., 2001 March;
98(3):319-26; Nishimoto N, et al, Anti-interleukin 6 receptor
antibody treatment in rheumatic disease, Ann Rheum Dis., 2000
November; 59 Suppl 1:i21-7; Tackey E, et al, Rationale for
interleukin-6 blockade in systemic lupus erythematosus, Lupus,
2004; 13(5):339-43; Finck B K, et al, Interleukin 6 promotes murine
lupus in NZB/NZW F1 mice, J Clin Invest., 1994 August; 94
(2):585-91; Kitani A, et al, Autostimulatory effects of IL-6 on
excessive B cell differentiation in patients with systemic lupus
erythematosus: analysis of IL-6 production and IL-6R expression,
Clin Exp Immunol., 1992 April; 88(1):75-83; Stuart R A, et al,
Elevated serum interleukin-6 levels associated with active disease
in systemic connective tissue disorders, Clin Exp Rheumatol., 1995
January-February; 13 (1):17-22; Mihara M, et al, IL-6 receptor
blockage inhibits the onset of autoimmune kidney disease in NZB/W
F1 mice, Clin Exp Immunol., 1998 June; 12(3):397-402; Woo P, et al,
Open label phase II trial of single, ascending doses of MRA in
Caucasian children with severe systemic juvenile idiopathic
arthritis: proof of principle of the efficacy of IL-6 receptor
blockade in this type of arthritis and demonstration of prolonged
clinical improvement, Arthritis Res Ther., 2005; 7(6):RI281-8. Epub
2005 Sep. 15; Yokota S, et al, Clinical study of tocilizumab in
children with systemic-onset juvenile idiopathic arthritis, Clin
Rev Allergy Immunol., 2005 June; 28(3):231-8; Yokota S, et al,
Therapeutic efficacy of humanized recombinant anti-interleukin-6
receptor antibody in children with systemic-onset juvenile
idiopathic arthritis, Arthritis Rheum., 2005 March; 52(3):818-25;
de Benedetti F, et al, Targeting the interleukin-6 receptor: a new
treatment for systemic juvenile idiopathic arthritis?, Arthritis
Rheum., 2005 March; 52(3):687-93; De Benedetti F, et al, Is
systemic juvenile rheumatoid arthritis an interleukin 6 mediated
disease?, J Rheumatol., 1998 February; 25(2):203-7; Ishihara K, et
al, IL-6 in autoimmune disease and chronic inflammatory
proliferative disease, Cytokine Growth Factor Rev., 2002
August-October; 13 (4-5):357-68; Gilhar A, et al, In vivo effects
of cytokines on psoriatic skin grafted on nude mice: involvement of
the tumor necrosis factor (TNF) receptor, Clin Exp Immunol., 1996
October; 106(1):134-42; Spadaro A, et al, Interleukin-6 and soluble
interleukin-2 receptor in psoriatic arthritis: correlations with
clinical and laboratory parameters, Clin Exp Rheumatol., 1996
July-August; 14 (4):413-6; Ameglio F, et al, Interleukin-6 and
tumor necrosis factor levels decrease in the suction blister fluids
of psoriatic patients during effective therapy, Dermatology, 1994;
189(4):359-63; Wendling D, et al, Combination therapy of anti-CD4
and anti-IL-6 monoclonal antibodies in a case of severe
spondylarthropathy, Br J Rheumatol., 1996 December; 35(12):1330;
Gratacos J, et al, Serum cytokines (IL-6, TNF-alpha, IL-1 beta and
IFN-gamma) in ankylosing spondylitis: a close correlation between
serum IL-6 and disease activity and severity, Br J Rheumatol., 1994
October; 33(10):927-31; Ito H., Treatment of Crohn's disease with
anti-IL-6 receptor antibody, J Gastroenterol., 2005 March; 40 Suppl
16:32-4; Ito H, et al, A pilot randomized trial of a human
anti-interleukin-6 receptor monoclonal antibody in active Crohn's
disease, Gastroenterology, 2004 April; 126(4):989-96; discussion
947; Ito H., IL-6 and Crohn's disease, Curr Drug Targets Inflamm
Allergy, 2003 June; 2(2):12530; Ito H, et al, Anti-IL-6 receptor
monoclonal antibody inhibits leukocyte recruitment and promotes
T-cell apoptosis in a murine model of Crohn's disease, J
Gastroenterol., 2002 November; 37 Suppl 14:56-61; Ito H.,
Anti-interleukin-6 therapy for Crohn's disease, Curr Pharm Des.,
2003; 9(4):295-305; Salvarani C, et al, Acute-phase reactants and
the risk of relapse/recurrence in polymyalgia rheumatica: a
prospective follow-up study, Arthritis Rheum., 2005 Feb. 15;
53(1):33-8; Roche N E, et al, Correlation of interleukin-6
production and disease activity in polymyalgia rheumatica and giant
cell arteritis, Arthritis Rheum., 1993 September; 36(9):1286-94;
Gupta M, et al, Cytokine modulation with immune gamma-globulin in
peripheral blood of normal children and its implications in
Kawasaki disease treatment, J Clin Immunol., 2001 May; 21(3):193-9;
Noris M, et al, Interleukin-6 and RANTES in Takayasu arteritis: a
guide for therapeutic decisions?, Circulation, 1999 Jul. 6;
100(1):55-60; Besbas N, et al, The role of cytokines in Henoch
Schonlein purpura, Scand J Rheumatol., 1997; 26(6):456-60; Hirohata
S, et al, Cerebrospinal fluid interleukin-6 in progressive
Neuro-Behcet's syndrome, Clin Immunol Immunopathol., 1997 January;
82(1):12-7; Yamakawa Y, et al, Interleukin-6 (IL-6) in patients
with Behcet's disease, J Dermatol Sci., 1996 March; 11(3):189-95;
Kim D S., Serum interleukin-6 in Kawasaki disease, Yonsei Med J.,
1992 June; 33(2):183-8; Lange, A., et al, Cytokines, adhesion
molecules (E-selectin and VCAM-1) and graft-versus-host disease,
Arch. Immunol Ther Exp., 1995, 43(2):99-105; Tanaka, J., et al,
Cytokine gene expression after allogeneic bone marrow
transplantation, Leuk. Lymphoma, 1995 16(5-6):413-418; Dickenson, A
M, et al, Predicting outcome in hematological stem cell
transplantation, Arch Immunol Ther Exp., 2002 50(6):371-8; Zeiser,
R, et al, Immunopathogenesis of acute graft-versus-host disease:
implications for novel preventive and therapeutic strategies, Ann
Hematol., 2004 83(9):551-65; Dickinson, A M, et al, Genetic
polymorphisms predicting the outcome of bone marrow transplants,
Br. J Haematol., 2004 127(5):479-90; and Scheinberg M A, et al,
Interleukin 6: a possible marker of disease activity in adult onset
Still's disease, Clin Exp Rheumatol., 1996 November-December; 14
(6):653-5, the disclosures of each of which are herein incorporated
by reference in their entireties.
[0659] In another embodiment of the invention, anti-IL-6 antibodies
described herein, or fragments or variants thereof, are useful for
ameliorating or reducing the symptoms of, or treating, or
preventing, diseases and disorders associated with the skeletal
system. Diseases and disorders associated with the skeletal system
include, but are not limited to, osteoarthritis, osteoporosis and
Paget's disease of bone. In a preferred embodiment of the
invention, humanized anti-IL-6 antibodies described herein, or
fragments or variants thereof, are useful for ameliorating or
reducing the symptoms of, or treating, or preventing,
osteoarthritis. See, for example, Malemud C J., Cytokines as
therapeutic targets for osteoarthritis, BioDrugs, 2004;
18(1):23-35; Westacott C I, et al, Cytokines in osteoarthritis:
mediators or markers of joint destruction?, Semin Arthritis Rheum.,
1996 February; 25(4):254-72; Sugiyama T., Involvement of
interleukin-6 and prostaglandin E2 in particular osteoporosis of
postmenopausal women with rheumatoid arthritis, J Bone Miner
Metab., 2001; 19(2):89-96; Abrahamsen B, et al, Cytokines and bone
loss in a 5-year longitudinal study--hormone replacement therapy
suppresses serum soluble interleukin-6 receptor and increases
interleukin-1-receptor antagonist: the Danish Osteoporosis
Prevention Study, J Bone Miner Res., 2000 August; 15(8):1545-54;
Straub R H, et al, Hormone replacement therapy and interrelation
between serum interleukin-6 and body mass index in postmenopausal
women: a population-based study, J Clin Endocrinol Metab., 2000
March; 85(3):1340-4; Manolagas S C, The role of IL-6 type cytokines
and their receptors in bone, Ann N Y Acad Sci., 1998 May 1;
840:194-204; Ershler W B, et al, Immunologic aspects of
osteoporosis, Dev Comp Immunol., 1997 November-December;
21(6):487-99; Jilka R L, et al, Increased osteoclast development
after estrogen loss: mediation by interleukin-6, Science, 1992 Jul.
3; 257(5066):88-91; Kallen K J, et al, New developments in IL-6
dependent biology and therapy: where do we stand and what are the
options?, Expert Opin Investig Drugs, 1999 September; 8(9):1327-49;
Neale S D, et al, The influence of serum cytokines and growth
factors on osteoclast formation in Paget's disease, QJM, 2002
April; 95 (4):233-40; Roodman G D, Osteoclast function In Paget's
disease and multiple myeloma, Bone, 1995 August; 17(2
Suppl):57S-61S; Hoyland J A, et al, Interleukin-6, IL-6 receptor,
and IL-6 nuclear factor gene expression in Paget's disease, J Bone
Miner Res., 1994 January; 9(1):75-80; and Roodman G D, et al,
Interleukin 6. A potential autocrine/paracrine factor in Paget's
disease of bone, J Clin Invest., 1992 January; 89(1):46-52; the
disclosures of each of which are herein incorporated by reference
in their entireties.
[0660] In another embodiment of the invention, anti-IL-6 antibodies
described herein, or fragments or variants thereof, are useful for
ameliorating or reducing the symptoms of, or treating, or
preventing, diseases and disorders associated with cancer. Diseases
and disorders associated with cancer include, but are not limited
to, Acanthoma, Acinic cell carcinoma, Acoustic neuroma, Acral
lentiginous melanoma, Acrospiroma, Acute eosinophilic leukemia,
Acute lymphoblastic leukemia, Acute megakaryoblastic leukemia,
Acute monocytic leukemia, Acute myeloblastic leukemia with
maturation, Acute myeloid dendritic cell leukemia, Acute myeloid
leukemia, Acute promyelocytic leukemia, Adamantinoma,
Adenocarcinoma, Adenoid cystic carcinoma, Adenoma, Adenomatoid
odontogenic tumor, Adrenocortical carcinoma, Adult T-cell leukemia,
Aggressive NK-cell leukemia, AIDS-Related Cancers, AIDS-related
lymphoma, Alveolar soft part sarcoma, Ameloblastic fibroma, Anal
cancer, Anaplastic large cell lymphoma, Anaplastic thyroid cancer,
Angioimmunoblastic T-cell lymphoma, Angiomyolipoma, Angiosarcoma,
Appendix cancer, Astrocytoma, Atypical teratoid rhabdoid tumor,
Basal cell carcinoma, Basal-like carcinoma, B-cell leukemia, B-cell
lymphoma, Bellini duct carcinoma, Biliary tract cancer, Bladder
cancer, Blastoma, Bone Cancer, Bone tumor, Brain Stem Glioma, Brain
Tumor, Breast Cancer, Brenner tumor, Bronchial Tumor,
Bronchioloalveolar carcinoma, Brown tumor, Burkitt's lymphoma,
Cancer of Unknown Primary Site, Carcinoid Tumor, Carcinoma,
Carcinoma in situ, Carcinoma of the penis, Carcinoma of Unknown
Primary Site, Carcinosarcoma, Castleman's Disease, Central Nervous
System Embryonal Tumor, Cerebellar Astrocytoma, Cerebral
Astrocytoma, Cervical Cancer, Cholangiocarcinoma, Chondroma,
Chondrosarcoma, Chordoma, Choriocarcinoma, Choroid plexus
papilloma, Chronic Lymphocytic Leukemia, Chronic monocytic
leukemia, Chronic myelogenous leukemia, Chronic Myeloproliferative
Disorder, Chronic neutrophilic leukemia, Clear-cell tumor, Colon
Cancer,
[0661] Colorectal cancer, Craniopharyngioma, Cutaneous T-cell
lymphoma, Degos disease, Dermatofibrosarcoma protuberans, Dermoid
cyst, Desmoplastic small round cell tumor, Diffuse large B cell
lymphoma, Dysembryoplastic neuroepithelial tumor, Embryonal
carcinoma, Endodermal sinus tumor, Endometrial cancer, Endometrial
Uterine Cancer, Endometrioid tumor, Enteropathy-associated T-cell
lymphoma, Ependymoblastoma, Ependymoma, Epithelioid sarcoma,
Erythroleukemia, Esophageal cancer, Esthesioneuroblastoma, Ewing
Family of Tumor, Ewing Family Sarcoma, Ewing's sarcoma,
Extracranial Germ Cell Tumor, Extragonadal Germ Cell Tumor,
Extrahepatic Bile Duct Cancer, Extramammary Paget's disease,
Fallopian tube cancer, Fetus in fetu, Fibroma, Fibrosarcoma,
Follicular lymphoma, Follicular thyroid cancer, Gallbladder Cancer,
Gallbladder cancer, Ganglioglioma, Ganglioneuroma, Gastric Cancer,
Gastric lymphoma, Gastrointestinal cancer, Gastrointestinal
Carcinoid Tumor, Gastrointestinal Stromal Tumor, Gastrointestinal
stromal tumor, Germ cell tumor, Germinoma, Gestational
choriocarcinoma, Gestational Trophoblastic Tumor, Giant cell tumor
of bone, Glioblastoma multiforme, Glioma, Gliomatosis cerebri,
Glomus tumor, Glucagonoma, Gonadoblastoma, Granulosa cell tumor,
Hairy Cell Leukemia, Hairy cell leukemia, Head and Neck Cancer,
Head and neck cancer, Heart cancer, Hemangioblastoma,
Hemangiopericytoma, Hemangiosarcoma, Hematological malignancy,
Hepatocellular carcinoma, Hepatosplenic T-cell lymphoma, Hereditary
breast-ovarian cancer syndrome, Hodgkin Lymphoma, Hodgkin's
lymphoma, Hypopharyngeal Cancer, Hypothalamic Glioma, Inflammatory
breast cancer, Intraocular Melanoma, Islet cell carcinoma, Islet
Cell Tumor, Juvenile myelomonocytic leukemia, Kaposi Sarcoma,
Kaposi's sarcoma, Kidney Cancer, Klatskin tumor, Krukenberg tumor,
Laryngeal Cancer, Laryngeal cancer, Lentigo maligna melanoma,
Leukemia, Leukemia, Lip and Oral Cavity Cancer, Liposarcoma, Lung
cancer, Luteoma, Lymphangioma, Lymphangiosarcoma,
Lymphoepithelioma, Lymphoid leukemia, Lymphoma, Macroglobulinemia,
Malignant Fibrous Histiocytoma, Malignant fibrous histiocytoma,
Malignant Fibrous Histiocytoma of Bone, Malignant Glioma, Malignant
Mesothelioma, Malignant peripheral nerve sheath tumor, Malignant
rhabdoid tumor, Malignant triton tumor, MALT lymphoma, Mantle cell
lymphoma, Mast cell leukemia, Mediastinal germ cell tumor,
Mediastinal tumor, Medullary thyroid cancer, Medulloblastoma,
Medulloblastoma, Medulloepithelioma, Melanoma, Melanoma,
Meningioma, Merkel Cell Carcinoma, Mesothelioma, Mesothelioma,
Metastatic Squamous Neck Cancer with Occult Primary, Metastatic
urothelial carcinoma, Mixed Mullerian tumor, Monocytic leukemia,
Mouth Cancer, Mucinous tumor, Multicentric Castleman's disease,
Multiple Endocrine Neoplasia Syndrome, Multiple Myeloma, Multiple
myeloma, Mycosis Fungoides, Mycosis fungoides, Myelodysplastic
Disease, Myelodysplastic Syndromes, Myeloid leukemia, Myeloid
sarcoma, Myeloproliferative Disease, Myxoma, Nasal Cavity Cancer,
Nasopharyngeal Cancer, Nasopharyngeal carcinoma, Neoplasm,
Neurinoma, Neuroblastoma, Neuroblastoma, Neurofibroma, Neuroma,
Nodular melanoma, Non-Hodgkin Lymphoma, Non-Hodgkin lymphoma,
Nonmelanoma Skin Cancer, Non-Small Cell Lung Cancer, Ocular
oncology, Oligoastrocytoma, Oligodendroglioma, Oncocytoma, Optic
nerve sheath meningioma, Oral Cancer, Oral cancer, Oropharyngeal
Cancer, Osteosarcoma, Osteosarcoma, Ovarian Cancer, Ovarian cancer,
Ovarian Epithelial Cancer, Ovarian Germ Cell Tumor, Ovarian Low
Malignant Potential Tumor, Paget's disease of the breast, Pancoast
tumor, Pancreatic Cancer, Pancreatic cancer, Papillary thyroid
cancer, Papillomatosis, Paraganglioma, Paranasal Sinus Cancer,
Parathyroid Cancer, Penile Cancer, Perivascular epithelioid cell
tumor, Pharyngeal Cancer, Pheochromocytoma, Pineal Parenchymal
Tumor of Intermediate Differentiation, Pineoblastoma, Pituicytoma,
Pituitary adenoma, Pituitary tumor, Plasma Cell Neoplasm,
Pleuropulmonary blastoma, Polyembryoma, Precursor T-lymphoblastic
lymphoma, Primary central nervous system lymphoma, Primary effusion
lymphoma, Primary Hepatocellular Cancer, Primary Liver Cancer,
Primary peritoneal cancer, Primitive neuroectodermal tumor,
Prostate cancer, Pseudomyxoma peritonei, Rectal Cancer, Renal cell
carcinoma, Respiratory Tract Carcinoma Involving the NUT Gene on
Chromosome 15, Retinoblastoma, Rhabdomyoma, Rhabdomyosarcoma,
Richter's transformation, Sacrococcygeal teratoma, Salivary Gland
Cancer, Sarcoma, Schwannomatosis, Sebaceous gland carcinoma,
Secondary neoplasm, Seminoma, Serous tumor, Sertoli-Leydig cell
tumor, Sex cord-stromal tumor, Sezary Syndrome, Signet ring cell
carcinoma, Skin Cancer, Small blue round cell tumor, Small cell
carcinoma, Small Cell Lung Cancer, Small cell lymphoma, Small
intestine cancer, Soft tissue sarcoma, Somatostatinoma, Soot wart,
Spinal Cord Tumor, Spinal tumor, Splenic marginal zone lymphoma,
Squamous cell carcinoma, Stomach cancer, Superficial spreading
melanoma, Supratentorial Primitive Neuroectodermal Tumor, Surface
epithelial-stromal tumor, Synovial sarcoma, T-cell acute
lymphoblastic leukemia, T-cell large granular lymphocyte leukemia,
T-cell leukemia, T-cell lymphoma, T-cell prolymphocytic leukemia,
Teratoma, Terminal lymphatic cancer, Testicular cancer, Thecoma,
Throat Cancer, Thymic Carcinoma, Thymoma, Thyroid cancer,
Transitional Cell Cancer of Renal Pelvis and Ureter, Transitional
cell carcinoma, Urachal cancer, Urethral cancer, Urogenital
neoplasm, Uterine sarcoma, Uveal melanoma, Vaginal Cancer, Verner
Morrison syndrome, Verrucous carcinoma, Visual Pathway Glioma,
Vulvar Cancer, Waldenstrom's macroglobulinemia, Warthin's tumor,
Wilms' tumor, or any combination thereof, as well as drug
resistance in cancer chemotherapy and cancer chemotherapy toxicity.
See, for example, Hirata T, et al, Humanized anti-interleukin-6
receptor monoclonal antibody induced apoptosis of fresh and cloned
human myeloma cells in vitro, Leuk Res., 2003 April; 27(4):343-9,
Bataille R, et al, Biologic effects of anti-interleukin-6 murine
monoclonal antibody in advanced multiple myeloma, Blood, 1995 Jul.
15; 86 (2):685-91; Goto H, et al, Mouse anti-human interleukin-6
receptor monoclonal antibody inhibits proliferation of fresh human
myeloma cells in vitro, Jpn J Cancer Res., 1994 September;
85(9):958-65; Klein B, et al, Murine anti-interleukin-6 monoclonal
antibody therapy for a patient with plasma cell leukemia, Blood,
1991 Sep. 1; 78(5):1198-204; Mauray S, et al, Epstein-Barr
virus-dependent lymphoproliferative disease: critical role of IL-6,
Eur J Immunol., 2000 July; 30(7):2065-73; Tsunenari T, et al, New
xenograft model of multiple myeloma and efficacy of a humanized
antibody against human interleukin-6 receptor, Blood, 1997 Sep. 15;
90(6):2437-44; Emilie D, et al, lnterleukin-6 production in
high-grade B lymphomas: correlation with the presence of malignant
immunoblasts in acquired immunodeficiency syndrome and in human
immunodeficiency virus-seronegative patients, Blood, 1992 Jul. 15;
80(2):498-504; Emilie D, et al, Administration of an
anti-interleukin-6 monoclonal antibody to patients with acquired
immunodeficiency syndrome and lymphoma: effect on lymphoma growth
and on B clinical Symptoms, Blood, 1994 Oct. 15; 84(8):2472-9;
Smith P C, et al, Anti-interleukin-6 monoclonal antibody induces
regression of human prostate cancer xenografts in nude mice,
Prostate, 2001 Jun. 15; 48(1):47-53; Smith P C, et al,
Interleukin-6 and prostate cancer progression, Cytokine Growth
Factor Rev., 2001 March; 12(1):33-40; Chung T D, et al,
Characterization of the role of IL-6 in the progression of prostate
cancer, Prostate, 1999 Feb. 15; 38(3):199-207; Okamoto M, et al,
Interleukin-6 as a paracrine and autocrine growth factor in human
prostatic carcinoma cells in vitro, Cancer Res., 1997 Jan. 1;
57(1):141-6; Reittie J E, et al, Interleukin-6 inhibits apoptosis
and tumor necrosis factor induced proliferation of B-chronic
lymphocytic leukemia, Leuk Lymphoma, 1996 June; 22(1-2):83-90,
follow 186, color plate VI; Sugiyama H, et al, The expression of
IL-6 and its related genes in acute leukemia, Leuk Lymphoma, 1996
March; 21(1-2):49-52; Bataille R, et al, Effects of an
anti-interleukin-6 (IL-6) murine monoclonal antibody in a patient
with acute monoblastic leukemia, Med Oncol Tumor Pharmacother.,
1993; 10(4):185-8; Kedar I, et al, Thalidomide reduces serum
C-reactive protein and interleukin-6 and induces response to IL-2
in a fraction of metastatic renal cell cancer patients who failed
IL-2-based therapy, Int J Cancer, 2004 Jun. 10; 110(2):260-5;
Angelo L S, Talpaz M, Kurzrock R, Autocrine interleukin-6
production in renal cell carcinoma: evidence for the involvement of
p53, Cancer Res., 2002 Feb. 1; 62(3):932-40; Nishimoto N, Humanized
anti-interleukin-6 receptor antibody treatment of multicentric
Castleman disease, Blood, 2005 Oct. 15; 106(8):2627-32, Epub 2005
Jul. 5; Katsume A, et al, Anti-interleukin 6 (IL-6) receptor
antibody suppresses Castleman's disease like symptoms emerged in
IL-6 transgenic mice, Cytokine, 2002 Dec. 21; 20(6):304-11;
Nishimoto N, et al, Improvement in Castleman's disease by humanized
anti-interleukin-6 receptor antibody therapy, Blood, 2000 Jan. 1;
95(1):56-61; Screpanti I, Inactivation of the IL-6 gene prevents
development of multicentric Castleman's disease in C/EBP
beta-deficient mice, J Exp Med., 1996 Oct. 1; 184(4):1561-6; Hsu S
M, et al, Expression of interleukin-6 in Castleman's disease, Hum
Pathol., 1993 August; 24(8):833-9; Yoshizaki K, et al, Pathogenic
significance of interleukin-6 (IL 6/BSF-2) in Castleman's disease,
Blood, 1989 September; 74(4):1360-7; Nilsson M B, et al,
Interleukin-6, secreted by human ovarian carcinoma cells, is a
potent proangiogenic cytokine, Cancer Res., 2005 Dec. 1;
65(23):10794-800; Toutirais O, et al, Constitutive expression of
TGF-beta1, interleukin-6 and interleukin-8 by tumor cells as a
major component of immune escape in human ovarian carcinoma, Eur
Cytokine Netw., 2003 October-December; 14(4):246-55; Obata N H, et
al, Effects of interleukin 6 on in vitro cell attachment, migration
and invasion of human ovarian carcinoma, Anticancer Res., 1997
January-February; 17 (1A):337-42; Dedoussis G V, et al, Endogenous
interleukin 6 conveys resistance to
cis-diamminedichloroplatinum-mediated apoptosis of the K562 human
leukemic cell line, Exp Cell Res., 1999 Jun. 15; 249(2):269-78;
Borsellino N, et al, Blocking signaling through the Gp130 receptor
chain by interleukin-6 and oncostatin M inhibits PC-3 cell growth
and sensitizes the tumor cells to etoposide and cisplatin-mediated
cytotoxicity, Cancer, 1999 Jan. 1; 85(1):134-44; Borsellino N, et
al, Endogenous interleukin 6 is a resistance factor for
cis-diamminedichloroplatinum and etoposide-mediated cytotoxicity of
human prostate carcinoma cell lines, Cancer Res., 1995 Oct. 15;
55(20):4633-9; Mizutani Y, et al, Sensitization of human renal cell
carcinoma cells to cis-diamminedichloroplatinum(II) by
anti-interleukin 6 monoclonal antibody or anti-interleukin 6
receptor monoclonal antibody; Cancer Res., 1995 Feb. 1;
55(3):590-6; Yusuf R Z, et al, Paclitaxel resistance: molecular
mechanisms and pharmacologic manipulation, Curr Cancer Drug
Targets, 2003 February; 3(1):1-19; Duan Z, et al, Overexpression of
IL-6 but not IL-8 increases paclitaxel resistance of U-20S human
osteosarcoma cells, Cytokine, 2002 Mar. 7; 17(5):234-42; Conze D,
et al, Autocrine production of interleukin 6 causes multidrug
resistance in breast cancer cells, Cancer Res., 2001 Dec. 15;
61(24):8851-8; Rossi J F, et al, Optimizing the use of
anti-interleukin-6 monoclonal antibody with dexamethasone and 140
mg/m2 of melphalan in multiple myeloma: results of a pilot study
including biological aspects, Bone Marrow Transplant, 2005
November; 36(9):771-9; and Tonini G, et al, Oxaliplatin may induce
cytokine-release syndrome in colorectal cancer patients, J Biol
Regul Homeost Agents, 2002 April-June; 16 (2):105-9; the
disclosures of each of which are herein incorporated by reference
in their entireties.
[0662] In another embodiment of the invention, anti-IL-6 antibodies
described herein, or fragments or variants thereof, are useful for
ameliorating or reducing the symptoms of, or treating, or
preventing, ischemic heart disease, atherosclerosis, obesity,
diabetes, asthma, multiple sclerosis, Alzheimer's disease,
cerebrovascular disease, fever, acute phase response, allergies,
anemia, anemia of inflammation (anemia of chronic disease),
hypertension, depression, depression associated with a chronic
illness, thrombosis, thrombocytosis, acute heart failure, metabolic
syndrome, miscarriage, obesity, chronic prostatitis,
glomerulonephritis, pelvic inflammatory disease, reperfusion
injury, and transplant rejection. See, for example, Tzoulaki I, et
al, C-reactive protein, interleukin-6, and soluble adhesion
molecules as predictors of progressive peripheral atherosclerosis
in the general population: Edinburgh Artery Study, Circulation,
2005 Aug. 16; 112(7):976-83, Epub 2005 Aug. 8; Rattazzi M, et al,
C-reactive protein and interleukin-6 in vascular disease: culprits
or passive bystanders?, J Hypertens., 2003 October;
21(10):1787-803; Ito T, et al, HMG-CoA reductase inhibitors reduce
interleukin-6 synthesis in human vascular smooth muscle cells,
Cardiovasc Drugs Ther., 2002 March; 16(2):121-6; Stenvinkel P, et
al, Mortality, malnutrition, and atherosclerosis in ESRD: what is
the role of interleukin-6?, Kidney Int Suppl., 2002 May;
(80):103-8; Yudkin J S, et al, Inflammation, obesity, stress and
coronary heart disease: is interleukin-6 the link?,
Atherosclerosis, 2000 February; 148(2):209-14; Huber S A, et al,
Interleukin-6 exacerbates early atherosclerosis in mice,
Arterioscler Thromb Vasc Biol., 1999 October; 19(10):2364-7; Kado
S, et al, Circulating levels of interleukin-6, its soluble receptor
and interleukin-6/interleukin-6 receptor complexes in patients with
type 2 diabetes mellitus, Acta Diabetol., 1999 June; 36(1-2):67-72;
Sukovich D A, et al, Expression of interleukin-6 in atherosclerotic
lesions of male ApoE-knockout mice: inhibition by 17beta-estradiol,
Arterioscler Thromb Vasc Biol., 1998 September; 8(9):1498-505;
Klover P J, et al, Interleukin-6 depletion selectively improves
hepatic insulin action in obesity, Endocrinology, 2005 August;
146(8):3417-27, Epub 2005 Apr. 21; Lee Y H, et al, The evolving
role of inflammation in obesity and the metabolic syndrome, Curr
Diab Rep., 2005 February; 5(1):70-5; Diamant M, et al, The
association between abdominal visceral fat and carotid stiffness is
mediated by circulating inflammatory markers in uncomplicated type
2 diabetes, J Clin Endocrinol Metab., 2005 March; 90(3):1495-501,
Epub 2004 Dec. 21; Bray G A, Medical consequences of obesity, J
Clin Endocrinol Metab., 2004 June; 89(6):2583 9; Klover P J, et al,
Chronic exposure to interleukin-6 causes hepatic insulin resistance
in mice, Diabetes, 2003 November; 52 (11):2784-9; Yudkin J S, et
al, Inflammation, obesity, stress and coronary heart disease: is
interleukin-6 the link?, Atherosclerosis, 2000 February;
148(2):209-14; Doganci A, et al, Pathological role of IL-6 in the
experimental allergic bronchial asthma in mice, Clin Rev Allergy
Immunol., 2005 June; 28(3):257-70; Doganci A, et al, The IL-6R
alpha chain controls lung CD4+CD25+ Treg development and function
during allergic airway inflammation in vivo, J Clin Invest., 2005
February; 115(2):313 25, (Erratum in: J Clin Invest., 2005 May;
115(5):1388, Lehr, Hans A [added]); Stelmasiak Z, et al, IL 6 and
sIL-6R concentration in the cerebrospinal fluid and serum of MS
patients, Med Sci Monit., 2001 September-October; 7(5):914-8;
Tilgner J, et al, Continuous interleukin-6 application in vivo via
macroencapsulation of interleukin-6-expressing COS-7 cells induces
massive gliosis, Glia, 2001 September; 35(3):234-45, Brunello A G,
et al, Astrocytic alterations in interleukin-6 Soluble
interleukin-6 receptor alpha double-transgenic mice, Am J Pathol.,
2000 November; 157(5):1485-93; Hampel H, et al, Pattern of
interleukin-6 receptor complex immunoreactivity between cortical
regions of rapid autopsy normal and Alzheimer's disease brain, Eur
Arch Psychiatry Clin Neurosci., 2005 August; 255(4):269-78, Epub
2004 Nov. 26; Cacquevel M, et al, Cytokines in neuroinflammation
and Alzheimer's disease, Curr Drug Targets, 2004 August;
5(6):529-34; Quintanilla R A, et al, Interleukin 6 induces
Alzheimer-type phosphorylation of tau protein by deregulating the
cdk5/p35 pathway, Exp Cell Res., 2004 Apr. 15; 295 (1):245-57;
Gadient R A, et al, Interleukin-6 (IL-6)--a molecule with both
beneficial and destructive potentials, Prog Neurobiol., 1997
August; 52(5):379-90; Hull M, et al, Occurrence of interleukin-6 in
cortical plaques of Alzheimer's disease patients may precede
transformation of diffuse into neuritic plaques, Ann N Y Acad Sci.,
1996 Jan. 17; 777:205-12; Rallidis L S, et al, Inflammatory markers
and in-hospital mortality in acute ischaemic stroke,
Atherosclerosis, 2005 Dec. 30; Emsley H C, et al, Interleukin-6 and
acute ischaemic stroke, Acta Neurol Scand., 2005 October;
112(4):273-4; Smith C J, et al, Peak plasma interleukin-6 and other
peripheral markers of inflammation in the first week of ischaemic
stroke correlate with brain infarct volume, stroke severity and
long-term outcome, BMC Neurol., 2004 Jan. 15; 4:2; Vila N, et al,
Proinflammatory cytokines and early neurological worsening in
ischemic stroke, Stroke, 2000 October; 31(10):2325-9; and Tarkowski
E, et al, Early intrathecal production of interleukin-6 predicts
the size of brain lesion in stroke, Stroke, 1995 August;
26(8):1393-8; the disclosures of each of which are herein
incorporated by reference in their entireties.
[0663] In another embodiment of the invention, anti-IL-6 antibodies
described herein, or fragments or variants thereof, are useful for
ameliorating or reducing the symptoms of, or treating, or
preventing, diseases and disorders associated with cytokine storm.
Diseases and disorders associated with cytokine storm include, but
are not limited to, graft versus host disease (GVHD), avian
influenza, smallpox, pandemic influenza, adult respiratory distress
syndrome (ARDS), severe acute respiratory syndrome (SARS), sepsis,
and systemic inflammatory response syndrome (SIRS). See, for
example, Cecil, R. L., Goldman, L., & Bennett, J. C. (2000).
Cecil textbook of medicine. Philadelphia: W.B. Saunders; Ferrara J
L, et al., Cytokine storm of graft-versus-host disease: a critical
effector role for interleukin-1, Transplant Proc. 1993 February;
25(1 Pt 2):1216-7; Osterholm M T, Preparing for the Next Pandemic,
N Engl J Med. 2005 May 5; 352(18):1839-42; Huang K J, et al., An
interferon-gamma-related cytokine storm in SARS patients, J Med
Virol. 2005 February; 75(2):185-94; and Cheung C Y, et al.,
Induction of proinflammatory cytokines in human macrophages by
influenza A (H5N1) viruses: a mechanism for the unusual severity of
human disease? Lancet. 2002 Dec. 7; 360(9348):1831-7.
[0664] In another embodiment of the invention, anti-IL-6 antibodies
described herein, or fragments or variants thereof, are useful as a
wakefulness aid.
[0665] Administration
[0666] In one embodiment of the invention, the anti-IL-6 antibodies
described herein, or IL-6 binding fragments or variants thereof, as
well as combinations of said antibody fragments or variants, are
administered to a subject at a concentration of between about 0.1
and 20 mg/kg, such as about 0.4 mg/kg, about 0.8 mg/kg, about 1.6
mg/kg, or about 4 mg/kg, of body weight of recipient subject. In a
preferred embodiment of the invention, the anti-IL-6 antibodies
described herein, or IL-6 binding fragments or variants thereof, as
well as combinations of said antibody fragments or variants, are
administered to a subject at a concentration of about 0.4 mg/kg of
body weight of recipient subject. In a preferred embodiment of the
invention, the anti-IL-6 antibodies described herein, or IL-6
binding fragments or variants thereof, as well as combinations of
said antibody fragments or variants, are administered to a
recipient subject with a frequency of once every twenty-six weeks
or less, such as once every sixteen weeks or less, once every eight
weeks or less, or once every four weeks, or less. In another
preferred embodiment of the invention, the anti-IL-6 antibodies
described herein, or IL-6 binding fragments or variants thereof, as
well as combinations thereof, are administered to a recipient
subject with a frequency at most once per period of approximately
one week, such as at most once per period of approximately two
weeks, such as at most once per period of approximately four weeks,
such as at most once per period of approximately eight weeks, such
as at most once per period of approximately twelve weeks, such as
at most once per period of approximately sixteen weeks, such as at
most once per period of approximately twenty-four weeks.
[0667] It is understood that the effective dosage may depend on
recipient subject attributes, such as, for example, age, gender,
pregnancy status, body mass index, lean body mass, condition or
conditions for which the composition is given, other health
conditions of the recipient subject that may affect metabolism or
tolerance of the composition, levels of IL-6 in the recipient
subject, and resistance to the composition (for example, arising
from the patient developing antibodies against the composition). A
person of skill in the art would be able to determine an effective
dosage and frequency of administration through routine
experimentation, for example guided by the disclosure herein and
the teachings in Goodman, L. S., Gilman, A., Brunton, L. L., Lazo,
J. S., & Parker, K. L. (2006). Goodman & Gilman's the
pharmacological basis of therapeutics. New York: McGraw-Hill;
Howland, R. D., Mycek, M. J., Harvey, R. A., Champe, P. C., &
Mycek, M. J. (2006). Pharmacology. Lippincott's illustrated
reviews. Philadelphia: Lippincott Williams & Wilkins; and
Golan, D. E. (2008). Principles of pharmacology: the
pathophysiologic basis of drug therapy. Philadelphia, Pa., [etc.]:
Lippincott Williams & Wilkins.
[0668] In another embodiment of the invention, the anti-IL-6
antibodies described herein, or IL-6 binding fragments or variants
thereof, as well as combinations of said antibody fragments or
variants, are administered to a subject in a pharmaceutical
formulation.
[0669] A "pharmaceutical composition" refers to a chemical or
biological composition suitable for administration to a mammal.
Such compositions may be specifically formulated for administration
via one or more of a number of routes, including but not limited to
buccal, epicutaneous, epidural, inhalation, intraarterial,
intracardial, intracerebroventricular, intradermal, intramuscular,
intranasal, intraocular, intraperitoneal, intraspinal, intrathecal,
intravenous, oral, parenteral, rectally via an enema or
suppository, subcutaneous, subdermal, sublingual, transdermal, and
transmucosal. In addition, administration can occur by means of
injection, powder, liquid, gel, drops, or other means of
administration.
[0670] In one embodiment of the invention, the anti-IL-6 antibodies
described herein, or IL-6 binding fragments or variants thereof, as
well as combinations of said antibody fragments or variants, may be
optionally administered in combination with one or more active
agents. Such active agents include analgesic, antipyretic,
anti-inflammatory, antibiotic, antiviral, and anti-cytokine agents.
Active agents include agonists, antagonists, and modulators of
TNF-.alpha., IL-2, IL-4, IL-6, IL-10, IL-12, IL-13, IL-18,
IFN-.alpha., IFN-.gamma., BAFF, CXCLI3, IP-I0, VEGF, EPO, EGF, HRG,
Hepatocyte Growth Factor (HGF), Hepcidin, including antibodies
reactive against any of the foregoing, and antibodies reactive
against any of their receptors. Active agents also include
2-Arylpropionic acids, Aceclofenac, Acemetacin, Acetylsalicylic
acid (Aspirin), Alclofenac, Alminoprofen, Amoxiprin, Ampyrone,
Arylalkanoic acids, Azapropazone, Benorylate/Benorilate,
Benoxaprofen, Bromfenac, Carprofen, Celecoxib, Choline magnesium
salicylate, Clofezone, COX-2 inhibitors, Dexibuprofen,
Dexketoprofen, Diclofenac, Diflunisal, Droxicam, Ethenzamide,
Etodolac, Etoricoxib, Faislamine, fenamic acids, Fenbufen,
Fenoprofen, Flufenamic acid, Flunoxaprofen, Flurbiprofen,
Ibuprofen, Ibuproxam, Indometacin, Indoprofen, Kebuzone,
Ketoprofen, Ketorolac, Lornoxicam, Loxoprofen, Lumiracoxib,
Magnesium salicylate, Meclofenamic acid, Mefenamic acid, Meloxicam,
Metamizole, Methyl salicylate, Mofebutazone, Nabumetone, Naproxen,
N-Arylanthranilic acids, Oxametacin, Oxaprozin, Oxicams,
Oxyphenbutazone, Parecoxib, Phenazone, Phenylbutazone,
Phenylbutazone, Piroxicam, Pirprofen, profens, Proglumetacin,
Pyrazolidine derivatives, Rofecoxib, Salicyl salicylate,
Salicylamide, Salicylates, Sulfinpyrazone, Sulindac, Suprofen,
Tenoxicam, Tiaprofenic acid, Tolfenamic acid, Tolmetin, and
Valdecoxib. Antibiotics include Amikacin, Aminoglycosides,
Amoxicillin, Ampicillin, Ansamycins, Arsphenamine, Azithromycin,
Azlocillin, Aztreonam, Bacitracin, Carbacephem, Carbapenems,
Carbenicillin, Cefaclor, Cefadroxil, Cefalexin, Cefalothin,
Cefalotin, Cefamandole, Cefazolin, Cefdinir, Cefditoren, Cefepime,
Cefixime, Cefoperazone, Cefotaxime, Cefoxitin, Cefpodoxime,
Cefprozil, Ceftazidime, Ceftibuten, Ceftizoxime, Ceftobiprole,
Ceftriaxone, Cefuroxime, Cephalosporins, Chloramphenicol,
Cilastatin, Ciprofloxacin, Clarithromycin, Clindamycin,
Cloxacillin, Colistin, Co-trimoxazole, Dalfopristin,
Demeclocycline, Dicloxacillin, Dirithromycin, Doripenem,
Doxycycline, Enoxacin, Ertapenem, Erythromycin, Ethambutol,
Flucloxacillin, Fosfomycin, Furazolidone, Fusidic acid,
Gatifloxacin, Geldanamycin, Gentamicin, Glycopeptides, Herbimycin,
Imipenem, Isoniazid, Kanamycin, Levofloxacin, Lincomycin,
Linezolid, Lomefloxacin, Loracarbef, Macrolides, Mafenide,
Meropenem, Meticillin, Metronidazole, Mezlocillin, Minocycline,
Monobactams, Moxifloxacin, Mupirocin, Nafcillin, Neomycin,
Netilmicin, Nitrofurantoin, Norfloxacin, Ofloxacin, Oxacillin,
Oxytetracycline, Paromomycin, Penicillin, Penicillins,
Piperacillin, Platensimycin, Polymyxin B, Polypeptides, Prontosil,
Pyrazinamide, Quinolones, Quinupristin, Rifampicin, Rifampin,
Roxithromycin, Spectinomycin, Streptomycin, Sulfacetamide,
Sulfamethizole, Sulfanilimide, Sulfasalazine, Sulfisoxazole,
Sulfonamides, Teicoplanin, Telithromycin, Tetracycline,
Tetracyclines, Ticarcillin, Tinidazole, Tobramycin, Trimethoprim,
Trimethoprim-Sulfamethoxazole, Troleandomycin, Trovafloxacin, and
Vancomycin. Active agents also include Aldosterone, Beclometasone,
Betamethasone, Corticosteroids, Cortisol, Cortisone acetate,
Deoxycorticosterone acetate, Dexamethasone, Fludrocortisone
acetate, Glucocorticoids, Hydrocortisone, Methylprednisolone,
Prednisolone, Prednisone, Steroids, and Triamcinolone. Antiviral
agents include abacavir, aciclovir, acyclovir, adefovir,
amantadine, amprenavir, an antiretroviral fixed dose combination,
an antiretroviral synergistic enhancer, arbidol, atazanavir,
atripla, brivudine, cidofovir, combivir, darunavir, delavirdine,
didanosine, docosanol, edoxudine, efavirenz, emtricitabine,
enfuvirtide, entecavir, entry inhibitors, famciclovir, fomivirsen,
fosamprenavir, foscarnet, fosfonet, fusion inhibitor, ganciclovir,
gardasil, ibacitabine, idoxuridine, imiquimod, imunovir, indinavir,
inosine, integrase inhibitor, interferon, interferon type I,
interferon type II, interferon type III, lamivudine, lopinavir,
loviride, maraviroc, MK-0518, moroxydine, nelfinavir, nevirapine,
nexavir, nucleoside analogues, oseltamivir, penciclovir, peramivir,
pleconaril, podophyllotoxin, protease inhibitor, reverse
transcriptase inhibitor, ribavirin, rimantadine, ritonavir,
saquinavir, stavudine, tenofovir, tenofovir disoproxil, tipranavir,
trifluridine, trizivir, tromantadine, truvada, valaciclovir,
valganciclovir, vicriviroc, vidarabine, viramidine, zalcitabine,
zanamivir, and zidovudine. Any suitable combination of these active
agents is also contemplated.
[0671] A "pharmaceutical excipient" or a "pharmaceutically
acceptable excipient" is a carrier, usually a liquid, in which an
active therapeutic agent is formulated. In one embodiment of the
invention, the active therapeutic agent is a humanized antibody
described herein, or one or more fragments or variants thereof. The
excipient generally does not provide any pharmacological activity
to the formulation, though it may provide chemical and/or
biological stability, and release characteristics. Exemplary
formulations can be found, for example, in Remington's
Pharmaceutical Sciences, 19.sup.th Ed., Grennaro, A., Ed., 1995
which is incorporated by reference.
[0672] As used herein "pharmaceutically acceptable carrier" or
"excipient" includes any and all solvents, dispersion media,
coatings, antibacterial and antifungal agents, isotonic and
absorption delaying agents that are physiologically compatible. In
one embodiment, the carrier is suitable for parenteral
administration. Alternatively, the carrier can be suitable for
intravenous, intraperitoneal, intramuscular, or sublingual
administration. Pharmaceutically acceptable carriers include
sterile aqueous solutions or dispersions and sterile powders for
the extemporaneous preparation of sterile injectable solutions or
dispersions. The use of such media and agents for pharmaceutically
active substances is well known in the art. Except insofar as any
conventional media or agent is incompatible with the active
compound, use thereof in the pharmaceutical compositions of the
invention is contemplated. Supplementary active compounds can also
be incorporated into the compositions.
[0673] Pharmaceutical compositions typically must be sterile and
stable under the conditions of manufacture and storage. The
invention contemplates that the pharmaceutical composition is
present in lyophilized form. The composition can be formulated as a
solution, microemulsion, liposome, or other ordered structure
suitable to high drug concentration. The carrier can be a solvent
or dispersion medium containing, for example, water, ethanol,
polyol (for example, glycerol, propylene glycol, and liquid
polyethylene glycol), and suitable mixtures thereof. The invention
further contemplates the inclusion of a stabilizer in the
pharmaceutical composition.
[0674] In many cases, it will be preferable to include isotonic
agents, for example, sugars, polyalcohols such as mannitol,
sorbitol, or sodium chloride in the composition. Prolonged
absorption of the injectable compositions can be brought about by
including in the composition an agent which delays absorption, for
example, monostearate salts and gelatin. Moreover, the alkaline
polypeptide can be formulated in a time release formulation, for
example in a composition which includes a slow release polymer. The
active compounds can be prepared with carriers that will protect
the compound against rapid release, such as a controlled release
formulation, including implants and microencapsulated delivery
systems. Biodegradable, biocompatible polymers can be used, such as
ethylene vinyl acetate, polyanhydrides, polyglycolic acid,
collagen, polyorthoesters, polylactic acid and polylactic,
polyglycolic copolymers (PLG). Many methods for the preparation of
such formulations are known to those skilled in the art.
[0675] For each of the recited embodiments, the compounds can be
administered by a variety of dosage forms. Any
biologically-acceptable dosage form known to persons of ordinary
skill in the art, and combinations thereof, are contemplated.
Examples of such dosage forms include, without limitation,
reconstitutable powders, elixirs, liquids, solutions, suspensions,
emulsions, powders, granules, particles, microparticles,
dispersible granules, cachets, inhalants, aerosol inhalants,
patches, particle inhalants, implants, depot implants, injectables
(including subcutaneous, intramuscular, intravenous, and
intradermal), infusions, and combinations thereof.
[0676] The above description of various illustrated embodiments of
the invention is not intended to be exhaustive or to limit the
invention to the precise form disclosed. While specific embodiments
of, and examples for, the invention are described herein for
illustrative purposes, various equivalent modifications are
possible within the scope of the invention, as those skilled in the
relevant art will recognize. The teachings provided herein of the
invention can be applied to other purposes, other than the examples
described above.
[0677] These and other changes can be made to the invention in
light of the above detailed description. In general, in the
following claims, the terms used should not be construed to limit
the invention to the specific embodiments disclosed in the
specification and the claims. Accordingly, the invention is not
limited by the disclosure, but instead the scope of the invention
is to be determined entirely by the following claims.
[0678] The invention may be practiced in ways other than those
particularly described in the foregoing description and examples.
Numerous modifications and variations of the invention are possible
in light of the above teachings and, therefore, are within the
scope of the appended claims.
[0679] Certain teachings related to methods for obtaining a clonal
population of antigen-specific B cells were disclosed in U.S.
Provisional patent application No. 60/801,412, filed May 19, 2006,
the disclosure of which is herein incorporated by reference in its
entirety.
[0680] Certain teachings related to humanization of rabbit-derived
monoclonal antibodies and preferred sequence modifications to
maintain antigen binding affinity were disclosed in International
application Ser. No. 12/124,723, corresponding to Attorney Docket
No. 67858.704001, entitled "Novel Rabbit Antibody Humanization
Method and Humanized Rabbit Antibodies", filed May 21, 2008, the
disclosure of which is herein incorporated by reference in its
entirety.
[0681] Certain teachings related to producing antibodies or
fragments thereof using mating competent yeast and corresponding
methods were disclosed in U.S. patent application Ser. No.
11/429,053, filed May 8, 2006, (U.S. Patent Application Publication
No. US2006/0270045), the disclosure of which is herein incorporated
by reference in its entirety.
[0682] Certain teachings related to anti-IL-6 antibodies, methods
of producing antibodies or fragments thereof using mating competent
yeast and corresponding methods were disclosed in U.S. provisional
patent application No. 60/924,550, filed May 21, 2007, the
disclosure of which is herein incorporated by reference in its
entirety.
[0683] Certain anti-IL-6 antibody polynucleotides and polypeptides
are disclosed in the sequence listing accompanying this patent
application filing, and the disclosure of said sequence listing is
herein incorporated by reference in its entirety.
[0684] The entire disclosure of each document cited herein
(including patents, patent applications, journal articles,
abstracts, manuals, books, or other disclosures) is herein
incorporated by reference in its entirety.
[0685] The following examples are put forth so as to provide those
of ordinary skill in the art with a complete disclosure and
description of how to make and use the subject invention, and are
not intended to limit the scope of what is regarded as the
invention. Efforts have been made to ensure accuracy with respect
to the numbers used (e.g. amounts, temperature, concentrations,
etc.) but some experimental errors and deviations should be allowed
for. Unless otherwise indicated, parts are parts by weight,
molecular weight is average molecular weight, temperature is in
degrees centigrade; and pressure is at or near atmospheric.
EXAMPLES
[0686] In the following examples, the term "AbI" refers to an
antibody containing the light chain sequence of SEQ ID NO: 702 and
the heavy chain sequence of SEQ ID NO: 704, except where the
context indicates otherwise.
Example 1 Production of Enriched Antigen-Specific B Cell Antibody
Culture
[0687] Panels of antibodies are derived by immunizing traditional
antibody host animals to exploit the native immune response to a
target antigen of interest. Typically, the host used for
immunization is a rabbit or other host that produces antibodies
using a similar maturation process and provides for a population of
antigen-specific B cells producing antibodies of comparable
diversity, e.g., epitopic diversity. The initial antigen
immunization can be conducted using complete Freund's adjuvant
(CFA), and the subsequent boosts effected with incomplete adjuvant.
At about 50-60 days after immunization, preferably at day 55,
antibody titers are tested, and the Antibody Selection (ABS)
process is initiated if appropriate titers are established. The two
key criteria for ABS initiation are potent antigen recognition and
function-modifying activity in the polyclonal sera.
[0688] At the time positive antibody titers are established,
animals are sacrificed and B cell sources isolated. These sources
include: the spleen, lymph nodes, bone marrow, and peripheral blood
mononuclear cells (PBMCs). Single cell suspensions are generated,
and the cell suspensions are washed to make them compatible for low
temperature long term storage. The cells are then typically
frozen.
[0689] To initiate the antibody identification process, a small
fraction of the frozen cell suspensions are thawed, washed, and
placed in tissue culture media. These suspensions are then mixed
with a biotinylated form of the antigen that was used to generate
the animal immune response, and antigen-specific cells are
recovered using the Miltenyi magnetic bead cell selection
methodology. Specific enrichment is conducted using streptavidin
beads. The enriched population is recovered and progressed in the
next phase of specific B cell isolation.
Example 2 Production of Clonal, Antigen-Specific B Cell-Containing
Culture
[0690] Enriched B cells produced according to Example 1 are then
plated at varying cell densities per well in a 96 well microtiter
plate. Generally, this is at 50, 100, 250, or 500 cells per well
with 10 plates per group. The media is supplemented with 4%
activated rabbit T cell conditioned media along with 50K frozen
irradiated EL4B feeder cells. These cultures are left undisturbed
for 5-7 days at which time supernatant-containing secreted antibody
is collected and evaluated for target properties in a separate
assay setting. The remaining supernatant is left intact, and the
plate is frozen at -70.degree. C. Under these conditions, the
culture process typically results in wells containing a mixed cell
population that comprises a clonal population of antigen-specific B
cells, i.e., a single well will only contain a single monoclonal
antibody specific to the desired antigen.
Example 3 Screening of Antibody Supernatants for Monoclonal
Antibody of Desired Specificity and/or Functional Properties
[0691] Antibody-containing supernatants derived from the well
containing a clonal antigen-specific B cell population produced
according to Example 2 are initially screened for antigen
recognition using ELISA methods. This includes selective antigen
immobilization (e.g., biotinylated antigen capture by streptavidin
coated plate), non-specific antigen plate coating, or
alternatively, through an antigen build-up strategy (e.g.,
selective antigen capture followed by binding partner addition to
generate a heteromeric protein-antigen complex). Antigen-positive
well supernatants are then optionally tested in a
function-modifying assay that is strictly dependant on the ligand.
One such example is an in vitro protein-protein interaction assay
that recreates the natural interaction of the antigen ligand with
recombinant receptor protein. Alternatively, a cell-based response
that is ligand dependent and easily monitored (e.g., proliferation
response) is utilized. Supernatant that displays significant
antigen recognition and potency is deemed a positive well. Cells
derived from the original positive well are then transitioned to
the antibody recovery phase.
Example 4 Recovery of Single, Antibody-Producing B Cell of Desired
Antigen Specificity
[0692] Cells are isolated from a well that contains a clonal
population of antigen-specific B cells (produced according to
Example 2 or 3), which secrete a single antibody sequence. The
isolated cells are then assayed to isolate a single,
antibody-secreting cell. Dynal streptavidin beads are coated with
biotinylated target antigen under buffered medium to prepare
antigen-containing microbeads compatible with cell viability. Next
antigen-loaded beads, antibody-producing cells from the positive
well, and a fluorescein isothiocyanate (FITC)-labeled anti-host
H&L IgG antibody (as noted, the host can be any mammalian host,
e.g., rabbit, mouse, rat, etc.) are incubated together at
37.degree. C. This mixture is then re-pipetted in aliquots onto a
glass slide such that each aliquot has on average a single,
antibody-producing B-cell. The antigen-specific, antibody-secreting
cells are then detected through fluorescence microscopy. Secreted
antibody is locally concentrated onto the adjacent beads due to the
bound antigen and provides localization information based on the
strong fluorescent signal. Antibody-secreting cells are identified
via FITC detection of antibody-antigen complexes formed adjacent to
the secreting cell. The single cell found in the center of this
complex is then recovered using a micromanipulator. The cell is
snap-frozen in an eppendorf PCR tube for storage at -80.degree. C.
until antibody sequence recovery is initiated.
Example 5 Isolation of Antibody Sequences from Antigen-Specific B
Cell
[0693] Antibody sequences are recovered using a combined RT-PCR
based method from a single isolated B-cell produced according to
Example 4 or an antigenic specific B cell isolated from the clonal
B cell population obtained according to Example 2. Primers are
designed to anneal in conserved and constant regions of the target
immunoglobulin genes (heavy and light), such as rabbit
immunoglobulin sequences, and a two-step nested PCR recovery step
is used to obtain the antibody sequence. Amplicons from each well
are analyzed for recovery and size integrity. The resulting
fragments are then digested with AluI to fingerprint the sequence
clonality. Identical sequences display a common fragmentation
pattern in their electrophoretic analysis. Significantly, this
common fragmentation pattern which proves cell clonality is
generally observed even in the wells originally plated up to 1000
cells/well. The original heavy and light chain amplicon fragments
are then restriction enzyme digested with HindIII and XhoI or
HindIII and BsiWI to prepare the respective pieces of DNA for
cloning. The resulting digestions are then ligated into an
expression vector and transformed into bacteria for plasmid
propagation and production. Colonies are selected for sequence
characterization.
Example 6 Recombinant Production of Monoclonal Antibody of Desired
Antigen Specificity and/or Functional Properties
[0694] Correct full-length antibody sequences for each well
containing a single monoclonal antibody is established and miniprep
DNA is prepared using Qiagen solid-phase methodology. This DNA is
then used to transfect mammalian cells to produce recombinant
full-length antibody. Crude antibody product is tested for antigen
recognition and functional properties to confirm the original
characteristics are found in the recombinant antibody protein.
Where appropriate, large-scale transient mammalian transfections
are completed, and antibody is purified through Protein A affinity
chromatography. Kd is assessed using standard methods (e.g.,
Biacore) as well as IC50 in a potency assay.
Example 7 Preparation of Antibodies that Bind Human IL-6
[0695] By using the antibody selection protocol described herein,
one can generate an extensive panel of antibodies. The antibodies
have high affinity towards IL-6 (single to double digit pM Kd) and
demonstrate potent antagonism of IL-6 in multiple cell-based
screening systems (T1165 and HepG2). Furthermore, the collection of
antibodies display distinct modes of antagonism toward IL-6-driven
processes.
[0696] Immunization Strategy
[0697] Rabbits were immunized with huIL-6 (R&R). Immunization
consisted of a first subcutaneous (sc) injection of 100 .mu.g in
complete Freund's adjuvant (CFA) (Sigma) followed by two boosts,
two weeks apart, of 50 .mu.g each in incomplete Freund's adjuvant
(IFA) (Sigma). Animals were bled on day 55, and serum titers were
determined by ELISA (antigen recognition) and by non-radioactive
proliferation assay (Promega) using the T1165 cell line.
[0698] Antibody Selection Titer Assessment
[0699] Antigen recognition was determined by coating Immulon 4
plates (Thermo) with 1 .mu.g/ml of huIL-6 (50 .mu.l/well) in
phosphate buffered saline (PBS, Hyclone) overnight at 4.degree. C.
On the day of the assay, plates were washed 3 times with PBS/Tween
20 (PB ST tablets, Calbiochem). Plates were then blocked with 200
.mu.l/well of 0.5% fish skin gelatin (FSG, Sigma) in PBS for 30
minutes at 37.degree. C. Blocking solution was removed, and plates
were blotted. Serum samples were made (bleeds and pre-bleeds) at a
starting dilution of 1:100 (all dilutions were made in FSG 50
.mu.l/well) followed by 1:10 dilutions across the plate (column 12
was left blank for background control). Plates were incubated for
30 minutes at 37.degree. C. Plates were washed 3 times with
PBS/Tween 20. Goat anti-rabbit FC-HRP (Pierce) diluted 1:5000 was
added to all wells (50 .mu.l/well), and plates were incubated for
30 minutes at 37.degree. C. Plates were washed as described above.
50 .mu.l/well of TMB-Stable stop (Fitzgerald Industries) was added
to plates, and color was allowed to develop, generally for 3 to 5
minutes. The development reaction was stopped with 50 .mu.l/well
0.5 M HCl. Plates were read at 450 nm. Optical density (OD) versus
dilution was plotted using Graph Pad Prizm software, and titers
were determined.
[0700] Functional Titer Assessment
[0701] The functional activity of the samples was determined by a
T1165 proliferation assay. T1165 cells were routinely maintained in
modified RPMI medium (Hyclone) supplemented with Hepes, sodium
pyruvate, sodium bicarbonate, L-glutamine, high glucose,
penicillin/streptomycin, 10% heat inactivated fetal bovine serum
(FBS) (all supplements from Hyclone), 2-mercaptoethanol (Sigma),
and 10 ng/ml of hulL-6 (R&D). On the day of the assay, cell
viability was determined by trypan blue (Invitrogen), and cells
were seeded at a fixed density of 20,000 cells/well. Prior to
seeding, cells were washed twice in the medium described above
without human-IL-6 (by centrifuging at 13000 rpm for 5 minutes and
discarding the supernatant). After the last wash, cells were
resuspended in the same medium used for washing in a volume
equivalent to 50 .mu.l/well. Cells were set aside at room
temperature.
[0702] In a round-bottom, 96-well plate (Costar), serum samples
were added starting at 1:100, followed by a 1:10 dilution across
the plate (columns 2 to 10) at 30 .mu.l/well in replicates of 5
(rows B to F: dilution made in the medium described above with no
huIL-6). Column 11 was medium only for IL-6 control. 30 .mu.l/well
of huIL-6 at 4.times. concentration of the final EC50
(concentration previously determined) were added to all wells
(hulL-6 was diluted in the medium described above). Wells were
incubated for 1 hour at 37.degree. C. to allow antibody binding to
occur. After 1 hour, 50 .mu.l/well of antibody-antigen (Ab-Ag)
complex were transferred to a flat-bottom, 96-well plate (Costar)
following the plate map format laid out in the round-bottom plate.
On Row G, 50 .mu.l/well of medium were added to all wells (columns
2 to 11) for background control. 50 .mu.l/well of the cell
suspension set aside were added to all wells (columns 2 to 11, rows
B to G). On Columns 1 and 12 and on rows A and H, 200 .mu.l/well of
medium was added to prevent evaporation of test wells and to
minimize edge effect. Plates were incubated for 72 h at 37.degree.
C. in 4% CO2. At 72 h, 20 .mu.l/well of CellTiter96 (Promega)
reagents was added to all test wells per manufacturer protocol, and
plates were incubated for 2 h at 37.degree. C. At 2 h, plates were
gently mixed on an orbital shaker to disperse cells and to allow
homogeneity in the test wells. Plates were read at 490 nm
wavelength. Optical density (OD) versus dilution was plotted using
Graph Pad Prizm software, and functional titer was determined. A
positive assay control plate was conducted as described above using
MAB2061 (R&D Systems) at a starting concentration of 1 .mu.g/ml
(final concentration) followed by 1:3 dilutions across the
plate.
[0703] Tissue Harvesting
[0704] Once acceptable titers were established, the rabbit(s) were
sacrificed. Spleen, lymph nodes, and whole blood were harvested and
processed as follows:
[0705] Spleen and lymph nodes were processed into a single cell
suspension by disassociating the tissue and pushing through sterile
wire mesh at 70 .mu.m (Fisher) with a plunger of a 20 cc syringe.
Cells were collected in the modified RPMI medium described above
without hulL-6, but with low glucose. Cells were washed twice by
centrifugation. After the last wash, cell density was determined by
trypan blue. Cells were centrifuged at 1500 rpm for 10 minutes; the
supernatant was discarded. Cells were resuspended in the
appropriate volume of 10% dimethyl sulfoxide (DMSO, Sigma) in FBS
(Hyclone) and dispensed at 1 ml/vial. Vials were then stored at
-70.degree. C. for 24 h prior to being placed in a liquid nitrogen
(LN2) tank for long-term storage.
[0706] Peripheral blood mononuclear cells (PBMCs) were isolated by
mixing whole blood with equal parts of the low glucose medium
described above without FBS. 35 ml of the whole blood mixture was
carefully layered onto 8 ml of Lympholyte Rabbit (Cedarlane) into a
45 ml conical tube (Corning) and centrifuged 30 minutes at 2500 rpm
at room temperature without brakes. After centrifugation, the PBMC
layers were carefully removed using a glass Pasteur pipette (VWR),
combined, and placed into a clean 50 ml vial. Cells were washed
twice with the modified medium described above by centrifugation at
1500 rpm for 10 minutes at room temperature, and cell density was
determined by trypan blue staining. After the last wash, cells were
resuspended in an appropriate volume of 10% DMSO/FBS medium and
frozen as described above.
[0707] B Cell Culture
[0708] On the day of setting up B cell culture, PBMC, splenocyte,
or lymph node vials were thawed for use. Vials were removed from
LN2 tank and placed in a 37.degree. C. water bath until thawed.
Contents of vials were transferred into 15 ml conical centrifuge
tube (Corning) and 10 ml of modified RPMI described above was
slowly added to the tube. Cells were centrifuged for 5 minutes at
1.5K rpm, and the supernatant was discarded. Cells were resuspended
in 10 ml of fresh media. Cell density and viability was determined
by trypan blue. Cells were washed again and resuspended at 1E07
cells/80 ul medium. Biotinylated huIL-6 (B huIL-6) was added to the
cell suspension at the final concentration of 3 ug/mL and incubated
for 30 minutes at 4.degree. C. Unbound B huIL-6 was removed with
two 10 ml washes of phosphate-buffered (PBF):Ca/Mg free PBS
(Hyclone), 2 mM ethylenediamine tetraacetic acid (EDTA), 0.5%
bovine serum albumin (BSA) (Sigma-biotin free). After the second
wash, cells were resuspended at 1E07 cells/80 .mu.l PBF. 20 .mu.l
of MACS.RTM. streptavidin beads (Milteni)/10E7 cells were added to
the cell suspension. Cells were incubated at 4.degree. C. for 15
minutes. Cells were washed once with 2 ml of PBF/10E7 cells. After
washing, the cells were resuspended at 1E08 cells/500 .mu.l of PBF
and set aside. A MACS.RTM. MS column (Milteni) was pre-rinsed with
500 ml of PBF on a magnetic stand (Milteni). Cell suspension was
applied to the column through a pre-filter, and unbound fraction
was collected. The column was washed with 1.5 ml of PBF buffer. The
column was removed from the magnet stand and placed onto a clean,
sterile 5 ml Polypropylene Falcon tube. 1 ml of PBF buffer was
added to the top of the column, and positive selected cells were
collected. The yield and viability of positive and negative cell
fraction was determined by trypan blue staining. Positive selection
yielded an average of 1% of the starting cell concentration.
[0709] A pilot cell screen was established to provide information
on seeding levels for the culture. Three 10-plate groups (a total
of 30 plates) were seeded at 50, 100, and 200 enriched B
cells/well. In addition, each well contained 50K cells/well of
irradiated EL-4.B5 cells (5,000 Rads) and an appropriate level of T
cell supernatant (ranging from 1-5% depending on preparation) in
high glucose modified RPMI medium at a final volume of 250
.mu.l/well. Cultures were incubated for 5 to 7 days at 37.degree.
C. in 4% CO2.
[0710] Identification of Selective Antibody Secreting B Cells
[0711] Cultures were tested for antigen recognition and functional
activity between days 5 and 7.
[0712] Antigen Recognition Screening
[0713] The ELISA format used is as described above except 50 pi of
supernatant from the B cell cultures (BCC) wells (all 30 plates)
was used as the source of the antibody. The conditioned medium was
transferred to antigen-coated plates. After positive wells were
identified, the supernatant was removed and transferred to a
96-well master plate(s). The original culture plates were then
frozen by removing all the supernatant except 40 .mu.l/well and
adding 60 .mu.l/well of 16% DMSO in FBS. Plates were wrapped in
paper towels to slow freezing and placed at -70.degree. C.
[0714] Functional Activity Screening
[0715] Master plates were then screened for functional activity in
the T1165 proliferation assay as described before, except row B was
media only for background control, row C was media+IL-6 for
positive proliferation control, and rows D-G and columns 2-11 were
the wells from the BCC (50 .mu.l/well, single points). 40 .mu.l of
IL-6 was added to all wells except the media row at 2.5 times the
EC50 concentration determined for the assay. After 1 h incubation,
the Ab/Ag complex was transferred to a tissue culture (TC) treated,
96-well, flat-bottom plate. 20 .mu.l of cell suspension in modified
RPMI medium without hulL-6 (T1165 at 20,000 cells/well) was added
to all wells (100 .mu.l final volume per well). Background was
subtracted, and observed OD values were transformed into % of
inhibition.
[0716] B Cell Recovery
[0717] Plates containing wells of interest were removed from
-70.degree. C., and the cells from each well were recovered with
5-200 .mu.l washes of medium/well. The washes were pooled in a 1.5
ml sterile centrifuge tube, and cells were pelleted for 2 minutes
at 1500 rpm.
[0718] The tube was inverted, the spin repeated, and the
supernatant carefully removed. Cells were resuspended in 100
.mu.l/tube of medium. 100 .mu.l biotinylated IL-6 coated
streptavidin M280 dynabeads (Invitrogen) and 16 .mu.l of goat
anti-rabbit H&L IgG-FITC diluted 1:100 in medium was added to
the cell suspension.
[0719] 20 .mu.l of cell/beads/FITC suspension was removed, and 5
.mu.l droplets were prepared on a glass slide (Corning) previously
treated with Sigmacote (Sigma), 35 to 40 droplets/slide. An
impermeable barrier of parafin oil (JT Baker) was added to submerge
the droplets, and the slide was incubated for 90 minutes at
37.degree. C., 4% CO2 in the dark.
[0720] Specific B cells that produce antibody can be identified by
the fluorescent ring around them due to antibody secretion,
recognition of the bead-associated biotinylated antigen, and
subsequent detection by the fluorescent-IgG detection reagent. Once
a cell of interest was identified, the cell in the center of the
fluorescent ring was recovered via a micromanipulator (Eppendorf).
The single cell synthesizing and exporting the antibody was
transferred into a 250 .mu.l microcentrifuge tube and placed in dry
ice. After recovering all cells of interest, these were transferred
to -70.degree. C. for long-term storage.
Example 8 Yeast Cell Expression
[0721] Antibody Genes:
[0722] Genes were cloned and constructed that directed the
synthesis of a chimeric humanized rabbit monoclonal antibody.
[0723] Expression Vector:
[0724] The vector contains the following functional components: 1)
a mutant ColE1 origin of replication, which facilitates the
replication of the plasmid vector in cells of the bacterium
Escherichia coli; 2) a bacterial Sh ble gene, which confers
resistance to the antibiotic Zeocin and serves as the selectable
marker for transformations of both E. coli and P. pastoris; 3) an
expression cassette composed of the glyceraldehyde dehydrogenase
gene (GAP gene) promoter, fused to sequences encoding the
Saccharomyces cerevisiae alpha mating factor pre pro secretion
leader sequence, followed by sequences encoding a P. pastoris
transcriptional termination signal from the P. pastoris alcohol
oxidase I gene (AOX1). The Zeocin resistance marker gene provides a
means of enrichment for strains that contain multiple integrated
copies of an expression vector in a strain by selecting for
transformants that are resistant to higher levels of Zeocin.
[0725] P. pastoris strains: P. pastoris strains met1, lys3, ura3
and ade1 may be used. Although any two complementing sets of
auxotrophic strains could be used for the construction and
maintenance of diploid strains, these two strains are especially
suited for this method for two reasons. First, they grow more
slowly than diploid strains that are the result of their mating or
fusion. Thus, if a small number of haploid ade1 or ura3 cells
remain present in a culture or arise through meiosis or other
mechanism, the diploid strain should outgrow them in culture.
[0726] The second is that it is easy to monitor the sexual state of
these strains since diploid Ade+ colonies arising from their mating
are a normal white or cream color, whereas cells of any strains
that are haploid ade1 mutants will form a colony with a distinct
pink color. In addition, any strains that are haploid ura3 mutants
are resistant to the drug 5-fluoro-orotic acid (FOA) and can be
sensitively identified by plating samples of a culture on minimal
medium+uracil plates with FOA. On these plates, only
uracil-requiring ura3 mutant (presumably haploid) strains can grow
and form colonies. Thus, with haploid parent strains marked with
ade1 and ura3, one can readily monitor the sexual state of the
resulting antibody-producing diploid strains (haploid versus
diploid).
[0727] Methods
[0728] Construction of pGAPZ-Alpha Expression Vectors for
Transcription of Light and Heavy Chain Antibody Genes.
[0729] The humanized light and heavy chain fragments were cloned
into the pGAPZ expression vectors through a PCR directed process.
The recovered humanized constructs were subjected to amplification
under standard KOD polymerase (Novagen) kit conditions ((1)
94.degree. C., 2 minutes; (2) 94.degree. C., 30 seconds (3)
55.degree. C., 30 seconds; (4) 72.degree. C., 30 seconds-cycling
through steps 2-4 for 35 times; (5) 72.degree. C. 2 minutes)
employing the following primers (1) light chain forward
TABLE-US-00011 AGCGCTTATTCCGCTATCCAGATGACCCAGTC-
the AfeI site is single underlined. The end of the HSA signal
sequence is double underlined, followed by the sequence for the
mature variable light chain (not underlined); the reverse
CGTACGTTTGATTTCCACCTTG.
[0730] Variable light chain reverse primer. BsiWI site is
underlined, followed by the reverse complement for the 3' end of
the variable light chain. Upon restriction enzyme digest with AfeI
and BsiWI this enable insertion in-frame with the pGAPZ vector
using the human HAS leader sequence in frame with the human kapp
light chain constant region for export. (2) A similar strategy is
performed for the heavy chain. The forward primer employed is
TABLE-US-00012 AGCGCTTATTCCGAGGTGCAGCTGGTGGAGTC.
[0731] The AfeI site is single underlined. The end of the HSA
signal sequence is double underlined, followed by the sequence for
the mature variable heavy chain (not underlined). The reverse heavy
chain primer is CTCGAGACGGTGACGAGGGT. The XhoI site is underlined,
followed by the reverse complement for the 3' end of the variable
heavy chain. This enables cloning of the heavy chain in-frame with
IgG-.gamma.1 CH1-CH2-CH3 region previous inserted within pGAPZ
using a comparable directional cloning strategy.
[0732] Transformation of Expression Vectors into Haploid Ade1 Ura3,
Met1 and Lys3 Host Strains of P. pastoris.
[0733] All methods used for transformation of haploid P. pastoris
strains and genetic manipulation of the P. pastoris sexual cycle
are as described in Higgins, D. R., and Cregg, J. M., Eds. 1998.
Pichia Protocols. Methods in Molecular Biology. Humana Press,
Totowa, N.J.
[0734] Prior to transformation, each expression vector is
linearized within the GAP promoter sequences with AvrII to direct
the integration of the vectors into the GAP promoter locus of the
P. pastoris genome. Samples of each vector are then individually
transformed into electrocompetent cultures of the ade1, ura3, met1
and lys3 strains by electroporation and successful transformants
are selected on YPD Zeocin plates by their resistance to this
antibiotic. Resulting colonies are selected, streaked for single
colonies on YPD Zeocin plates and then examined for the presence of
the antibody gene insert by a PCR assay on genomic DNA extracted
from each strain for the proper antibody gene insert and/or by the
ability of each strain to synthesize an antibody chain by a colony
lift/immunoblot method (Wung et al. Biotechniques 21 808-812
(1996). Haploid ade1, met1 and lys3 strains expressing one of the
three heavy chain constructs are collected for diploid
constructions along with haploid ura3 strain expressing light chain
gene. The haploid expressing heavy chain genes are mated with the
appropriate light chain haploid ura3 to generate diploid secreting
protein.
[0735] Mating of haploid strains synthesizing a single antibody
chain and selection of diploid derivatives synthesizing tetrameric
functional antibodies. To mate P. pastoris haploid strains, each
ade1 (or met1 or lys3) heavy chain producing strain to be crossed
is streaked across a rich YPD plate and the ura3 light chain
producing strain is streaked across a second YPD plate (.about.10
streaks per plate). After one or two days incubation at 30.degree.
C., cells from one plate containing heavy chain strains and one
plate containing ura3 light chain strains are transferred to a
sterile velvet cloth on a replica-plating block in a cross hatched
pattern so that each heavy chain strain contain a patch of cells
mixed with each light chain strain. The cross-streaked replica
plated cells are then transferred to a mating plate and incubated
at 25.degree. C. to stimulate the initiation of mating between
strains. After two days, the cells on the mating plates are
transferred again to a sterile velvet on a replica-plating block
and then transferred to minimal medium plates. These plates are
incubated at 30.degree. C. for three days to allow for the
selective growth of colonies of prototrophic diploid strains.
Colonies that arose are picked and streaked onto a second minimal
medium plate to single colony isolate and purify each diploid
strain. The resulting diploid cell lines are then examined for
antibody production.
[0736] Putative diploid strains are tested to demonstrate that they
are diploid and contain both expression vectors for antibody
production. For diploidy, samples of a strain are spread on mating
plates to stimulate them to go through meiosis and form spores.
Haploid spore products are collected and tested for phenotype. If a
significant percentage of the resulting spore products are single
or double auxotrophs it may be concluded that the original strain
must have been diploid. Diploid strains are examined for the
presence of both antibody genes by extracting genomic DNA from each
and utilizing this DNA in PCR reactions specific for each gene.
[0737] Fusion of haploid strains synthesizing a single antibody
chain and selection of diploid derivatives synthesizing tetrameric
functional antibodies. As an alternative to the mating procedure
described above, individual cultures of single-chain antibody
producing haploid ade1 and ura3 strains are spheroplasted and their
resulting spheroplasts fused using polyethylene glycol/CaCl.sub.2.
The fused haploid strains are then embedded in agar containing 1 M
sorbitol and minimal medium to allow diploid strains to regenerate
their cell wall and grow into visible colonies. Resulting colonies
are picked from the agar, streaked onto a minimal medium plate, and
the plates are incubated for two days at 30.degree. C. to generate
colonies from single cells of diploid cell lines. The resulting
putative diploid cell lines are then examined for diploidy and
antibody production as described above.
[0738] Purification and analysis of antibodies. A diploid strain
for the production of full length antibody is derived through the
mating of met1 light chain and lys3 heavy chain using the methods
described above. Culture media from shake-flask or fermenter
cultures of diploid P. pastoris expression strains are collected
and examined for the presence of antibody protein via SDS-PAGE and
immunoblotting using antibodies directed against heavy and light
chains of human IgG, or specifically against the heavy chain of
IgG.
[0739] To purify the yeast secreted antibodies, clarified media
from antibody producing cultures are passed through a protein A
column and after washing with 20 mM sodium phosphate, pH 7.0,
binding buffer, protein A bound protein is eluted using 0.1 M
glycine HCl buffer, pH 3.0. Fractions containing the most total
protein are examined by Coomasie blue strained SDS-PAGE and
immunoblotting for antibody protein. Antibody is characterized
using the ELISA described above for IL-6 recognition.
[0740] Assay for Antibody Activity.
[0741] The recombinant yeast-derived humanized antibody is
evaluated for functional activity through the IL-6 driven T1165
cell proliferation assay and IL-6 stimulated HepG2 haptoglobin
assay described above.
Example 9 Acute Phase Response Neutralization by Intravenous
Administration of Anti-IL-6 Antibody Ab1
[0742] Human IL-6 can provoke an acute phase response in rats, and
one of the major acute phase proteins that is stimulated in the rat
is .alpha.-2 macroglobulin (A2M). A study was designed to assess
the dose of antibody Ab1 required to ablate the A2M response to a
single s.c. injection of 100 .mu.g of human IL-6 given one hour
after different doses (0.03, 0.1, 0.3, 1, and 3 mg/kg) of antibody
Ab1 administered intravenously (n=10 rats/dose level) or polyclonal
human IgG1 as the control (n=10 rats). Plasma was recovered and the
A2M was quantitated via a commercial sandwich ELISA kit (ICL Inc.,
Newberg Oreg.; cat. no. --E-25A2M). The endpoint was the difference
in the plasma concentration of A2M at the 24 hour time point
(post-Ab1). The results are presented in FIG. 4.
[0743] The ID50 for antibody Ab1 was 0.1 mg/kg with complete
suppression of the A2M response at the 0.3 mg/kg. This firmly
establishes in vivo neutralization of human IL-6 can be
accomplished by antibody Ab1.
Example 10 RXF393 Cachexia Model Study 1
[0744] Introduction
[0745] The human renal cell cancer cell line, RXF393 produces
profound weight loss when transplanted into athymic nude mice.
Weight loss begins around day 15 after transplantation with 80% of
all animals losing at least 30% of their total body weight by day
18-20 after transplantation. RXF393 secretes human IL-6 and the
plasma concentration of human IL-6 in these animals is very high at
around 10 ng/ml. Human IL-6 can bind murine soluble IL-6 receptor
and activate IL-6 responses in the mouse. Human IL-6 is
approximately 10 times less potent than murine IL-6 at activating
IL-6 responses in the mouse. The objectives of this study were to
determine the effect of antibody Ab1, on survival, body weight,
serum amyloid A protein, hematology parameters, and tumor growth in
athymic nude mice transplanted with the human renal cell cancer
cell line, RXF393.
[0746] Methods
[0747] Eighty, 6 week old, male athymic nude mice were implanted
with RXF393 tumor fragments (30-40 mg) subcutaneously in the right
flank. Animals were then divided into eight groups of ten mice.
Three groups were given either antibody Ab1 at 3 mg/kg, 10 mg/kg,
or 30 mg/kg intravenously weekly on day 1, day 8, day 15 and day 22
after transplantation (progression groups). Another three groups
were given either antibody Ab1 at 3 mg/kg, or 10 mg/kg, or 30 mg/kg
intravenously weekly on day 8, day 15 and day 22 after
transplantation (regression groups). Finally, one control group was
given polyclonal human IgG 30 mg/kg and a second control group was
given phosphate buffered saline intravenously weekly on day 1, day
8, day 15 and day 22 after transplantation.
[0748] Animals were euthanized at either day 28, when the tumor
reached 4,000 mm.sup.3 or if they became debilitated (>30% loss
of body weight). Animals were weighed on days 1, 6 and then daily
from days 9 to 28 after transplantation. Mean Percent Body Weight
(MPBW) was used as the primary parameter to monitor weight loss
during the study. It was calculated as follows: (Body Weight-Tumor
Weight)/Baseline Body Weight.times.100. Tumor weight was measured
on days 1, 6, 9, 12, 15, 18, 22, 25 and 28 after transplantation.
Blood was taken under anesthesia from five mice in each group on
days 5 and 13 and all ten mice in each group when euthanized (day
28 in most cases). Blood was analyzed for hematology and serum
amyloid A protein (SAA) concentration. An additional group of 10
non-tumor bearing 6 week old, athymic nude male mice had blood
samples taken for hematology and SAA concentration estimation to
act as a baseline set of values.
[0749] Results--Survival
[0750] No animals were euthanized or died in any of the antibody
Ab1 groups prior to the study termination date of day 28. In the
two control groups, 15 animals (7/9 in the polyclonal human IgG
group and 8/10 in the phosphate buffered saline group) were found
dead or were euthanized because they were very debilitated (>30%
loss of body weight). Median survival time in both control groups
was 20 days.
[0751] The survival curves for the two control groups and the
antibody Ab1 progression (dosed from day 1 of the study) groups are
presented in FIG. 5.
[0752] The survival curves for the two control groups and the
antibody Ab1 regression (dosed from day 8 of the study) groups are
presented in FIG. 6.
[0753] There was a statistically significant difference between the
survival curves for the polyclonal human IgG (p=0.0038) and
phosphate buffered saline (p=0.0003) control groups and the
survival curve for the six antibody Ab1 groups. There was no
statistically significant difference between the two control groups
(p=0.97).
[0754] Results--Tumor Size
[0755] Tumor size in surviving mice was estimated by palpation. For
the first 15 days of the study, none of the mice in any group were
found dead or were euthenized, and so comparison of tumor sizes
between groups on these days was free from sampling bias. No
difference in tumor size was observed between the antibody Ab1
progression or regression groups and the control groups through day
15. Comparison of the tumor size between surviving mice in the
control and treatment groups subsequent to the onset of mortality
in the controls (on day 15) was not undertaken because tumor size
the surviving control mice was presumed to be biased and
accordingly the results of such comparison would not be
meaningful.
[0756] As administration of antibody Ab1 promoted survival without
any apparent reduction in tumor size, elevated serum IL-6 may
contribute to mortality through mechanisms independent of tumor
growth. These observations supports the hypothesis that antibody
Ab1 can promote cancer patient survivability without directly
affecting tumor growth, possibly by enhancing general patient
well-being.
[0757] Results--Weight Loss
[0758] Mean Percent Body Weight (MPBW) (.+-.SEM) versus time is
shown in FIG. 27. Compared to controls, mice dosed with Ab1 were
protected from weight loss. On day 18, MPBW in control mice was
75%, corresponding to an average weight loss of 25%. In contrast,
on the same day, MPBW in Ab-1 treatment groups was minimally
changed (between 97% and 103%). There was a statistically
significant difference between the MPBW curves for the controls
(receiving polyclonal human IgG or PBS) and the 10 mg/kg dosage
group (p<0.0001) or 3 mg/kg and 30 mg/kg dosage groups
(p<0.0005). There was no statistically significant difference
between the two control groups.
[0759] Representative photographs of control and Ab1-treated mice
(FIG. 28) illustrate the emaciated condition of the control mice,
compared to the normal appearance of the Ab1-treated mouse, at the
end of the study (note externally visible tumor sites in right
flank).
[0760] These results suggest that Ab1 may be useful to prevent or
treat cachexia caused by elevated IL-6 in humans.
[0761] Results--Plasma Serum Amyloid A
[0762] The mean (.+-.SEM) plasma serum amyloid A concentration
versus time for the two control groups and the antibody Ab1
progression (dosed from day 1 of the study) and regression (dosed
from day 8 of the study) groups are presented in Table 5 and
graphically in FIG. 32.
TABLE-US-00013 TABLE 5 Mean Plasma SAA - antibody Ab1, all groups
versus control groups Mean Plasma Mean Plasma Mean Plasma SAA .+-.
SEM SAA .+-. SEM SAA .+-. SEM Day 5 Day 13 Terminal Bleed
(.mu.g/ml) (.mu.g/ml) (.mu.g/ml) Polyclonal IgG iv 675 .+-. 240
3198 .+-. 628 13371 .+-. 2413 weekly from day 1 (n = 5) (n = 4) (n
= 4) PBS iv weekly 355 .+-. 207 4844 .+-. 1126 15826 .+-. 802 from
day 1 (n = 5) (n = 5) (n = 3) Ab1 30 mg/kg iv 246 .+-. 100 2979
.+-. 170 841 .+-. 469 weekly from day 1 (n = 5) (n = 5) (n = 10)
Ab1 10 mg/kg iv 3629 .+-. 624 3096 .+-. 690 996 .+-. 348 weekly
from day 1 (n = 5) (n = 5) (n = 10) Ab1 3 mg/kg iv 106 .+-. 9 1623
.+-. 595 435 .+-. 70 weekly from day 1 (n = 5) (n = 4) (n = 9) Ab1
30 mg/kg iv 375 .+-. 177 1492 .+-. 418 498 .+-. 83 weekly from day
8 (n = 5) (n = 4) (n = 9) Ab1 10 mg/kg iv 487 .+-. 170 1403 .+-.
187 396 .+-. 58 weekly from day 8 (n = 5) (n = 5) (n = 10) Ab1 3
mg/kg iv 1255 .+-. 516 466 .+-. 157 685 .+-. 350 weekly from day 8
(n = 5) (n = 5) (n = 5)
[0763] SAA is up-regulated via the stimulation of hIL-6 and this
response is directly correlated with circulating levels of hIL-6
derived from the implanted tumor. The surrogate marker provides an
indirect readout for active hIL-6. Thus in the two treatment groups
described above there are significantly decreased levels of SAA due
to the neutralization of tumor-derived hIL-6. This further supports
the contention that antibody Ab1 displays in vivo efficacy.
Example 11 RXF393 Cachexia Model Study 2
[0764] Introduction
[0765] A second study was performed in the RXF-393 cachexia model
where treatment with antibody Ab1 was started at a later stage
(days 10 and 13 post-transplantation) and with a more prolonged
treatment phase (out to 49 days post transplantation). The dosing
interval with antibody Ab1 was shortened to 3 days from 7 and also
daily food consumption was measured. There was also an attempt to
standardize the tumor sizes at the time of initiating dosing with
antibody Ab1.
[0766] Methods
[0767] Eighty, 6 week old, male athymic nude mice were implanted
with RXF393 tumor fragments (30-40 mg) subcutaneously in the right
flank. 20 mice were selected whose tumors had reached between
270-320 mg in size and divided into two groups. One group received
antibody Ab1 at 10 mg/kg i.v. every three days and the other group
received polyclonal human IgG 10 mg/kg every 3 days from that
time-point (day 10 after transplantation). Another 20 mice were
selected when their tumor size had reached 400-527 mg in size and
divided into two groups. One group received antibody Ab1 at 10
mg/kg i.v. every three days and the other group received polyclonal
human IgG 10 mg/kg every 3 days from that time-point (day 13 after
transplantation). The remaining 40 mice took no further part in the
study and were euthanized at either day 49, when the tumor reached
4,000 mm.sup.3 or if they became very debilitated (>30% loss of
body weight).
[0768] Animals were weighed every 3-4 days from day 1 to day 49
after transplantation. Mean Percent Body Weight (MPBW) was used as
the primary parameter to monitor weight loss during the study. It
was calculated as follows: ((Body Weight-Tumor Weight)/Baseline
Body Weight).times.100. Tumor weight was measured every 3-4 days
from day 5 to day 49 after transplantation. Food consumption was
measured (amount consumed in 24 hours by weight (g) by each
treatment group) every day from day 10 for the 270-320 mg tumor
groups and day 13 for the 400-527 mg tumor groups.
[0769] Results--Survival
[0770] The survival curves for antibody Ab1 at 10 mg/kg i.v. every
three days (270-320 mg tumor size) and for the polyclonal human IgG
10 mg/kg i.v. every three days (270-320 mg tumor size) are
presented in FIG. 7.
[0771] Median survival for the antibody Ab1 at 10 mg/kg i.v. every
three days (270-320 mg tumor size) was 46 days and for the
polyclonal human IgG at 10 mg/kg i.v. every three days (270-320 mg
tumor size) was 32.5 days (p=0.0071).
[0772] The survival curves for the antibody Ab1 at 10 mg/kg i.v.
every three days (400-527 mg tumor size) and for the polyclonal
human IgG at 10 mg/kg i.v. every three days (400-527 mg tumor size)
are presented in FIG. 8. Median survival for the antibody Ab1 at 10
mg/kg i.v. every three days (400-527 mg tumor size) was 46.5 days
and for the polyclonal human IgG at 10 mg/kg i.v. every three days
(400-527 mg tumor size) was 27 days (p=0.0481).
Example 12 Multi-Dose Pharmacokinetic Evaluation of Antibody Ab1 in
Non-Human Primates
[0773] Antibody Ab1 was dosed in a single bolus infusion to a
single male and single female cynomologus monkey in phosphate
buffered saline. Plasma samples were removed at fixed time
intervals and the level of antibody Ab1 was quantitated through of
the use of an antigen capture ELISA assay. Biotinylated IL-6 (50
.mu.l of 3 .mu.g/mL) was captured on Streptavidin coated 96 well
microtiter plates. The plates were washed and blocked with 0.5%
Fish skin gelatin. Appropriately diluted plasma samples were added
and incubated for 1 hour at room temperature. The supernatants
removed and an anti-hFc-HRP conjugated secondary antibody applied
and left at room temperature.
[0774] The plates were then aspirated and TMB added to visualize
the amount of antibody. The specific levels were then determined
through the use of a standard curve. A second dose of antibody Ab1
was administered at day 35 to the same two cynomologus monkeys and
the experiment replicated using an identical sampling plan. The
resulting concentrations are then plot vs. time as show in FIG.
9.
[0775] This humanized full length aglycosylated antibody expressed
and purified Pichia pastoris displays comparable characteristics to
mammalian expressed protein. In addition, multiple doses of this
product display reproducible half-lives inferring that this
production platform does not generate products that display
enhanced immunogenicity.
Example 13 Octet Mechanistic Characterization of Antibody
Proteins
[0776] IL-6 signaling is dependent upon interactions between IL-6
and two receptors, IL-6R1 (CD126) and gp130 (IL-6 signal
transducer). To determine the antibody mechanism of action,
mechanistic studies were performed using bio-layer interferometry
with an Octet QK instrument (ForteBio; Menlo Park, Calif.). Studies
were performed in two different configurations. In the first
orientation, biotinylated IL-6 (R&D systems part number
206-IL-001MG/CF, biotinylated using Pierce EZ-link
sulfo-NHS-LC-LC-biotin product number 21338 according to
manufacturer's protocols) was initially bound to a streptavidin
coated biosensor (ForteBio part number 18-5006). Binding is
monitored as an increase in signal.
[0777] The IL-6 bound to the sensor was then incubated either with
the antibody in question or diluent solution alone. The sensor was
then incubated with soluble IL-6R1 (R&D systems product number
227-SR-025/CF) molecule. If the IL-6R1 molecule failed to bind, the
antibody was deemed to block IL-6/IL-6R1 interactions. These
complexes were incubated with gp130 (R&D systems 228-GP-010/CF)
in the presence of IL-6R1 for stability purposes. If gp130 did not
bind, it was concluded that the antibody blocked gp130 interactions
with IL-6.
[0778] In the second orientation, the antibody was bound to a
biosensor coated with an anti-human IgG1 Fc-specific reagent
(ForteBio part number 18-5001). The IL-6 was bound to the
immobilized antibody and the sensor was incubated with IL-6R1. If
the IL-6R1 did not interact with the IL-6, then it was concluded
that the IL-6 binding antibody blocked IL-6/IL-6R1 interactions. In
those situations where antibody/IL-6/IL-6R1 was observed, the
complex was incubated with gp130 in the presence of IL-6R1. If
gp130 did not interact, then it was concluded that the antibody
blocked IL-6/gp130 interactions. All studies were performed in a
200 .mu.L final volume, at 30 C and 1000 rpms. For these studies,
all proteins were diluted using ForteBio's sample diluent buffer
(part number 18-5028).
[0779] Results are presented in FIG. 10 (A-E) and FIG. 11.
Example 14 Peptide Mapping
[0780] In order to determine the epitope recognized by Ab1 on human
IL-6, the antibody was employed in a western-blot based assay. The
form of human IL-6 utilized in this example had a sequence of 183
amino acids in length (shown below). A 57-member library of
overlapping 15 amino acid peptides encompassing this sequence was
commercially synthesized and covalently bound to a PepSpots
nitrocellulose membrane (JPT Peptide technologies, Berlin,
Germany). The sequences of the overlapping 15 amino acid peptides
is shown in FIG. 12 and correspond to SEQ ID NOs: 590-646. Blots
were prepared and probed according to the manufacturer's
recommendations.
[0781] Briefly, blots were pre-wet in methanol, rinsed in PBS, and
blocked for over 2 hours in 10% non-fat milk in PBS/0.05% Tween
(Blocking Solution). The Ab1 antibody was used at 1 mg/ml final
dilution, and the HRP-conjugated Mouse Anti-Human-Kappa secondary
antibody (Southern BioTech #9220-05) was used at a 1:5000 dilution.
Antibody dilutions/incubations were performed in blocking solution.
Blots were developed using Amersham ECL advance reagents (GE#
RPN2135) and chemiluminescent signal documented using a CCD camera
(Alphalnnotec). The results of the blots is shown in FIG. 13 and
FIG. 14.
[0782] The sequence of the form of human IL-6 utilized to generate
peptide library is set forth:
TABLE-US-00014 (SEQ ID NO: 1)
VPPGEDSKDVAAPHRQPLTSSERIDKQIRYILDGISALRKETCNKSNMCE
SSKEALAENNLNLPKMAEKDGCFQSGFNEETCLVKIITGLLEFEVYLEYL
QNRFESSEEQARAVQMSTKVLIQFLQKKAKNLDAITTPDPTTNASLLTKL
QAQNQWLQDMTTHLILRSFKEFLQSSLRALRQM.
Example 15 Ab1 has High Affinity for IL-6
[0783] Surface plasmon resonance was used to measure association
rate (K.sub.a), dissociation rate (K.sub.d) and dissociation
constant (K.sub.D) for Ab1 to IL-6 from rat, mouse, dog, human, and
cynomolgus monkey at 25.degree. C. (FIG. 15A). The dissociation
constant for human IL-6 was 4 pM, indicating very high affinity. As
expected, affinity generally decreased with phylogenetic distance
from human. The dissociation constants of Ab1 for IL-6 of
cynomolgus monkey, rat, and mouse were 31 pM, 1.4 nM, and 0.4 nM,
respectively. Ab1 affinity for dog IL-6 below the limit of
quantitation of the experiment.
[0784] The high affinity of Ab1 for mouse, rat, and cynomolgus
monkey IL-6 suggest that Ab1 may be used to inhibit IL-6 of these
species. This hypothesis was tested using a cell proliferation
assay. In brief, each species's IL-6 was used to stimulate
proliferation of T1165 cells, and the concentration at which Ab1
could inhibit 50% of proliferation (IC50) was measured. Inhibition
was consistent with the measured dissociation constants (FIG. 15B).
These results demonstrate that Ab1 can inhibit the native IL-6 of
these species, and suggest the use of these organisms for in vitro
or in vivo modeling of IL-6 inhibition by Ab1.
Example 16 Multi-Dose Pharmacokinetic Evaluation of Antibody Ab1 in
Healthy Human Volunteers
[0785] Antibody Ab1 was dosed in a single bolus infusion in
histidine and sorbitol to healthy human volunteers. Dosages of 1
mg, 3 mg, 10 mg, 30 mg or 100 mg were administered to each
individual in dosage groups containing five to six individuals.
Plasma samples were removed at fixed time intervals for up to
twelve weeks. Human plasma was collected via venipuncture into a
vacuum collection tube containing EDTA. Plasma was separated and
used to assess the circulating levels of Ab1 using a monoclonal
antibody specific for Ab1, as follows. A 96 well microtiter plate
was coated overnight with the monoclonal antibody specific for Ab1
in 1.times.PBS overnight at 4.degree. C. The remaining steps were
conducted at room temperature. The wells were aspirated and
subsequently blocked using 0.5% Fish Skin Gelatin (FSG) (Sigma) in
1.times.PBS for 60 minutes. Human plasma samples were then added
and incubated for 60 minutes, then aspirated, then 50 ul of 1
.mu.g/mL biotinylated IL-6 was then added to each well and
incubated for 60 minutes. The wells were aspirated, and 50 .mu.L
streptavidin-HRP (Pharmingen), diluted 1:5,000 in 0.5% FSG/PBS, was
added and incubated for 45 minutes. Development was conducted using
standard methods employing TMB for detection. Levels were then
determined via comparison to a standard curve prepared in a
comparable format.
[0786] Average plasma concentration of Ab1 for each dosage group
versus time is shown in FIG. 16. Mean AUC and C.sub.max increased
linearly with dosage (FIG. 17 and FIG. 18, respectively). For
dosages of 30 mg and above, the average Ab1 half-life in each
dosage group was between approximately 25 and 30 days (FIG.
19).
Example 17 Pharmacokinetics of Ab1 in Patients with Advanced
Cancer
[0787] Antibody Ab1 was dosed in a single bolus infusion in
phosphate buffered saline to five individuals with advanced cancer.
Each individual received a dosage of 80 mg (n=2) or 160 mg (n=3) of
Ab1. Plasma samples were drawn weekly, and the level of antibody
Ab1 was quantitated as in Example 16.
[0788] Average plasma concentration of Ab1 in these individuals as
a function of time is shown in FIG. 20. The average Ab1 half-life
was approximately 31 days.
Example 18 Unprecedented Half-Life of Ab1
[0789] Overall, the average half-life of Ab1 was approximately 31
days in humans (for dosages of 10 mg and above), and approximately
15-21 days in cynomolgus monkey. The Ab1 half-life in humans and
cynomolgus monkeys are unprecedented when compared with the
half-lives of other anti-IL-6 antibodies (FIG. 21). As described
above, Ab1 was derived from humanization of a rabbit antibody, and
is produced from Pichia pastoris in an aglycosylated form. These
characteristics results in an antibody with very low immunogenicity
in humans. Moreover, the lack of glycosylation prevents Ab1 from
interacting with the Fc receptor or complement. Without intent to
be limited by theory, it is believed that the unprecedent half-life
of Ab1 is at least partially attributable to the humanization and
lack of glycosylation. The particular sequence and/or structure of
the antigen binding surfaces may also contribute to Ab1's
half-life.
Example 19 Ab1 Effect on Hemoglobin Concentration, Plasma Lipid
Concentration, and Neutrophil Counts in Patients with Advanced
Cancer
[0790] Antibody Ab1 was dosed in a single bolus infusion in
phosphate buffered saline to eight individuals with advanced cancer
(NSCLC, colorectal cancer, cholangiocarcinoma, or mesothelioma).
Each individual received a dosage of 80 mg, 160 mg, or 320 mg of
Ab1. Blood samples were removed just prior to infusion and at fixed
time intervals for six weeks, and the hemoglobin concentration,
plasma lipid concentration, and neutrophil counts were determined.
Average hemoglobin concentration rose slightly (FIG. 22), as did
total cholesterol and triglycerides (FIG. 23), while mean
neutrophil counts fell slightly (FIG. 24).
[0791] These results further demonstrate some of the beneficial
effects of administration of Ab1 to chronically ill individuals.
Because IL-6 is the main cytokine responsible for the anemia of
chronic disease (including cancer-related anemia), neutralization
of IL-6 by Ab1 increases hemoglobin concentration in these
individuals. Similarly, as IL-6 is centrally important in
increasing neutrophil counts in inflammation, the observed slight
reduction in neutrophil counts further confirms that Ab1 inhibits
IL-6. Finally, IL-6 causes anorexia as well as cachexia in these
patients; neutralization of IL-6 by Ab1 results in the return of
appetite and reversal of cachexia. The increase in plasma lipid
concentrations reflect the improved nutritional status of the
patients. Taken together, these results further demonstrate that
Ab1 effectively reverses these adverse consequences of IL-6 in
these patients.
Example 20 Ab1 Suppresses Serum CRP in Healthy Volunteers and in
Patients with Advanced Cancer
[0792] Introduction
[0793] Serum CRP concentrations have been identified as a strong
prognostic indicator in patients with certain forms of cancer. For
example, Hashimoto et al. performed univariate and multivariate
analysis of preoperative serum CRP concentrations in patients with
hepatocellular carcinoma in order to identify factors affecting
survival and disease recurrence (Hashimoto, K., et al., Cancer,
103(9):1856-1864 (2005)). Patients were classified into two groups,
those with serum CRP levels >1.0 mg/dl ("the CRP positive
group") and those with serum CRP levels <1.0 mg/dl ("the CRP
negative group"). The authors identified "a significant correlation
between preoperative serum CRP level and tumor size." Id.
Furthermore, the authors found that "[t]he overall survival and
recurrence-free survival rates in the CRP-positive group were
significantly lower compared with the rates in the CRP-negative
group." Id. The authors concluded that the preoperative CRP level
of patients is an independent and significant predictive indicator
or poor prognosis and early recurrence in patients with
hepatocellular carcinoma.
[0794] Similar correlations have been identified by other
investigators. For example, Karakiewicz et al. determined that
serum CRP was an independent and informative predictor of renal
cell carcinoma-specific mortality (Karakiewicz, P. I., et al.,
Cancer, 110(6):1241-1247 (2007)). Accordingly, there remains a need
in the art for methods and/or treatments that reduce serum
C-Reactive Protein (CRP) concentrations in cancer patients, and
particularly those with advanced cancers.
[0795] Methods
[0796] Healthy volunteers received a single 1-hour intravenous (IV)
infusion of either 100 mgs (5 patients), 30 mgs (5 patients), 10
mgs (6 patients), 3 mgs (6 patients) or 1 mg (6 patients) of the
Ab1 monoclonal antibody, while another 14 healthy volunteers
received intravenous placebo. Comparatively, 2 patients with
advanced forms of colorectal cancer received a single 1-hour
intravenous (IV) infusion of 80 mgs of the Ab1 monoclonal antibody.
No further dosages of the Ab1 monoclonal antibody were administered
to the test population.
[0797] Patients were evaluated prior to administration of the
dosage, and thereafter on a weekly basis for at least 5 weeks post
dose. At the time of each evaluation, patients were screened for
serum CRP concentration.
[0798] Results
[0799] Healthy Volunteers
[0800] As noted above, serum CRP levels are a marker of
inflammation; accordingly, baseline CRP levels are typically low in
healthy individuals. The low baseline CRP levels can make a further
reduction in CRP levels difficult to detect. Nonetheless, a
substantial reduction in serum CRP concentrations was detectable in
healthy volunteers receiving all concentrations of the Ab1
monoclonal antibody, compared to controls (FIG. 25). The reduction
in serum CRP levels was rapid, occurring within one week of
antibody administration, and prolonged, continuing at least through
the final measurement was taken (8 or 12 weeks from antibody
administration).
[0801] Cancer Patients
[0802] Five advanced cancer patients (colorectal cancer,
cholangiocarcinoma, or NSCLC) having elevated serum CRP levels were
dosed with 80 mg or 160 mg of Ab1. Serum CRP levels were greatly
reduced in these patients (FIG. 26A). The reduction in serum CRP
levels was rapid, with 90% of the decrease occurring within one
week of Ab1 administration, and prolonged, continuing at least
until the final measurement was taken (up to twelve weeks). The CRP
levels of two representative individuals are shown in FIG. 26B. In
those individuals, the CRP levels were lowered to below the normal
reference range (less than 5-6 mg/1) within one week. Thus,
administration of Ab1 to advanced cancer patients can cause a rapid
and sustained suppression of serum CRP levels.
Example 21 Ab1 Improved Muscular Strength, Improved Weight, and
Reduced Fatigue in Patients with Advanced Cancer
[0803] Introduction
[0804] Weight loss and fatigue (and accompanying muscular weakness)
are very common symptoms of patients with advanced forms of cancer,
and these symptoms can worsen as the cancer continues to progress.
Fatigue, weight loss and muscular weakness can have significant
negative effects on the recovery of patients with advanced forms of
cancer, for example by disrupting lifestyles and relationships and
affecting the willingness or ability of patients to continue cancer
treatments. Known methods of addressing fatigue, weight loss and
muscular weakness include regular routines of fitness and exercise,
methods of conserving the patient's energy, and treatments that
address anemia-induced fatigue and muscular weakness. Nevertheless,
there remains a need in the art for methods and/or treatments that
improve fatigue, weight loss and muscular weakness in cancer
patients.
[0805] Methods
[0806] Four patients with advanced forms of cancer (colorectal
cancer (2), NSCLC (1), cholangiocarcinoma (1) received a single
1-hour intravenous (IV) infusion of either 80 mgs or 160 mgs of the
Ab1 monoclonal antibody. No further dosages of the Ab1 monoclonal
antibody were administered to the test population.
[0807] Patients were evaluated prior to administration of the
dosage, and thereafter for at least 6 weeks post dose. At the time
of each evaluation, patients were screened for the following: a.)
any change in weight; b.) fatigue as measured using the Facit-F
Fatigue Subscale questionnaire a medically recognized test for
evaluating fatigue (See, e.g., Cella, D., Lai, J. S., Chang, C. H.,
Peterman, A., & Slavin, M. (2002). Fatigue in cancer patients
compared with fatigue in the general population. Cancer, 94(2),
528-538; Cella, D., Eton, D. T., Lai, F J-S., Peterman, A. H &
Merkel, D. E. (2002). Combining anchor and distribution based
methods to derive minimal clinically important differences on the
Functional Assessment of Cancer Therapy anemia and fatigue scales.
Journal of Pain & Symptom Management, 24 (6) 547-561.); and
hand-grip strength (a medically recognized test for evaluating
muscle strength, typically employing a handgrip dynamometer).
[0808] Results
[0809] Weight Change
[0810] The averaged data for both dosage concentrations (80 mgs and
160 mgs) of the Ab1 monoclonal antibody demonstrated an increase of
about 2 kilograms of weight per patient over the period of 6 weeks
(FIG. 29).
[0811] Fatigue
[0812] The averaged data for both dosage concentrations (80 mgs and
160 mgs) of the Ab1 monoclonal antibody demonstrated an increase in
the mean Facit-F FS subscale score of at least about 10 points in
the patient population over the period of 6 weeks (FIG. 30).
[0813] Hand-Grip Strength
[0814] The averaged data for both dosage concentrations (80 mgs and
160 mgs) of the Ab1 monoclonal antibody demonstrated an increase in
the mean hand-grip strength of at least about 10 percent in the
patient population over the period of 6 weeks (FIG. 31).
Example 22 Ab1 for Prevention of Thrombosis
[0815] Prior studies have shown that administration of an anti-IL-6
antibody can cause decreased platelet counts. Emilie, D. et al.,
Blood, 84(8):2472-9 (1994); Blay et al., Int J Cancer, 72(3):424-30
(1997). These results have apparently been viewed as an indicator
of potential danger, because further decreases in platelet counts
could cause complications such as bleeding. However, Applicants
have now discerned that inhibiting IL-6 restores a normal
coagulation profile, which Applicants predict will prevent
thrombosis. Decreased platelet counts resulting from inhibition of
IL-6 is not a sign of potential danger but rather reflects the
beneficial restoration of normal coagulation.
[0816] The mechanism by which normal coagulation is restored is
believed to result from the interplay between IL-6 and the acute
phase reaction. In response to elevated IL-6 levels, as for example
in a cancer patient, the liver produces acute phase proteins. These
acute phase proteins include coagulation factors, such as Factor
II, Factor V, Factor VIII, Factor IX, Factor XI, Factor XII,
F/fibrin degradation products, thrombin-antithrombin III complex,
fibrinogen, plasminogen, prothrombin, and von Willebrand factor.
This increase in coagulation factors may be measured directly, or
may be inferred from functional measurements of clotting ability.
Antagonists of IL-6, such as AbI, suppresses acute phase proteins,
e.g., Serum Amyloid A (see FIG. 32 and Example I0). Applicants now
predict that this suppression of acute phase proteins will restore
the normal coagulation profile, and thereby prevent thrombosis. The
restoration of normal coagulation may cause a slight drop in
platelet counts, but the patient will nonetheless retain normal
coagulation ability and thus will not have an increased risk of
bleeding. Such a treatment will represent a vast improvement over
the available anticoagulation therapies whose usefulness is limited
by the risk of adverse side-effects, such as major bleeding.
[0817] Applicants contemplate that the same beneficial effects of
inhibiting IL-6 will be obtained regardless of the method of
inhibition. Suitable methods of inhibiting IL-6 include
administration of anti-IL-6 antibodies, antisense therapy, soluble
IL-6 receptor, etc. either individually or in combinations.
Example 23 Ab1 Increases Plasma Albumin Concentration in Patients
with Advanced Cancer
[0818] Introduction
[0819] Serum albumin concentrations are recognized as predictive
indicators of survival and/or recovery success of cancer patients.
Hypoalbumenia correlates strongly with poor patient performance in
numerous forms of cancer. For example, in one study no patients
undergoing systemic chemotherapy for metastatic pancreatic
adenocarcinoma and having serum albumin levels less than 3.5 g/dL
successfully responded to systemic chemotherapy (Fujishiro, M., et
al., Hepatogastroenterology, 47(36):1744-46 (2000)). The authors
conclude that "[p]atients with . . . hypoalbuminemia . . . might be
inappropriate candidates for systemic chemotherapy and might be
treated with other experimental approaches or supportive care."
Id.
[0820] Similarly, Senior and Maroni state that "[t]he recent
appreciation that hypoalbuminemia is the most powerful predictor of
mortality in end-stage renal disease highlights the critical
importance of ensuring adequate protein intake in this patient
population." (J. R. Senior and B. J. Maroni, Am. Soc. Nutr. Sci.,
129:313S-314S (1999)).
[0821] In at least one study, attempts to recitfy hypoalbuminemia
in 27 patients with metastatic cancer by daily intravenous albumin
infusion of 20 g until normal serum albumin levels (>3.5 g/dl)
were achieved had little success. The authors note that "[a]lbumin
infusion for the advanced stage cancer patients has limited value
in clinical practice. Patients with PS 4 and hypoalbuminemia have
poorer prognosis." (Demirkazik, A., et al., Proc. Am. Soc. Clin.
Oncol., 21:Abstr 2892 (2002)).
[0822] Accordingly, there remains a need in the art for methods
and/or treatments that improve serum albumin concentrations in
cancer patients and address hypoalbuminemic states in cancer
patients, particularly those with advanced cancers.
[0823] Methods
[0824] Four patients with advanced forms of cancer (colorectal
cancer (2), NSCLC (1), cholangiocarcinoma (1) received a single
1-hour intravenous (IV) infusion of either 80 mgs or 160 mgs of the
Ab1 monoclonal antibody. No further dosages of the Ab1 monoclonal
antibody were administered to the test population.
[0825] Patients were evaluated prior to administration of the
dosage, and thereafter for at least 6 weeks post dose. At the time
of each evaluation, patients were screened for plasma albumin
concentration.
[0826] Results
[0827] The averaged data for both dosage concentrations (80 mgs and
160 mgs) of the Ab1 monoclonal antibody demonstrated an increase of
about 5 g/L of plasma albumin concentration per patient over the
period of 6 weeks (FIG. 33).
Sequence CWU 1
1
7281183PRTHomo sapiens 1Val Pro Pro Gly Glu Asp Ser Lys Asp Val Ala
Ala Pro His Arg Gln 1 5 10 15 Pro Leu Thr Ser Ser Glu Arg Ile Asp
Lys Gln Ile Arg Tyr Ile Leu 20 25 30 Asp Gly Ile Ser Ala Leu Arg
Lys Glu Thr Cys Asn Lys Ser Asn Met 35 40 45 Cys Glu Ser Ser Lys
Glu Ala Leu Ala Glu Asn Asn Leu Asn Leu Pro 50 55 60 Lys Met Ala
Glu Lys Asp Gly Cys Phe Gln Ser Gly Phe Asn Glu Glu 65 70 75 80 Thr
Cys Leu Val Lys Ile Ile Thr Gly Leu Leu Glu Phe Glu Val Tyr 85 90
95 Leu Glu Tyr Leu Gln Asn Arg Phe Glu Ser Ser Glu Glu Gln Ala Arg
100 105 110 Ala Val Gln Met Ser Thr Lys Val Leu Ile Gln Phe Leu Gln
Lys Lys 115 120 125 Ala Lys Asn Leu Asp Ala Ile Thr Thr Pro Asp Pro
Thr Thr Asn Ala 130 135 140 Ser Leu Leu Thr Lys Leu Gln Ala Gln Asn
Gln Trp Leu Gln Asp Met 145 150 155 160 Thr Thr His Leu Ile Leu Arg
Ser Phe Lys Glu Phe Leu Gln Ser Ser 165 170 175 Leu Arg Ala Leu Arg
Gln Met 180 2163PRTOryctolagus cuniculus 2Met Asp Thr Arg Ala Pro
Thr Gln Leu Leu Gly Leu Leu Leu Leu Trp 1 5 10 15 Leu Pro Gly Ala
Arg Cys Ala Tyr Asp Met Thr Gln Thr Pro Ala Ser 20 25 30 Val Ser
Ala Ala Val Gly Gly Thr Val Thr Ile Lys Cys Gln Ala Ser 35 40 45
Gln Ser Ile Asn Asn Glu Leu Ser Trp Tyr Gln Gln Lys Pro Gly Gln 50
55 60 Arg Pro Lys Leu Leu Ile Tyr Arg Ala Ser Thr Leu Ala Ser Gly
Val 65 70 75 80 Ser Ser Arg Phe Lys Gly Ser Gly Ser Gly Thr Glu Phe
Thr Leu Thr 85 90 95 Ile Ser Asp Leu Glu Cys Ala Asp Ala Ala Thr
Tyr Tyr Cys Gln Gln 100 105 110 Gly Tyr Ser Leu Arg Asn Ile Asp Asn
Ala Phe Gly Gly Gly Thr Glu 115 120 125 Val Val Val Lys Arg Thr Val
Ala Ala Pro Ser Val Phe Ile Phe Pro 130 135 140 Pro Ser Asp Glu Gln
Leu Lys Ser Gly Thr Ala Ser Val Val Cys Leu 145 150 155 160 Leu Asn
Asn 3166PRTOryctolagus cuniculus 3Met Glu Thr Gly Leu Arg Trp Leu
Leu Leu Val Ala Val Leu Lys Gly 1 5 10 15 Val Gln Cys Gln Ser Leu
Glu Glu Ser Gly Gly Arg Leu Val Thr Pro 20 25 30 Gly Thr Pro Leu
Thr Leu Thr Cys Thr Ala Ser Gly Phe Ser Leu Ser 35 40 45 Asn Tyr
Tyr Val Thr Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu 50 55 60
Trp Ile Gly Ile Ile Tyr Gly Ser Asp Glu Thr Ala Tyr Ala Thr Trp 65
70 75 80 Ala Ile Gly Arg Phe Thr Ile Ser Lys Thr Ser Thr Thr Val
Asp Leu 85 90 95 Lys Met Thr Ser Leu Thr Ala Ala Asp Thr Ala Thr
Tyr Phe Cys Ala 100 105 110 Arg Asp Asp Ser Ser Asp Trp Asp Ala Lys
Phe Asn Leu Trp Gly Gln 115 120 125 Gly Thr Leu Val Thr Val Ser Ser
Ala Ser Thr Lys Gly Pro Ser Val 130 135 140 Phe Pro Leu Ala Pro Ser
Ser Lys Ser Thr Ser Gly Gly Thr Ala Ala 145 150 155 160 Leu Gly Cys
Leu Val Lys 165 411PRTOryctolagus cuniculus 4Gln Ala Ser Gln Ser
Ile Asn Asn Glu Leu Ser 1 5 10 57PRTOryctolagus cuniculus 5Arg Ala
Ser Thr Leu Ala Ser 1 5 612PRTOryctolagus cuniculus 6Gln Gln Gly
Tyr Ser Leu Arg Asn Ile Asp Asn Ala 1 5 10 75PRTOryctolagus
cuniculus 7Asn Tyr Tyr Val Thr 1 5 816PRTOryctolagus cuniculus 8Ile
Ile Tyr Gly Ser Asp Glu Thr Ala Tyr Ala Thr Trp Ala Ile Gly 1 5 10
15 912PRTOryctolagus cuniculus 9Asp Asp Ser Ser Asp Trp Asp Ala Lys
Phe Asn Leu 1 5 10 10491DNAOryctolagus cuniculus 10atggacacga
gggcccccac tcagctgctg gggctcctgc tgctctggct cccaggtgcc 60agatgtgcct
atgatatgac ccagactcca gcctcggtgt ctgcagctgt gggaggcaca
120gtcaccatca agtgccaggc cagtcagagc attaacaatg aattatcctg
gtatcagcag 180aaaccagggc agcgtcccaa gctcctgatc tatagggcat
ccactctggc atctggggtc 240tcatcgcggt tcaaaggcag tggatctggg
acagagttca ctctcaccat cagcgacctg 300gagtgtgccg atgctgccac
ttactactgt caacagggtt atagtctgag gaatattgat 360aatgctttcg
gcggagggac cgaggtggtg gtcaaacgta cggtagcggc cccatctgtc
420ttcatcttcc cgccatctga tgagcagttg aaatctggaa ctgcctctgt
tgtgtgcctg 480ctgaataact t 49111499DNAOryctolagus cuniculus
11atggagactg ggctgcgctg gcttctcctg gtcgctgtgc tcaaaggtgt ccagtgtcag
60tcgctggagg agtccggggg tcgcctggtc acgcctggga cacccctgac actcacctgc
120acagcctctg gattctccct cagtaactac tacgtgacct gggtccgcca
ggctccaggg 180aaggggctgg aatggatcgg aatcatttat ggtagtgatg
aaacggccta cgcgacctgg 240gcgataggcc gattcaccat ctccaaaacc
tcgaccacgg tggatctgaa aatgaccagt 300ctgacagccg cggacacggc
cacctatttc tgtgccagag atgatagtag tgactgggat 360gcaaaattta
acttgtgggg ccaaggcacc ctggtcaccg tctcgagcgc ctccaccaag
420ggcccatcgg tcttccccct ggcaccctcc tccaagagca cctctggggg
cacagcggcc 480ctgggctgcc tggtcaagg 4991233DNAOryctolagus cuniculus
12caggccagtc agagcattaa caatgaatta tcc 331321DNAOryctolagus
cuniculus 13agggcatcca ctctggcatc t 211436DNAOryctolagus cuniculus
14caacagggtt atagtctgag gaatattgat aatgct 361515DNAOryctolagus
cuniculus 15aactactacg tgacc 151648DNAOryctolagus cuniculus
16atcatttatg gtagtgatga aacggcctac gcgacctggg cgataggc
481736DNAOryctolagus cuniculus 17gatgatagta gtgactggga tgcaaaattt
aacttg 3618109PRTOryctolagus cuniculus 18Glu Val Gln Leu Val Glu
Ser Gly Gly Gly Leu Val Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu
Ser Cys Ala Ala Ser Gly Phe Ser Leu Ser Asn Tyr 20 25 30 Tyr Val
Thr Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45
Gly Ile Ile Tyr Gly Ser Asp Glu Thr Ala Tyr Ala Thr Trp Ala Ile 50
55 60 Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr
Leu 65 70 75 80 Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr
Tyr Cys Ala 85 90 95 Arg Asp Asp Ser Ser Asp Trp Asp Ala Lys Phe
Asn Leu 100 105 19109PRTOryctolagus cuniculus 19Glu Val Gln Leu Val
Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg
Leu Ser Cys Ala Ala Ser Gly Phe Ser Leu Ser Asn Tyr 20 25 30 Tyr
Val Thr Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40
45 Gly Ile Ile Tyr Gly Ser Asp Glu Thr Ala Tyr Ala Thr Ser Ala Ile
50 55 60 Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu
Tyr Leu 65 70 75 80 Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val
Tyr Tyr Cys Ala 85 90 95 Arg Asp Asp Ser Ser Asp Trp Asp Ala Lys
Phe Asn Leu 100 105 2099PRTOryctolagus cuniculus 20Ile Gln Met Thr
Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly Asp 1 5 10 15 Arg Val
Thr Ile Thr Cys Gln Ala Ser Gln Ser Ile Asn Asn Glu Leu 20 25 30
Ser Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile Tyr 35
40 45 Arg Ala Ser Thr Leu Ala Ser Gly Val Pro Ser Arg Phe Ser Gly
Ser 50 55 60 Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu
Gln Pro Asp 65 70 75 80 Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Gly Tyr
Ser Leu Arg Asn Ile 85 90 95 Asp Asn Ala 21170PRTOryctolagus
cuniculus 21Met Asp Thr Arg Ala Pro Thr Gln Leu Leu Gly Leu Leu Leu
Leu Trp 1 5 10 15 Leu Pro Gly Ala Arg Cys Ala Tyr Asp Met Thr Gln
Thr Pro Ala Ser 20 25 30 Val Glu Val Ala Val Gly Gly Thr Val Thr
Ile Asn Cys Gln Ala Ser 35 40 45 Glu Thr Ile Tyr Ser Trp Leu Ser
Trp Tyr Gln Gln Lys Pro Gly Gln 50 55 60 Pro Pro Lys Leu Leu Ile
Tyr Gln Ala Ser Asp Leu Ala Ser Gly Val 65 70 75 80 Pro Ser Arg Phe
Ser Gly Ser Gly Ala Gly Thr Glu Tyr Thr Leu Thr 85 90 95 Ile Ser
Gly Val Gln Cys Asp Asp Ala Ala Thr Tyr Tyr Cys Gln Gln 100 105 110
Gly Tyr Ser Gly Ser Asn Val Asp Asn Val Phe Gly Gly Gly Thr Glu 115
120 125 Val Val Val Lys Arg Thr Val Ala Ala Pro Ser Val Phe Ile Phe
Pro 130 135 140 Pro Ser Asp Glu Gln Leu Lys Ser Gly Thr Ala Ser Val
Val Cys Leu 145 150 155 160 Leu Asn Asn Phe Tyr Pro Arg Glu Ala Lys
165 170 22167PRTOryctolagus cuniculus 22Met Glu Thr Gly Leu Arg Trp
Leu Leu Leu Val Ala Val Leu Lys Gly 1 5 10 15 Val Gln Cys Gln Glu
Gln Leu Lys Glu Ser Gly Gly Arg Leu Val Thr 20 25 30 Pro Gly Thr
Pro Leu Thr Leu Thr Cys Thr Ala Ser Gly Phe Ser Leu 35 40 45 Asn
Asp His Ala Met Gly Trp Val Arg Gln Ala Pro Gly Lys Gly Leu 50 55
60 Glu Tyr Ile Gly Phe Ile Asn Ser Gly Gly Ser Ala Arg Tyr Ala Ser
65 70 75 80 Trp Ala Glu Gly Arg Phe Thr Ile Ser Arg Thr Ser Thr Thr
Val Asp 85 90 95 Leu Lys Met Thr Ser Leu Thr Thr Glu Asp Thr Ala
Thr Tyr Phe Cys 100 105 110 Val Arg Gly Gly Ala Val Trp Ser Ile His
Ser Phe Asp Pro Trp Gly 115 120 125 Pro Gly Thr Leu Val Thr Val Ser
Ser Ala Ser Thr Lys Gly Pro Ser 130 135 140 Val Phe Pro Leu Ala Pro
Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala 145 150 155 160 Ala Leu Gly
Cys Leu Val Lys 165 2311PRTOryctolagus cuniculus 23Gln Ala Ser Glu
Thr Ile Tyr Ser Trp Leu Ser 1 5 10 247PRTOryctolagus cuniculus
24Gln Ala Ser Asp Leu Ala Ser 1 5 2512PRTOryctolagus cuniculus
25Gln Gln Gly Tyr Ser Gly Ser Asn Val Asp Asn Val 1 5 10
265PRTOryctolagus cuniculus 26Asp His Ala Met Gly 1 5
2716PRTOryctolagus cuniculus 27Phe Ile Asn Ser Gly Gly Ser Ala Arg
Tyr Ala Ser Trp Ala Glu Gly 1 5 10 15 2812PRTOryctolagus cuniculus
28Gly Gly Ala Val Trp Ser Ile His Ser Phe Asp Pro 1 5 10
29511DNAOryctolagus cuniculus 29atggacacga gggcccccac tcagctgctg
gggctcctgc tgctctggct cccaggtgcc 60agatgtgcct atgatatgac ccagactcca
gcctctgtgg aggtagctgt gggaggcaca 120gtcaccatca attgccaggc
cagtgagacc atttacagtt ggttatcctg gtatcagcag 180aagccagggc
agcctcccaa gctcctgatc taccaggcat ccgatctggc atctggggtc
240ccatcgcgat tcagcggcag tggggctggg acagagtaca ctctcaccat
cagcggcgtg 300cagtgtgacg atgctgccac ttactactgt caacagggtt
atagtggtag taatgttgat 360aatgttttcg gcggagggac cgaggtggtg
gtcaaacgta cggtagcggc cccatctgtc 420ttcatcttcc cgccatctga
tgagcagttg aaatctggaa ctgcctctgt tgtgtgcctg 480ctgaataact
tctatcccag agaggccaaa g 51130501DNAOryctolagus cuniculus
30atggagactg ggctgcgctg gcttctcctg gtcgctgtgc tcaaaggtgt ccagtgtcag
60gagcagctga aggagtccgg gggtcgcctg gtcacgcctg ggacacccct gacacttacc
120tgcacagcct ctggattctc cctcaatgac catgcaatgg gctgggtccg
ccaggctcca 180gggaaggggc tggaatacat cggattcatt aatagtggtg
gtagcgcacg ctacgcgagc 240tgggcagaag gccgattcac catctccaga
acctcgacca cggtggatct gaaaatgacc 300agtctgacaa ccgaggacac
ggccacctat ttctgtgtca gagggggtgc tgtttggagt 360attcatagtt
ttgatccctg gggcccaggg accctggtca ccgtctcgag cgcctccacc
420aagggcccat cggtcttccc cctggcaccc tcctccaaga gcacctctgg
gggcacagcg 480gccctgggct gcctggtcaa g 5013133DNAOryctolagus
cuniculus 31caggccagtg agaccattta cagttggtta tcc
333221DNAOryctolagus cuniculus 32caggcatccg atctggcatc t
213336DNAOryctolagus cuniculus 33caacagggtt atagtggtag taatgttgat
aatgtt 363415DNAOryctolagus cuniculus 34gaccatgcaa tgggc
153548DNAOryctolagus cuniculus 35ttcattaata gtggtggtag cgcacgctac
gcgagctggg cagaaggc 483636DNAOryctolagus cuniculus 36gggggtgctg
tttggagtat tcatagtttt gatccc 3637165PRTOryctolagus cuniculus 37Met
Asp Thr Arg Ala Pro Thr Gln Leu Leu Gly Leu Leu Leu Leu Trp 1 5 10
15 Leu Pro Gly Ala Thr Phe Ala Ala Val Leu Thr Gln Thr Pro Ser Pro
20 25 30 Val Ser Ala Ala Val Gly Gly Thr Val Ser Ile Ser Cys Gln
Ala Ser 35 40 45 Gln Ser Val Tyr Asp Asn Asn Tyr Leu Ser Trp Phe
Gln Gln Lys Pro 50 55 60 Gly Gln Pro Pro Lys Leu Leu Ile Tyr Gly
Ala Ser Thr Leu Ala Ser 65 70 75 80 Gly Val Pro Ser Arg Phe Val Gly
Ser Gly Ser Gly Thr Gln Phe Thr 85 90 95 Leu Thr Ile Thr Asp Val
Gln Cys Asp Asp Ala Ala Thr Tyr Tyr Cys 100 105 110 Ala Gly Val Tyr
Asp Asp Asp Ser Asp Asn Ala Phe Gly Gly Gly Thr 115 120 125 Glu Val
Val Val Lys Arg Thr Val Ala Ala Pro Ser Val Phe Ile Phe 130 135 140
Pro Pro Ser Asp Glu Gln Leu Lys Ser Gly Thr Ala Ser Val Val Cys 145
150 155 160 Leu Leu Asn Asn Phe 165 38166PRTOryctolagus cuniculus
38Met Glu Thr Gly Leu Arg Trp Leu Leu Leu Val Ala Val Leu Lys Gly 1
5 10 15 Val Gln Cys Gln Ser Leu Glu Glu Ser Gly Gly Arg Leu Val Thr
Pro 20 25 30 Gly Thr Pro Leu Thr Leu Thr Cys Thr Ala Ser Gly Phe
Ser Leu Ser 35 40 45 Val Tyr Tyr Met Asn Trp Val Arg Gln Ala Pro
Gly Lys Gly Leu Glu 50 55 60 Trp Ile Gly Phe Ile Thr Met Ser Asp
Asn Ile Asn Tyr Ala Ser Trp 65 70 75 80 Ala Lys Gly Arg Phe Thr Ile
Ser Lys Thr Ser Thr Thr Val Asp Leu 85 90 95 Lys Met Thr Ser Pro
Thr Thr Glu Asp Thr Ala Thr Tyr Phe Cys Ala 100 105 110 Arg Ser Arg
Gly Trp Gly Thr Met Gly Arg Leu Asp Leu Trp Gly Pro 115 120 125 Gly
Thr Leu Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val 130 135
140 Phe Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala Ala
145 150 155 160 Leu Gly Cys Leu Val Lys 165 3913PRTOryctolagus
cuniculus 39Gln Ala Ser Gln Ser Val Tyr Asp Asn Asn Tyr Leu Ser 1 5
10 407PRTOryctolagus cuniculus 40Gly Ala Ser Thr Leu Ala Ser 1 5
4111PRTOryctolagus cuniculus 41Ala Gly Val Tyr Asp Asp Asp Ser Asp
Asn Ala 1 5 10 425PRTOryctolagus cuniculus 42Val Tyr Tyr Met Asn 1
5 4316PRTOryctolagus cuniculus 43Phe Ile Thr Met Ser Asp Asn Ile
Asn Tyr Ala Ser Trp Ala Lys Gly 1 5 10 15 4412PRTOryctolagus
cuniculus 44Ser Arg Gly Trp Gly Thr Met
Gly Arg Leu Asp Leu 1 5 10 45496DNAOryctolagus cuniculus
45atggacacga gggcccccac tcagctgctg gggctcctgc tgctctggct cccaggtgcc
60acatttgccg ccgtgctgac ccagactcca tctcccgtgt ctgcagctgt gggaggcaca
120gtcagcatca gttgccaggc cagtcagagt gtttatgaca acaactactt
atcctggttt 180cagcagaaac cagggcagcc tcccaagctc ctgatctatg
gtgcatccac tctggcatct 240ggggtcccat cgcggttcgt gggcagtgga
tctgggacac agttcactct caccatcaca 300gacgtgcagt gtgacgatgc
tgccacttac tattgtgcag gcgtttatga tgatgatagt 360gataatgcct
tcggcggagg gaccgaggtg gtggtcaaac gtacggtagc ggccccatct
420gtcttcatct tcccgccatc tgatgagcag ttgaaatctg gaactgcctc
tgttgtgtgc 480ctgctgaata acttct 49646499DNAOryctolagus cuniculus
46atggagactg ggctgcgctg gcttctcctg gtggctgtgc tcaaaggtgt ccagtgtcag
60tcgctggagg agtccggggg tcgcctggtc acccctggga cacccctgac actcacctgc
120acagcctctg gattctccct cagtgtctac tacatgaact gggtccgcca
ggctccaggg 180aaggggctgg aatggatcgg attcattaca atgagtgata
atataaatta cgcgagctgg 240gcgaaaggcc gattcaccat ctccaaaacc
tcgaccacgg tggatctgaa aatgaccagt 300ccgacaaccg aggacacggc
cacctatttc tgtgccagga gtcgtggctg gggtacaatg 360ggtcggttgg
atctctgggg cccaggcacc ctcgtcaccg tctcgagcgc ctccaccaag
420ggcccatcgg tcttccccct ggcaccctcc tccaagagca cctctggggg
cacagcggcc 480ctgggctgcc tggtcaagg 4994739DNAOryctolagus cuniculus
47caggccagtc agagtgttta tgacaacaac tacttatcc 394821DNAOryctolagus
cuniculus 48ggtgcatcca ctctggcatc t 214933DNAOryctolagus cuniculus
49gcaggcgttt atgatgatga tagtgataat gcc 335015DNAOryctolagus
cuniculus 50gtctactaca tgaac 155148DNAOryctolagus cuniculus
51ttcattacaa tgagtgataa tataaattac gcgagctggg cgaaaggc
485236DNAOryctolagus cuniculus 52agtcgtggct ggggtacaat gggtcggttg
gatctc 3653164PRTOryctolagus cuniculus 53Met Asp Thr Arg Ala Pro
Thr Gln Leu Leu Gly Leu Leu Leu Leu Trp 1 5 10 15 Leu Pro Gly Ala
Ile Cys Asp Pro Val Leu Thr Gln Thr Pro Ser Pro 20 25 30 Val Ser
Ala Pro Val Gly Gly Thr Val Ser Ile Ser Cys Gln Ala Ser 35 40 45
Gln Ser Val Tyr Glu Asn Asn Tyr Leu Ser Trp Phe Gln Gln Lys Pro 50
55 60 Gly Gln Pro Pro Lys Leu Leu Ile Tyr Gly Ala Ser Thr Leu Asp
Ser 65 70 75 80 Gly Val Pro Ser Arg Phe Lys Gly Ser Gly Ser Gly Thr
Gln Phe Thr 85 90 95 Leu Thr Ile Thr Asp Val Gln Cys Asp Asp Ala
Ala Thr Tyr Tyr Cys 100 105 110 Ala Gly Val Tyr Asp Asp Asp Ser Asp
Asp Ala Phe Gly Gly Gly Thr 115 120 125 Glu Val Val Val Lys Arg Thr
Val Ala Ala Pro Ser Val Phe Ile Phe 130 135 140 Pro Pro Ser Asp Glu
Gln Leu Lys Ser Gly Thr Ala Ser Val Val Cys 145 150 155 160 Leu Leu
Asn Asn 54167PRTOryctolagus cuniculus 54Met Glu Thr Gly Leu Arg Trp
Leu Leu Leu Val Ala Val Leu Lys Gly 1 5 10 15 Val Gln Cys Gln Glu
Gln Leu Lys Glu Ser Gly Gly Gly Leu Val Thr 20 25 30 Pro Gly Gly
Thr Leu Thr Leu Thr Cys Thr Ala Ser Gly Phe Ser Leu 35 40 45 Asn
Ala Tyr Tyr Met Asn Trp Val Arg Gln Ala Pro Gly Lys Gly Leu 50 55
60 Glu Trp Ile Gly Phe Ile Thr Leu Asn Asn Asn Val Ala Tyr Ala Asn
65 70 75 80 Trp Ala Lys Gly Arg Phe Thr Phe Ser Lys Thr Ser Thr Thr
Val Asp 85 90 95 Leu Lys Met Thr Ser Pro Thr Pro Glu Asp Thr Ala
Thr Tyr Phe Cys 100 105 110 Ala Arg Ser Arg Gly Trp Gly Ala Met Gly
Arg Leu Asp Leu Trp Gly 115 120 125 His Gly Thr Leu Val Thr Val Ser
Ser Ala Ser Thr Lys Gly Pro Ser 130 135 140 Val Phe Pro Leu Ala Pro
Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala 145 150 155 160 Ala Leu Gly
Cys Leu Val Lys 165 5513PRTOryctolagus cuniculus 55Gln Ala Ser Gln
Ser Val Tyr Glu Asn Asn Tyr Leu Ser 1 5 10 567PRTOryctolagus
cuniculus 56Gly Ala Ser Thr Leu Asp Ser 1 5 5711PRTOryctolagus
cuniculus 57Ala Gly Val Tyr Asp Asp Asp Ser Asp Asp Ala 1 5 10
585PRTOryctolagus cuniculus 58Ala Tyr Tyr Met Asn 1 5
5916PRTOryctolagus cuniculus 59Phe Ile Thr Leu Asn Asn Asn Val Ala
Tyr Ala Asn Trp Ala Lys Gly 1 5 10 15 6012PRTOryctolagus cuniculus
60Ser Arg Gly Trp Gly Ala Met Gly Arg Leu Asp Leu 1 5 10
61494DNAOryctolagus cuniculus 61atggacacga gggcccccac tcagctgctg
gggctcctgc tgctctggct cccaggtgcc 60atatgtgacc ctgtgctgac ccagactcca
tctcccgtat ctgcacctgt gggaggcaca 120gtcagcatca gttgccaggc
cagtcagagt gtttatgaga acaactattt atcctggttt 180cagcagaaac
cagggcagcc tcccaagctc ctgatctatg gtgcatccac tctggattct
240ggggtcccat cgcggttcaa aggcagtgga tctgggacac agttcactct
caccattaca 300gacgtgcagt gtgacgatgc tgccacttac tattgtgcag
gcgtttatga tgatgatagt 360gatgatgcct tcggcggagg gaccgaggtg
gtggtcaaac gtacggtagc ggccccatct 420gtcttcatct tcccgccatc
tgatgagcag ttgaaatctg gaactgcctc tgttgtgtgc 480ctgctgaata actt
49462502DNAOryctolagus cuniculus 62atggagactg ggctgcgctg gcttctcctg
gtggctgtgc tcaaaggtgt ccagtgtcag 60gagcagctga aggagtccgg aggaggcctg
gtaacgcctg gaggaaccct gacactcacc 120tgcacagcct ctggattctc
cctcaatgcc tactacatga actgggtccg ccaggctcca 180gggaaggggc
tggaatggat cggattcatt actctgaata ataatgtagc ttacgcgaac
240tgggcgaaag gccgattcac cttctccaaa acctcgacca cggtggatct
gaaaatgacc 300agtccgacac ccgaggacac ggccacctat ttctgtgcca
ggagtcgtgg ctggggtgca 360atgggtcggt tggatctctg gggccatggc
accctggtca ccgtctcgag cgcctccacc 420aagggcccat cggtcttccc
cctggcaccc tcctccaaga gcacctctgg gggcacagcg 480gccctgggct
gcctggtcaa gg 5026339DNAOryctolagus cuniculus 63caggccagtc
agagtgttta tgagaacaac tatttatcc 396421DNAOryctolagus cuniculus
64ggtgcatcca ctctggattc t 216533DNAOryctolagus cuniculus
65gcaggcgttt atgatgatga tagtgatgat gcc 336615DNAOryctolagus
cuniculus 66gcctactaca tgaac 156748DNAOryctolagus cuniculus
67ttcattactc tgaataataa tgtagcttac gcgaactggg cgaaaggc
486836DNAOryctolagus cuniculus 68agtcgtggct ggggtgcaat gggtcggttg
gatctc 3669164PRTOryctolagus cuniculus 69Met Asp Thr Arg Ala Pro
Thr Gln Leu Leu Gly Leu Leu Leu Leu Trp 1 5 10 15 Leu Pro Gly Ala
Thr Phe Ala Gln Val Leu Thr Gln Thr Pro Ser Pro 20 25 30 Val Ser
Ala Ala Val Gly Gly Thr Val Thr Ile Asn Cys Gln Ala Ser 35 40 45
Gln Ser Val Asp Asp Asn Asn Trp Leu Gly Trp Tyr Gln Gln Lys Arg 50
55 60 Gly Gln Pro Pro Lys Tyr Leu Ile Tyr Ser Ala Ser Thr Leu Ala
Ser 65 70 75 80 Gly Val Pro Ser Arg Phe Lys Gly Ser Gly Ser Gly Thr
Gln Phe Thr 85 90 95 Leu Thr Ile Ser Asp Leu Glu Cys Asp Asp Ala
Ala Thr Tyr Tyr Cys 100 105 110 Ala Gly Gly Phe Ser Gly Asn Ile Phe
Ala Phe Gly Gly Gly Thr Glu 115 120 125 Val Val Val Lys Arg Thr Val
Ala Ala Pro Ser Val Phe Ile Phe Pro 130 135 140 Pro Ser Asp Glu Gln
Leu Lys Ser Gly Thr Ala Ser Val Val Cys Leu 145 150 155 160 Leu Asn
Asn Phe 70164PRTOryctolagus cuniculus 70Met Glu Thr Gly Leu Arg Trp
Leu Leu Leu Val Ala Val Leu Lys Gly 1 5 10 15 Val Gln Cys Gln Ser
Val Glu Glu Ser Gly Gly Arg Leu Val Thr Pro 20 25 30 Gly Thr Pro
Leu Thr Leu Thr Cys Thr Val Ser Gly Phe Ser Leu Ser 35 40 45 Ser
Tyr Ala Met Ser Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu 50 55
60 Trp Ile Gly Ile Ile Gly Gly Phe Gly Thr Thr Tyr Tyr Ala Thr Trp
65 70 75 80 Ala Lys Gly Arg Phe Thr Ile Ser Lys Thr Ser Thr Thr Val
Asp Leu 85 90 95 Arg Ile Thr Ser Pro Thr Thr Glu Asp Thr Ala Thr
Tyr Phe Cys Ala 100 105 110 Arg Gly Gly Pro Gly Asn Gly Gly Asp Ile
Trp Gly Gln Gly Thr Leu 115 120 125 Val Thr Val Ser Ser Ala Ser Thr
Lys Gly Pro Ser Val Phe Pro Leu 130 135 140 Ala Pro Ser Ser Lys Ser
Thr Ser Gly Gly Thr Ala Ala Leu Gly Cys 145 150 155 160 Leu Val Lys
Asp 7113PRTOryctolagus cuniculus 71Gln Ala Ser Gln Ser Val Asp Asp
Asn Asn Trp Leu Gly 1 5 10 727PRTOryctolagus cuniculus 72Ser Ala
Ser Thr Leu Ala Ser 1 5 7310PRTOryctolagus cuniculus 73Ala Gly Gly
Phe Ser Gly Asn Ile Phe Ala 1 5 10 745PRTOryctolagus cuniculus
74Ser Tyr Ala Met Ser 1 5 7516PRTOryctolagus cuniculus 75Ile Ile
Gly Gly Phe Gly Thr Thr Tyr Tyr Ala Thr Trp Ala Lys Gly 1 5 10 15
769PRTOryctolagus cuniculus 76Gly Gly Pro Gly Asn Gly Gly Asp Ile 1
5 77493DNAOryctolagus cuniculus 77atggacacga gggcccccac tcagctgctg
gggctcctgc tgctctggct cccaggtgcc 60acatttgccc aagtgctgac ccagactcca
tcgcctgtgt ctgcagctgt gggaggcaca 120gtcaccatca actgccaggc
cagtcagagt gttgatgata acaactggtt aggctggtat 180cagcagaaac
gagggcagcc tcccaagtac ctgatctatt ctgcatccac tctggcatct
240ggggtcccat cgcggttcaa aggcagtgga tctgggacac agttcactct
caccatcagc 300gacctggagt gtgacgatgc tgccacttac tactgtgcag
gcggttttag tggtaatatc 360tttgctttcg gcggagggac cgaggtggtg
gtcaaacgta cggtagcggc cccatctgtc 420ttcatcttcc cgccatctga
tgagcagttg aaatctggaa ctgcctctgt tgtgtgcctg 480ctgaataact tct
49378493DNAOryctolagus cuniculus 78atggagactg ggctgcgctg gcttctcctg
gtcgctgtgc tcaaaggtgt ccagtgtcag 60tcggtggagg agtccggggg tcgcctggtc
acgcctggga cacccctgac actcacctgc 120acagtctctg gcttctccct
cagtagctat gcaatgagct gggtccgcca ggctccagga 180aaggggctgg
agtggatcgg aatcattggt ggttttggta ccacatacta cgcgacctgg
240gcgaaaggcc gattcaccat ctccaaaacc tcgaccacgg tggatctgag
aatcaccagt 300ccgacaaccg aggacacggc cacctatttc tgtgccagag
gtggtcctgg taatggtggt 360gacatctggg gccaagggac cctggtcacc
gtctcgagcg cctccaccaa gggcccatcg 420gtcttccccc tggcaccctc
ctccaagagc acctctgggg gcacagcggc cctgggctgc 480ctggtcaagg act
4937939DNAOryctolagus cuniculus 79caggccagtc agagtgttga tgataacaac
tggttaggc 398021DNAOryctolagus cuniculus 80tctgcatcca ctctggcatc t
218130DNAOryctolagus cuniculus 81gcaggcggtt ttagtggtaa tatctttgct
308215DNAOryctolagus cuniculus 82agctatgcaa tgagc
158348DNAOryctolagus cuniculus 83atcattggtg gttttggtac cacatactac
gcgacctggg cgaaaggc 488427DNAOryctolagus cuniculus 84ggtggtcctg
gtaatggtgg tgacatc 2785164PRTOryctolagus cuniculus 85Met Asp Thr
Arg Ala Pro Thr Gln Leu Leu Gly Leu Leu Leu Leu Trp 1 5 10 15 Leu
Pro Gly Ala Thr Phe Ala Ala Val Leu Thr Gln Thr Pro Ser Pro 20 25
30 Val Ser Val Pro Val Gly Gly Thr Val Thr Ile Lys Cys Gln Ser Ser
35 40 45 Gln Ser Val Tyr Asn Asn Phe Leu Ser Trp Tyr Gln Gln Lys
Pro Gly 50 55 60 Gln Pro Pro Lys Leu Leu Ile Tyr Gln Ala Ser Lys
Leu Ala Ser Gly 65 70 75 80 Val Pro Asp Arg Phe Ser Gly Ser Gly Ser
Gly Thr Gln Phe Thr Leu 85 90 95 Thr Ile Ser Gly Val Gln Cys Asp
Asp Ala Ala Thr Tyr Tyr Cys Leu 100 105 110 Gly Gly Tyr Asp Asp Asp
Ala Asp Asn Ala Phe Gly Gly Gly Thr Glu 115 120 125 Val Val Val Lys
Arg Thr Val Ala Ala Pro Ser Val Phe Ile Phe Pro 130 135 140 Pro Ser
Asp Glu Gln Leu Lys Ser Gly Thr Ala Ser Val Val Cys Leu 145 150 155
160 Leu Asn Asn Phe 86170PRTOryctolagus cuniculus 86Met Glu Thr Gly
Leu Arg Trp Leu Leu Leu Val Ala Val Leu Lys Gly 1 5 10 15 Val Gln
Cys Gln Ser Val Glu Glu Ser Gly Gly Arg Leu Val Thr Pro 20 25 30
Gly Thr Pro Leu Thr Leu Thr Cys Thr Val Ser Gly Ile Asp Leu Ser 35
40 45 Asp Tyr Ala Met Ser Trp Val Arg Gln Ala Pro Gly Lys Gly Leu
Glu 50 55 60 Trp Ile Gly Ile Ile Tyr Ala Gly Ser Gly Ser Thr Trp
Tyr Ala Ser 65 70 75 80 Trp Ala Lys Gly Arg Phe Thr Ile Ser Lys Thr
Ser Thr Thr Val Asp 85 90 95 Leu Lys Ile Thr Ser Pro Thr Thr Glu
Asp Thr Ala Thr Tyr Phe Cys 100 105 110 Ala Arg Asp Gly Tyr Asp Asp
Tyr Gly Asp Phe Asp Arg Leu Asp Leu 115 120 125 Trp Gly Pro Gly Thr
Leu Val Thr Val Ser Ser Ala Ser Thr Lys Gly 130 135 140 Pro Ser Val
Phe Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly 145 150 155 160
Thr Ala Ala Leu Gly Cys Leu Val Lys Asp 165 170 8712PRTOryctolagus
cuniculus 87Gln Ser Ser Gln Ser Val Tyr Asn Asn Phe Leu Ser 1 5 10
887PRTOryctolagus cuniculus 88Gln Ala Ser Lys Leu Ala Ser 1 5
8911PRTOryctolagus cuniculus 89Leu Gly Gly Tyr Asp Asp Asp Ala Asp
Asn Ala 1 5 10 905PRTOryctolagus cuniculus 90Asp Tyr Ala Met Ser 1
5 9117PRTOryctolagus cuniculus 91Ile Ile Tyr Ala Gly Ser Gly Ser
Thr Trp Tyr Ala Ser Trp Ala Lys 1 5 10 15 Gly 9214PRTOryctolagus
cuniculus 92Asp Gly Tyr Asp Asp Tyr Gly Asp Phe Asp Arg Leu Asp Leu
1 5 10 93492DNAOryctolagus cuniculus 93atggacacga gggcccccac
tcagctgctg gggctcctgc tgctctggct cccaggtgcc 60acatttgcag ccgtgctgac
ccagacacca tcgcccgtgt ctgtacctgt gggaggcaca 120gtcaccatca
agtgccagtc cagtcagagt gtttataata atttcttatc gtggtatcag
180cagaaaccag ggcagcctcc caagctcctg atctaccagg catccaaact
ggcatctggg 240gtcccagata ggttcagcgg cagtggatct gggacacagt
tcactctcac catcagcggc 300gtgcagtgtg acgatgctgc cacttactac
tgtctaggcg gttatgatga tgatgctgat 360aatgctttcg gcggagggac
cgaggtggtg gtcaaacgta cggtagcggc cccatctgtc 420ttcatcttcc
cgccatctga tgagcagttg aaatctggaa ctgcctctgt tgtgtgcctg
480ctgaataact tc 49294511DNAOryctolagus cuniculus 94atggagactg
ggctgcgctg gcttctcctg gtcgctgtgc tcaaaggtgt ccagtgtcag 60tcggtggagg
agtccggggg tcgcctggtc acgcctggga cacccctgac gctcacctgc
120acagtctctg gaatcgacct cagtgactat gcaatgagct gggtccgcca
ggctccaggg 180aaggggctgg aatggatcgg aatcatttat gctggtagtg
gtagcacatg gtacgcgagc 240tgggcgaaag gccgattcac catctccaaa
acctcgacca cggtggatct gaaaatcacc 300agtccgacaa ccgaggacac
ggccacctat ttctgtgcca gagatggata cgatgactat 360ggtgatttcg
atcgattgga tctctggggc ccaggcaccc tcgtcaccgt ctcgagcgcc
420tccaccaagg gcccatcggt cttccccctg gcaccctcct ccaagagcac
ctctgggggc 480acagcggccc tgggctgcct ggtcaaggac t
5119536DNAOryctolagus cuniculus 95cagtccagtc agagtgttta taataatttc
ttatcg 369621DNAOryctolagus cuniculus 96caggcatcca aactggcatc t
219733DNAOryctolagus cuniculus 97ctaggcggtt atgatgatga tgctgataat
gct 339815DNAOryctolagus cuniculus 98gactatgcaa tgagc
159951DNAOryctolagus cuniculus 99atcatttatg ctggtagtgg
tagcacatgg
tacgcgagct gggcgaaagg c 5110042DNAOryctolagus cuniculus
100gatggatacg atgactatgg tgatttcgat cgattggatc tc
42101164PRTOryctolagus cuniculus 101Met Asp Thr Arg Ala Pro Thr Gln
Leu Leu Gly Leu Leu Leu Leu Trp 1 5 10 15 Leu Pro Gly Ala Arg Cys
Ala Tyr Asp Met Thr Gln Thr Pro Ala Ser 20 25 30 Val Ser Ala Ala
Val Gly Gly Thr Val Thr Ile Lys Cys Gln Ala Ser 35 40 45 Gln Ser
Ile Asn Asn Glu Leu Ser Trp Tyr Gln Gln Lys Ser Gly Gln 50 55 60
Arg Pro Lys Leu Leu Ile Tyr Arg Ala Ser Thr Leu Ala Ser Gly Val 65
70 75 80 Ser Ser Arg Phe Lys Gly Ser Gly Ser Gly Thr Glu Phe Thr
Leu Thr 85 90 95 Ile Ser Asp Leu Glu Cys Ala Asp Ala Ala Thr Tyr
Tyr Cys Gln Gln 100 105 110 Gly Tyr Ser Leu Arg Asn Ile Asp Asn Ala
Phe Gly Gly Gly Thr Glu 115 120 125 Val Val Val Lys Arg Thr Val Ala
Ala Pro Ser Val Phe Ile Phe Pro 130 135 140 Pro Ser Asp Glu Gln Leu
Lys Ser Gly Thr Ala Ser Val Val Cys Leu 145 150 155 160 Leu Asn Asn
Phe 102166PRTOryctolagus cuniculus 102Met Glu Thr Gly Leu Arg Trp
Leu Leu Leu Val Ala Val Leu Ser Gly 1 5 10 15 Val Gln Cys Gln Ser
Leu Glu Glu Ser Gly Gly Arg Leu Val Thr Pro 20 25 30 Gly Thr Pro
Leu Thr Leu Thr Cys Thr Ala Ser Gly Phe Ser Leu Ser 35 40 45 Asn
Tyr Tyr Met Thr Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu 50 55
60 Trp Ile Gly Met Ile Tyr Gly Ser Asp Glu Thr Ala Tyr Ala Asn Trp
65 70 75 80 Ala Ile Gly Arg Phe Thr Ile Ser Lys Thr Ser Thr Thr Val
Asp Leu 85 90 95 Lys Met Thr Ser Leu Thr Ala Ala Asp Thr Ala Thr
Tyr Phe Cys Ala 100 105 110 Arg Asp Asp Ser Ser Asp Trp Asp Ala Lys
Phe Asn Leu Trp Gly Gln 115 120 125 Gly Thr Leu Val Thr Val Ser Ser
Ala Ser Thr Lys Gly Pro Ser Val 130 135 140 Phe Pro Leu Ala Pro Ser
Ser Lys Ser Thr Ser Gly Gly Thr Ala Ala 145 150 155 160 Leu Gly Cys
Leu Val Lys 165 10311PRTOryctolagus cuniculus 103Gln Ala Ser Gln
Ser Ile Asn Asn Glu Leu Ser 1 5 10 1047PRTOryctolagus cuniculus
104Arg Ala Ser Thr Leu Ala Ser 1 5 10512PRTOryctolagus cuniculus
105Gln Gln Gly Tyr Ser Leu Arg Asn Ile Asp Asn Ala 1 5 10
1065PRTOryctolagus cuniculus 106Asn Tyr Tyr Met Thr 1 5
10716PRTOryctolagus cuniculus 107Met Ile Tyr Gly Ser Asp Glu Thr
Ala Tyr Ala Asn Trp Ala Ile Gly 1 5 10 15 10812PRTOryctolagus
cuniculus 108Asp Asp Ser Ser Asp Trp Asp Ala Lys Phe Asn Leu 1 5 10
109492DNAOryctolagus cuniculus 109atggacacga gggcccccac tcagctgctg
gggctcctgc tgctctggct cccaggtgcc 60agatgtgcct atgatatgac ccagactcca
gcctcggtgt ctgcagctgt gggaggcaca 120gtcaccatca aatgccaggc
cagtcagagc attaacaatg aattatcctg gtatcagcag 180aaatcagggc
agcgtcccaa gctcctgatc tatagggcat ccactctggc atctggggtc
240tcatcgcggt tcaaaggcag tggatctggg acagagttca ctctcaccat
cagcgacctg 300gagtgtgccg atgctgccac ttactactgt caacagggtt
atagtctgag gaatattgat 360aatgctttcg gcggagggac cgaggtggtg
gtcaaacgta cggtagcggc cccatctgtc 420ttcatcttcc cgccatctga
tgagcagttg aaatctggaa ctgcctctgt tgtgtgcctg 480ctgaataact tc
492110499DNAOryctolagus cuniculus 110atggagactg ggctgcgctg
gcttctcctg gtcgctgtgc tctcaggtgt ccagtgtcag 60tcgctggagg agtccggggg
tcgcctggtc acgcctggga cacccctgac actcacctgc 120acagcctctg
gattctccct cagtaactac tacatgacct gggtccgcca ggctccaggg
180aaggggctgg aatggatcgg aatgatttat ggtagtgatg aaacagccta
cgcgaactgg 240gcgataggcc gattcaccat ctccaaaacc tcgaccacgg
tggatctgaa aatgaccagt 300ctgacagccg cggacacggc cacctatttc
tgtgccagag atgatagtag tgactgggat 360gcaaaattta acttgtgggg
ccaagggacc ctcgtcaccg tctcgagcgc ctccaccaag 420ggcccatcgg
tcttccccct ggcaccctcc tccaagagca cctctggggg cacagcggcc
480ctgggctgcc tggtcaagg 49911133DNAOryctolagus cuniculus
111caggccagtc agagcattaa caatgaatta tcc 3311221DNAOryctolagus
cuniculus 112agggcatcca ctctggcatc t 2111336DNAOryctolagus
cuniculus 113caacagggtt atagtctgag gaatattgat aatgct
3611415DNAOryctolagus cuniculus 114aactactaca tgacc
1511548DNAOryctolagus cuniculus 115atgatttatg gtagtgatga aacagcctac
gcgaactggg cgataggc 4811636DNAOryctolagus cuniculus 116gatgatagta
gtgactggga tgcaaaattt aacttg 36117109PRTOryctolagus cuniculus
117Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly
1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Ser Leu Ser
Asn Tyr 20 25 30 Tyr Met Thr Trp Val Arg Gln Ala Pro Gly Lys Gly
Leu Glu Trp Val 35 40 45 Gly Met Ile Tyr Gly Ser Asp Glu Thr Ala
Tyr Ala Asn Trp Ala Ile 50 55 60 Gly Arg Phe Thr Ile Ser Arg Asp
Asn Ser Lys Asn Thr Leu Tyr Leu 65 70 75 80 Gln Met Asn Ser Leu Arg
Ala Glu Asp Thr Ala Val Tyr Tyr Cys Ala 85 90 95 Arg Asp Asp Ser
Ser Asp Trp Asp Ala Lys Phe Asn Leu 100 105 118109PRTOryctolagus
cuniculus 118Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln
Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe
Ser Leu Ser Asn Tyr 20 25 30 Tyr Met Thr Trp Val Arg Gln Ala Pro
Gly Lys Gly Leu Glu Trp Val 35 40 45 Gly Met Ile Tyr Gly Ser Asp
Glu Thr Ala Tyr Ala Asn Ser Ala Ile 50 55 60 Gly Arg Phe Thr Ile
Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr Leu 65 70 75 80 Gln Met Asn
Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys Ala 85 90 95 Arg
Asp Asp Ser Ser Asp Trp Asp Ala Lys Phe Asn Leu 100 105
119100PRTOryctolagus cuniculus 119Asp Ile Gln Met Thr Gln Ser Pro
Ser Thr Leu Ser Ala Ser Val Gly 1 5 10 15 Asp Arg Val Thr Ile Thr
Cys Gln Ala Ser Gln Ser Ile Asn Asn Glu 20 25 30 Leu Ser Trp Tyr
Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile 35 40 45 Tyr Arg
Ala Ser Thr Leu Ala Ser Gly Val Pro Ser Arg Phe Ser Gly 50 55 60
Ser Gly Ser Gly Thr Glu Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro 65
70 75 80 Asp Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Gly Tyr Ser Leu
Arg Asn 85 90 95 Ile Asp Asn Ala 100 12016PRTOryctolagus cuniculus
120Ile Ile Tyr Gly Ser Asp Glu Thr Ala Tyr Ala Thr Ser Ala Ile Gly
1 5 10 15 12116PRTOryctolagus cuniculus 121Met Ile Tyr Gly Ser Asp
Glu Thr Ala Tyr Ala Asn Ser Ala Ile Gly 1 5 10 15
122123PRTOryctolagus cuniculus 122Met Asp Thr Arg Ala Pro Thr Gln
Leu Leu Gly Leu Leu Leu Leu Trp 1 5 10 15 Leu Pro Gly Ala Thr Phe
Ala Ala Val Leu Thr Gln Thr Pro Ser Pro 20 25 30 Val Ser Ala Ala
Val Gly Gly Thr Val Thr Ile Ser Cys Gln Ser Ser 35 40 45 Gln Ser
Val Gly Asn Asn Gln Asp Leu Ser Trp Phe Gln Gln Arg Pro 50 55 60
Gly Gln Pro Pro Lys Leu Leu Ile Tyr Glu Ile Ser Lys Leu Glu Ser 65
70 75 80 Gly Val Pro Ser Arg Phe Ser Gly Ser Gly Ser Gly Thr His
Phe Thr 85 90 95 Leu Thr Ile Ser Gly Val Gln Cys Asp Asp Ala Ala
Thr Tyr Tyr Cys 100 105 110 Leu Gly Gly Tyr Asp Asp Asp Ala Asp Asn
Ala 115 120 123128PRTOryctolagus cuniculus 123Met Glu Thr Gly Leu
Arg Trp Leu Leu Leu Val Ala Val Leu Lys Gly 1 5 10 15 Val Gln Cys
His Ser Val Glu Glu Ser Gly Gly Arg Leu Val Thr Pro 20 25 30 Gly
Thr Pro Leu Thr Leu Thr Cys Thr Val Ser Gly Phe Ser Leu Ser 35 40
45 Ser Arg Thr Met Ser Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu
50 55 60 Trp Ile Gly Tyr Ile Trp Ser Gly Gly Ser Thr Tyr Tyr Ala
Thr Trp 65 70 75 80 Ala Lys Gly Arg Phe Thr Ile Ser Lys Thr Ser Thr
Thr Val Asp Leu 85 90 95 Lys Ile Thr Ser Pro Thr Thr Glu Asp Thr
Ala Thr Tyr Phe Cys Ala 100 105 110 Arg Leu Gly Asp Thr Gly Gly His
Ala Tyr Ala Thr Arg Leu Asn Leu 115 120 125 12413PRTOryctolagus
cuniculus 124Gln Ser Ser Gln Ser Val Gly Asn Asn Gln Asp Leu Ser 1
5 10 1257PRTOryctolagus cuniculus 125Glu Ile Ser Lys Leu Glu Ser 1
5 12611PRTOryctolagus cuniculus 126Leu Gly Gly Tyr Asp Asp Asp Ala
Asp Asn Ala 1 5 10 1275PRTOryctolagus cuniculus 127Ser Arg Thr Met
Ser 1 5 12816PRTOryctolagus cuniculus 128Tyr Ile Trp Ser Gly Gly
Ser Thr Tyr Tyr Ala Thr Trp Ala Lys Gly 1 5 10 15
12915PRTOryctolagus cuniculus 129Leu Gly Asp Thr Gly Gly His Ala
Tyr Ala Thr Arg Leu Asn Leu 1 5 10 15 130369DNAOryctolagus
cuniculus 130atggacacga gggcccccac tcagctgctg gggctcctgc tgctctggct
cccaggtgcc 60acatttgcag ccgtgctgac ccagacacca tcacccgtgt ctgcagctgt
gggaggcaca 120gtcaccatca gttgccagtc cagtcagagt gttggtaata
accaggactt atcctggttt 180cagcagagac cagggcagcc tcccaagctc
ctgatctacg aaatatccaa actggaatct 240ggggtcccat cgcggttcag
cggcagtgga tctgggacac acttcactct caccatcagc 300ggcgtacagt
gtgacgatgc tgccacttac tactgtctag gcggttatga tgatgatgct 360gataatgct
369131384DNAOryctolagus cuniculus 131atggagactg ggctgcgctg
gcttctcctg gtcgctgtgc tcaaaggtgt ccagtgtcac 60tcggtggagg agtccggggg
tcgcctggtc acgcctggga cacccctgac actcacctgc 120acagtctctg
gattctccct cagtagtcgt acaatgtcct gggtccgcca ggctccaggg
180aaggggctgg agtggatcgg atacatttgg agtggtggta gcacatacta
cgcgacctgg 240gcgaaaggcc gattcaccat ctccaaaacc tcgaccacgg
tggatctgaa aatcaccagt 300ccgacaaccg aggacacggc cacctatttc
tgtgccagat tgggcgatac tggtggtcac 360gcttatgcta ctcgcttaaa tctc
38413239DNAOryctolagus cuniculus 132cagtccagtc agagtgttgg
taataaccag gacttatcc 3913321DNAOryctolagus cuniculus 133gaaatatcca
aactggaatc t 2113433DNAOryctolagus cuniculus 134ctaggcggtt
atgatgatga tgctgataat gct 3313515DNAOryctolagus cuniculus
135agtcgtacaa tgtcc 1513648DNAOryctolagus cuniculus 136tacatttgga
gtggtggtag cacatactac gcgacctggg cgaaaggc 4813745DNAOryctolagus
cuniculus 137ttgggcgata ctggtggtca cgcttatgct actcgcttaa atctc
45138123PRTOryctolagus cuniculus 138Met Asp Thr Arg Ala Pro Thr Gln
Leu Leu Gly Leu Leu Leu Leu Trp 1 5 10 15 Leu Pro Gly Ala Thr Phe
Ala Ala Val Leu Thr Gln Thr Pro Ser Ser 20 25 30 Val Ser Ala Ala
Val Gly Gly Thr Val Ser Ile Ser Cys Gln Ser Ser 35 40 45 Gln Ser
Val Tyr Ser Asn Lys Tyr Leu Ala Trp Tyr Gln Gln Lys Pro 50 55 60
Gly Gln Pro Pro Lys Leu Leu Ile Tyr Trp Thr Ser Lys Leu Ala Ser 65
70 75 80 Gly Ala Pro Ser Arg Phe Ser Gly Ser Gly Ser Gly Thr Gln
Phe Thr 85 90 95 Leu Thr Ile Ser Gly Val Gln Cys Asp Asp Ala Ala
Thr Tyr Tyr Cys 100 105 110 Leu Gly Ala Tyr Asp Asp Asp Ala Asp Asn
Ala 115 120 139126PRTOryctolagus cuniculus 139Met Glu Thr Gly Leu
Arg Trp Leu Leu Leu Val Ala Val Leu Lys Gly 1 5 10 15 Val Gln Cys
Gln Ser Val Glu Glu Ser Gly Gly Arg Leu Val Lys Pro 20 25 30 Asp
Glu Thr Leu Thr Leu Thr Cys Thr Ala Ser Gly Phe Ser Leu Glu 35 40
45 Gly Gly Tyr Met Thr Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu
50 55 60 Trp Ile Gly Ile Ser Tyr Asp Ser Gly Ser Thr Tyr Tyr Ala
Ser Trp 65 70 75 80 Ala Lys Gly Arg Phe Thr Ile Ser Lys Thr Ser Ser
Thr Thr Val Asp 85 90 95 Leu Lys Met Thr Ser Leu Thr Thr Glu Asp
Thr Ala Thr Tyr Phe Cys 100 105 110 Val Arg Ser Leu Lys Tyr Pro Thr
Val Thr Ser Asp Asp Leu 115 120 125 14013PRTOryctolagus cuniculus
140Gln Ser Ser Gln Ser Val Tyr Ser Asn Lys Tyr Leu Ala 1 5 10
1417PRTOryctolagus cuniculus 141Trp Thr Ser Lys Leu Ala Ser 1 5
14211PRTOryctolagus cuniculus 142Leu Gly Ala Tyr Asp Asp Asp Ala
Asp Asn Ala 1 5 10 1435PRTOryctolagus cuniculus 143Gly Gly Tyr Met
Thr 1 5 14416PRTOryctolagus cuniculus 144Ile Ser Tyr Asp Ser Gly
Ser Thr Tyr Tyr Ala Ser Trp Ala Lys Gly 1 5 10 15
14512PRTOryctolagus cuniculus 145Ser Leu Lys Tyr Pro Thr Val Thr
Ser Asp Asp Leu 1 5 10 146369DNAOryctolagus cuniculus 146atggacacga
gggcccccac tcagctgctg gggctcctgc tgctctggct cccaggtgcc 60acatttgcag
ccgtgctgac ccagacacca tcgtccgtgt ctgcagctgt gggaggcaca
120gtcagcatca gttgccagtc cagtcagagt gtttatagta ataagtacct
agcctggtat 180cagcagaaac cagggcagcc tcccaagctc ctgatctact
ggacatccaa actggcatct 240ggggccccat cacggttcag cggcagtgga
tctgggacac aattcactct caccatcagc 300ggcgtgcagt gtgacgatgc
tgccacttac tactgtctag gcgcttatga tgatgatgct 360gataatgct
369147378DNAOryctolagus cuniculus 147atggagactg ggctgcgctg
gcttctcctg gtcgctgtgc tcaaaggtgt ccagtgtcag 60tcggtggaag agtccggggg
tcgcctggtc aagcctgacg aaaccctgac actcacctgc 120acagcctctg
gattctccct ggagggcggc tacatgacct gggtccgcca ggctccaggg
180aaggggctgg aatggatcgg aatcagttat gatagtggta gcacatacta
cgcgagctgg 240gcgaaaggcc gattcaccat ctccaagacc tcgtcgacca
cggtggatct gaaaatgacc 300agtctgacaa ccgaggacac ggccacctat
ttctgcgtca gatcactaaa atatcctact 360gttacttctg atgacttg
37814839DNAOryctolagus cuniculus 148cagtccagtc agagtgttta
tagtaataag tacctagcc 3914921DNAOryctolagus cuniculus 149tggacatcca
aactggcatc t 2115033DNAOryctolagus cuniculus 150ctaggcgctt
atgatgatga tgctgataat gct 3315115DNAOryctolagus cuniculus
151ggcggctaca tgacc 1515248DNAOryctolagus cuniculus 152atcagttatg
atagtggtag cacatactac gcgagctggg cgaaaggc 4815336DNAOryctolagus
cuniculus 153tcactaaaat atcctactgt tacttctgat gacttg
36154123PRTOryctolagus cuniculus 154Met Asp Thr Arg Ala Pro Thr Gln
Leu Leu Gly Leu Leu Leu Leu Trp 1 5 10 15 Leu Pro Gly Ala Thr Phe
Ala Ala Val Leu Thr Gln Thr Pro Ser Pro 20 25 30 Val Ser Ala Ala
Val Gly Gly Thr Val Thr Ile Ser Cys Gln Ser Ser 35 40 45 Gln Ser
Val Tyr Asn Asn Asn Asp Leu Ala Trp Tyr Gln Gln Lys Pro 50 55 60
Gly Gln Pro Pro Lys Leu Leu Ile Tyr Tyr Ala Ser Thr Leu Ala Ser 65
70 75 80 Gly Val Pro Ser Arg
Phe Lys Gly Ser Gly Ser Gly Thr Gln Phe Thr 85 90 95 Leu Thr Ile
Ser Gly Val Gln Cys Asp Asp Ala Ala Ala Tyr Tyr Cys 100 105 110 Leu
Gly Gly Tyr Asp Asp Asp Ala Asp Asn Ala 115 120
155129PRTOryctolagus cuniculus 155Met Glu Thr Gly Leu Arg Trp Leu
Leu Leu Val Ala Val Leu Lys Gly 1 5 10 15 Val Gln Cys Gln Ser Val
Glu Glu Ser Gly Gly Arg Leu Val Thr Pro 20 25 30 Gly Thr Pro Leu
Thr Leu Thr Cys Thr Val Ser Gly Leu Ser Leu Ser 35 40 45 Ser Asn
Thr Ile Asn Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu 50 55 60
Trp Ile Gly Tyr Ile Trp Ser Gly Gly Ser Thr Tyr Tyr Ala Ser Trp 65
70 75 80 Val Asn Gly Arg Phe Thr Ile Ser Lys Thr Ser Thr Thr Val
Asp Leu 85 90 95 Lys Ile Thr Ser Pro Thr Thr Glu Asp Thr Ala Thr
Tyr Phe Cys Ala 100 105 110 Arg Gly Gly Tyr Ala Ser Gly Gly Tyr Pro
Tyr Ala Thr Arg Leu Asp 115 120 125 Leu 15613PRTOryctolagus
cuniculus 156Gln Ser Ser Gln Ser Val Tyr Asn Asn Asn Asp Leu Ala 1
5 10 1577PRTOryctolagus cuniculus 157Tyr Ala Ser Thr Leu Ala Ser 1
5 15811PRTOryctolagus cuniculus 158Leu Gly Gly Tyr Asp Asp Asp Ala
Asp Asn Ala 1 5 10 1595PRTOryctolagus cuniculus 159Ser Asn Thr Ile
Asn 1 5 16016PRTOryctolagus cuniculus 160Tyr Ile Trp Ser Gly Gly
Ser Thr Tyr Tyr Ala Ser Trp Val Asn Gly 1 5 10 15
16116PRTOryctolagus cuniculus 161Gly Gly Tyr Ala Ser Gly Gly Tyr
Pro Tyr Ala Thr Arg Leu Asp Leu 1 5 10 15 162369DNAOryctolagus
cuniculus 162atggacacga gggcccccac tcagctgctg gggctcctgc tgctctggct
cccaggtgcc 60acatttgcag ccgtgctgac ccagacacca tcacccgtgt ctgcagctgt
gggaggcaca 120gtcaccatca gttgccagtc cagtcagagt gtttataata
ataacgactt agcctggtat 180cagcagaaac cagggcagcc tcctaaactc
ctgatctatt atgcatccac tctggcatct 240ggggtcccat cgcggttcaa
aggcagtgga tctgggacac agttcactct caccatcagc 300ggcgtgcagt
gtgacgatgc tgccgcttac tactgtctag gcggttatga tgatgatgct 360gataatgct
369163387DNAOryctolagus cuniculus 163atggagactg ggctgcgctg
gcttctcctg gtcgctgtgc tcaaaggtgt ccagtgtcag 60tcggtggagg agtccggggg
tcgcctggtc acgcctggga cacccctgac actcacctgc 120acagtatctg
gattatccct cagtagcaat acaataaact gggtccgcca ggctccaggg
180aaggggctgg agtggatcgg atacatttgg agtggtggta gtacatacta
cgcgagctgg 240gtgaatggtc gattcaccat ctccaaaacc tcgaccacgg
tggatctgaa aatcaccagt 300ccgacaaccg aggacacggc cacctatttc
tgtgccagag ggggttacgc tagtggtggt 360tatccttatg ccactcggtt ggatctc
38716439DNAOryctolagus cuniculus 164cagtccagtc agagtgttta
taataataac gacttagcc 3916521DNAOryctolagus cuniculus 165tatgcatcca
ctctggcatc t 2116633DNAOryctolagus cuniculus 166ctaggcggtt
atgatgatga tgctgataat gct 3316715DNAOryctolagus cuniculus
167agcaatacaa taaac 1516848DNAOryctolagus cuniculus 168tacatttgga
gtggtggtag tacatactac gcgagctggg tgaatggt 4816948DNAOryctolagus
cuniculus 169gggggttacg ctagtggtgg ttatccttat gccactcggt tggatctc
48170123PRTOryctolagus cuniculus 170Met Asp Thr Arg Ala Pro Thr Gln
Leu Leu Gly Leu Leu Leu Leu Trp 1 5 10 15 Leu Pro Gly Ala Thr Phe
Ala Ala Val Leu Thr Gln Thr Pro Ser Ser 20 25 30 Val Ser Ala Ala
Val Gly Gly Thr Val Thr Ile Asn Cys Gln Ser Ser 35 40 45 Gln Ser
Val Tyr Asn Asn Asp Tyr Leu Ser Trp Tyr Gln Gln Arg Pro 50 55 60
Gly Gln Arg Pro Lys Leu Leu Ile Tyr Gly Ala Ser Lys Leu Ala Ser 65
70 75 80 Gly Val Pro Ser Arg Phe Lys Gly Ser Gly Ser Gly Lys Gln
Phe Thr 85 90 95 Leu Thr Ile Ser Gly Val Gln Cys Asp Asp Ala Ala
Thr Tyr Tyr Cys 100 105 110 Leu Gly Asp Tyr Asp Asp Asp Ala Asp Asn
Thr 115 120 171123PRTOryctolagus cuniculus 171Met Glu Thr Gly Leu
Arg Trp Leu Leu Leu Val Ala Val Leu Lys Gly 1 5 10 15 Val Gln Cys
Gln Ser Leu Glu Glu Ser Gly Gly Arg Leu Val Thr Pro 20 25 30 Gly
Thr Pro Leu Thr Leu Thr Cys Thr Val Ser Gly Phe Thr Leu Ser 35 40
45 Thr Asn Tyr Tyr Leu Ser Trp Val Arg Gln Ala Pro Gly Lys Gly Leu
50 55 60 Glu Trp Ile Gly Ile Ile Tyr Pro Ser Gly Asn Thr Tyr Cys
Ala Lys 65 70 75 80 Trp Ala Lys Gly Arg Phe Thr Ile Ser Lys Thr Ser
Ser Thr Thr Val 85 90 95 Asp Leu Lys Met Thr Ser Pro Thr Thr Glu
Asp Thr Ala Thr Tyr Phe 100 105 110 Cys Ala Arg Asn Tyr Gly Gly Asp
Glu Ser Leu 115 120 17213PRTOryctolagus cuniculus 172Gln Ser Ser
Gln Ser Val Tyr Asn Asn Asp Tyr Leu Ser 1 5 10 1737PRTOryctolagus
cuniculus 173Gly Ala Ser Lys Leu Ala Ser 1 5 17411PRTOryctolagus
cuniculus 174Leu Gly Asp Tyr Asp Asp Asp Ala Asp Asn Thr 1 5 10
1756PRTOryctolagus cuniculus 175Thr Asn Tyr Tyr Leu Ser 1 5
17616PRTOryctolagus cuniculus 176Ile Ile Tyr Pro Ser Gly Asn Thr
Tyr Cys Ala Lys Trp Ala Lys Gly 1 5 10 15 1778PRTOryctolagus
cuniculus 177Asn Tyr Gly Gly Asp Glu Ser Leu 1 5
178369DNAOryctolagus cuniculus 178atggacacga gggcccccac tcagctgctg
gggctcctgc tgctctggct cccaggtgcc 60acatttgcag ccgtgctgac ccagacacca
tcctccgtgt ctgcagctgt gggaggcaca 120gtcaccatca attgccagtc
cagtcagagt gtttataata acgactactt atcctggtat 180caacagaggc
cagggcaacg tcccaagctc ctaatctatg gtgcttccaa actggcatct
240ggggtcccgt cacggttcaa aggcagtgga tctgggaaac agtttactct
caccatcagc 300ggcgtgcagt gtgacgatgc tgccacttac tactgtctgg
gcgattatga tgatgatgct 360gataatact 369179369DNAOryctolagus
cuniculus 179atggagactg ggctgcgctg gcttctcctg gtcgctgtgc tcaaaggtgt
ccagtgtcag 60tcgctggagg agtccggggg tcgcctggtc acgcctggga cacccctgac
actcacttgc 120acagtctctg gattcaccct cagtaccaac tactacctga
gctgggtccg ccaggctcca 180gggaaggggc tagaatggat cggaatcatt
tatcctagtg gtaacacata ttgcgcgaag 240tgggcgaaag gccgattcac
catctccaaa acctcgtcga ccacggtgga tctgaaaatg 300accagtccga
caaccgagga cacagccacg tatttctgtg ccagaaatta tggtggtgat 360gaaagtttg
36918039DNAOryctolagus cuniculus 180cagtccagtc agagtgttta
taataacgac tacttatcc 3918121DNAOryctolagus cuniculus 181ggtgcttcca
aactggcatc t 2118233DNAOryctolagus cuniculus 182ctgggcgatt
atgatgatga tgctgataat act 3318318DNAOryctolagus cuniculus
183accaactact acctgagc 1818448DNAOryctolagus cuniculus
184atcatttatc ctagtggtaa cacatattgc gcgaagtggg cgaaaggc
4818524DNAOryctolagus cuniculus 185aattatggtg gtgatgaaag tttg
24186119PRTOryctolagus cuniculus 186Met Asp Thr Arg Ala Pro Thr Gln
Leu Leu Gly Leu Leu Leu Leu Trp 1 5 10 15 Leu Pro Gly Ala Arg Cys
Asp Val Val Met Thr Gln Thr Pro Ala Ser 20 25 30 Val Glu Ala Ala
Val Gly Gly Thr Val Thr Ile Lys Cys Gln Ala Ser 35 40 45 Glu Thr
Ile Gly Asn Ala Leu Ala Trp Tyr Gln Gln Lys Ser Gly Gln 50 55 60
Pro Pro Lys Leu Leu Ile Tyr Lys Ala Ser Lys Leu Ala Ser Gly Val 65
70 75 80 Pro Ser Arg Phe Lys Gly Ser Gly Ser Gly Thr Glu Tyr Thr
Leu Thr 85 90 95 Ile Ser Asp Leu Glu Cys Ala Asp Ala Ala Thr Tyr
Tyr Cys Gln Trp 100 105 110 Cys Tyr Phe Gly Asp Ser Val 115
187128PRTOryctolagus cuniculus 187Met Glu Thr Gly Leu Arg Trp Leu
Leu Leu Val Thr Val Leu Lys Gly 1 5 10 15 Val Gln Cys Gln Glu Gln
Leu Val Glu Ser Gly Gly Gly Leu Val Gln 20 25 30 Pro Glu Gly Ser
Leu Thr Leu Thr Cys Thr Ala Ser Gly Phe Asp Phe 35 40 45 Ser Ser
Gly Tyr Tyr Met Cys Trp Val Arg Gln Ala Pro Gly Lys Gly 50 55 60
Leu Glu Trp Ile Ala Cys Ile Phe Thr Ile Thr Thr Asn Thr Tyr Tyr 65
70 75 80 Ala Ser Trp Ala Lys Gly Arg Phe Thr Ile Ser Lys Thr Ser
Ser Thr 85 90 95 Thr Val Thr Leu Gln Met Thr Ser Leu Thr Ala Ala
Asp Thr Ala Thr 100 105 110 Tyr Leu Cys Ala Arg Gly Ile Tyr Ser Asp
Asn Asn Tyr Tyr Ala Leu 115 120 125 18811PRTOryctolagus cuniculus
188Gln Ala Ser Glu Thr Ile Gly Asn Ala Leu Ala 1 5 10
1897PRTOryctolagus cuniculus 189Lys Ala Ser Lys Leu Ala Ser 1 5
1909PRTOryctolagus cuniculus 190Gln Trp Cys Tyr Phe Gly Asp Ser Val
1 5 1916PRTOryctolagus cuniculus 191Ser Gly Tyr Tyr Met Cys 1 5
19217PRTOryctolagus cuniculus 192Cys Ile Phe Thr Ile Thr Thr Asn
Thr Tyr Tyr Ala Ser Trp Ala Lys 1 5 10 15 Gly 19311PRTOryctolagus
cuniculus 193Gly Ile Tyr Ser Asp Asn Asn Tyr Tyr Ala Leu 1 5 10
194357DNAOryctolagus cuniculus 194atggacacga gggcccccac tcagctgctg
gggctcctgc tgctctggct cccaggtgcc 60agatgtgatg ttgtgatgac ccagactcca
gcctccgtgg aggcagctgt gggaggcaca 120gtcaccatca agtgccaggc
cagtgagacc attggcaatg cattagcctg gtatcagcag 180aaatcagggc
agcctcccaa gctcctgatc tacaaggcat ccaaactggc atctggggtc
240ccatcgcggt tcaaaggcag tggatctggg acagagtaca ctctcaccat
cagcgacctg 300gagtgtgccg atgctgccac ttactactgt caatggtgtt
attttggtga tagtgtt 357195384DNAOryctolagus cuniculus 195atggagactg
ggctgcgctg gcttctcctg gtcactgtgc tcaaaggtgt ccagtgtcag 60gagcagctgg
tggagtccgg gggaggcctg gtccagcctg agggatccct gacactcacc
120tgcacagcct ctggattcga cttcagtagc ggctactaca tgtgctgggt
ccgccaggct 180ccagggaagg ggctggagtg gatcgcgtgt attttcacta
ttactactaa cacttactac 240gcgagctggg cgaaaggccg attcaccatc
tccaagacct cgtcgaccac ggtgactctg 300caaatgacca gtctgacagc
cgcggacacg gccacctatc tctgtgcgag agggatttat 360tctgataata
attattatgc cttg 38419633DNAOryctolagus cuniculus 196caggccagtg
agaccattgg caatgcatta gcc 3319721DNAOryctolagus cuniculus
197aaggcatcca aactggcatc t 2119827DNAOryctolagus cuniculus
198caatggtgtt attttggtga tagtgtt 2719918DNAOryctolagus cuniculus
199agcggctact acatgtgc 1820051DNAOryctolagus cuniculus
200tgtattttca ctattactac taacacttac tacgcgagct gggcgaaagg c
5120133DNAOryctolagus cuniculus 201gggatttatt ctgataataa ttattatgcc
ttg 33202119PRTOryctolagus cuniculus 202Met Asp Thr Arg Ala Pro Thr
Gln Leu Leu Gly Leu Leu Leu Leu Trp 1 5 10 15 Leu Pro Gly Ala Arg
Cys Asp Val Val Met Thr Gln Thr Pro Ala Ser 20 25 30 Val Glu Ala
Ala Val Gly Gly Thr Val Thr Ile Lys Cys Gln Ala Ser 35 40 45 Glu
Ser Ile Gly Asn Ala Leu Ala Trp Tyr Gln Gln Lys Pro Gly Gln 50 55
60 Pro Pro Lys Leu Leu Ile Tyr Lys Ala Ser Thr Leu Ala Ser Gly Val
65 70 75 80 Pro Ser Arg Phe Ser Gly Ser Gly Ser Gly Thr Glu Phe Thr
Leu Thr 85 90 95 Ile Ser Gly Val Gln Cys Ala Asp Ala Ala Ala Tyr
Tyr Cys Gln Trp 100 105 110 Cys Tyr Phe Gly Asp Ser Val 115
203128PRTOryctolagus cuniculus 203Met Glu Thr Gly Leu Arg Trp Leu
Leu Leu Val Ala Val Leu Lys Gly 1 5 10 15 Val Gln Cys Gln Gln Gln
Leu Val Glu Ser Gly Gly Gly Leu Val Lys 20 25 30 Pro Gly Ala Ser
Leu Thr Leu Thr Cys Lys Ala Ser Gly Phe Ser Phe 35 40 45 Ser Ser
Gly Tyr Tyr Met Cys Trp Val Arg Gln Ala Pro Gly Lys Gly 50 55 60
Leu Glu Ser Ile Ala Cys Ile Phe Thr Ile Thr Asp Asn Thr Tyr Tyr 65
70 75 80 Ala Asn Trp Ala Lys Gly Arg Phe Thr Ile Ser Lys Pro Ser
Ser Pro 85 90 95 Thr Val Thr Leu Gln Met Thr Ser Leu Thr Ala Ala
Asp Thr Ala Thr 100 105 110 Tyr Phe Cys Ala Arg Gly Ile Tyr Ser Thr
Asp Asn Tyr Tyr Ala Leu 115 120 125 20411PRTOryctolagus cuniculus
204Gln Ala Ser Glu Ser Ile Gly Asn Ala Leu Ala 1 5 10
2057PRTOryctolagus cuniculus 205Lys Ala Ser Thr Leu Ala Ser 1 5
2069PRTOryctolagus cuniculus 206Gln Trp Cys Tyr Phe Gly Asp Ser Val
1 5 2076PRTOryctolagus cuniculus 207Ser Gly Tyr Tyr Met Cys 1 5
20817PRTOryctolagus cuniculus 208Cys Ile Phe Thr Ile Thr Asp Asn
Thr Tyr Tyr Ala Asn Trp Ala Lys 1 5 10 15 Gly 20911PRTOryctolagus
cuniculus 209Gly Ile Tyr Ser Thr Asp Asn Tyr Tyr Ala Leu 1 5 10
210357DNAOryctolagus cuniculus 210atggacacga gggcccccac tcagctgctg
gggctcctgc tgctctggct cccaggtgcc 60agatgtgatg ttgtgatgac ccagactcca
gcctccgtgg aggcagctgt gggaggcaca 120gtcaccatca agtgccaggc
cagtgagagc attggcaatg cattagcctg gtatcagcag 180aaaccagggc
agcctcccaa gctcctgatc tacaaggcat ccactctggc atctggggtc
240ccatcgcggt tcagcggcag tggatctggg acagagttca ctctcaccat
cagcggcgtg 300cagtgtgccg atgctgccgc ttactactgt caatggtgtt
attttggtga tagtgtt 357211384DNAOryctolagus cuniculus 211atggagactg
ggctgcgctg gcttctcctg gtcgctgtgc tcaaaggtgt ccagtgtcag 60cagcagctgg
tggagtccgg gggaggcctg gtcaagccgg gggcatccct gacactcacc
120tgcaaagcct ctggattctc cttcagtagc ggctactaca tgtgctgggt
ccgccaggct 180ccagggaagg ggctggagtc gatcgcatgc atttttacta
ttactgataa cacttactac 240gcgaactggg cgaaaggccg attcaccatc
tccaagccct cgtcgcccac ggtgactctg 300caaatgacca gtctgacagc
cgcggacacg gccacctatt tctgtgcgag ggggatttat 360tctactgata
attattatgc cttg 38421233DNAOryctolagus cuniculus 212caggccagtg
agagcattgg caatgcatta gcc 3321321DNAOryctolagus cuniculus
213aaggcatcca ctctggcatc t 2121427DNAOryctolagus cuniculus
214caatggtgtt attttggtga tagtgtt 2721518DNAOryctolagus cuniculus
215agcggctact acatgtgc 1821651DNAOryctolagus cuniculus
216tgcattttta ctattactga taacacttac tacgcgaact gggcgaaagg c
5121733DNAOryctolagus cuniculus 217gggatttatt ctactgataa ttattatgcc
ttg 33218123PRTOryctolagus cuniculus 218Met Asp Thr Arg Ala Pro Thr
Gln Leu Leu Gly Leu Leu Leu Leu Trp 1 5 10 15 Leu Pro Gly Ala Arg
Cys Asp Val Val Met Thr Gln Thr Pro Ala Ser 20 25 30 Val Glu Ala
Ala Val Gly Gly Thr Val Thr Ile Lys Cys Gln Ala Ser 35 40 45 Gln
Ser Val Ser Ser Tyr Leu Asn Trp Tyr Gln Gln Lys Pro Gly Gln 50 55
60 Pro Pro Lys Leu Leu Ile Tyr Arg Ala Ser Thr Leu Glu Ser Gly Val
65 70 75 80 Pro Ser Arg Phe Lys Gly Ser Gly Ser Gly Thr Glu Phe Thr
Leu Thr 85 90 95 Ile Ser Asp Leu Glu Cys Ala Asp Ala Ala Thr Tyr
Tyr Cys Gln Cys 100 105 110 Thr Tyr Gly Thr Ser Ser Ser Tyr Gly Ala
Ala 115 120 219133PRTOryctolagus cuniculus 219Met Glu Thr Gly Leu
Arg Trp Leu Leu Leu Val Ala Val Leu Lys Gly 1 5
10 15 Val Gln Cys Gln Ser Val Glu Glu Ser Gly Gly Arg Leu Val Thr
Pro 20 25 30 Gly Thr Pro Leu Thr Leu Thr Cys Thr Val Ser Gly Ile
Ser Leu Ser 35 40 45 Ser Asn Ala Ile Ser Trp Val Arg Gln Ala Pro
Gly Lys Gly Leu Glu 50 55 60 Trp Ile Gly Ile Ile Ser Tyr Ser Gly
Thr Thr Tyr Tyr Ala Ser Trp 65 70 75 80 Ala Lys Gly Arg Phe Thr Ile
Ser Lys Thr Ser Ser Thr Thr Val Asp 85 90 95 Leu Lys Ile Thr Ser
Pro Thr Thr Glu Asp Thr Ala Thr Tyr Phe Cys 100 105 110 Ala Arg Asp
Asp Pro Thr Thr Val Met Val Met Leu Ile Pro Phe Gly 115 120 125 Ala
Gly Met Asp Leu 130 22011PRTOryctolagus cuniculus 220Gln Ala Ser
Gln Ser Val Ser Ser Tyr Leu Asn 1 5 10 2217PRTOryctolagus cuniculus
221Arg Ala Ser Thr Leu Glu Ser 1 5 22213PRTOryctolagus cuniculus
222Gln Cys Thr Tyr Gly Thr Ser Ser Ser Tyr Gly Ala Ala 1 5 10
2235PRTOryctolagus cuniculus 223Ser Asn Ala Ile Ser 1 5
22416PRTOryctolagus cuniculus 224Ile Ile Ser Tyr Ser Gly Thr Thr
Tyr Tyr Ala Ser Trp Ala Lys Gly 1 5 10 15 22519PRTOryctolagus
cuniculus 225Asp Asp Pro Thr Thr Val Met Val Met Leu Ile Pro Phe
Gly Ala Gly 1 5 10 15 Met Asp Leu 226369DNAOryctolagus cuniculus
226atggacacga gggcccccac tcagctgctg gggctcctgc tgctctggct
cccaggtgcc 60agatgtgatg ttgtgatgac ccagactcca gcctccgtgg aggcagctgt
gggaggcaca 120gtcaccatca agtgccaggc cagtcagagc gttagtagct
acttaaactg gtatcagcag 180aaaccagggc agcctcccaa gctcctgatc
tacagggcat ccactctgga atctggggtc 240ccatcgcggt tcaaaggcag
tggatctggg acagagttca ctctcaccat cagcgacctg 300gagtgtgccg
atgctgccac ttactactgt caatgtactt atggtactag tagtagttat 360ggtgctgct
369227399DNAOryctolagus cuniculus 227atggagactg ggctgcgctg
gcttctcctg gtcgctgtgc tcaaaggtgt ccagtgtcag 60tcggtggagg agtccggggg
tcgcctggtc acgcctggga cacccctgac actcacctgc 120accgtctctg
gtatctccct cagtagcaat gcaataagct gggtccgcca ggctccaggg
180aaggggctgg aatggatcgg aatcattagt tatagtggta ccacatacta
cgcgagctgg 240gcgaaaggcc gattcaccat ctccaaaacc tcgtcgacca
cggtggatct gaaaatcact 300agtccgacaa ccgaggacac ggccacctac
ttctgtgcca gagatgaccc tacgacagtt 360atggttatgt tgataccttt
tggagccggc atggacctc 39922833DNAOryctolagus cuniculus 228caggccagtc
agagcgttag tagctactta aac 3322921DNAOryctolagus cuniculus
229agggcatcca ctctggaatc t 2123039DNAOryctolagus cuniculus
230caatgtactt atggtactag tagtagttat ggtgctgct 3923115DNAOryctolagus
cuniculus 231agcaatgcaa taagc 1523248DNAOryctolagus cuniculus
232atcattagtt atagtggtac cacatactac gcgagctggg cgaaaggc
4823357DNAOryctolagus cuniculus 233gatgacccta cgacagttat ggttatgttg
ataccttttg gagccggcat ggacctc 57234125PRTOryctolagus cuniculus
234Met Asp Thr Arg Ala Pro Thr Gln Leu Leu Gly Leu Leu Leu Leu Trp
1 5 10 15 Leu Pro Gly Ala Thr Phe Ala Gln Val Leu Thr Gln Thr Ala
Ser Pro 20 25 30 Val Ser Ala Ala Val Gly Gly Thr Val Thr Ile Asn
Cys Gln Ala Ser 35 40 45 Gln Ser Val Tyr Lys Asn Asn Tyr Leu Ser
Trp Tyr Gln Gln Lys Pro 50 55 60 Gly Gln Pro Pro Lys Gly Leu Ile
Tyr Ser Ala Ser Thr Leu Asp Ser 65 70 75 80 Gly Val Pro Leu Arg Phe
Ser Gly Ser Gly Ser Gly Thr Gln Phe Thr 85 90 95 Leu Thr Ile Ser
Asp Val Gln Cys Asp Asp Ala Ala Thr Tyr Tyr Cys 100 105 110 Leu Gly
Ser Tyr Asp Cys Ser Ser Gly Asp Cys Tyr Ala 115 120 125
235119PRTOryctolagus cuniculus 235Met Glu Thr Gly Leu Arg Trp Leu
Leu Leu Val Ala Val Leu Lys Gly 1 5 10 15 Val Gln Cys Gln Ser Leu
Glu Glu Ser Gly Gly Asp Leu Val Lys Pro 20 25 30 Glu Gly Ser Leu
Thr Leu Thr Cys Thr Ala Ser Gly Phe Ser Phe Ser 35 40 45 Ser Tyr
Trp Met Cys Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu 50 55 60
Trp Ile Ala Cys Ile Val Thr Gly Asn Gly Asn Thr Tyr Tyr Ala Asn 65
70 75 80 Trp Ala Lys Gly Arg Phe Thr Ile Ser Lys Thr Ser Ser Thr
Thr Val 85 90 95 Thr Leu Gln Met Thr Ser Leu Thr Ala Ala Asp Thr
Ala Thr Tyr Phe 100 105 110 Cys Ala Lys Ala Tyr Asp Leu 115
23613PRTOryctolagus cuniculus 236Gln Ala Ser Gln Ser Val Tyr Lys
Asn Asn Tyr Leu Ser 1 5 10 2377PRTOryctolagus cuniculus 237Ser Ala
Ser Thr Leu Asp Ser 1 5 23813PRTOryctolagus cuniculus 238Leu Gly
Ser Tyr Asp Cys Ser Ser Gly Asp Cys Tyr Ala 1 5 10
2395PRTOryctolagus cuniculus 239Ser Tyr Trp Met Cys 1 5
24017PRTOryctolagus cuniculus 240Cys Ile Val Thr Gly Asn Gly Asn
Thr Tyr Tyr Ala Asn Trp Ala Lys 1 5 10 15 Gly 2414PRTOryctolagus
cuniculus 241Ala Tyr Asp Leu 1 242375DNAOryctolagus cuniculus
242atggacacga gggcccccac tcagctgctg gggctcctgc tgctctggct
cccaggtgcc 60acatttgccc aagtgctgac ccagactgca tcgcccgtgt ctgcagctgt
gggaggcaca 120gtcaccatca actgccaggc cagtcagagt gtttataaga
acaactactt atcctggtat 180cagcagaaac cagggcagcc tcccaaaggc
ctgatctatt ctgcatcgac tctagattct 240ggggtcccat tgcggttcag
cggcagtgga tctgggacac agttcactct caccatcagc 300gacgtgcagt
gtgacgatgc tgccacttac tactgtctag gcagttatga ttgtagtagt
360ggtgattgtt atgct 375243357DNAOryctolagus cuniculus 243atggagactg
ggctgcgctg gcttctcctg gtcgctgtgc tcaaaggtgt ccagtgtcag 60tcgttggagg
agtccggggg agacctggtc aagcctgagg gatccctgac actcacctgc
120acagcctctg gattctcctt cagtagctac tggatgtgct gggtccgcca
ggctccaggg 180aaggggctgg agtggatcgc atgcattgtt actggtaatg
gtaacactta ctacgcgaac 240tgggcgaaag gccgattcac catctccaaa
acctcgtcga ccacggtgac tctgcaaatg 300accagtctga cagccgcgga
cacggccacc tatttttgtg cgaaagccta tgacttg 35724439DNAOryctolagus
cuniculus 244caggccagtc agagtgttta taagaacaac tacttatcc
3924521DNAOryctolagus cuniculus 245tctgcatcga ctctagattc t
2124639DNAOryctolagus cuniculus 246ctaggcagtt atgattgtag tagtggtgat
tgttatgct 3924715DNAOryctolagus cuniculus 247agctactgga tgtgc
1524851DNAOryctolagus cuniculus 248tgcattgtta ctggtaatgg taacacttac
tacgcgaact gggcgaaagg c 5124912DNAOryctolagus cuniculus
249gcctatgact tg 12250123PRTOryctolagus cuniculus 250Met Asp Thr
Arg Ala Pro Thr Gln Leu Leu Gly Leu Leu Leu Leu Trp 1 5 10 15 Leu
Pro Gly Ser Thr Phe Ala Ala Val Leu Thr Gln Thr Pro Ser Pro 20 25
30 Val Ser Ala Ala Val Gly Gly Thr Val Ser Ile Ser Cys Gln Ala Ser
35 40 45 Gln Ser Val Tyr Asp Asn Asn Tyr Leu Ser Trp Tyr Gln Gln
Lys Pro 50 55 60 Gly Gln Pro Pro Lys Leu Leu Ile Tyr Gly Ala Ser
Thr Leu Ala Ser 65 70 75 80 Gly Val Pro Ser Arg Phe Lys Gly Thr Gly
Ser Gly Thr Gln Phe Thr 85 90 95 Leu Thr Ile Thr Asp Val Gln Cys
Asp Asp Ala Ala Thr Tyr Tyr Cys 100 105 110 Ala Gly Val Phe Asn Asp
Asp Ser Asp Asp Ala 115 120 251125PRTOryctolagus cuniculus 251Met
Glu Thr Gly Leu Arg Trp Leu Leu Leu Val Ala Val Pro Lys Gly 1 5 10
15 Val Gln Cys Gln Ser Leu Glu Glu Ser Gly Gly Arg Leu Val Thr Pro
20 25 30 Gly Thr Pro Leu Thr Leu Thr Cys Thr Leu Ser Gly Phe Ser
Leu Ser 35 40 45 Ala Tyr Tyr Met Ser Trp Val Arg Gln Ala Pro Gly
Lys Gly Leu Glu 50 55 60 Trp Ile Gly Phe Ile Thr Leu Ser Asp His
Ile Ser Tyr Ala Arg Trp 65 70 75 80 Ala Lys Gly Arg Phe Thr Ile Ser
Lys Thr Ser Thr Thr Val Asp Leu 85 90 95 Lys Met Thr Ser Pro Thr
Thr Glu Asp Thr Ala Thr Tyr Phe Cys Ala 100 105 110 Arg Ser Arg Gly
Trp Gly Ala Met Gly Arg Leu Asp Leu 115 120 125 25213PRTOryctolagus
cuniculus 252Gln Ala Ser Gln Ser Val Tyr Asp Asn Asn Tyr Leu Ser 1
5 10 2537PRTOryctolagus cuniculus 253Gly Ala Ser Thr Leu Ala Ser 1
5 25411PRTOryctolagus cuniculus 254Ala Gly Val Phe Asn Asp Asp Ser
Asp Asp Ala 1 5 10 2555PRTOryctolagus cuniculus 255Ala Tyr Tyr Met
Ser 1 5 25616PRTOryctolagus cuniculus 256Phe Ile Thr Leu Ser Asp
His Ile Ser Tyr Ala Arg Trp Ala Lys Gly 1 5 10 15
25712PRTOryctolagus cuniculus 257Ser Arg Gly Trp Gly Ala Met Gly
Arg Leu Asp Leu 1 5 10 258369DNAOryctolagus cuniculus 258atggacacga
gggcccccac tcagctgctg gggctcctgc tgctctggct cccaggttcc 60acatttgccg
ccgtgctgac ccagactcca tctcccgtgt ctgcagctgt gggaggcaca
120gtcagcatca gttgccaggc cagtcagagt gtttatgaca acaactattt
atcctggtat 180cagcagaaac caggacagcc tcccaagctc ctgatctatg
gtgcatccac tctggcatct 240ggggtcccat cgcggttcaa aggcacggga
tctgggacac agttcactct caccatcaca 300gacgtgcagt gtgacgatgc
tgccacttac tattgtgcag gcgtttttaa tgatgatagt 360gatgatgcc
369259375DNAOryctolagus cuniculus 259atggagactg ggctgcgctg
gcttctcctg gtcgctgtgc ccaaaggtgt ccagtgtcag 60tcgctggagg agtccggggg
tcgcctggtc acgcctggga cacccctgac actcacctgc 120acactctctg
gattctccct cagtgcatac tatatgagct gggtccgcca ggctccaggg
180aaggggctgg aatggatcgg attcattact ctgagtgatc atatatctta
cgcgaggtgg 240gcgaaaggcc gattcaccat ctccaaaacc tcgaccacgg
tggatctgaa aatgaccagt 300ccgacaaccg aggacacggc cacctatttc
tgtgccagga gtcgtggctg gggtgcaatg 360ggtcggttgg atctc
37526039DNAOryctolagus cuniculus 260caggccagtc agagtgttta
tgacaacaac tatttatcc 3926121DNAOryctolagus cuniculus 261ggtgcatcca
ctctggcatc t 2126233DNAOryctolagus cuniculus 262gcaggcgttt
ttaatgatga tagtgatgat gcc 3326315DNAOryctolagus cuniculus
263gcatactata tgagc 1526448DNAOryctolagus cuniculus 264ttcattactc
tgagtgatca tatatcttac gcgaggtggg cgaaaggc 4826536DNAOryctolagus
cuniculus 265agtcgtggct ggggtgcaat gggtcggttg gatctc
36266123PRTOryctolagus cuniculus 266Met Asp Thr Arg Ala Pro Thr Gln
Leu Leu Gly Leu Leu Leu Leu Trp 1 5 10 15 Leu Pro Gly Ala Thr Phe
Ala Ala Val Leu Thr Gln Thr Pro Ser Pro 20 25 30 Val Ser Ala Ala
Val Gly Gly Thr Val Thr Ile Ser Cys Gln Ala Ser 35 40 45 Gln Ser
Val Tyr Asn Asn Lys Asn Leu Ala Trp Tyr Gln Gln Lys Ser 50 55 60
Gly Gln Pro Pro Lys Leu Leu Ile Tyr Trp Ala Ser Thr Leu Ala Ser 65
70 75 80 Gly Val Ser Ser Arg Phe Ser Gly Ser Gly Ser Gly Thr Gln
Phe Thr 85 90 95 Leu Thr Val Ser Gly Val Gln Cys Asp Asp Ala Ala
Thr Tyr Tyr Cys 100 105 110 Leu Gly Val Phe Asp Asp Asp Ala Asp Asn
Ala 115 120 267121PRTOryctolagus cuniculus 267Met Glu Thr Gly Leu
Arg Trp Leu Leu Leu Val Ala Val Leu Lys Gly 1 5 10 15 Val Gln Cys
Gln Ser Val Glu Glu Ser Gly Gly Arg Leu Val Thr Pro 20 25 30 Gly
Thr Pro Leu Thr Leu Thr Cys Thr Ala Ser Gly Phe Ser Leu Ser 35 40
45 Ser Tyr Ser Met Thr Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu
50 55 60 Tyr Ile Gly Val Ile Gly Thr Ser Gly Ser Thr Tyr Tyr Ala
Thr Trp 65 70 75 80 Ala Lys Gly Arg Phe Thr Ile Ser Arg Thr Ser Thr
Thr Val Ala Leu 85 90 95 Lys Ile Thr Ser Pro Thr Thr Glu Asp Thr
Ala Thr Tyr Phe Cys Val 100 105 110 Arg Ser Leu Ser Ser Ile Thr Phe
Leu 115 120 26813PRTOryctolagus cuniculus 268Gln Ala Ser Gln Ser
Val Tyr Asn Asn Lys Asn Leu Ala 1 5 10 2697PRTOryctolagus cuniculus
269Trp Ala Ser Thr Leu Ala Ser 1 5 27011PRTOryctolagus cuniculus
270Leu Gly Val Phe Asp Asp Asp Ala Asp Asn Ala 1 5 10
2715PRTOryctolagus cuniculus 271Ser Tyr Ser Met Thr 1 5
27216PRTOryctolagus cuniculus 272Val Ile Gly Thr Ser Gly Ser Thr
Tyr Tyr Ala Thr Trp Ala Lys Gly 1 5 10 15 2738PRTOryctolagus
cuniculus 273Ser Leu Ser Ser Ile Thr Phe Leu 1 5
274369DNAOryctolagus cuniculus 274atggacacga gggcccccac tcagctgctg
gggctcctgc tgctctggct cccaggtgcc 60acattcgcag ccgtgctgac ccagacacca
tcgcccgtgt ctgcggctgt gggaggcaca 120gtcaccatca gttgccaggc
cagtcagagt gtttataaca acaaaaattt agcctggtat 180cagcagaaat
cagggcagcc tcccaagctc ctgatctact gggcatccac tctggcatct
240ggggtctcat cgcggttcag cggcagtgga tctgggacac agttcactct
caccgtcagc 300ggcgtgcagt gtgacgatgc tgccacttac tactgtctag
gcgtttttga tgatgatgct 360gataatgct 369275363DNAOryctolagus
cuniculus 275atggagactg ggctgcgctg gcttctcctg gtcgctgtgc tcaaaggtgt
ccaatgtcag 60tcggtggagg agtccggggg tcgcctggtc acgcctggga cacccctgac
actcacctgc 120acagcctctg gattctccct cagtagctac tccatgacct
gggtccgcca ggctccaggg 180aaggggctgg aatatatcgg agtcattggt
actagtggta gcacatacta cgcgacctgg 240gcgaaaggcc gattcaccat
ctccagaacc tcgaccacgg tggctctgaa aatcaccagt 300ccgacaaccg
aggacacggc cacctatttc tgtgtcagga gtctttcttc tattactttc 360ttg
36327639DNAOryctolagus cuniculus 276caggccagtc agagtgttta
taacaacaaa aatttagcc 3927721DNAOryctolagus cuniculus 277tgggcatcca
ctctggcatc t 2127833DNAOryctolagus cuniculus 278ctaggcgttt
ttgatgatga tgctgataat gct 3327915DNAOryctolagus cuniculus
279agctactcca tgacc 1528048DNAOryctolagus cuniculus 280gtcattggta
ctagtggtag cacatactac gcgacctggg cgaaaggc 4828124DNAOryctolagus
cuniculus 281agtctttctt ctattacttt cttg 24282120PRTOryctolagus
cuniculus 282Met Asp Thr Arg Ala Pro Thr Gln Leu Leu Gly Leu Leu
Leu Leu Trp 1 5 10 15 Leu Pro Gly Ala Arg Cys Ala Phe Glu Leu Thr
Gln Thr Pro Ala Ser 20 25 30 Val Glu Ala Ala Val Gly Gly Thr Val
Thr Ile Asn Cys Gln Ala Ser 35 40 45 Gln Asn Ile Tyr Arg Tyr Leu
Ala Trp Tyr Gln Gln Lys Pro Gly Gln 50 55 60 Pro Pro Lys Phe Leu
Ile Tyr Leu Ala Ser Thr Leu Ala Ser Gly Val 65 70 75 80 Pro Ser Arg
Phe Lys Gly Ser Gly Ser Gly Thr Glu Phe Thr Leu Thr 85 90 95 Ile
Ser Asp Leu Glu Cys Ala Asp Ala Ala Thr Tyr Tyr Cys Gln Ser 100 105
110 Tyr Tyr Ser Ser Asn Ser Val Ala 115 120 283128PRTOryctolagus
cuniculus 283Met Glu Thr Gly Leu Arg Trp Leu Leu Leu Val Ala Val
Leu Lys Gly 1 5 10 15 Val Gln Cys Gln Glu Gln Leu Val Glu Ser Gly
Gly Asp Leu Val Gln 20 25 30 Pro Glu Gly Ser Leu Thr Leu Thr Cys
Thr Ala Ser Glu Leu Asp Phe 35 40 45 Ser Ser Gly Tyr Trp Ile Cys
Trp Val Arg Gln Val Pro Gly Lys Gly 50
55 60 Leu Glu Trp Ile Gly Cys Ile Tyr Thr Gly Ser Ser Gly Ser Thr
Phe 65 70 75 80 Tyr Ala Ser Trp Ala Lys Gly Arg Phe Thr Ile Ser Lys
Thr Ser Ser 85 90 95 Thr Thr Val Thr Leu Gln Met Thr Ser Leu Thr
Ala Ala Asp Thr Ala 100 105 110 Thr Tyr Phe Cys Ala Arg Gly Tyr Ser
Gly Phe Gly Tyr Phe Lys Leu 115 120 125 28411PRTOryctolagus
cuniculus 284Gln Ala Ser Gln Asn Ile Tyr Arg Tyr Leu Ala 1 5 10
2857PRTOryctolagus cuniculus 285Leu Ala Ser Thr Leu Ala Ser 1 5
28610PRTOryctolagus cuniculus 286Gln Ser Tyr Tyr Ser Ser Asn Ser
Val Ala 1 5 10 2876PRTOryctolagus cuniculus 287Ser Gly Tyr Trp Ile
Cys 1 5 28818PRTOryctolagus cuniculus 288Cys Ile Tyr Thr Gly Ser
Ser Gly Ser Thr Phe Tyr Ala Ser Trp Ala 1 5 10 15 Lys Gly
28910PRTOryctolagus cuniculus 289Gly Tyr Ser Gly Phe Gly Tyr Phe
Lys Leu 1 5 10 290360DNAOryctolagus cuniculus 290atggacacga
gggcccccac tcagctgctg gggctcctgc tgctctggct cccaggtgcc 60agatgtgcat
tcgaattgac ccagactcca gcctccgtgg aggcagctgt gggaggcaca
120gtcaccatca attgccaggc cagtcagaac atttatagat acttagcctg
gtatcagcag 180aaaccagggc agcctcccaa gttcctgatc tatctggcat
ctactctggc atctggggtc 240ccatcgcggt ttaaaggcag tggatctggg
acagagttca ctctcaccat cagcgacctg 300gagtgtgccg atgctgccac
ttactactgt caaagttatt atagtagtaa tagtgtcgct 360291384DNAOryctolagus
cuniculus 291atggagactg ggctgcgctg gcttctcctg gtcgctgtgc tcaaaggtgt
ccagtgtcag 60gagcagctgg tggagtccgg gggagacctg gtccagcctg agggatccct
gacactcacc 120tgcacagctt ctgagttaga cttcagtagc ggctactgga
tatgctgggt ccgccaggtt 180ccagggaagg ggctggagtg gatcggatgc
atttatactg gtagtagtgg tagcactttt 240tacgcgagtt gggcgaaagg
ccgattcacc atctccaaaa cctcgtcgac cacggtgact 300ctgcaaatga
ccagtctgac agccgcggac acggccacct atttctgtgc gagaggttat
360agtggctttg gttactttaa gttg 38429233DNAOryctolagus cuniculus
292caggccagtc agaacattta tagatactta gcc 3329321DNAOryctolagus
cuniculus 293ctggcatcta ctctggcatc t 2129430DNAOryctolagus
cuniculus 294caaagttatt atagtagtaa tagtgtcgct 3029518DNAOryctolagus
cuniculus 295agcggctact ggatatgc 1829654DNAOryctolagus cuniculus
296tgcatttata ctggtagtag tggtagcact ttttacgcga gttgggcgaa aggc
5429730DNAOryctolagus cuniculus 297ggttatagtg gctttggtta ctttaagttg
30298122PRTOryctolagus cuniculus 298Met Asp Thr Arg Ala Pro Thr Gln
Leu Leu Gly Leu Leu Leu Leu Trp 1 5 10 15 Leu Pro Gly Ala Arg Cys
Ala Tyr Asp Met Thr Gln Thr Pro Ala Ser 20 25 30 Val Glu Val Ala
Val Gly Gly Thr Val Thr Ile Lys Cys Gln Ala Ser 35 40 45 Glu Asp
Ile Tyr Arg Leu Leu Ala Trp Tyr Gln Gln Lys Pro Gly Gln 50 55 60
Pro Pro Lys Leu Leu Ile Tyr Asp Ser Ser Asp Leu Ala Ser Gly Val 65
70 75 80 Pro Ser Arg Phe Lys Gly Ser Gly Ser Gly Thr Glu Phe Thr
Leu Ala 85 90 95 Ile Ser Gly Val Gln Cys Asp Asp Ala Ala Thr Tyr
Tyr Cys Gln Gln 100 105 110 Ala Trp Ser Tyr Ser Asp Ile Asp Asn Ala
115 120 299123PRTOryctolagus cuniculus 299Met Glu Thr Gly Leu Arg
Trp Leu Leu Leu Val Ala Val Leu Lys Gly 1 5 10 15 Val Gln Cys Gln
Ser Val Glu Glu Ser Gly Gly Arg Leu Val Thr Pro 20 25 30 Gly Thr
Pro Leu Thr Leu Thr Cys Thr Ala Ser Gly Phe Ser Leu Ser 35 40 45
Ser Tyr Tyr Met Ser Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu 50
55 60 Trp Ile Gly Ile Ile Thr Thr Ser Gly Asn Thr Phe Tyr Ala Ser
Trp 65 70 75 80 Ala Lys Gly Arg Leu Thr Ile Ser Arg Thr Ser Thr Thr
Val Asp Leu 85 90 95 Lys Ile Thr Ser Pro Thr Thr Glu Asp Thr Ala
Thr Tyr Phe Cys Ala 100 105 110 Arg Thr Ser Asp Ile Phe Tyr Tyr Arg
Asn Leu 115 120 30011PRTOryctolagus cuniculus 300Gln Ala Ser Glu
Asp Ile Tyr Arg Leu Leu Ala 1 5 10 3017PRTOryctolagus cuniculus
301Asp Ser Ser Asp Leu Ala Ser 1 5 30212PRTOryctolagus cuniculus
302Gln Gln Ala Trp Ser Tyr Ser Asp Ile Asp Asn Ala 1 5 10
3035PRTOryctolagus cuniculus 303Ser Tyr Tyr Met Ser 1 5
30416PRTOryctolagus cuniculus 304Ile Ile Thr Thr Ser Gly Asn Thr
Phe Tyr Ala Ser Trp Ala Lys Gly 1 5 10 15 30510PRTOryctolagus
cuniculus 305Thr Ser Asp Ile Phe Tyr Tyr Arg Asn Leu 1 5 10
306366DNAOryctolagus cuniculus 306atggacacga gggcccccac tcagctgctg
gggctcctgc tgctctggct cccaggtgcc 60agatgtgcct atgatatgac ccagactcca
gcctctgtgg aggtagctgt gggaggcaca 120gtcaccatca agtgccaggc
cagtgaggac atttataggt tattggcctg gtatcaacag 180aaaccagggc
agcctcccaa gctcctgatc tatgattcat ccgatctggc atctggggtc
240ccatcgcggt tcaaaggcag tggatctggg acagagttca ctctcgccat
cagcggtgtg 300cagtgtgacg atgctgccac ttactactgt caacaggctt
ggagttatag tgatattgat 360aatgct 366307369DNAOryctolagus cuniculus
307atggagactg ggctgcgctg gcttctcctg gtcgctgtgc tcaaaggtgt
ccagtgtcag 60tcggtggagg agtccggggg tcgcctggtc acgccgggga cacccctgac
actcacctgc 120acagcctctg gattctccct cagtagctac tacatgagct
gggtccgcca ggctccaggg 180aaggggctgg aatggatcgg aatcattact
actagtggta atacatttta cgcgagctgg 240gcgaaaggcc ggctcaccat
ctccagaacc tcgaccacgg tggatctgaa aatcaccagt 300ccgacaaccg
aggacacggc cacctatttc tgtgccagaa cttctgatat tttttattat 360cgtaacttg
36930833DNAOryctolagus cuniculus 308caggccagtg aggacattta
taggttattg gcc 3330921DNAOryctolagus cuniculus 309gattcatccg
atctggcatc t 2131036DNAOryctolagus cuniculus 310caacaggctt
ggagttatag tgatattgat aatgct 3631115DNAOryctolagus cuniculus
311agctactaca tgagc 1531248DNAOryctolagus cuniculus 312atcattacta
ctagtggtaa tacattttac gcgagctggg cgaaaggc 4831330DNAOryctolagus
cuniculus 313acttctgata ttttttatta tcgtaacttg
30314123PRTOryctolagus cuniculus 314Met Asp Thr Arg Ala Pro Thr Gln
Leu Leu Gly Leu Leu Leu Leu Trp 1 5 10 15 Leu Pro Gly Ala Thr Phe
Ala Ala Val Leu Thr Gln Thr Ala Ser Pro 20 25 30 Val Ser Ala Ala
Val Gly Ala Thr Val Thr Ile Asn Cys Gln Ser Ser 35 40 45 Gln Ser
Val Tyr Asn Asp Met Asp Leu Ala Trp Phe Gln Gln Lys Pro 50 55 60
Gly Gln Pro Pro Lys Leu Leu Ile Tyr Ser Ala Ser Thr Leu Ala Ser 65
70 75 80 Gly Val Pro Ser Arg Phe Ser Gly Ser Gly Ser Gly Thr Glu
Phe Thr 85 90 95 Leu Thr Ile Ser Gly Val Gln Cys Asp Asp Ala Ala
Thr Tyr Tyr Cys 100 105 110 Leu Gly Ala Phe Asp Asp Asp Ala Asp Asn
Thr 115 120 315129PRTOryctolagus cuniculus 315Met Glu Thr Gly Leu
Arg Trp Leu Leu Leu Val Ala Val Leu Lys Gly 1 5 10 15 Val Gln Cys
Gln Ser Val Glu Glu Ser Gly Gly Arg Leu Val Thr Pro 20 25 30 Gly
Thr Pro Leu Thr Leu Thr Cys Thr Val Ser Gly Phe Ser Leu Thr 35 40
45 Arg His Ala Ile Thr Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu
50 55 60 Trp Ile Gly Cys Ile Trp Ser Gly Gly Ser Thr Tyr Tyr Ala
Thr Trp 65 70 75 80 Ala Lys Gly Arg Phe Thr Ile Ser Lys Thr Ser Thr
Thr Val Asp Leu 85 90 95 Arg Ile Thr Ser Pro Thr Thr Glu Asp Thr
Ala Thr Tyr Phe Cys Ala 100 105 110 Arg Val Ile Gly Asp Thr Ala Gly
Tyr Ala Tyr Phe Thr Gly Leu Asp 115 120 125 Leu 31613PRTOryctolagus
cuniculus 316Gln Ser Ser Gln Ser Val Tyr Asn Asp Met Asp Leu Ala 1
5 10 3177PRTOryctolagus cuniculus 317Ser Ala Ser Thr Leu Ala Ser 1
5 31811PRTOryctolagus cuniculus 318Leu Gly Ala Phe Asp Asp Asp Ala
Asp Asn Thr 1 5 10 3195PRTOryctolagus cuniculus 319Arg His Ala Ile
Thr 1 5 32016PRTOryctolagus cuniculus 320Cys Ile Trp Ser Gly Gly
Ser Thr Tyr Tyr Ala Thr Trp Ala Lys Gly 1 5 10 15
32116PRTOryctolagus cuniculus 321Val Ile Gly Asp Thr Ala Gly Tyr
Ala Tyr Phe Thr Gly Leu Asp Leu 1 5 10 15 322369DNAOryctolagus
cuniculus 322atggacacga gggcccccac tcagctgctg gggctcctgc tgctctggct
cccaggtgcc 60acgtttgcag ccgtgctgac ccagactgca tcacccgtgt ctgccgctgt
gggagccaca 120gtcaccatca actgccagtc cagtcagagt gtttataatg
acatggactt agcctggttt 180cagcagaaac cagggcagcc tcccaagctc
ctgatctatt ctgcatccac tctggcatct 240ggggtcccat cgcggttcag
cggcagtgga tctgggacag agttcactct caccatcagc 300ggcgtgcagt
gtgacgatgc tgccacttac tactgtctag gcgcttttga tgatgatgct 360gataatact
369323387DNAOryctolagus cuniculus 323atggagactg ggctgcgctg
gcttctcctg gtcgctgtgc tcaaaggtgt ccagtgtcag 60tcggtggagg agtccggggg
tcgcctggtc acgcctggga cacccctgac actcacctgc 120acagtctctg
gattctccct cactaggcat gcaataacct gggtccgcca ggctccaggg
180aaggggctgg aatggatcgg atgcatttgg agtggtggta gcacatacta
cgcgacctgg 240gcgaaaggcc gattcaccat ctccaaaacc tcgaccacgg
tggatctcag aatcaccagt 300ccgacaaccg aggacacggc cacctacttc
tgtgccagag tcattggcga tactgctggt 360tatgcttatt ttacggggct tgacttg
38732439DNAOryctolagus cuniculus 324cagtccagtc agagtgttta
taatgacatg gacttagcc 3932521DNAOryctolagus cuniculus 325tctgcatcca
ctctggcatc t 2132633DNAOryctolagus cuniculus 326ctaggcgctt
ttgatgatga tgctgataat act 3332715DNAOryctolagus cuniculus
327aggcatgcaa taacc 1532848DNAOryctolagus cuniculus 328tgcatttgga
gtggtggtag cacatactac gcgacctggg cgaaaggc 4832948DNAOryctolagus
cuniculus 329gtcattggcg atactgctgg ttatgcttat tttacggggc ttgacttg
48330121PRTOryctolagus cuniculus 330Met Asp Thr Arg Ala Pro Thr Gln
Leu Leu Gly Leu Leu Leu Leu Trp 1 5 10 15 Leu Pro Gly Ala Arg Cys
Ala Tyr Asp Met Thr Gln Thr Pro Ala Ser 20 25 30 Val Glu Val Ala
Val Gly Gly Thr Val Thr Ile Lys Cys Gln Ala Ser 35 40 45 Gln Ser
Val Tyr Asn Trp Leu Ser Trp Tyr Gln Gln Lys Pro Gly Gln 50 55 60
Pro Pro Lys Leu Leu Ile Tyr Thr Ala Ser Ser Leu Ala Ser Gly Val 65
70 75 80 Pro Ser Arg Phe Ser Gly Ser Gly Ser Gly Thr Glu Phe Thr
Leu Thr 85 90 95 Ile Ser Gly Val Glu Cys Ala Asp Ala Ala Thr Tyr
Tyr Cys Gln Gln 100 105 110 Gly Tyr Thr Ser Asp Val Asp Asn Val 115
120 331130PRTOryctolagus cuniculus 331Met Glu Thr Gly Leu Arg Trp
Leu Leu Leu Val Ala Val Leu Lys Gly 1 5 10 15 Val Gln Cys Gln Ser
Leu Glu Glu Ala Gly Gly Arg Leu Val Thr Pro 20 25 30 Gly Thr Pro
Leu Thr Leu Thr Cys Thr Val Ser Gly Ile Asp Leu Ser 35 40 45 Ser
Tyr Ala Met Gly Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu 50 55
60 Tyr Ile Gly Ile Ile Ser Ser Ser Gly Ser Thr Tyr Tyr Ala Thr Trp
65 70 75 80 Ala Lys Gly Arg Phe Thr Ile Ser Gln Ala Ser Ser Thr Thr
Val Asp 85 90 95 Leu Lys Ile Thr Ser Pro Thr Thr Glu Asp Ser Ala
Thr Tyr Phe Cys 100 105 110 Ala Arg Gly Gly Ala Gly Ser Gly Gly Val
Trp Leu Leu Asp Gly Phe 115 120 125 Asp Pro 130 33211PRTOryctolagus
cuniculus 332Gln Ala Ser Gln Ser Val Tyr Asn Trp Leu Ser 1 5 10
3337PRTOryctolagus cuniculus 333Thr Ala Ser Ser Leu Ala Ser 1 5
33411PRTOryctolagus cuniculus 334Gln Gln Gly Tyr Thr Ser Asp Val
Asp Asn Val 1 5 10 3355PRTOryctolagus cuniculus 335Ser Tyr Ala Met
Gly 1 5 33616PRTOryctolagus cuniculus 336Ile Ile Ser Ser Ser Gly
Ser Thr Tyr Tyr Ala Thr Trp Ala Lys Gly 1 5 10 15
33716PRTOryctolagus cuniculus 337Gly Gly Ala Gly Ser Gly Gly Val
Trp Leu Leu Asp Gly Phe Asp Pro 1 5 10 15 338363DNAOryctolagus
cuniculus 338atggacacga gggcccccac tcagctgctg gggctcctgc tgctctggct
cccaggtgcc 60agatgtgcct atgatatgac ccagactcca gcctctgtgg aggtagctgt
gggaggcaca 120gtcaccatca agtgccaggc cagtcagagt gtttataatt
ggttatcctg gtatcagcag 180aaaccagggc agcctcccaa gctcctgatc
tatactgcat ccagtctggc atctggggtc 240ccatcgcggt tcagtggcag
tggatctggg acagagttca ctctcaccat cagcggcgtg 300gagtgtgccg
atgctgccac ttactactgt caacagggtt atactagtga tgttgataat 360gtt
363339390DNAOryctolagus cuniculus 339atggagactg ggctgcgctg
gcttctcctg gtcgctgtgc tcaaaggtgt ccagtgtcag 60tcgctggagg aggccggggg
tcgcctggtc acgcctggga cacccctgac actcacctgc 120acagtctctg
gaatcgacct cagtagctat gcaatgggct gggtccgcca ggctccaggg
180aaggggctgg aatacatcgg aatcattagt agtagtggta gcacatacta
cgcgacctgg 240gcgaaaggcc gattcaccat ctcacaagcc tcgtcgacca
cggtggatct gaaaattacc 300agtccgacaa ccgaggactc ggccacatat
ttctgtgcca gagggggtgc tggtagtggt 360ggtgtttggc tgcttgatgg
ttttgatccc 39034033DNAOryctolagus cuniculus 340caggccagtc
agagtgttta taattggtta tcc 3334121DNAOryctolagus cuniculus
341actgcatcca gtctggcatc t 2134233DNAOryctolagus cuniculus
342caacagggtt atactagtga tgttgataat gtt 3334315DNAOryctolagus
cuniculus 343agctatgcaa tgggc 1534448DNAOryctolagus cuniculus
344atcattagta gtagtggtag cacatactac gcgacctggg cgaaaggc
4834548DNAOryctolagus cuniculus 345gggggtgctg gtagtggtgg tgtttggctg
cttgatggtt ttgatccc 48346123PRTOryctolagus cuniculus 346Met Asp Thr
Arg Ala Pro Thr Gln Leu Leu Gly Leu Leu Leu Leu Trp 1 5 10 15 Leu
Pro Gly Ala Lys Cys Ala Asp Val Val Met Thr Gln Thr Pro Ala 20 25
30 Ser Val Ser Ala Ala Val Gly Gly Thr Val Thr Ile Asn Cys Gln Ala
35 40 45 Ser Glu Asn Ile Tyr Asn Trp Leu Ala Trp Tyr Gln Gln Lys
Pro Gly 50 55 60 Gln Pro Pro Lys Leu Leu Ile Tyr Thr Val Gly Asp
Leu Ala Ser Gly 65 70 75 80 Val Ser Ser Arg Phe Lys Gly Ser Gly Ser
Gly Thr Glu Phe Thr Leu 85 90 95 Thr Ile Ser Asp Leu Glu Cys Ala
Asp Ala Ala Thr Tyr Tyr Cys Gln 100 105 110 Gln Gly Tyr Ser Ser Ser
Tyr Val Asp Asn Val 115 120 347130PRTOryctolagus cuniculus 347Met
Glu Thr Gly Leu Arg Trp Leu Leu Leu Val Ala Val Leu Lys Gly 1 5 10
15 Val Gln Cys Gln Glu Gln Leu Lys Glu Ser Gly Gly Arg Leu Val Thr
20 25 30 Pro Gly Thr Pro Leu Thr Leu Thr Cys Thr Val Ser Gly Phe
Ser Leu 35 40 45 Asn Asp Tyr Ala Val Gly Trp Phe Arg Gln Ala Pro
Gly Lys Gly Leu 50 55 60 Glu Trp Ile Gly Tyr Ile Arg Ser Ser Gly
Thr Thr Ala Tyr Ala Thr 65 70 75 80 Trp Ala Lys Gly Arg Phe Thr Ile
Ser
Ala Thr Ser Thr Thr Val Asp 85 90 95 Leu Lys Ile Thr Ser Pro Thr
Thr Glu Asp Thr Ala Thr Tyr Phe Cys 100 105 110 Ala Arg Gly Gly Ala
Gly Ser Ser Gly Val Trp Ile Leu Asp Gly Phe 115 120 125 Ala Pro 130
34811PRTOryctolagus cuniculus 348Gln Ala Ser Glu Asn Ile Tyr Asn
Trp Leu Ala 1 5 10 3497PRTOryctolagus cuniculus 349Thr Val Gly Asp
Leu Ala Ser 1 5 35012PRTOryctolagus cuniculus 350Gln Gln Gly Tyr
Ser Ser Ser Tyr Val Asp Asn Val 1 5 10 3515PRTOryctolagus cuniculus
351Asp Tyr Ala Val Gly 1 5 35216PRTOryctolagus cuniculus 352Tyr Ile
Arg Ser Ser Gly Thr Thr Ala Tyr Ala Thr Trp Ala Lys Gly 1 5 10 15
35316PRTOryctolagus cuniculus 353Gly Gly Ala Gly Ser Ser Gly Val
Trp Ile Leu Asp Gly Phe Ala Pro 1 5 10 15 354369DNAOryctolagus
cuniculus 354atggacacga gggcccccac tcagctgctg gggctcctgc tgctctggct
cccaggtgcc 60aaatgtgccg atgttgtgat gacccagact ccagcctccg tgtctgcagc
tgtgggaggc 120acagtcacca tcaattgcca ggccagtgag aacatttata
attggttagc ctggtatcag 180cagaaaccag ggcagcctcc caagctcctg
atctatactg taggcgatct ggcatctggg 240gtctcatcgc ggttcaaagg
cagtggatct gggacagagt tcactctcac catcagcgac 300ctggagtgtg
ccgatgctgc cacttactat tgtcaacagg gttatagtag tagttatgtt 360gataatgtt
369355390DNAOryctolagus cuniculus 355atggagactg ggctgcgctg
gcttctcctg gtcgctgtgc tcaaaggtgt ccagtgtcag 60gagcagctga aggagtccgg
gggtcgcctg gtcacgcctg ggacacccct gacactcacc 120tgcacagtct
ctggattctc cctcaatgac tatgcagtgg gctggttccg ccaggctcca
180gggaaggggc tggaatggat cggatacatt cgtagtagtg gtaccacagc
ctacgcgacc 240tgggcgaaag gccgattcac catctccgct acctcgacca
cggtggatct gaaaatcacc 300agtccgacaa ccgaggacac ggccacctat
ttctgtgcca gagggggtgc tggtagtagt 360ggtgtgtgga tccttgatgg
ttttgctccc 39035633DNAOryctolagus cuniculus 356caggccagtg
agaacattta taattggtta gcc 3335721DNAOryctolagus cuniculus
357actgtaggcg atctggcatc t 2135836DNAOryctolagus cuniculus
358caacagggtt atagtagtag ttatgttgat aatgtt 3635915DNAOryctolagus
cuniculus 359gactatgcag tgggc 1536048DNAOryctolagus cuniculus
360tacattcgta gtagtggtac cacagcctac gcgacctggg cgaaaggc
4836148DNAOryctolagus cuniculus 361gggggtgctg gtagtagtgg tgtgtggatc
cttgatggtt ttgctccc 48362121PRTOryctolagus cuniculus 362Met Asp Thr
Arg Ala Pro Thr Gln Leu Leu Gly Leu Leu Leu Leu Trp 1 5 10 15 Leu
Pro Gly Ala Thr Phe Ala Gln Val Leu Thr Gln Thr Pro Ser Ser 20 25
30 Val Ser Ala Ala Val Gly Gly Thr Val Thr Ile Asn Cys Gln Ala Ser
35 40 45 Gln Ser Val Tyr Gln Asn Asn Tyr Leu Ser Trp Phe Gln Gln
Lys Pro 50 55 60 Gly Gln Pro Pro Lys Leu Leu Ile Tyr Gly Ala Ala
Thr Leu Ala Ser 65 70 75 80 Gly Val Pro Ser Arg Phe Lys Gly Ser Gly
Ser Gly Thr Gln Phe Thr 85 90 95 Leu Thr Ile Ser Asp Leu Glu Cys
Asp Asp Ala Ala Thr Tyr Tyr Cys 100 105 110 Ala Gly Ala Tyr Arg Asp
Val Asp Ser 115 120 363130PRTOryctolagus cuniculus 363Met Glu Thr
Gly Leu Arg Trp Leu Leu Leu Val Ala Val Leu Lys Gly 1 5 10 15 Val
Gln Cys Gln Ser Leu Glu Glu Ser Gly Gly Asp Leu Val Lys Pro 20 25
30 Gly Ala Ser Leu Thr Leu Thr Cys Thr Ala Ser Gly Phe Ser Phe Thr
35 40 45 Ser Thr Tyr Tyr Ile Tyr Trp Val Arg Gln Ala Pro Gly Lys
Gly Leu 50 55 60 Glu Trp Ile Ala Cys Ile Asp Ala Gly Ser Ser Gly
Ser Thr Tyr Tyr 65 70 75 80 Ala Thr Trp Val Asn Gly Arg Phe Thr Ile
Ser Lys Thr Ser Ser Thr 85 90 95 Thr Val Thr Leu Gln Met Thr Ser
Leu Thr Ala Ala Asp Thr Ala Thr 100 105 110 Tyr Phe Cys Ala Lys Trp
Asp Tyr Gly Gly Asn Val Gly Trp Gly Tyr 115 120 125 Asp Leu 130
36413PRTOryctolagus cuniculus 364Gln Ala Ser Gln Ser Val Tyr Gln
Asn Asn Tyr Leu Ser 1 5 10 3657PRTOryctolagus cuniculus 365Gly Ala
Ala Thr Leu Ala Ser 1 5 3669PRTOryctolagus cuniculus 366Ala Gly Ala
Tyr Arg Asp Val Asp Ser 1 5 3676PRTOryctolagus cuniculus 367Ser Thr
Tyr Tyr Ile Tyr 1 5 36818PRTOryctolagus cuniculus 368Cys Ile Asp
Ala Gly Ser Ser Gly Ser Thr Tyr Tyr Ala Thr Trp Val 1 5 10 15 Asn
Gly 36913PRTOryctolagus cuniculus 369Trp Asp Tyr Gly Gly Asn Val
Gly Trp Gly Tyr Asp Leu 1 5 10 370363DNAOryctolagus cuniculus
370atggacacga gggcccccac tcagctgctg gggctcctgc tgctctggct
cccaggtgcc 60acatttgctc aagtgctgac ccagactcca tcctccgtgt ctgcagctgt
gggaggcaca 120gtcaccatca attgccaggc cagtcagagt gtttatcaga
acaactactt atcctggttt 180cagcagaaac cagggcagcc tcccaagctc
ctgatctatg gtgcggccac tctggcatct 240ggggtcccat cgcggttcaa
aggcagtgga tctgggacac agttcactct caccatcagc 300gacctggagt
gtgacgatgc tgccacttac tactgtgcag gcgcttatag ggatgtggat 360tct
363371390DNAOryctolagus cuniculus 371atggagactg ggctgcgctg
gcttctcctg gtcgctgtgc tcaaaggtgt ccagtgtcag 60tcgttggagg agtccggggg
agacctggtc aagcctgggg catccctgac actcacctgc 120acagcctctg
gattctcctt tactagtacc tactacatct actgggtccg ccaggctcca
180gggaaggggc tggagtggat cgcatgtatt gatgctggta gtagtggtag
cacttactac 240gcgacctggg tgaatggccg attcaccatc tccaaaacct
cgtcgaccac ggtgactctg 300caaatgacca gtctgacagc cgcggacacg
gccacctatt tctgtgcgaa atgggattat 360ggtggtaatg ttggttgggg
ttatgacttg 39037239DNAOryctolagus cuniculus 372caggccagtc
agagtgttta tcagaacaac tacttatcc 3937321DNAOryctolagus cuniculus
373ggtgcggcca ctctggcatc t 2137427DNAOryctolagus cuniculus
374gcaggcgctt atagggatgt ggattct 2737518DNAOryctolagus cuniculus
375agtacctact acatctac 1837654DNAOryctolagus cuniculus
376tgtattgatg ctggtagtag tggtagcact tactacgcga cctgggtgaa tggc
5437739DNAOryctolagus cuniculus 377tgggattatg gtggtaatgt tggttggggt
tatgacttg 39378120PRTOryctolagus cuniculus 378Met Asp Thr Arg Ala
Pro Thr Gln Leu Leu Gly Leu Leu Leu Leu Trp 1 5 10 15 Leu Pro Gly
Ala Arg Cys Ala Phe Glu Leu Thr Gln Thr Pro Ser Ser 20 25 30 Val
Glu Ala Ala Val Gly Gly Thr Val Thr Ile Lys Cys Gln Ala Ser 35 40
45 Gln Ser Ile Ser Ser Tyr Leu Ala Trp Tyr Gln Gln Lys Pro Gly Gln
50 55 60 Pro Pro Lys Phe Leu Ile Tyr Arg Ala Ser Thr Leu Ala Ser
Gly Val 65 70 75 80 Pro Ser Arg Phe Lys Gly Ser Gly Ser Gly Thr Glu
Phe Thr Leu Thr 85 90 95 Ile Ser Asp Leu Glu Cys Ala Asp Ala Ala
Thr Tyr Tyr Cys Gln Ser 100 105 110 Tyr Tyr Asp Ser Val Ser Asn Pro
115 120 379127PRTOryctolagus cuniculus 379Met Glu Thr Gly Leu Arg
Trp Leu Leu Leu Val Ala Val Leu Lys Gly 1 5 10 15 Val Gln Cys Gln
Ser Leu Glu Glu Ser Gly Gly Asp Leu Val Lys Pro 20 25 30 Glu Gly
Ser Leu Thr Leu Thr Cys Lys Ala Ser Gly Leu Asp Leu Gly 35 40 45
Thr Tyr Trp Phe Met Cys Trp Val Arg Gln Ala Pro Gly Lys Gly Leu 50
55 60 Glu Trp Ile Ala Cys Ile Tyr Thr Gly Ser Ser Gly Ser Thr Phe
Tyr 65 70 75 80 Ala Ser Trp Val Asn Gly Arg Phe Thr Ile Ser Lys Thr
Ser Ser Thr 85 90 95 Thr Val Thr Leu Gln Met Thr Ser Leu Thr Ala
Ala Asp Thr Ala Thr 100 105 110 Tyr Phe Cys Ala Arg Gly Tyr Ser Gly
Tyr Gly Tyr Phe Lys Leu 115 120 125 38011PRTOryctolagus cuniculus
380Gln Ala Ser Gln Ser Ile Ser Ser Tyr Leu Ala 1 5 10
3817PRTOryctolagus cuniculus 381Arg Ala Ser Thr Leu Ala Ser 1 5
38210PRTOryctolagus cuniculus 382Gln Ser Tyr Tyr Asp Ser Val Ser
Asn Pro 1 5 10 3836PRTOryctolagus cuniculus 383Thr Tyr Trp Phe Met
Cys 1 5 38418PRTOryctolagus cuniculus 384Cys Ile Tyr Thr Gly Ser
Ser Gly Ser Thr Phe Tyr Ala Ser Trp Val 1 5 10 15 Asn Gly
38510PRTOryctolagus cuniculus 385Gly Tyr Ser Gly Tyr Gly Tyr Phe
Lys Leu 1 5 10 386360DNAOryctolagus cuniculus 386atggacacga
gggcccccac tcagctgctg gggctcctgc tgctctggct cccaggtgcc 60agatgtgcat
tcgaattgac ccagactcca tcctccgtgg aggcagctgt gggaggcaca
120gtcaccatca agtgccaggc cagtcagagc attagtagtt acttagcctg
gtatcagcag 180aaaccagggc agcctcccaa gttcctgatc tacagggcgt
ccactctggc atctggggtc 240ccatcgcgat tcaaaggcag tggatctggg
acagagttca ctctcaccat cagcgacctg 300gagtgtgccg atgctgccac
ttactactgt caaagctatt atgatagtgt ttcaaatcct 360387381DNAOryctolagus
cuniculus 387atggagactg ggctgcgctg gcttctcctg gtcgctgtgc tcaaaggtgt
ccagtgtcag 60tcgttggagg agtccggggg agacctggtc aagcctgagg gatccctgac
actcacctgc 120aaagcctctg gactcgacct cggtacctac tggttcatgt
gctgggtccg ccaggctcca 180gggaaggggc tggagtggat cgcttgtatt
tatactggta gtagtggttc cactttctac 240gcgagctggg tgaatggccg
attcaccatc tccaaaacct cgtcgaccac ggtgactctg 300caaatgacca
gtctgacagc cgcggacacg gccacttatt tttgtgcgag aggttatagt
360ggttatggtt attttaagtt g 38138833DNAOryctolagus cuniculus
388caggccagtc agagcattag tagttactta gcc 3338921DNAOryctolagus
cuniculus 389agggcgtcca ctctggcatc t 2139030DNAOryctolagus
cuniculus 390caaagctatt atgatagtgt ttcaaatcct 3039118DNAOryctolagus
cuniculus 391acctactggt tcatgtgc 1839254DNAOryctolagus cuniculus
392tgtatttata ctggtagtag tggttccact ttctacgcga gctgggtgaa tggc
5439330DNAOryctolagus cuniculus 393ggttatagtg gttatggtta ttttaagttg
30394124PRTOryctolagus cuniculus 394Met Asp Thr Arg Ala Pro Thr Gln
Leu Leu Gly Leu Leu Leu Leu Trp 1 5 10 15 Leu Pro Gly Val Thr Phe
Ala Ile Glu Met Thr Gln Ser Pro Phe Ser 20 25 30 Val Ser Ala Ala
Val Gly Gly Thr Val Ser Ile Ser Cys Gln Ala Ser 35 40 45 Gln Ser
Val Tyr Lys Asn Asn Gln Leu Ser Trp Tyr Gln Gln Lys Ser 50 55 60
Gly Gln Pro Pro Lys Leu Leu Ile Tyr Gly Ala Ser Ala Leu Ala Ser 65
70 75 80 Gly Val Pro Ser Arg Phe Lys Gly Ser Gly Ser Gly Thr Glu
Phe Thr 85 90 95 Leu Thr Ile Ser Asp Val Gln Cys Asp Asp Ala Ala
Thr Tyr Tyr Cys 100 105 110 Ala Gly Ala Ile Thr Gly Ser Ile Asp Thr
Asp Gly 115 120 395130PRTOryctolagus cuniculus 395Met Glu Thr Gly
Leu Arg Trp Leu Leu Leu Val Ala Val Leu Lys Gly 1 5 10 15 Val Gln
Cys Gln Ser Leu Glu Glu Ser Gly Gly Asp Leu Val Lys Pro 20 25 30
Gly Ala Ser Leu Thr Leu Thr Cys Thr Thr Ser Gly Phe Ser Phe Ser 35
40 45 Ser Ser Tyr Phe Ile Cys Trp Val Arg Gln Ala Pro Gly Lys Gly
Leu 50 55 60 Glu Trp Ile Ala Cys Ile Tyr Gly Gly Asp Gly Ser Thr
Tyr Tyr Ala 65 70 75 80 Ser Trp Ala Lys Gly Arg Phe Thr Ile Ser Lys
Thr Ser Ser Thr Thr 85 90 95 Val Thr Leu Gln Met Thr Ser Leu Thr
Ala Ala Asp Thr Ala Thr Tyr 100 105 110 Phe Cys Ala Arg Glu Trp Ala
Tyr Ser Gln Gly Tyr Phe Gly Ala Phe 115 120 125 Asp Leu 130
39613PRTOryctolagus cuniculus 396Gln Ala Ser Gln Ser Val Tyr Lys
Asn Asn Gln Leu Ser 1 5 10 3977PRTOryctolagus cuniculus 397Gly Ala
Ser Ala Leu Ala Ser 1 5 39812PRTOryctolagus cuniculus 398Ala Gly
Ala Ile Thr Gly Ser Ile Asp Thr Asp Gly 1 5 10 3996PRTOryctolagus
cuniculus 399Ser Ser Tyr Phe Ile Cys 1 5 40017PRTOryctolagus
cuniculus 400Cys Ile Tyr Gly Gly Asp Gly Ser Thr Tyr Tyr Ala Ser
Trp Ala Lys 1 5 10 15 Gly 40114PRTOryctolagus cuniculus 401Glu Trp
Ala Tyr Ser Gln Gly Tyr Phe Gly Ala Phe Asp Leu 1 5 10
402372DNAOryctolagus cuniculus 402atggacacga gggcccccac tcagctgctg
gggctcctgc tgctctggct cccaggtgtc 60acatttgcca tcgaaatgac ccagagtcca
ttctccgtgt ctgcagctgt gggaggcaca 120gtcagcatca gttgccaggc
cagtcagagt gtttataaga acaaccaatt atcctggtat 180cagcagaaat
cagggcagcc tcccaagctc ctgatctatg gtgcatcggc tctggcatct
240ggggtcccat cgcggttcaa aggcagtgga tctgggacag agttcactct
caccatcagc 300gacgtgcagt gtgacgatgc tgccacttac tactgtgcag
gcgctattac tggtagtatt 360gatacggatg gt 372403390DNAOryctolagus
cuniculus 403atggagactg ggctgcgctg gcttctcctg gtcgctgtgc tcaaaggtgt
ccagtgtcag 60tcgttggagg agtccggggg agacctggtc aagcctgggg catccctgac
actcacctgc 120acaacttctg gattctcctt cagtagcagc tacttcattt
gctgggtccg ccaggctcca 180gggaaggggc tggagtggat cgcatgcatt
tatggtggtg atggcagcac atactacgcg 240agctgggcga aaggccgatt
caccatctcc aaaacctcgt cgaccacggt gacgctgcaa 300atgaccagtc
tgacagccgc ggacacggcc acctatttct gtgcgagaga atgggcatat
360agtcaaggtt attttggtgc ttttgatctc 39040439DNAOryctolagus
cuniculus 404caggccagtc agagtgttta taagaacaac caattatcc
3940521DNAOryctolagus cuniculus 405ggtgcatcgg ctctggcatc t
2140636DNAOryctolagus cuniculus 406gcaggcgcta ttactggtag tattgatacg
gatggt 3640718DNAOryctolagus cuniculus 407agcagctact tcatttgc
1840851DNAOryctolagus cuniculus 408tgcatttatg gtggtgatgg cagcacatac
tacgcgagct gggcgaaagg c 5140942DNAOryctolagus cuniculus
409gaatgggcat atagtcaagg ttattttggt gcttttgatc tc
42410124PRTOryctolagus cuniculus 410Met Asp Thr Arg Ala Pro Thr Gln
Leu Leu Gly Leu Leu Leu Leu Trp 1 5 10 15 Leu Pro Gly Ala Arg Cys
Asp Val Val Met Thr Gln Thr Pro Ala Ser 20 25 30 Val Glu Ala Ala
Val Gly Gly Thr Val Thr Ile Lys Cys Gln Ala Ser 35 40 45 Glu Asp
Ile Ser Ser Tyr Leu Ala Trp Tyr Gln Gln Lys Pro Gly Gln 50 55 60
Pro Pro Lys Leu Leu Ile Tyr Ala Ala Ser Asn Leu Glu Ser Gly Val 65
70 75 80 Ser Ser Arg Phe Lys Gly Ser Gly Ser Gly Thr Glu Tyr Thr
Leu Thr 85 90 95 Ile Ser Asp Leu Glu Cys Ala Asp Ala Ala Thr Tyr
Tyr Cys Gln Cys 100 105 110 Thr Tyr Gly Thr Ile Ser Ile Ser Asp Gly
Asn Ala 115 120 411124PRTOryctolagus cuniculus 411Met Glu Thr Gly
Leu Arg Trp Leu Leu Leu Val Ala Val Leu Lys Gly 1 5 10 15 Val Gln
Cys Gln Ser Val Glu Glu Ser Gly Gly Arg Leu Val Thr Pro 20 25 30
Gly Thr Pro Leu Thr Leu Thr Cys Thr Val Ser Gly Phe Ser Leu Ser 35
40 45 Ser Tyr Phe Met Thr Trp Val Arg Gln Ala Pro Gly Glu Gly Leu
Glu 50 55 60 Tyr Ile Gly Phe Ile Asn Pro Gly Gly Ser Ala Tyr Tyr
Ala Ser Trp 65 70 75 80 Val Lys Gly Arg Phe Thr Ile Ser Lys Ser Ser
Thr Thr Val Asp Leu 85
90 95 Lys Ile Thr Ser Pro Thr Thr Glu Asp Thr Ala Thr Tyr Phe Cys
Ala 100 105 110 Arg Val Leu Ile Val Ser Tyr Gly Ala Phe Thr Ile 115
120 41211PRTOryctolagus cuniculus 412Gln Ala Ser Glu Asp Ile Ser
Ser Tyr Leu Ala 1 5 10 4137PRTOryctolagus cuniculus 413Ala Ala Ser
Asn Leu Glu Ser 1 5 41414PRTOryctolagus cuniculus 414Gln Cys Thr
Tyr Gly Thr Ile Ser Ile Ser Asp Gly Asn Ala 1 5 10
4155PRTOryctolagus cuniculus 415Ser Tyr Phe Met Thr 1 5
41616PRTOryctolagus cuniculus 416Phe Ile Asn Pro Gly Gly Ser Ala
Tyr Tyr Ala Ser Trp Val Lys Gly 1 5 10 15 41711PRTOryctolagus
cuniculus 417Val Leu Ile Val Ser Tyr Gly Ala Phe Thr Ile 1 5 10
418372DNAOryctolagus cuniculus 418atggacacga gggcccccac tcagctgctg
gggctcctgc tgctctggct cccaggtgcc 60agatgtgatg ttgtgatgac ccagactcca
gcctccgtgg aggcagctgt gggaggcaca 120gtcaccatca agtgccaggc
cagtgaggat attagtagct acttagcctg gtatcagcag 180aaaccagggc
agcctcccaa gctcctgatc tatgctgcat ccaatctgga atctggggtc
240tcatcgcgat tcaaaggcag tggatctggg acagagtaca ctctcaccat
cagcgacctg 300gagtgtgccg atgctgccac ctattactgt caatgtactt
atggtactat ttctattagt 360gatggtaatg ct 372419372DNAOryctolagus
cuniculus 419atggagactg ggctgcgctg gcttctcctg gtcgctgtgc tcaaaggtgt
ccaatgtcag 60tcggtggagg agtccggggg tcgcctggtc acgcctggga cacccctgac
actcacctgc 120acagtctctg gattctccct cagtagctac ttcatgacct
gggtccgcca ggctccaggg 180gaggggctgg aatacatcgg attcattaat
cctggtggta gcgcttacta cgcgagctgg 240gtgaaaggcc gattcaccat
ctccaagtcc tcgaccacgg tagatctgaa aatcaccagt 300ccgacaaccg
aggacacggc cacctatttc tgtgccaggg ttctgattgt ttcttatgga
360gcctttacca tc 37242033DNAOryctolagus cuniculus 420caggccagtg
aggatattag tagctactta gcc 3342121DNAOryctolagus cuniculus
421gctgcatcca atctggaatc t 2142242DNAOryctolagus cuniculus
422caatgtactt atggtactat ttctattagt gatggtaatg ct
4242315DNAOryctolagus cuniculus 423agctacttca tgacc
1542448DNAOryctolagus cuniculus 424ttcattaatc ctggtggtag cgcttactac
gcgagctggg tgaaaggc 4842533DNAOryctolagus cuniculus 425gttctgattg
tttcttatgg agcctttacc atc 33426124PRTOryctolagus cuniculus 426Met
Asp Thr Arg Ala Pro Thr Gln Leu Leu Gly Leu Leu Leu Leu Trp 1 5 10
15 Leu Pro Gly Ala Arg Cys Asp Val Val Met Thr Gln Thr Pro Ala Ser
20 25 30 Val Ser Ala Ala Val Gly Gly Thr Val Thr Ile Lys Cys Gln
Ala Ser 35 40 45 Glu Asp Ile Glu Ser Tyr Leu Ala Trp Tyr Gln Gln
Lys Pro Gly Gln 50 55 60 Pro Pro Lys Leu Leu Ile Tyr Gly Ala Ser
Asn Leu Glu Ser Gly Val 65 70 75 80 Ser Ser Arg Phe Lys Gly Ser Gly
Ser Gly Thr Glu Phe Thr Leu Thr 85 90 95 Ile Ser Asp Leu Glu Cys
Ala Asp Ala Ala Thr Tyr Tyr Cys Gln Cys 100 105 110 Thr Tyr Gly Ile
Ile Ser Ile Ser Asp Gly Asn Ala 115 120 427124PRTOryctolagus
cuniculus 427Met Glu Thr Gly Leu Arg Trp Leu Leu Leu Val Ala Val
Leu Lys Gly 1 5 10 15 Val Gln Cys Gln Ser Val Glu Glu Ser Gly Gly
Arg Leu Val Thr Pro 20 25 30 Gly Thr Pro Leu Thr Leu Thr Cys Thr
Val Ser Gly Phe Ser Leu Ser 35 40 45 Ser Tyr Phe Met Thr Trp Val
Arg Gln Ala Pro Gly Glu Gly Leu Glu 50 55 60 Tyr Ile Gly Phe Met
Asn Thr Gly Asp Asn Ala Tyr Tyr Ala Ser Trp 65 70 75 80 Ala Lys Gly
Arg Phe Thr Ile Ser Lys Thr Ser Thr Thr Val Asp Leu 85 90 95 Lys
Ile Thr Ser Pro Thr Thr Glu Asp Thr Ala Thr Tyr Phe Cys Ala 100 105
110 Arg Val Leu Val Val Ala Tyr Gly Ala Phe Asn Ile 115 120
42811PRTOryctolagus cuniculus 428Gln Ala Ser Glu Asp Ile Glu Ser
Tyr Leu Ala 1 5 10 4297PRTOryctolagus cuniculus 429Gly Ala Ser Asn
Leu Glu Ser 1 5 43014PRTOryctolagus cuniculus 430Gln Cys Thr Tyr
Gly Ile Ile Ser Ile Ser Asp Gly Asn Ala 1 5 10 4315PRTOryctolagus
cuniculus 431Ser Tyr Phe Met Thr 1 5 43216PRTOryctolagus cuniculus
432Phe Met Asn Thr Gly Asp Asn Ala Tyr Tyr Ala Ser Trp Ala Lys Gly
1 5 10 15 43311PRTOryctolagus cuniculus 433Val Leu Val Val Ala Tyr
Gly Ala Phe Asn Ile 1 5 10 434372DNAOryctolagus cuniculus
434atggacacga gggcccccac tcagctgctg gggctcctgc tgctctggct
cccaggtgcc 60agatgtgatg ttgtgatgac ccagactcca gcctccgtgt ctgcagctgt
gggaggcaca 120gtcaccatca agtgccaggc cagtgaggac attgaaagct
atctagcctg gtatcagcag 180aaaccagggc agcctcccaa gctcctgatc
tatggtgcat ccaatctgga atctggggtc 240tcatcgcggt tcaaaggcag
tggatctggg acagagttca ctctcaccat cagcgacctg 300gagtgtgccg
atgctgccac ttactattgt caatgcactt atggtattat tagtattagt
360gatggtaatg ct 372435372DNAOryctolagus cuniculus 435atggagactg
ggctgcgctg gcttctcctg gtcgctgtgc tcaaaggtgt ccagtgtcag 60tcggtggagg
agtccggggg tcgcctggtc acgcctggga cacccctgac actcacctgc
120acagtgtctg gattctccct cagtagctac ttcatgacct gggtccgcca
ggctccaggg 180gaggggctgg aatacatcgg attcatgaat actggtgata
acgcatacta cgcgagctgg 240gcgaaaggcc gattcaccat ctccaaaacc
tcgaccacgg tggatctgaa aatcaccagt 300ccgacaaccg aggacacggc
cacctatttc tgtgccaggg ttcttgttgt tgcttatgga 360gcctttaaca tc
37243633DNAOryctolagus cuniculus 436caggccagtg aggacattga
aagctatcta gcc 3343721DNAOryctolagus cuniculus 437ggtgcatcca
atctggaatc t 2143842DNAOryctolagus cuniculus 438caatgcactt
atggtattat tagtattagt gatggtaatg ct 4243915DNAOryctolagus cuniculus
439agctacttca tgacc 1544048DNAOryctolagus cuniculus 440ttcatgaata
ctggtgataa cgcatactac gcgagctggg cgaaaggc 4844133DNAOryctolagus
cuniculus 441gttcttgttg ttgcttatgg agcctttaac atc
33442124PRTOryctolagus cuniculus 442Met Asp Thr Arg Ala Pro Thr Gln
Leu Leu Gly Leu Leu Leu Leu Trp 1 5 10 15 Leu Pro Gly Ala Thr Phe
Ala Ala Val Leu Thr Gln Thr Pro Ser Pro 20 25 30 Val Ser Glu Pro
Val Gly Gly Thr Val Ser Ile Ser Cys Gln Ser Ser 35 40 45 Lys Ser
Val Met Asn Asn Asn Tyr Leu Ala Trp Tyr Gln Gln Lys Pro 50 55 60
Gly Gln Pro Pro Lys Leu Leu Ile Tyr Gly Ala Ser Asn Leu Ala Ser 65
70 75 80 Gly Val Pro Ser Arg Phe Ser Gly Ser Gly Ser Gly Thr Gln
Phe Thr 85 90 95 Leu Thr Ile Ser Asp Val Gln Cys Asp Asp Ala Ala
Thr Tyr Tyr Cys 100 105 110 Gln Gly Gly Tyr Thr Gly Tyr Ser Asp His
Gly Thr 115 120 443127PRTOryctolagus cuniculus 443Met Glu Thr Gly
Leu Arg Trp Leu Leu Leu Val Ala Val Leu Lys Gly 1 5 10 15 Val Gln
Cys Gln Ser Val Glu Glu Ser Gly Gly Arg Leu Val Lys Pro 20 25 30
Asp Glu Thr Leu Thr Leu Thr Cys Thr Val Ser Gly Ile Asp Leu Ser 35
40 45 Ser Tyr Pro Met Asn Trp Val Arg Gln Ala Pro Gly Lys Gly Leu
Glu 50 55 60 Trp Ile Gly Phe Ile Asn Thr Gly Gly Thr Ile Val Tyr
Ala Ser Trp 65 70 75 80 Ala Lys Gly Arg Phe Thr Ile Ser Lys Thr Ser
Thr Thr Val Asp Leu 85 90 95 Lys Met Thr Ser Pro Thr Thr Glu Asp
Thr Ala Thr Tyr Phe Cys Ala 100 105 110 Arg Gly Ser Tyr Val Ser Ser
Gly Tyr Ala Tyr Tyr Phe Asn Val 115 120 125 44413PRTOryctolagus
cuniculus 444Gln Ser Ser Lys Ser Val Met Asn Asn Asn Tyr Leu Ala 1
5 10 4457PRTOryctolagus cuniculus 445Gly Ala Ser Asn Leu Ala Ser 1
5 44612PRTOryctolagus cuniculus 446Gln Gly Gly Tyr Thr Gly Tyr Ser
Asp His Gly Thr 1 5 10 4475PRTOryctolagus cuniculus 447Ser Tyr Pro
Met Asn 1 5 44816PRTOryctolagus cuniculus 448Phe Ile Asn Thr Gly
Gly Thr Ile Val Tyr Ala Ser Trp Ala Lys Gly 1 5 10 15
44914PRTOryctolagus cuniculus 449Gly Ser Tyr Val Ser Ser Gly Tyr
Ala Tyr Tyr Phe Asn Val 1 5 10 450372DNAOryctolagus cuniculus
450atggacacga gggcccccac tcagctgctg gggctcctgc tgctctggct
cccaggtgcc 60acatttgccg ccgtgctgac ccagactcca tctcccgtgt ctgaacctgt
gggaggcaca 120gtcagcatca gttgccagtc cagtaagagt gttatgaata
acaactactt agcctggtat 180cagcagaaac cagggcagcc tcccaagctc
ctgatctatg gtgcatccaa tctggcatct 240ggggtcccat cacggttcag
cggcagtgga tctgggacac agttcactct caccatcagc 300gacgtgcagt
gtgacgatgc tgccacttac tactgtcaag gcggttatac tggttatagt
360gatcatggga ct 372451381DNAOryctolagus cuniculus 451atggagactg
ggctgcgctg gcttctcctg gtcgctgtgc tcaaaggtgt ccagtgtcag 60tcggtggagg
agtccggggg tcgcctggtc aagcctgacg aaaccctgac actcacctgc
120acagtctctg gaatcgacct cagtagctat ccaatgaact gggtccgcca
ggctccaggg 180aaggggctgg aatggatcgg attcattaat actggtggta
ccatagtcta cgcgagctgg 240gcaaaaggcc gattcaccat ctccaaaacc
tcgaccacgg tggatctgaa aatgaccagt 300ccgacaaccg aggacacggc
cacctatttc tgtgccagag gcagttatgt ttcatctggt 360tatgcctact
attttaatgt c 38145239DNAOryctolagus cuniculus 452cagtccagta
agagtgttat gaataacaac tacttagcc 3945321DNAOryctolagus cuniculus
453ggtgcatcca atctggcatc t 2145436DNAOryctolagus cuniculus
454caaggcggtt atactggtta tagtgatcat gggact 3645515DNAOryctolagus
cuniculus 455agctatccaa tgaac 1545648DNAOryctolagus cuniculus
456ttcattaata ctggtggtac catagtctac gcgagctggg caaaaggc
4845742DNAOryctolagus cuniculus 457ggcagttatg tttcatctgg ttatgcctac
tattttaatg tc 42458121PRTOryctolagus cuniculus 458Met Asp Thr Arg
Ala Pro Thr Gln Leu Leu Gly Leu Leu Leu Leu Trp 1 5 10 15 Leu Pro
Gly Ala Thr Phe Ala Ala Val Leu Thr Gln Thr Pro Ser Pro 20 25 30
Val Ser Ala Ala Val Gly Gly Thr Val Ser Ile Ser Cys Gln Ser Ser 35
40 45 Gln Ser Val Tyr Asn Asn Asn Trp Leu Ser Trp Phe Gln Gln Lys
Pro 50 55 60 Gly Gln Pro Pro Lys Leu Leu Ile Tyr Lys Ala Ser Thr
Leu Ala Ser 65 70 75 80 Gly Val Pro Ser Arg Phe Lys Gly Ser Gly Ser
Gly Thr Gln Phe Thr 85 90 95 Leu Thr Ile Ser Asp Val Gln Cys Asp
Asp Val Ala Thr Tyr Tyr Cys 100 105 110 Ala Gly Gly Tyr Leu Asp Ser
Val Ile 115 120 459126PRTOryctolagus cuniculus 459Met Glu Thr Gly
Leu Arg Trp Leu Leu Leu Val Ala Val Leu Lys Gly 1 5 10 15 Val Gln
Cys Gln Ser Val Glu Glu Ser Gly Gly Arg Leu Val Thr Pro 20 25 30
Gly Thr Pro Leu Thr Leu Thr Cys Thr Val Ser Gly Phe Ser Leu Ser 35
40 45 Thr Tyr Ser Ile Asn Trp Val Arg Gln Ala Pro Gly Lys Gly Leu
Glu 50 55 60 Trp Ile Gly Ile Ile Ala Asn Ser Gly Thr Thr Phe Tyr
Ala Asn Trp 65 70 75 80 Ala Lys Gly Arg Phe Thr Val Ser Lys Thr Ser
Thr Thr Val Asp Leu 85 90 95 Lys Ile Thr Ser Pro Thr Thr Glu Asp
Thr Ala Thr Tyr Phe Cys Ala 100 105 110 Arg Glu Ser Gly Met Tyr Asn
Glu Tyr Gly Lys Phe Asn Ile 115 120 125 46013PRTOryctolagus
cuniculus 460Gln Ser Ser Gln Ser Val Tyr Asn Asn Asn Trp Leu Ser 1
5 10 4617PRTOryctolagus cuniculus 461Lys Ala Ser Thr Leu Ala Ser 1
5 4629PRTOryctolagus cuniculus 462Ala Gly Gly Tyr Leu Asp Ser Val
Ile 1 5 4635PRTOryctolagus cuniculus 463Thr Tyr Ser Ile Asn 1 5
46416PRTOryctolagus cuniculus 464Ile Ile Ala Asn Ser Gly Thr Thr
Phe Tyr Ala Asn Trp Ala Lys Gly 1 5 10 15 46513PRTOryctolagus
cuniculus 465Glu Ser Gly Met Tyr Asn Glu Tyr Gly Lys Phe Asn Ile 1
5 10 466363DNAOryctolagus cuniculus 466atggacacga gggcccccac
tcagctgctg gggctcctgc tgctctggct cccaggtgcc 60acatttgccg ccgtgctgac
ccagactcca tctcccgtgt ctgcagctgt gggaggcaca 120gtcagcatca
gttgccagtc cagtcagagt gtttataata acaactggtt atcctggttt
180cagcagaaac cagggcagcc tcccaagctc ctgatctaca aggcatccac
tctggcatct 240ggggtcccat cgcggttcaa aggcagtgga tctgggacac
agttcactct caccatcagc 300gacgtgcagt gtgacgatgt tgccacttac
tactgtgcgg gcggttatct tgatagtgtt 360att 363467378DNAOryctolagus
cuniculus 467atggagactg ggctgcgctg gcttctcctg gtcgctgtgc tcaaaggtgt
ccagtgtcag 60tcggtggagg agtccggggg tcgcctggtc acgcctggga cacccctgac
actcacctgc 120acagtctctg gattctccct cagtacctat tcaataaact
gggtccgcca ggctccaggg 180aagggcctgg aatggatcgg aatcattgct
aatagtggta ccacattcta cgcgaactgg 240gcgaaaggcc gattcaccgt
ctccaaaacc tcgaccacgg tggatctgaa aatcaccagt 300ccgacaaccg
aggacacggc cacctatttc tgtgccagag agagtggaat gtacaatgaa
360tatggtaaat ttaacatc 37846839DNAOryctolagus cuniculus
468cagtccagtc agagtgttta taataacaac tggttatcc 3946921DNAOryctolagus
cuniculus 469aaggcatcca ctctggcatc t 2147027DNAOryctolagus
cuniculus 470gcgggcggtt atcttgatag tgttatt 2747115DNAOryctolagus
cuniculus 471acctattcaa taaac 1547248DNAOryctolagus cuniculus
472atcattgcta atagtggtac cacattctac gcgaactggg cgaaaggc
4847339DNAOryctolagus cuniculus 473gagagtggaa tgtacaatga atatggtaaa
tttaacatc 39474122PRTOryctolagus cuniculus 474Met Asp Thr Arg Ala
Pro Thr Gln Leu Leu Gly Leu Leu Leu Leu Trp 1 5 10 15 Leu Pro Gly
Ala Arg Cys Ala Ser Asp Met Thr Gln Thr Pro Ser Ser 20 25 30 Val
Ser Ala Ala Val Gly Gly Thr Val Thr Ile Asn Cys Gln Ala Ser 35 40
45 Glu Asn Ile Tyr Ser Phe Leu Ala Trp Tyr Gln Gln Lys Pro Gly Gln
50 55 60 Pro Pro Lys Leu Leu Ile Phe Lys Ala Ser Thr Leu Ala Ser
Gly Val 65 70 75 80 Ser Ser Arg Phe Lys Gly Ser Gly Ser Gly Thr Gln
Phe Thr Leu Thr 85 90 95 Ile Ser Asp Leu Glu Cys Asp Asp Ala Ala
Thr Tyr Tyr Cys Gln Gln 100 105 110 Gly Ala Thr Val Tyr Asp Ile Asp
Asn Asn 115 120 475128PRTOryctolagus cuniculus 475Met Glu Thr Gly
Leu Arg Trp Leu Leu Leu Val Ala Val Leu Lys Gly 1 5 10 15 Val Gln
Cys Gln Ser Leu Glu Glu Ser Gly Gly Arg Leu Val Thr Pro 20 25 30
Gly Thr Pro Leu Thr Leu Thr Cys Thr Val Ser Gly Ile Asp Leu Ser 35
40 45 Ala Tyr Ala Met Ile Trp Val Arg Gln Ala Pro Gly Glu Gly Leu
Glu 50 55 60 Trp Ile Thr Ile Ile Tyr Pro Asn Gly Ile Thr Tyr Tyr
Ala Asn Trp 65 70 75 80 Ala Lys Gly Arg Phe Thr Val Ser Lys Thr Ser
Thr Ala Met Asp Leu 85 90 95 Lys Ile Thr Ser Pro Thr Thr Glu Asp
Thr Ala Thr Tyr Phe Cys Ala 100 105 110 Arg Asp Ala Glu Ser Ser Lys
Asn Ala Tyr Trp Gly Tyr Phe Asn Val 115 120
125 47611PRTOryctolagus cuniculus 476Gln Ala Ser Glu Asn Ile Tyr
Ser Phe Leu Ala 1 5 10 4777PRTOryctolagus cuniculus 477Lys Ala Ser
Thr Leu Ala Ser 1 5 47812PRTOryctolagus cuniculus 478Gln Gln Gly
Ala Thr Val Tyr Asp Ile Asp Asn Asn 1 5 10 4795PRTOryctolagus
cuniculus 479Ala Tyr Ala Met Ile 1 5 48016PRTOryctolagus cuniculus
480Ile Ile Tyr Pro Asn Gly Ile Thr Tyr Tyr Ala Asn Trp Ala Lys Gly
1 5 10 15 48115PRTOryctolagus cuniculus 481Asp Ala Glu Ser Ser Lys
Asn Ala Tyr Trp Gly Tyr Phe Asn Val 1 5 10 15 482366DNAOryctolagus
cuniculus 482atggacacga gggcccccac tcagctgctg gggctcctgc tgctctggct
cccaggtgcc 60agatgtgcct ctgatatgac ccagactcca tcctccgtgt ctgcagctgt
gggaggcaca 120gtcaccatca attgccaggc cagtgagaac atttatagct
ttttggcctg gtatcagcag 180aaaccagggc agcctcccaa gctcctgatc
ttcaaggctt ccactctggc atctggggtc 240tcatcgcggt tcaaaggcag
tggatctggg acacagttca ctctcaccat cagcgacctg 300gagtgtgacg
atgctgccac ttactactgt caacagggtg ctactgtgta tgatattgat 360aataat
366483384DNAOryctolagus cuniculus 483atggagactg ggctgcgctg
gcttctcctg gtcgctgtgc tcaaaggtgt ccagtgtcag 60tcgctggagg agtccggggg
tcgcctggtc acgcctggga cacccctgac actcacctgc 120acagtttctg
gaatcgacct cagtgcctat gcaatgatct gggtccgcca ggctccaggg
180gaggggctgg aatggatcac aatcatttat cctaatggta tcacatacta
cgcgaactgg 240gcgaaaggcc gattcaccgt ctccaaaacc tcgaccgcga
tggatctgaa aatcaccagt 300ccgacaaccg aggacacggc cacctatttc
tgtgccagag atgcagaaag tagtaagaat 360gcttattggg gctactttaa cgtc
38448433DNAOryctolagus cuniculus 484caggccagtg agaacattta
tagctttttg gcc 3348521DNAOryctolagus cuniculus 485aaggcttcca
ctctggcatc t 2148636DNAOryctolagus cuniculus 486caacagggtg
ctactgtgta tgatattgat aataat 3648715DNAOryctolagus cuniculus
487gcctatgcaa tgatc 1548848DNAOryctolagus cuniculus 488atcatttatc
ctaatggtat cacatactac gcgaactggg cgaaaggc 4848945DNAOryctolagus
cuniculus 489gatgcagaaa gtagtaagaa tgcttattgg ggctacttta acgtc
45490122PRTOryctolagus cuniculus 490Met Asp Thr Arg Ala Pro Thr Gln
Leu Leu Gly Leu Leu Leu Leu Trp 1 5 10 15 Leu Pro Gly Ala Arg Cys
Ala Ser Asp Met Thr Gln Thr Pro Ser Ser 20 25 30 Val Ser Ala Ala
Val Gly Gly Thr Val Thr Ile Asn Cys Gln Ala Ser 35 40 45 Glu Asn
Ile Tyr Ser Phe Leu Ala Trp Tyr Gln Gln Lys Pro Gly Gln 50 55 60
Pro Pro Lys Leu Leu Ile Phe Arg Ala Ser Thr Leu Ala Ser Gly Val 65
70 75 80 Ser Ser Arg Phe Lys Gly Ser Gly Ser Gly Thr Gln Phe Thr
Leu Thr 85 90 95 Ile Ser Asp Leu Glu Cys Asp Asp Ala Ala Thr Tyr
Tyr Cys Gln Gln 100 105 110 Gly Ala Thr Val Tyr Asp Ile Asp Asn Asn
115 120 491128PRTOryctolagus cuniculus 491Met Glu Thr Gly Leu Arg
Trp Leu Leu Leu Val Ala Val Leu Lys Gly 1 5 10 15 Val Gln Cys Gln
Ser Leu Glu Glu Ser Gly Gly Arg Leu Val Thr Pro 20 25 30 Gly Thr
Pro Leu Thr Leu Thr Cys Thr Val Ser Gly Ile Asp Leu Ser 35 40 45
Ala Tyr Ala Met Ile Trp Val Arg Gln Ala Pro Gly Glu Gly Leu Glu 50
55 60 Trp Ile Thr Ile Ile Tyr Pro Asn Gly Ile Thr Tyr Tyr Ala Asn
Trp 65 70 75 80 Ala Lys Gly Arg Phe Thr Val Ser Lys Thr Ser Thr Ala
Met Asp Leu 85 90 95 Lys Ile Thr Ser Pro Thr Thr Glu Asp Thr Ala
Thr Tyr Phe Cys Ala 100 105 110 Arg Asp Ala Glu Ser Ser Lys Asn Ala
Tyr Trp Gly Tyr Phe Asn Val 115 120 125 49211PRTOryctolagus
cuniculus 492Gln Ala Ser Glu Asn Ile Tyr Ser Phe Leu Ala 1 5 10
4937PRTOryctolagus cuniculus 493Arg Ala Ser Thr Leu Ala Ser 1 5
49412PRTOryctolagus cuniculus 494Gln Gln Gly Ala Thr Val Tyr Asp
Ile Asp Asn Asn 1 5 10 4955PRTOryctolagus cuniculus 495Ala Tyr Ala
Met Ile 1 5 49616PRTOryctolagus cuniculus 496Ile Ile Tyr Pro Asn
Gly Ile Thr Tyr Tyr Ala Asn Trp Ala Lys Gly 1 5 10 15
49715PRTOryctolagus cuniculus 497Asp Ala Glu Ser Ser Lys Asn Ala
Tyr Trp Gly Tyr Phe Asn Val 1 5 10 15 498366DNAOryctolagus
cuniculus 498atggacacga gggcccccac tcagctgctg gggctcctgc tgctctggct
cccaggtgcc 60agatgtgcct ctgatatgac ccagactcca tcctccgtgt ctgcagctgt
gggaggcaca 120gtcaccatca attgccaggc cagtgagaac atttatagct
ttttggcctg gtatcagcag 180aaaccagggc agcctcccaa gctcctgatc
ttcagggctt ccactctggc atctggggtc 240tcatcgcggt tcaaaggcag
tggatctggg acacagttca ctctcaccat cagcgacctg 300gagtgtgacg
atgctgccac ttactactgt caacagggtg ctactgtgta tgatattgat 360aataat
366499384DNAOryctolagus cuniculus 499atggagactg ggctgcgctg
gcttctcctg gtcgctgtgc tcaaaggtgt ccagtgtcag 60tcgctggagg agtccggggg
tcgcctggtc acgcctggga cacccctgac actcacctgc 120acagtttctg
gaatcgacct cagtgcctat gcaatgatct gggtccgcca ggctccaggg
180gaggggctgg aatggatcac aatcatttat cctaatggta tcacatacta
cgcgaactgg 240gcgaaaggcc gattcaccgt ctccaaaacc tcgaccgcga
tggatctgaa aatcaccagt 300ccgacaaccg aggacacggc cacctatttc
tgtgccagag atgcagaaag tagtaagaat 360gcttattggg gctactttaa cgtc
38450033DNAOryctolagus cuniculus 500caggccagtg agaacattta
tagctttttg gcc 3350121DNAOryctolagus cuniculus 501agggcttcca
ctctggcatc t 2150236DNAOryctolagus cuniculus 502caacagggtg
ctactgtgta tgatattgat aataat 3650315DNAOryctolagus cuniculus
503gcctatgcaa tgatc 1550448DNAOryctolagus cuniculus 504atcatttatc
ctaatggtat cacatactac gcgaactggg cgaaaggc 4850545DNAOryctolagus
cuniculus 505gatgcagaaa gtagtaagaa tgcttattgg ggctacttta acgtc
45506124PRTOryctolagus cuniculus 506Met Asp Thr Arg Ala Pro Thr Gln
Leu Leu Gly Leu Leu Leu Leu Trp 1 5 10 15 Leu Pro Gly Ala Thr Phe
Ala Ile Glu Met Thr Gln Thr Pro Ser Pro 20 25 30 Val Ser Ala Ala
Val Gly Gly Thr Val Thr Ile Asn Cys Gln Ala Ser 35 40 45 Glu Ser
Val Phe Asn Asn Met Leu Ser Trp Tyr Gln Gln Lys Pro Gly 50 55 60
His Ser Pro Lys Leu Leu Ile Tyr Asp Ala Ser Asp Leu Ala Ser Gly 65
70 75 80 Val Pro Ser Arg Phe Lys Gly Ser Gly Ser Gly Thr Gln Phe
Thr Leu 85 90 95 Thr Ile Ser Gly Val Glu Cys Asp Asp Ala Ala Thr
Tyr Tyr Cys Ala 100 105 110 Gly Tyr Lys Ser Asp Ser Asn Asp Gly Asp
Asn Val 115 120 507123PRTOryctolagus cuniculus 507Met Glu Thr Gly
Leu Arg Trp Leu Leu Leu Val Ala Val Leu Lys Gly 1 5 10 15 Val Gln
Cys Gln Ser Leu Glu Glu Ser Gly Gly Arg Leu Val Thr Pro 20 25 30
Gly Thr Pro Leu Thr Leu Thr Cys Thr Val Ser Gly Phe Ser Leu Asn 35
40 45 Arg Asn Ser Ile Thr Trp Val Arg Gln Ala Pro Gly Glu Gly Leu
Glu 50 55 60 Trp Ile Gly Ile Ile Thr Gly Ser Gly Arg Thr Tyr Tyr
Ala Asn Trp 65 70 75 80 Ala Lys Gly Arg Phe Thr Ile Ser Lys Thr Ser
Thr Thr Val Asp Leu 85 90 95 Lys Met Thr Ser Pro Thr Thr Glu Asp
Thr Ala Thr Tyr Phe Cys Ala 100 105 110 Arg Gly His Pro Gly Leu Gly
Ser Gly Asn Ile 115 120 50812PRTOryctolagus cuniculus 508Gln Ala
Ser Glu Ser Val Phe Asn Asn Met Leu Ser 1 5 10 5097PRTOryctolagus
cuniculus 509Asp Ala Ser Asp Leu Ala Ser 1 5 51013PRTOryctolagus
cuniculus 510Ala Gly Tyr Lys Ser Asp Ser Asn Asp Gly Asp Asn Val 1
5 10 5115PRTOryctolagus cuniculus 511Arg Asn Ser Ile Thr 1 5
51216PRTOryctolagus cuniculus 512Ile Ile Thr Gly Ser Gly Arg Thr
Tyr Tyr Ala Asn Trp Ala Lys Gly 1 5 10 15 51310PRTOryctolagus
cuniculus 513Gly His Pro Gly Leu Gly Ser Gly Asn Ile 1 5 10
514372DNAOryctolagus cuniculus 514atggacacga gggcccccac tcagctgctg
gggctcctgc tgctctggct cccaggtgcc 60acatttgcca ttgaaatgac ccagactcca
tcccccgtgt ctgccgctgt gggaggcaca 120gtcaccatca attgccaggc
cagtgagagt gtttttaata atatgttatc ctggtatcag 180cagaaaccag
ggcactctcc taagctcctg atctatgatg catccgatct ggcatctggg
240gtcccatcgc ggttcaaagg cagtggatct gggacacagt tcactctcac
catcagtggc 300gtggagtgtg acgatgctgc cacttactat tgtgcagggt
ataaaagtga tagtaatgat 360ggcgataatg tt 372515369DNAOryctolagus
cuniculus 515atggagactg ggctgcgctg gcttctcctg gtcgctgtgc tcaaaggtgt
ccagtgtcag 60tcgctggagg agtccggggg tcgcctggtc acgcctggga cacccctgac
actcacctgc 120acagtctctg gattctccct caacaggaat tcaataacct
gggtccgcca ggctccaggg 180gaggggctgg aatggatcgg aatcattact
ggtagtggta gaacgtacta cgcgaactgg 240gcaaaaggcc gattcaccat
ctccaaaacc tcgaccacgg tggatctgaa aatgaccagt 300ccgacaaccg
aggacacggc cacctatttc tgtgccagag gccatcctgg tcttggtagt 360ggtaacatc
36951636DNAOryctolagus cuniculus 516caggccagtg agagtgtttt
taataatatg ttatcc 3651721DNAOryctolagus cuniculus 517gatgcatccg
atctggcatc t 2151839DNAOryctolagus cuniculus 518gcagggtata
aaagtgatag taatgatggc gataatgtt 3951915DNAOryctolagus cuniculus
519aggaattcaa taacc 1552048DNAOryctolagus cuniculus 520atcattactg
gtagtggtag aacgtactac gcgaactggg caaaaggc 4852130DNAOryctolagus
cuniculus 521ggccatcctg gtcttggtag tggtaacatc
30522121PRTOryctolagus cuniculus 522Met Asp Thr Arg Ala Pro Thr Gln
Leu Leu Gly Leu Leu Leu Leu Trp 1 5 10 15 Leu Pro Gly Ala Thr Phe
Ala Gln Val Leu Thr Gln Thr Ala Ser Ser 20 25 30 Val Ser Ala Ala
Val Gly Gly Thr Val Thr Ile Asn Cys Gln Ser Ser 35 40 45 Gln Ser
Val Tyr Asn Asn Tyr Leu Ser Trp Tyr Gln Gln Lys Pro Gly 50 55 60
Gln Pro Pro Lys Leu Leu Ile Tyr Thr Ala Ser Ser Leu Ala Ser Gly 65
70 75 80 Val Pro Ser Arg Phe Lys Gly Ser Gly Ser Gly Thr Gln Phe
Thr Leu 85 90 95 Thr Ile Ser Glu Val Gln Cys Asp Asp Ala Ala Thr
Tyr Tyr Cys Gln 100 105 110 Gly Tyr Tyr Ser Gly Pro Ile Ile Thr 115
120 523122PRTOryctolagus cuniculus 523Met Glu Thr Gly Leu Arg Trp
Leu Leu Leu Val Ala Val Leu Lys Gly 1 5 10 15 Val Gln Cys Gln Ser
Leu Glu Glu Ser Gly Gly Arg Leu Val Thr Pro 20 25 30 Gly Thr Pro
Leu Thr Leu Thr Cys Thr Ala Ser Gly Phe Ser Leu Asn 35 40 45 Asn
Tyr Tyr Ile Gln Trp Val Arg Gln Ala Pro Gly Glu Gly Leu Glu 50 55
60 Trp Ile Gly Ile Ile Tyr Ala Gly Gly Ser Ala Tyr Tyr Ala Thr Trp
65 70 75 80 Ala Asn Gly Arg Phe Thr Ile Ala Lys Thr Ser Ser Thr Thr
Val Asp 85 90 95 Leu Lys Met Thr Ser Leu Thr Thr Glu Asp Thr Ala
Thr Tyr Phe Cys 100 105 110 Ala Arg Gly Thr Phe Asp Gly Tyr Glu Leu
115 120 52412PRTOryctolagus cuniculus 524Gln Ser Ser Gln Ser Val
Tyr Asn Asn Tyr Leu Ser 1 5 10 5257PRTOryctolagus cuniculus 525Thr
Ala Ser Ser Leu Ala Ser 1 5 52610PRTOryctolagus cuniculus 526Gln
Gly Tyr Tyr Ser Gly Pro Ile Ile Thr 1 5 10 5275PRTOryctolagus
cuniculus 527Asn Tyr Tyr Ile Gln 1 5 52816PRTOryctolagus cuniculus
528Ile Ile Tyr Ala Gly Gly Ser Ala Tyr Tyr Ala Thr Trp Ala Asn Gly
1 5 10 15 5298PRTOryctolagus cuniculus 529Gly Thr Phe Asp Gly Tyr
Glu Leu 1 5 530363DNAOryctolagus cuniculus 530atggacacga gggcccccac
tcagctgctg gggctcctgc tgctctggct cccaggtgcc 60acatttgcgc aagtgctgac
ccagactgca tcgtccgtgt ctgcagctgt gggaggcaca 120gtcaccatca
attgccagtc cagtcagagt gtttataata actacttatc ctggtatcag
180cagaaaccag ggcagcctcc caagctcctg atctatactg catccagcct
ggcatctggg 240gtcccatcgc ggttcaaagg cagtggatct gggacacagt
tcactctcac catcagcgaa 300gtgcagtgtg acgatgctgc cacttactac
tgtcaaggct attatagtgg tcctataatt 360act 363531366DNAOryctolagus
cuniculus 531atggagactg ggctgcgctg gcttctcctg gtcgctgtgc tcaaaggtgt
ccagtgtcag 60tcgctggagg agtccggggg tcgcctggtc acgcctggga cacccctgac
actcacctgc 120acagcctctg gattctccct caataactac tacatacaat
gggtccgcca ggctccaggg 180gaggggctgg aatggatcgg gatcatttat
gctggtggta gcgcatacta cgcgacctgg 240gcaaacggcc gattcaccat
cgccaaaacc tcgtcgacca cggtggatct gaagatgacc 300agtctgacaa
ccgaggacac ggccacctat ttctgtgcca gagggacatt tgatggttat 360gagttg
36653236DNAOryctolagus cuniculus 532cagtccagtc agagtgttta
taataactac ttatcc 3653321DNAOryctolagus cuniculus 533actgcatcca
gcctggcatc t 2153430DNAOryctolagus cuniculus 534caaggctatt
atagtggtcc tataattact 3053515DNAOryctolagus cuniculus 535aactactaca
tacaa 1553648DNAOryctolagus cuniculus 536atcatttatg ctggtggtag
cgcatactac gcgacctggg caaacggc 4853724DNAOryctolagus cuniculus
537gggacatttg atggttatga gttg 24538122PRTOryctolagus cuniculus
538Met Asp Thr Arg Ala Pro Thr Gln Leu Leu Gly Leu Leu Leu Leu Trp
1 5 10 15 Leu Pro Gly Ala Thr Phe Ala Gln Val Leu Thr Gln Thr Pro
Ser Pro 20 25 30 Val Ser Val Pro Val Gly Asp Thr Val Thr Ile Ser
Cys Gln Ser Ser 35 40 45 Glu Ser Val Tyr Ser Asn Asn Leu Leu Ser
Trp Tyr Gln Gln Lys Pro 50 55 60 Gly Gln Pro Pro Lys Leu Leu Ile
Tyr Arg Ala Ser Asn Leu Ala Ser 65 70 75 80 Gly Val Pro Ser Arg Phe
Lys Gly Ser Gly Ser Gly Thr Gln Phe Thr 85 90 95 Leu Thr Ile Ser
Gly Ala Gln Cys Asp Asp Ala Ala Thr Tyr Tyr Cys 100 105 110 Gln Gly
Tyr Tyr Ser Gly Val Ile Asn Ser 115 120 539124PRTOryctolagus
cuniculus 539Met Glu Thr Gly Leu Arg Trp Leu Leu Leu Val Ala Val
Leu Lys Gly 1 5 10 15 Val Gln Cys Gln Ser Val Glu Glu Ser Gly Gly
Arg Leu Val Thr Pro 20 25 30 Gly Thr Pro Leu Thr Leu Thr Cys Thr
Val Ser Gly Phe Ser Leu Ser 35 40 45 Ser Tyr Phe Met Ser Trp Val
Arg Gln Ala Pro Gly Glu Gly Leu Glu 50 55 60 Tyr Ile Gly Phe Ile
Asn Pro Gly Gly Ser Ala Tyr Tyr Ala Ser Trp 65 70 75 80 Ala Ser Gly
Arg Leu Thr Ile Ser Lys Thr Ser Thr Thr Val Asp Leu 85 90 95 Lys
Ile Thr Ser Pro Thr Thr Glu Asp Thr Ala Thr Tyr Phe Cys Ala 100 105
110 Arg Ile Leu Ile Val Ser Tyr Gly Ala Phe Thr Ile 115 120
54013PRTOryctolagus cuniculus 540Gln Ser Ser Glu Ser Val Tyr Ser
Asn Asn Leu Leu Ser 1 5 10 5417PRTOryctolagus cuniculus 541Arg Ala
Ser Asn Leu Ala Ser 1 5 54210PRTOryctolagus cuniculus 542Gln Gly
Tyr Tyr Ser Gly Val Ile Asn Ser 1 5 10
5435PRTOryctolagus cuniculus 543Ser Tyr Phe Met Ser 1 5
54416PRTOryctolagus cuniculus 544Phe Ile Asn Pro Gly Gly Ser Ala
Tyr Tyr Ala Ser Trp Ala Ser Gly 1 5 10 15 54511PRTOryctolagus
cuniculus 545Ile Leu Ile Val Ser Tyr Gly Ala Phe Thr Ile 1 5 10
546366DNAOryctolagus cuniculus 546atggacacga gggcccccac tcagctgctg
gggctcctgc tgctctggct cccaggtgcc 60acatttgccc aagtgctgac ccagactcca
tcccctgtgt ctgtccctgt gggagacaca 120gtcaccatca gttgccagtc
cagtgagagc gtttatagta ataacctctt atcctggtat 180cagcagaaac
cagggcagcc tcccaagctc ctgatctaca gggcatccaa tctggcatct
240ggtgtcccat cgcggttcaa aggcagtgga tctgggacac agttcactct
caccatcagc 300ggcgcacagt gtgacgatgc tgccacttac tactgtcaag
gctattatag tggtgtcatt 360aatagt 366547372DNAOryctolagus cuniculus
547atggagactg ggctgcgctg gcttctcctg gtcgctgtgc tcaaaggtgt
ccagtgtcag 60tcggtggagg agtccggggg tcgcctggtc acgcctggga cacccctgac
actcacctgc 120acagtgtctg gattctccct cagtagctac ttcatgagct
gggtccgcca ggctccaggg 180gaggggctgg aatacatcgg attcattaat
cctggtggta gcgcatacta cgcgagctgg 240gcgagtggcc gactcaccat
ctccaaaacc tcgaccacgg tagatctgaa aatcaccagt 300ccgacaaccg
aggacacggc cacctatttc tgtgccagga ttcttattgt ttcttatgga
360gcctttacca tc 37254839DNAOryctolagus cuniculus 548cagtccagtg
agagcgttta tagtaataac ctcttatcc 3954921DNAOryctolagus cuniculus
549agggcatcca atctggcatc t 2155030DNAOryctolagus cuniculus
550caaggctatt atagtggtgt cattaatagt 3055115DNAOryctolagus cuniculus
551agctacttca tgagc 1555248DNAOryctolagus cuniculus 552ttcattaatc
ctggtggtag cgcatactac gcgagctggg cgagtggc 4855333DNAOryctolagus
cuniculus 553attcttattg tttcttatgg agcctttacc atc
33554122PRTOryctolagus cuniculus 554Met Asp Thr Arg Ala Pro Thr Gln
Leu Leu Gly Leu Leu Leu Leu Trp 1 5 10 15 Leu Pro Gly Ala Arg Cys
Ala Tyr Asp Met Thr Gln Thr Pro Ala Ser 20 25 30 Val Glu Val Ala
Val Gly Gly Thr Val Thr Ile Lys Cys Gln Ala Thr 35 40 45 Glu Ser
Ile Gly Asn Glu Leu Ser Trp Tyr Gln Gln Lys Pro Gly Gln 50 55 60
Ala Pro Lys Leu Leu Ile Tyr Ser Ala Ser Thr Leu Ala Ser Gly Val 65
70 75 80 Pro Ser Arg Phe Lys Gly Ser Gly Ser Gly Thr Gln Phe Thr
Leu Thr 85 90 95 Ile Thr Gly Val Glu Cys Asp Asp Ala Ala Thr Tyr
Tyr Cys Gln Gln 100 105 110 Gly Tyr Ser Ser Ala Asn Ile Asp Asn Ala
115 120 555128PRTOryctolagus cuniculus 555Met Glu Thr Gly Leu Arg
Trp Leu Leu Leu Val Ala Val Leu Lys Gly 1 5 10 15 Val Gln Cys Gln
Ser Leu Glu Glu Ser Gly Gly Arg Leu Val Thr Pro 20 25 30 Gly Thr
Pro Leu Thr Leu Thr Cys Thr Val Ser Gly Phe Ser Leu Ser 35 40 45
Lys Tyr Tyr Met Ser Trp Val Arg Gln Ala Pro Glu Lys Gly Leu Lys 50
55 60 Tyr Ile Gly Tyr Ile Asp Ser Thr Thr Val Asn Thr Tyr Tyr Ala
Thr 65 70 75 80 Trp Ala Arg Gly Arg Phe Thr Ile Ser Lys Thr Ser Thr
Thr Val Asp 85 90 95 Leu Lys Ile Thr Ser Pro Thr Ser Glu Asp Thr
Ala Thr Tyr Phe Cys 100 105 110 Ala Arg Gly Ser Thr Tyr Phe Thr Asp
Gly Gly His Arg Leu Asp Leu 115 120 125 55611PRTOryctolagus
cuniculus 556Gln Ala Thr Glu Ser Ile Gly Asn Glu Leu Ser 1 5 10
5577PRTOryctolagus cuniculus 557Ser Ala Ser Thr Leu Ala Ser 1 5
55812PRTOryctolagus cuniculus 558Gln Gln Gly Tyr Ser Ser Ala Asn
Ile Asp Asn Ala 1 5 10 5595PRTOryctolagus cuniculus 559Lys Tyr Tyr
Met Ser 1 5 56017PRTOryctolagus cuniculus 560Tyr Ile Asp Ser Thr
Thr Val Asn Thr Tyr Tyr Ala Thr Trp Ala Arg 1 5 10 15 Gly
56114PRTOryctolagus cuniculus 561Gly Ser Thr Tyr Phe Thr Asp Gly
Gly His Arg Leu Asp Leu 1 5 10 562366DNAOryctolagus cuniculus
562atggacacga gggcccccac tcagctgctg gggctcctgc tgctctggct
cccaggtgcc 60agatgtgcct atgatatgac ccagactcca gcctctgtgg aggtagctgt
gggaggcaca 120gtcaccatca agtgccaggc cactgagagc attggcaatg
agttatcctg gtatcagcag 180aaaccagggc aggctcccaa gctcctgatc
tattctgcat ccactctggc atctggggtc 240ccatcgcggt tcaaaggcag
tggatctggg acacagttca ctctcaccat caccggcgtg 300gagtgtgatg
atgctgccac ttactactgt caacagggtt atagtagtgc taatattgat 360aatgct
366563384DNAOryctolagus cuniculus 563atggagactg ggctgcgctg
gcttctcctg gtcgctgtgc tcaaaggtgt ccagtgtcag 60tcgctggagg agtccggggg
tcgcctggtc acgcctggga cacccctgac actcacctgc 120accgtctctg
gattctccct cagtaagtac tacatgagct gggtccgcca ggctccagag
180aaggggctga aatacatcgg atacattgat agtactactg ttaatacata
ctacgcgacc 240tgggcgagag gccgattcac catctccaaa acctcgacca
cggtggatct gaagatcacc 300agtccgacaa gtgaggacac ggccacctat
ttctgtgcca gaggaagtac ttattttact 360gatggaggcc atcggttgga tctc
38456433DNAOryctolagus cuniculus 564caggccactg agagcattgg
caatgagtta tcc 3356521DNAOryctolagus cuniculus 565tctgcatcca
ctctggcatc t 2156636DNAOryctolagus cuniculus 566caacagggtt
atagtagtgc taatattgat aatgct 3656715DNAOryctolagus cuniculus
567aagtactaca tgagc 1556851DNAOryctolagus cuniculus 568tacattgata
gtactactgt taatacatac tacgcgacct gggcgagagg c 5156942DNAOryctolagus
cuniculus 569ggaagtactt attttactga tggaggccat cggttggatc tc
42570122PRTOryctolagus cuniculus 570Met Asp Thr Arg Ala Pro Thr Gln
Leu Leu Gly Leu Leu Leu Leu Trp 1 5 10 15 Leu Pro Gly Ala Arg Cys
Ala Tyr Asp Met Thr Gln Thr Pro Ala Ser 20 25 30 Val Glu Val Ala
Val Gly Gly Thr Val Thr Ile Lys Cys Gln Ala Thr 35 40 45 Glu Ser
Ile Gly Asn Glu Leu Ser Trp Tyr Gln Gln Lys Pro Gly Gln 50 55 60
Ala Pro Lys Leu Leu Ile Tyr Ser Ala Ser Thr Leu Ala Ser Gly Val 65
70 75 80 Pro Ser Arg Phe Lys Gly Ser Gly Ser Gly Thr Gln Phe Thr
Leu Thr 85 90 95 Ile Thr Gly Val Glu Cys Asp Asp Ala Ala Thr Tyr
Tyr Cys Gln Gln 100 105 110 Gly Tyr Ser Ser Ala Asn Ile Asp Asn Ala
115 120 571124PRTOryctolagus cuniculus 571Met Glu Thr Gly Leu Arg
Trp Leu Leu Leu Val Ala Val Leu Lys Gly 1 5 10 15 Val Gln Cys Gln
Ser Leu Glu Glu Ser Gly Gly Arg Leu Val Thr Pro 20 25 30 Gly Thr
Pro Leu Thr Leu Thr Cys Thr Val Ser Gly Phe Ser Leu Ser 35 40 45
Thr Tyr Asn Met Gly Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu 50
55 60 Trp Ile Gly Ser Ile Thr Ile Asp Gly Arg Thr Tyr Tyr Ala Ser
Trp 65 70 75 80 Ala Lys Gly Arg Phe Thr Val Ser Lys Ser Ser Thr Thr
Val Asp Leu 85 90 95 Lys Met Thr Ser Leu Thr Thr Gly Asp Thr Ala
Thr Tyr Phe Cys Ala 100 105 110 Arg Ile Leu Ile Val Ser Tyr Gly Ala
Phe Thr Ile 115 120 57211PRTOryctolagus cuniculus 572Gln Ala Thr
Glu Ser Ile Gly Asn Glu Leu Ser 1 5 10 5737PRTOryctolagus cuniculus
573Ser Ala Ser Thr Leu Ala Ser 1 5 57412PRTOryctolagus cuniculus
574Gln Gln Gly Tyr Ser Ser Ala Asn Ile Asp Asn Ala 1 5 10
5755PRTOryctolagus cuniculus 575Thr Tyr Asn Met Gly 1 5
57616PRTOryctolagus cuniculus 576Ser Ile Thr Ile Asp Gly Arg Thr
Tyr Tyr Ala Ser Trp Ala Lys Gly 1 5 10 15 57711PRTOryctolagus
cuniculus 577Ile Leu Ile Val Ser Tyr Gly Ala Phe Thr Ile 1 5 10
578366DNAOryctolagus cuniculus 578atggacacga gggcccccac tcagctgctg
gggctcctgc tgctctggct cccaggtgcc 60agatgtgcct atgatatgac ccagactcca
gcctctgtgg aggtagctgt gggaggcaca 120gtcaccatca agtgccaggc
cactgagagc attggcaatg agttatcctg gtatcagcag 180aaaccagggc
aggctcccaa gctcctgatc tattctgcat ccactctggc atctggggtc
240ccatcgcggt tcaaaggcag tggatctggg acacagttca ctctcaccat
caccggcgtg 300gagtgtgatg atgctgccac ttactactgt caacagggtt
atagtagtgc taatattgat 360aatgct 366579372DNAOryctolagus cuniculus
579atggagactg ggctgcgctg gcttctcctg gtcgctgtgc tcaaaggtgt
ccagtgtcag 60tcgctggagg agtccggggg tcgcctggta acgcctggga cacccctgac
actcacctgc 120acagtctctg gattctccct cagtacctac aacatgggct
gggtccgcca ggctccaggg 180aaggggctgg aatggatcgg aagtattact
attgatggtc gcacatacta cgcgagctgg 240gcgaaaggcc gattcaccgt
ctccaaaagc tcgaccacgg tggatctgaa aatgaccagt 300ctgacaaccg
gggacacggc cacctatttc tgtgccagga ttcttattgt ttcttatggg
360gcctttacca tc 37258033DNAOryctolagus cuniculus 580caggccactg
agagcattgg caatgagtta tcc 3358121DNAOryctolagus cuniculus
581tctgcatcca ctctggcatc t 2158236DNAOryctolagus cuniculus
582caacagggtt atagtagtgc taatattgat aatgct 3658315DNAOryctolagus
cuniculus 583acctacaaca tgggc 1558448DNAOryctolagus cuniculus
584agtattacta ttgatggtcg cacatactac gcgagctggg cgaaaggc
4858533DNAOryctolagus cuniculus 585attcttattg tttcttatgg ggcctttacc
atc 33586105PRTArtificialKappa constant domain 586Val Ala Ala Pro
Ser Val Phe Ile Phe Pro Pro Ser Asp Glu Gln Leu 1 5 10 15 Lys Ser
Gly Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe Tyr Pro 20 25 30
Arg Glu Ala Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln Ser Gly 35
40 45 Asn Ser Gln Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser Thr
Tyr 50 55 60 Ser Leu Ser Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr
Glu Lys His 65 70 75 80 Lys Val Tyr Ala Cys Glu Val Thr His Gln Gly
Leu Ser Ser Pro Val 85 90 95 Thr Lys Ser Phe Asn Arg Gly Glu Cys
100 105 587315DNAArtificialKappa constant domain 587gtggctgcac
catctgtctt catcttcccg ccatctgatg agcagttgaa atctggaact 60gcctctgttg
tgtgcctgct gaataacttc tatcccagag aggccaaagt acagtggaag
120gtggataacg ccctccaatc gggtaactcc caggagagtg tcacagagca
ggacagcaag 180gacagcacct acagcctcag cagcaccctg acgctgagca
aagcagacta cgagaaacac 240aaagtctacg cctgcgaagt cacccatcag
ggcctgagct cgcccgtcac aaagagcttc 300aacaggggag agtgt
315588330PRTArtificialGamma-1 constant domain 588Ala Ser Thr Lys
Gly Pro Ser Val Phe Pro Leu Ala Pro Ser Ser Lys 1 5 10 15 Ser Thr
Ser Gly Gly Thr Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr 20 25 30
Phe Pro Glu Pro Val Thr Val Ser Trp Asn Ser Gly Ala Leu Thr Ser 35
40 45 Gly Val His Thr Phe Pro Ala Val Leu Gln Ser Ser Gly Leu Tyr
Ser 50 55 60 Leu Ser Ser Val Val Thr Val Pro Ser Ser Ser Leu Gly
Thr Gln Thr 65 70 75 80 Tyr Ile Cys Asn Val Asn His Lys Pro Ser Asn
Thr Lys Val Asp Lys 85 90 95 Arg Val Glu Pro Lys Ser Cys Asp Lys
Thr His Thr Cys Pro Pro Cys 100 105 110 Pro Ala Pro Glu Leu Leu Gly
Gly Pro Ser Val Phe Leu Phe Pro Pro 115 120 125 Lys Pro Lys Asp Thr
Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys 130 135 140 Val Val Val
Asp Val Ser His Glu Asp Pro Glu Val Lys Phe Asn Trp 145 150 155 160
Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu 165
170 175 Glu Gln Tyr Ala Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val
Leu 180 185 190 His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys
Val Ser Asn 195 200 205 Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile
Ser Lys Ala Lys Gly 210 215 220 Gln Pro Arg Glu Pro Gln Val Tyr Thr
Leu Pro Pro Ser Arg Glu Glu 225 230 235 240 Met Thr Lys Asn Gln Val
Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr 245 250 255 Pro Ser Asp Ile
Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn 260 265 270 Asn Tyr
Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe 275 280 285
Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn 290
295 300 Val Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn His Tyr
Thr 305 310 315 320 Gln Lys Ser Leu Ser Leu Ser Pro Gly Lys 325 330
589990DNAArtificialGamma-1 constant domain 589gcctccacca agggcccatc
ggtcttcccc ctggcaccct cctccaagag cacctctggg 60ggcacagcgg ccctgggctg
cctggtcaag gactacttcc ccgaaccggt gacggtgtcg 120tggaactcag
gcgccctgac cagcggcgtg cacaccttcc cggctgtcct acagtcctca
180ggactctact ccctcagcag cgtggtgacc gtgccctcca gcagcttggg
cacccagacc 240tacatctgca acgtgaatca caagcccagc aacaccaagg
tggacaagag agttgagccc 300aaatcttgtg acaaaactca cacatgccca
ccgtgcccag cacctgaact cctgggggga 360ccgtcagtct tcctcttccc
cccaaaaccc aaggacaccc tcatgatctc ccggacccct 420gaggtcacat
gcgtggtggt ggacgtgagc cacgaagacc ctgaggtcaa gttcaactgg
480tacgtggacg gcgtggaggt gcataatgcc aagacaaagc cgcgggagga
gcagtacgcc 540agcacgtacc gtgtggtcag cgtcctcacc gtcctgcacc
aggactggct gaatggcaag 600gagtacaagt gcaaggtctc caacaaagcc
ctcccagccc ccatcgagaa aaccatctcc 660aaagccaaag ggcagccccg
agaaccacag gtgtacaccc tgcccccatc ccgggaggag 720atgaccaaga
accaggtcag cctgacctgc ctggtcaaag gcttctatcc cagcgacatc
780gccgtggagt gggagagcaa tgggcagccg gagaacaact acaagaccac
gcctcccgtg 840ctggactccg acggctcctt cttcctctac agcaagctca
ccgtggacaa gagcaggtgg 900cagcagggga acgtcttctc atgctccgtg
atgcatgagg ctctgcacaa ccactacacg 960cagaagagcc tctccctgtc
tccgggtaaa 99059015PRTHomo sapiens 590Val Pro Pro Gly Glu Asp Ser
Lys Asp Val Ala Ala Pro His Arg 1 5 10 15 59115PRTHomo sapiens
591Gly Glu Asp Ser Lys Asp Val Ala Ala Pro His Arg Gln Pro Leu 1 5
10 15 59215PRTHomo sapiens 592Ser Lys Asp Val Ala Ala Pro His Arg
Gln Pro Leu Thr Ser Ser 1 5 10 15 59315PRTHomo sapiens 593Val Ala
Ala Pro His Arg Gln Pro Leu Thr Ser Ser Glu Arg Ile 1 5 10 15
59415PRTHomo sapiens 594Pro His Arg Gln Pro Leu Thr Ser Ser Glu Arg
Ile Asp Lys Gln 1 5 10 15 59515PRTHomo sapiens 595Gln Pro Leu Thr
Ser Ser Glu Arg Ile Asp Lys Gln Ile Arg Tyr 1 5 10 15 59615PRTHomo
sapiens 596Thr Ser Ser Glu Arg Ile Asp Lys Gln Ile Arg Tyr Ile Leu
Asp 1 5 10 15 59715PRTHomo sapiens 597Glu Arg Ile Asp Lys Gln Ile
Arg Tyr Ile Leu Asp Gly Ile Ser 1 5 10 15 59815PRTHomo sapiens
598Asp Lys Gln Ile Arg Tyr Ile Leu Asp Gly Ile Ser Ala Leu Arg 1 5
10 15 59915PRTHomo sapiens 599Ile Arg Tyr Ile Leu Asp Gly Ile Ser
Ala Leu Arg Lys Glu Thr 1 5 10 15 60015PRTHomo sapiens 600Ile Leu
Asp Gly Ile Ser Ala Leu Arg Lys Glu Thr Cys Asn Lys 1 5 10 15
60115PRTHomo sapiens 601Gly Ile Ser Ala Leu Arg Lys Glu Thr Cys Asn
Lys Ser Asn Met 1 5 10 15 60215PRTHomo sapiens 602Ala Leu Arg Lys
Glu Thr Cys Asn Lys Ser Asn
Met Cys Glu Ser 1 5 10 15 60315PRTHomo sapiens 603Lys Glu Thr Cys
Asn Lys Ser Asn Met Cys Glu Ser Ser Lys Glu 1 5 10 15 60415PRTHomo
sapiens 604Cys Asn Lys Ser Asn Met Cys Glu Ser Ser Lys Glu Ala Leu
Ala 1 5 10 15 60515PRTHomo sapiens 605Ser Asn Met Cys Glu Ser Ser
Lys Glu Ala Leu Ala Glu Asn Asn 1 5 10 15 60615PRTHomo sapiens
606Cys Glu Ser Ser Lys Glu Ala Leu Ala Glu Asn Asn Leu Asn Leu 1 5
10 15 60715PRTHomo sapiens 607Ser Lys Glu Ala Leu Ala Glu Asn Asn
Leu Asn Leu Pro Lys Met 1 5 10 15 60815PRTHomo sapiens 608Ala Leu
Ala Glu Asn Asn Leu Asn Leu Pro Lys Met Ala Glu Lys 1 5 10 15
60915PRTHomo sapiens 609Glu Asn Asn Leu Asn Leu Pro Lys Met Ala Glu
Lys Asp Gly Cys 1 5 10 15 61015PRTHomo sapiens 610Leu Asn Leu Pro
Lys Met Ala Glu Lys Asp Gly Cys Phe Gln Ser 1 5 10 15 61115PRTHomo
sapiens 611Pro Lys Met Ala Glu Lys Asp Gly Cys Phe Gln Ser Gly Phe
Asn 1 5 10 15 61215PRTHomo sapiens 612Ala Glu Lys Asp Gly Cys Phe
Gln Ser Gly Phe Asn Glu Glu Thr 1 5 10 15 61315PRTHomo sapiens
613Asp Gly Cys Phe Gln Ser Gly Phe Asn Glu Glu Thr Cys Leu Val 1 5
10 15 61415PRTHomo sapiens 614Phe Gln Ser Gly Phe Asn Glu Glu Thr
Cys Leu Val Lys Ile Ile 1 5 10 15 61515PRTHomo sapiens 615Gly Phe
Asn Glu Glu Thr Cys Leu Val Lys Ile Ile Thr Gly Leu 1 5 10 15
61615PRTHomo sapiens 616Glu Glu Thr Cys Leu Val Lys Ile Ile Thr Gly
Leu Leu Glu Phe 1 5 10 15 61715PRTHomo sapiens 617Cys Leu Val Lys
Ile Ile Thr Gly Leu Leu Glu Phe Glu Val Tyr 1 5 10 15 61815PRTHomo
sapiens 618Lys Ile Ile Thr Gly Leu Leu Glu Phe Glu Val Tyr Leu Glu
Tyr 1 5 10 15 61915PRTHomo sapiens 619Thr Gly Leu Leu Glu Phe Glu
Val Tyr Leu Glu Tyr Leu Gln Asn 1 5 10 15 62015PRTHomo sapiens
620Leu Glu Phe Glu Val Tyr Leu Glu Tyr Leu Gln Asn Arg Phe Glu 1 5
10 15 62115PRTHomo sapiens 621Glu Val Tyr Leu Glu Tyr Leu Gln Asn
Arg Phe Glu Ser Ser Glu 1 5 10 15 62215PRTHomo sapiens 622Leu Glu
Tyr Leu Gln Asn Arg Phe Glu Ser Ser Glu Glu Gln Ala 1 5 10 15
62315PRTHomo sapiens 623Leu Gln Asn Arg Phe Glu Ser Ser Glu Glu Gln
Ala Arg Ala Val 1 5 10 15 62415PRTHomo sapiens 624Arg Phe Glu Ser
Ser Glu Glu Gln Ala Arg Ala Val Gln Met Ser 1 5 10 15 62515PRTHomo
sapiens 625Ser Ser Glu Glu Gln Ala Arg Ala Val Gln Met Ser Thr Lys
Val 1 5 10 15 62615PRTHomo sapiens 626Glu Gln Ala Arg Ala Val Gln
Met Ser Thr Lys Val Leu Ile Gln 1 5 10 15 62715PRTHomo sapiens
627Arg Ala Val Gln Met Ser Thr Lys Val Leu Ile Gln Phe Leu Gln 1 5
10 15 62815PRTHomo sapiens 628Gln Met Ser Thr Lys Val Leu Ile Gln
Phe Leu Gln Lys Lys Ala 1 5 10 15 62915PRTHomo sapiens 629Thr Lys
Val Leu Ile Gln Phe Leu Gln Lys Lys Ala Lys Asn Leu 1 5 10 15
63015PRTHomo sapiens 630Leu Ile Gln Phe Leu Gln Lys Lys Ala Lys Asn
Leu Asp Ala Ile 1 5 10 15 63115PRTHomo sapiens 631Phe Leu Gln Lys
Lys Ala Lys Asn Leu Asp Ala Ile Thr Thr Pro 1 5 10 15 63215PRTHomo
sapiens 632Lys Lys Ala Lys Asn Leu Asp Ala Ile Thr Thr Pro Asp Pro
Thr 1 5 10 15 63315PRTHomo sapiens 633Lys Asn Leu Asp Ala Ile Thr
Thr Pro Asp Pro Thr Thr Asn Ala 1 5 10 15 63415PRTHomo sapiens
634Asp Ala Ile Thr Thr Pro Asp Pro Thr Thr Asn Ala Ser Leu Leu 1 5
10 15 63515PRTHomo sapiens 635Thr Thr Pro Asp Pro Thr Thr Asn Ala
Ser Leu Leu Thr Lys Leu 1 5 10 15 63615PRTHomo sapiens 636Asp Pro
Thr Thr Asn Ala Ser Leu Leu Thr Lys Leu Gln Ala Gln 1 5 10 15
63715PRTHomo sapiens 637Thr Asn Ala Ser Leu Leu Thr Lys Leu Gln Ala
Gln Asn Gln Trp 1 5 10 15 63815PRTHomo sapiens 638Ser Leu Leu Thr
Lys Leu Gln Ala Gln Asn Gln Trp Leu Gln Asp 1 5 10 15 63915PRTHomo
sapiens 639Thr Lys Leu Gln Ala Gln Asn Gln Trp Leu Gln Asp Met Thr
Thr 1 5 10 15 64015PRTHomo sapiens 640Gln Ala Gln Asn Gln Trp Leu
Gln Asp Met Thr Thr His Leu Ile 1 5 10 15 64115PRTHomo sapiens
641Asn Gln Trp Leu Gln Asp Met Thr Thr His Leu Ile Leu Arg Ser 1 5
10 15 64215PRTHomo sapiens 642Leu Gln Asp Met Thr Thr His Leu Ile
Leu Arg Ser Phe Lys Glu 1 5 10 15 64315PRTHomo sapiens 643Met Thr
Thr His Leu Ile Leu Arg Ser Phe Lys Glu Phe Leu Gln 1 5 10 15
64415PRTHomo sapiens 644His Leu Ile Leu Arg Ser Phe Lys Glu Phe Leu
Gln Ser Ser Leu 1 5 10 15 64515PRTHomo sapiens 645Leu Arg Ser Phe
Lys Glu Phe Leu Gln Ser Ser Leu Arg Ala Leu 1 5 10 15 64615PRTHomo
sapiens 646Phe Lys Glu Phe Leu Gln Ser Ser Leu Arg Ala Leu Arg Gln
Met 1 5 10 15 647111PRTOryctolagus cuniculus 647Ala Tyr Asp Met Thr
Gln Thr Pro Ala Ser Val Ser Ala Ala Val Gly 1 5 10 15 Gly Thr Val
Thr Ile Lys Cys Gln Ala Ser Gln Ser Ile Asn Asn Glu 20 25 30 Leu
Ser Trp Tyr Gln Gln Lys Pro Gly Gln Arg Pro Lys Leu Leu Ile 35 40
45 Tyr Arg Ala Ser Thr Leu Ala Ser Gly Val Ser Ser Arg Phe Lys Gly
50 55 60 Ser Gly Ser Gly Thr Glu Phe Thr Leu Thr Ile Ser Asp Leu
Glu Cys 65 70 75 80 Ala Asp Ala Ala Thr Tyr Tyr Cys Gln Gln Gly Tyr
Ser Leu Arg Asn 85 90 95 Ile Asp Asn Ala Phe Gly Gly Gly Thr Glu
Val Val Val Lys Arg 100 105 110 64888PRTHomo sapiens 648Ala Ile Gln
Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly 1 5 10 15 Asp
Arg Val Thr Ile Thr Cys Arg Ala Ser Gln Gly Ile Arg Asn Asp 20 25
30 Leu Gly Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile
35 40 45 Tyr Ala Ala Ser Ser Leu Gln Ser Gly Val Pro Ser Arg Phe
Ser Gly 50 55 60 Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser
Ser Leu Gln Pro 65 70 75 80 Glu Asp Phe Ala Thr Tyr Tyr Cys 85
64988PRTHomo sapiens 649Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu
Ser Ala Ser Val Gly 1 5 10 15 Asp Arg Val Thr Ile Thr Cys Arg Ala
Ser Gln Gly Ile Ser Asn Tyr 20 25 30 Leu Ala Trp Tyr Gln Gln Lys
Pro Gly Lys Val Pro Lys Leu Leu Ile 35 40 45 Tyr Ala Ala Ser Thr
Leu Gln Ser Gly Val Pro Ser Arg Phe Ser Gly 50 55 60 Ser Gly Ser
Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro 65 70 75 80 Glu
Asp Val Ala Thr Tyr Tyr Cys 85 65088PRTHomo sapiens 650Asp Ile Gln
Met Thr Gln Ser Pro Ser Thr Leu Ser Ala Ser Val Gly 1 5 10 15 Asp
Arg Val Thr Ile Thr Cys Arg Ala Ser Gln Ser Ile Ser Ser Trp 20 25
30 Leu Ala Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile
35 40 45 Tyr Lys Ala Ser Ser Leu Glu Ser Gly Val Pro Ser Arg Phe
Ser Gly 50 55 60 Ser Gly Ser Gly Thr Glu Phe Thr Leu Thr Ile Ser
Ser Leu Gln Pro 65 70 75 80 Asp Asp Phe Ala Thr Tyr Tyr Cys 85
651111PRTArtificialHumanized antibody sequence 651Ala Ile Gln Met
Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly 1 5 10 15 Asp Arg
Val Thr Ile Thr Cys Gln Ala Ser Gln Ser Ile Asn Asn Glu 20 25 30
Leu Ser Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile 35
40 45 Tyr Arg Ala Ser Thr Leu Ala Ser Gly Val Pro Ser Arg Phe Ser
Gly 50 55 60 Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser
Leu Gln Pro 65 70 75 80 Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Gly
Tyr Ser Leu Arg Asn 85 90 95 Ile Asp Asn Ala Phe Gly Gly Gly Thr
Lys Val Glu Ile Lys Arg 100 105 110 652117PRTOryctolagus cuniculus
652Gln Ser Leu Glu Glu Ser Gly Gly Arg Leu Val Thr Pro Gly Thr Pro
1 5 10 15 Leu Thr Leu Thr Cys Thr Ala Ser Gly Phe Ser Leu Ser Asn
Tyr Tyr 20 25 30 Val Thr Trp Val Arg Gln Ala Pro Gly Lys Gly Leu
Glu Trp Ile Gly 35 40 45 Ile Ile Tyr Gly Ser Asp Glu Thr Ala Tyr
Ala Thr Trp Ala Ile Gly 50 55 60 Arg Phe Thr Ile Ser Lys Thr Ser
Thr Thr Val Asp Leu Lys Met Thr 65 70 75 80 Ser Leu Thr Ala Ala Asp
Thr Ala Thr Tyr Phe Cys Ala Arg Asp Asp 85 90 95 Ser Ser Asp Trp
Asp Ala Lys Phe Asn Leu Trp Gly Gln Gly Thr Leu 100 105 110 Val Thr
Val Ser Ser 115 65397PRTHomo sapiens 653Glu Val Gln Leu Val Glu Ser
Gly Gly Gly Leu Val Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser
Cys Ala Ala Ser Gly Phe Thr Val Ser Ser Asn 20 25 30 Tyr Met Ser
Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45 Ser
Val Ile Tyr Ser Gly Gly Ser Thr Tyr Tyr Ala Asp Ser Val Lys 50 55
60 Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr Leu
65 70 75 80 Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr
Cys Ala 85 90 95 Arg 65497PRTHomo sapiens 654Glu Val Gln Leu Val
Glu Ser Gly Gly Gly Leu Ile Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg
Leu Ser Cys Ala Ala Ser Gly Phe Thr Val Ser Ser Asn 20 25 30 Tyr
Met Ser Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40
45 Ser Val Ile Tyr Ser Gly Gly Ser Thr Tyr Tyr Ala Asp Ser Val Lys
50 55 60 Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu
Tyr Leu 65 70 75 80 Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val
Tyr Tyr Cys Ala 85 90 95 Arg 65598PRTHomo sapiens 655Glu Val Gln
Leu Leu Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly 1 5 10 15 Ser
Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Ser Tyr 20 25
30 Ala Met Ser Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val
35 40 45 Ser Val Ile Tyr Ser Gly Gly Ser Ser Thr Tyr Tyr Ala Asp
Ser Val 50 55 60 Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys
Asn Thr Leu Tyr 65 70 75 80 Leu Gln Met Asn Ser Leu Arg Ala Glu Asp
Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Lys
656120PRTArtificialHumanized antibody sequence 656Glu Val Gln Leu
Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly 1 5 10 15 Ser Leu
Arg Leu Ser Cys Ala Ala Ser Gly Phe Ser Leu Ser Asn Tyr 20 25 30
Tyr Val Thr Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35
40 45 Gly Ile Ile Tyr Gly Ser Asp Glu Thr Ala Tyr Ala Thr Trp Ala
Ile 50 55 60 Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr
Leu Tyr Leu 65 70 75 80 Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala
Val Tyr Tyr Cys Ala 85 90 95 Arg Asp Asp Ser Ser Asp Trp Asp Ala
Lys Phe Asn Leu Trp Gly Gln 100 105 110 Gly Thr Leu Val Thr Val Ser
Ser 115 120 657120PRTArtificialHumanized antibody sequence 657Glu
Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly 1 5 10
15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Ser Leu Ser Asn Tyr
20 25 30 Tyr Val Thr Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu
Trp Val 35 40 45 Gly Ile Ile Tyr Gly Ser Asp Glu Thr Ala Tyr Ala
Thr Ser Ala Ile 50 55 60 Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser
Lys Asn Thr Leu Tyr Leu 65 70 75 80 Gln Met Asn Ser Leu Arg Ala Glu
Asp Thr Ala Val Tyr Tyr Cys Ala 85 90 95 Arg Asp Asp Ser Ser Asp
Trp Asp Ala Lys Phe Asn Leu Trp Gly Gln 100 105 110 Gly Thr Leu Val
Thr Val Ser Ser 115 120 658166PRTOryctolagus cuniculus 658Met Glu
Thr Gly Leu Arg Trp Leu Leu Leu Val Ala Val Leu Lys Gly 1 5 10 15
Val Gln Cys Gln Ser Leu Glu Glu Ser Gly Gly Arg Leu Val Thr Pro 20
25 30 Gly Thr Pro Leu Thr Leu Thr Cys Thr Ala Ser Gly Phe Ser Leu
Ser 35 40 45 Asn Tyr Tyr Val Thr Trp Val Arg Gln Ala Pro Gly Lys
Gly Leu Glu 50 55 60 Trp Ile Gly Ile Ile Tyr Gly Ser Asp Glu Thr
Ala Tyr Ala Thr Ser 65 70 75 80 Ala Ile Gly Arg Phe Thr Ile Ser Lys
Thr Ser Thr Thr Val Asp Leu 85 90 95 Lys Met Thr Ser Leu Thr Ala
Ala Asp Thr Ala Thr Tyr Phe Cys Ala 100 105 110 Arg Asp Asp Ser Ser
Asp Trp Asp Ala Lys Phe Asn Leu Trp Gly Gln 115 120 125 Gly Thr Leu
Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val 130 135 140 Phe
Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala Ala 145 150
155 160 Leu Gly Cys Leu Val Lys 165 65916PRTOryctolagus cuniculus
659Ile Ile Tyr Gly Ser Asp Glu Thr Ala Tyr Ala Thr Ser Ala Ile Gly
1 5 10 15 660122PRTOryctolagus cuniculus 660Met Asp Thr Arg Ala Pro
Thr Gln Leu Leu Gly Leu Leu Leu Leu Trp 1 5 10 15 Leu Pro Gly Ala
Arg Cys Ala Tyr Asp Met Thr Gln Thr Pro Ala Ser 20 25 30 Val Ser
Ala Ala Val Gly Gly Thr Val Thr Ile Lys Cys Gln Ala Ser 35 40 45
Gln Ser Ile Asn Asn Glu Leu Ser Trp Tyr Gln Gln Lys Pro Gly Gln 50
55 60 Arg Pro Lys Leu Leu Ile Tyr Arg Ala Ser Thr Leu Ala Ser Gly
Val 65 70 75 80 Ser Ser Arg Phe Lys Gly Ser Gly Ser Gly Thr Glu Phe
Thr Leu Thr 85 90 95 Ile Ser Asp Leu
Glu Cys Ala Asp Ala Ala Thr Tyr Tyr Cys Gln Gln 100 105 110 Gly Tyr
Ser Leu Arg Asn Ile Asp Asn Ala 115 120 661125PRTOryctolagus
cuniculus 661Met Glu Thr Gly Leu Arg Trp Leu Leu Leu Val Ala Val
Leu Lys Gly 1 5 10 15 Val Gln Cys Gln Ser Leu Glu Glu Ser Gly Gly
Arg Leu Val Thr Pro 20 25 30 Gly Thr Pro Leu Thr Leu Thr Cys Thr
Ala Ser Gly Phe Ser Leu Ser 35 40 45 Asn Tyr Tyr Val Thr Trp Val
Arg Gln Ala Pro Gly Lys Gly Leu Glu 50 55 60 Trp Ile Gly Ile Ile
Tyr Gly Ser Asp Glu Thr Ala Tyr Ala Thr Trp 65 70 75 80 Ala Ile Gly
Arg Phe Thr Ile Ser Lys Thr Ser Thr Thr Val Asp Leu 85 90 95 Lys
Met Thr Ser Leu Thr Ala Ala Asp Thr Ala Thr Tyr Phe Cys Ala 100 105
110 Arg Asp Asp Ser Ser Asp Trp Asp Ala Lys Phe Asn Leu 115 120 125
662366DNAOryctolagus cuniculus 662atggacacga gggcccccac tcagctgctg
gggctcctgc tgctctggct cccaggtgcc 60agatgtgcct atgatatgac ccagactcca
gcctcggtgt ctgcagctgt gggaggcaca 120gtcaccatca agtgccaggc
cagtcagagc attaacaatg aattatcctg gtatcagcag 180aaaccagggc
agcgtcccaa gctcctgatc tatagggcat ccactctggc atctggggtc
240tcatcgcggt tcaaaggcag tggatctggg acagagttca ctctcaccat
cagcgacctg 300gagtgtgccg atgctgccac ttactactgt caacagggtt
atagtctgag gaatattgat 360aatgct 366663375DNAOryctolagus cuniculus
663atggagactg ggctgcgctg gcttctcctg gtcgctgtgc tcaaaggtgt
ccagtgtcag 60tcgctggagg agtccggggg tcgcctggtc acgcctggga cacccctgac
actcacctgc 120acagcctctg gattctccct cagtaactac tacgtgacct
gggtccgcca ggctccaggg 180aaggggctgg aatggatcgg aatcatttat
ggtagtgatg aaacggccta cgcgacctgg 240gcgataggcc gattcaccat
ctccaaaacc tcgaccacgg tggatctgaa aatgaccagt 300ctgacagccg
cggacacggc cacctatttc tgtgccagag atgatagtag tgactgggat
360gcaaaattta acttg 375664450PRTOryctolagus cuniculus 664Glu Val
Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly 1 5 10 15
Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Ser Leu Ser Asn Tyr 20
25 30 Tyr Val Thr Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp
Val 35 40 45 Gly Ile Ile Tyr Gly Ser Asp Glu Thr Ala Tyr Ala Thr
Trp Ala Ile 50 55 60 Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys
Asn Thr Leu Tyr Leu 65 70 75 80 Gln Met Asn Ser Leu Arg Ala Glu Asp
Thr Ala Val Tyr Tyr Cys Ala 85 90 95 Arg Asp Asp Ser Ser Asp Trp
Asp Ala Lys Phe Asn Leu Trp Gly Gln 100 105 110 Gly Thr Leu Val Thr
Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val 115 120 125 Phe Pro Leu
Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala Ala 130 135 140 Leu
Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser 145 150
155 160 Trp Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala
Val 165 170 175 Leu Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val
Thr Val Pro 180 185 190 Ser Ser Ser Leu Gly Thr Gln Thr Tyr Ile Cys
Asn Val Asn His Lys 195 200 205 Pro Ser Asn Thr Lys Val Asp Lys Arg
Val Glu Pro Lys Ser Cys Asp 210 215 220 Lys Thr His Thr Cys Pro Pro
Cys Pro Ala Pro Glu Leu Leu Gly Gly 225 230 235 240 Pro Ser Val Phe
Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile 245 250 255 Ser Arg
Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His Glu 260 265 270
Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His 275
280 285 Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Ala Ser Thr Tyr
Arg 290 295 300 Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu
Asn Gly Lys 305 310 315 320 Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala
Leu Pro Ala Pro Ile Glu 325 330 335 Lys Thr Ile Ser Lys Ala Lys Gly
Gln Pro Arg Glu Pro Gln Val Tyr 340 345 350 Thr Leu Pro Pro Ser Arg
Asp Glu Leu Thr Lys Asn Gln Val Ser Leu 355 360 365 Thr Cys Leu Val
Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp 370 375 380 Glu Ser
Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val 385 390 395
400 Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp
405 410 415 Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val
Met His 420 425 430 Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu
Ser Leu Ser Pro 435 440 445 Gly Lys 450 665450PRTOryctolagus
cuniculus 665Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln
Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe
Ser Leu Ser Asn Tyr 20 25 30 Tyr Val Thr Trp Val Arg Gln Ala Pro
Gly Lys Gly Leu Glu Trp Val 35 40 45 Gly Ile Ile Tyr Gly Ser Asp
Glu Thr Ala Tyr Ala Thr Ser Ala Ile 50 55 60 Gly Arg Phe Thr Ile
Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr Leu 65 70 75 80 Gln Met Asn
Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys Ala 85 90 95 Arg
Asp Asp Ser Ser Asp Trp Asp Ala Lys Phe Asn Leu Trp Gly Gln 100 105
110 Gly Thr Leu Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val
115 120 125 Phe Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr
Ala Ala 130 135 140 Leu Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro
Val Thr Val Ser 145 150 155 160 Trp Asn Ser Gly Ala Leu Thr Ser Gly
Val His Thr Phe Pro Ala Val 165 170 175 Leu Gln Ser Ser Gly Leu Tyr
Ser Leu Ser Ser Val Val Thr Val Pro 180 185 190 Ser Ser Ser Leu Gly
Thr Gln Thr Tyr Ile Cys Asn Val Asn His Lys 195 200 205 Pro Ser Asn
Thr Lys Val Asp Lys Arg Val Glu Pro Lys Ser Cys Asp 210 215 220 Lys
Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly Gly 225 230
235 240 Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met
Ile 245 250 255 Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val
Ser His Glu 260 265 270 Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp
Gly Val Glu Val His 275 280 285 Asn Ala Lys Thr Lys Pro Arg Glu Glu
Gln Tyr Ala Ser Thr Tyr Arg 290 295 300 Val Val Ser Val Leu Thr Val
Leu His Gln Asp Trp Leu Asn Gly Lys 305 310 315 320 Glu Tyr Lys Cys
Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu 325 330 335 Lys Thr
Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr 340 345 350
Thr Leu Pro Pro Ser Arg Asp Glu Leu Thr Lys Asn Gln Val Ser Leu 355
360 365 Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu
Trp 370 375 380 Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr
Pro Pro Val 385 390 395 400 Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr
Ser Lys Leu Thr Val Asp 405 410 415 Lys Ser Arg Trp Gln Gln Gly Asn
Val Phe Ser Cys Ser Val Met His 420 425 430 Glu Ala Leu His Asn His
Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro 435 440 445 Gly Lys 450
666216PRTOryctolagus cuniculus 666Ile Gln Met Thr Gln Ser Pro Ser
Ser Leu Ser Ala Ser Val Gly Asp 1 5 10 15 Arg Val Thr Ile Thr Cys
Gln Ala Ser Gln Ser Ile Asn Asn Glu Leu 20 25 30 Ser Trp Tyr Gln
Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile Tyr 35 40 45 Arg Ala
Ser Thr Leu Ala Ser Gly Val Pro Ser Arg Phe Ser Gly Ser 50 55 60
Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro Asp 65
70 75 80 Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Gly Tyr Ser Leu Arg
Asn Ile 85 90 95 Asp Asn Ala Phe Gly Gly Gly Thr Lys Val Glu Ile
Lys Arg Thr Val 100 105 110 Ala Ala Pro Ser Val Phe Ile Phe Pro Pro
Ser Asp Glu Gln Leu Lys 115 120 125 Ser Gly Thr Ala Ser Val Val Cys
Leu Leu Asn Asn Phe Tyr Pro Arg 130 135 140 Glu Ala Lys Val Gln Trp
Lys Val Asp Asn Ala Leu Gln Ser Gly Asn 145 150 155 160 Ser Gln Glu
Ser Val Thr Glu Gln Asp Ser Lys Asp Ser Thr Tyr Ser 165 170 175 Leu
Ser Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu Lys His Lys 180 185
190 Val Tyr Ala Cys Glu Val Thr His Gln Gly Leu Ser Ser Pro Val Thr
195 200 205 Lys Ser Phe Asn Arg Gly Glu Cys 210 215
667122PRTOryctolagus cuniculus 667Met Asp Thr Arg Ala Pro Thr Gln
Leu Leu Gly Leu Leu Leu Leu Trp 1 5 10 15 Leu Pro Gly Ala Arg Cys
Ala Tyr Asp Met Thr Gln Thr Pro Ala Ser 20 25 30 Val Glu Val Ala
Val Gly Gly Thr Val Thr Ile Asn Cys Gln Ala Ser 35 40 45 Glu Thr
Ile Tyr Ser Trp Leu Ser Trp Tyr Gln Gln Lys Pro Gly Gln 50 55 60
Pro Pro Lys Leu Leu Ile Tyr Gln Ala Ser Asp Leu Ala Ser Gly Val 65
70 75 80 Pro Ser Arg Phe Ser Gly Ser Gly Ala Gly Thr Glu Tyr Thr
Leu Thr 85 90 95 Ile Ser Gly Val Gln Cys Asp Asp Ala Ala Thr Tyr
Tyr Cys Gln Gln 100 105 110 Gly Tyr Ser Gly Ser Asn Val Asp Asn Val
115 120 668126PRTOryctolagus cuniculus 668Met Glu Thr Gly Leu Arg
Trp Leu Leu Leu Val Ala Val Leu Lys Gly 1 5 10 15 Val Gln Cys Gln
Glu Gln Leu Lys Glu Ser Gly Gly Arg Leu Val Thr 20 25 30 Pro Gly
Thr Pro Leu Thr Leu Thr Cys Thr Ala Ser Gly Phe Ser Leu 35 40 45
Asn Asp His Ala Met Gly Trp Val Arg Gln Ala Pro Gly Lys Gly Leu 50
55 60 Glu Tyr Ile Gly Phe Ile Asn Ser Gly Gly Ser Ala Arg Tyr Ala
Ser 65 70 75 80 Trp Ala Glu Gly Arg Phe Thr Ile Ser Arg Thr Ser Thr
Thr Val Asp 85 90 95 Leu Lys Met Thr Ser Leu Thr Thr Glu Asp Thr
Ala Thr Tyr Phe Cys 100 105 110 Val Arg Gly Gly Ala Val Trp Ser Ile
His Ser Phe Asp Pro 115 120 125 669366DNAOryctolagus cuniculus
669atggacacga gggcccccac tcagctgctg gggctcctgc tgctctggct
cccaggtgcc 60agatgtgcct atgatatgac ccagactcca gcctctgtgg aggtagctgt
gggaggcaca 120gtcaccatca attgccaggc cagtgagacc atttacagtt
ggttatcctg gtatcagcag 180aagccagggc agcctcccaa gctcctgatc
taccaggcat ccgatctggc atctggggtc 240ccatcgcgat tcagcggcag
tggggctggg acagagtaca ctctcaccat cagcggcgtg 300cagtgtgacg
atgctgccac ttactactgt caacagggtt atagtggtag taatgttgat 360aatgtt
366670378DNAOryctolagus cuniculus 670atggagactg ggctgcgctg
gcttctcctg gtcgctgtgc tcaaaggtgt ccagtgtcag 60gagcagctga aggagtccgg
gggtcgcctg gtcacgcctg ggacacccct gacacttacc 120tgcacagcct
ctggattctc cctcaatgac catgcaatgg gctgggtccg ccaggctcca
180gggaaggggc tggaatacat cggattcatt aatagtggtg gtagcgcacg
ctacgcgagc 240tgggcagaag gccgattcac catctccaga acctcgacca
cggtggatct gaaaatgacc 300agtctgacaa ccgaggacac ggccacctat
ttctgtgtca gagggggtgc tgtttggagt 360attcatagtt ttgatccc
378671123PRTOryctolagus cuniculus 671Met Asp Thr Arg Ala Pro Thr
Gln Leu Leu Gly Leu Leu Leu Leu Trp 1 5 10 15 Leu Pro Gly Ala Thr
Phe Ala Ala Val Leu Thr Gln Thr Pro Ser Pro 20 25 30 Val Ser Ala
Ala Val Gly Gly Thr Val Ser Ile Ser Cys Gln Ala Ser 35 40 45 Gln
Ser Val Tyr Asp Asn Asn Tyr Leu Ser Trp Phe Gln Gln Lys Pro 50 55
60 Gly Gln Pro Pro Lys Leu Leu Ile Tyr Gly Ala Ser Thr Leu Ala Ser
65 70 75 80 Gly Val Pro Ser Arg Phe Val Gly Ser Gly Ser Gly Thr Gln
Phe Thr 85 90 95 Leu Thr Ile Thr Asp Val Gln Cys Asp Asp Ala Ala
Thr Tyr Tyr Cys 100 105 110 Ala Gly Val Tyr Asp Asp Asp Ser Asp Asn
Ala 115 120 672125PRTOryctolagus cuniculus 672Met Glu Thr Gly Leu
Arg Trp Leu Leu Leu Val Ala Val Leu Lys Gly 1 5 10 15 Val Gln Cys
Gln Ser Leu Glu Glu Ser Gly Gly Arg Leu Val Thr Pro 20 25 30 Gly
Thr Pro Leu Thr Leu Thr Cys Thr Ala Ser Gly Phe Ser Leu Ser 35 40
45 Val Tyr Tyr Met Asn Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu
50 55 60 Trp Ile Gly Phe Ile Thr Met Ser Asp Asn Ile Asn Tyr Ala
Ser Trp 65 70 75 80 Ala Lys Gly Arg Phe Thr Ile Ser Lys Thr Ser Thr
Thr Val Asp Leu 85 90 95 Lys Met Thr Ser Pro Thr Thr Glu Asp Thr
Ala Thr Tyr Phe Cys Ala 100 105 110 Arg Ser Arg Gly Trp Gly Thr Met
Gly Arg Leu Asp Leu 115 120 125 673369DNAOryctolagus cuniculus
673atggacacga gggcccccac tcagctgctg gggctcctgc tgctctggct
cccaggtgcc 60acatttgccg ccgtgctgac ccagactcca tctcccgtgt ctgcagctgt
gggaggcaca 120gtcagcatca gttgccaggc cagtcagagt gtttatgaca
acaactactt atcctggttt 180cagcagaaac cagggcagcc tcccaagctc
ctgatctatg gtgcatccac tctggcatct 240ggggtcccat cgcggttcgt
gggcagtgga tctgggacac agttcactct caccatcaca 300gacgtgcagt
gtgacgatgc tgccacttac tattgtgcag gcgtttatga tgatgatagt 360gataatgcc
369674375DNAOryctolagus cuniculus 674atggagactg ggctgcgctg
gcttctcctg gtggctgtgc tcaaaggtgt ccagtgtcag 60tcgctggagg agtccggggg
tcgcctggtc acccctggga cacccctgac actcacctgc 120acagcctctg
gattctccct cagtgtctac tacatgaact gggtccgcca ggctccaggg
180aaggggctgg aatggatcgg attcattaca atgagtgata atataaatta
cgcgagctgg 240gcgaaaggcc gattcaccat ctccaaaacc tcgaccacgg
tggatctgaa aatgaccagt 300ccgacaaccg aggacacggc cacctatttc
tgtgccagga gtcgtggctg gggtacaatg 360ggtcggttgg atctc
375675123PRTOryctolagus cuniculus 675Met Asp Thr Arg Ala Pro Thr
Gln Leu Leu Gly Leu Leu Leu Leu Trp 1 5 10 15 Leu Pro Gly Ala Ile
Cys Asp Pro Val Leu Thr Gln Thr Pro Ser Pro 20 25 30 Val Ser Ala
Pro Val Gly Gly Thr Val Ser Ile Ser Cys Gln Ala Ser 35 40 45 Gln
Ser Val Tyr Glu Asn Asn Tyr Leu Ser Trp Phe Gln Gln Lys Pro 50 55
60 Gly Gln Pro Pro Lys Leu Leu Ile Tyr Gly Ala Ser Thr Leu Asp Ser
65 70 75 80 Gly Val Pro Ser Arg Phe Lys Gly Ser Gly Ser
Gly Thr Gln Phe Thr 85 90 95 Leu Thr Ile Thr Asp Val Gln Cys Asp
Asp Ala Ala Thr Tyr Tyr Cys 100 105 110 Ala Gly Val Tyr Asp Asp Asp
Ser Asp Asp Ala 115 120 676126PRTOryctolagus cuniculus 676Met Glu
Thr Gly Leu Arg Trp Leu Leu Leu Val Ala Val Leu Lys Gly 1 5 10 15
Val Gln Cys Gln Glu Gln Leu Lys Glu Ser Gly Gly Gly Leu Val Thr 20
25 30 Pro Gly Gly Thr Leu Thr Leu Thr Cys Thr Ala Ser Gly Phe Ser
Leu 35 40 45 Asn Ala Tyr Tyr Met Asn Trp Val Arg Gln Ala Pro Gly
Lys Gly Leu 50 55 60 Glu Trp Ile Gly Phe Ile Thr Leu Asn Asn Asn
Val Ala Tyr Ala Asn 65 70 75 80 Trp Ala Lys Gly Arg Phe Thr Phe Ser
Lys Thr Ser Thr Thr Val Asp 85 90 95 Leu Lys Met Thr Ser Pro Thr
Pro Glu Asp Thr Ala Thr Tyr Phe Cys 100 105 110 Ala Arg Ser Arg Gly
Trp Gly Ala Met Gly Arg Leu Asp Leu 115 120 125
677369DNAOryctolagus cuniculus 677atggacacga gggcccccac tcagctgctg
gggctcctgc tgctctggct cccaggtgcc 60atatgtgacc ctgtgctgac ccagactcca
tctcccgtat ctgcacctgt gggaggcaca 120gtcagcatca gttgccaggc
cagtcagagt gtttatgaga acaactattt atcctggttt 180cagcagaaac
cagggcagcc tcccaagctc ctgatctatg gtgcatccac tctggattct
240ggggtcccat cgcggttcaa aggcagtgga tctgggacac agttcactct
caccattaca 300gacgtgcagt gtgacgatgc tgccacttac tattgtgcag
gcgtttatga tgatgatagt 360gatgatgcc 369678378DNAOryctolagus
cuniculus 678atggagactg ggctgcgctg gcttctcctg gtggctgtgc tcaaaggtgt
ccagtgtcag 60gagcagctga aggagtccgg aggaggcctg gtaacgcctg gaggaaccct
gacactcacc 120tgcacagcct ctggattctc cctcaatgcc tactacatga
actgggtccg ccaggctcca 180gggaaggggc tggaatggat cggattcatt
actctgaata ataatgtagc ttacgcgaac 240tgggcgaaag gccgattcac
cttctccaaa acctcgacca cggtggatct gaaaatgacc 300agtccgacac
ccgaggacac ggccacctat ttctgtgcca ggagtcgtgg ctggggtgca
360atgggtcggt tggatctc 378679122PRTOryctolagus cuniculus 679Met Asp
Thr Arg Ala Pro Thr Gln Leu Leu Gly Leu Leu Leu Leu Trp 1 5 10 15
Leu Pro Gly Ala Thr Phe Ala Gln Val Leu Thr Gln Thr Pro Ser Pro 20
25 30 Val Ser Ala Ala Val Gly Gly Thr Val Thr Ile Asn Cys Gln Ala
Ser 35 40 45 Gln Ser Val Asp Asp Asn Asn Trp Leu Gly Trp Tyr Gln
Gln Lys Arg 50 55 60 Gly Gln Pro Pro Lys Tyr Leu Ile Tyr Ser Ala
Ser Thr Leu Ala Ser 65 70 75 80 Gly Val Pro Ser Arg Phe Lys Gly Ser
Gly Ser Gly Thr Gln Phe Thr 85 90 95 Leu Thr Ile Ser Asp Leu Glu
Cys Asp Asp Ala Ala Thr Tyr Tyr Cys 100 105 110 Ala Gly Gly Phe Ser
Gly Asn Ile Phe Ala 115 120 680122PRTOryctolagus cuniculus 680Met
Glu Thr Gly Leu Arg Trp Leu Leu Leu Val Ala Val Leu Lys Gly 1 5 10
15 Val Gln Cys Gln Ser Val Glu Glu Ser Gly Gly Arg Leu Val Thr Pro
20 25 30 Gly Thr Pro Leu Thr Leu Thr Cys Thr Val Ser Gly Phe Ser
Leu Ser 35 40 45 Ser Tyr Ala Met Ser Trp Val Arg Gln Ala Pro Gly
Lys Gly Leu Glu 50 55 60 Trp Ile Gly Ile Ile Gly Gly Phe Gly Thr
Thr Tyr Tyr Ala Thr Trp 65 70 75 80 Ala Lys Gly Arg Phe Thr Ile Ser
Lys Thr Ser Thr Thr Val Asp Leu 85 90 95 Arg Ile Thr Ser Pro Thr
Thr Glu Asp Thr Ala Thr Tyr Phe Cys Ala 100 105 110 Arg Gly Gly Pro
Gly Asn Gly Gly Asp Ile 115 120 681366DNAOryctolagus cuniculus
681atggacacga gggcccccac tcagctgctg gggctcctgc tgctctggct
cccaggtgcc 60acatttgccc aagtgctgac ccagactcca tcgcctgtgt ctgcagctgt
gggaggcaca 120gtcaccatca actgccaggc cagtcagagt gttgatgata
acaactggtt aggctggtat 180cagcagaaac gagggcagcc tcccaagtac
ctgatctatt ctgcatccac tctggcatct 240ggggtcccat cgcggttcaa
aggcagtgga tctgggacac agttcactct caccatcagc 300gacctggagt
gtgacgatgc tgccacttac tactgtgcag gcggttttag tggtaatatc 360tttgct
366682366DNAOryctolagus cuniculus 682atggagactg ggctgcgctg
gcttctcctg gtcgctgtgc tcaaaggtgt ccagtgtcag 60tcggtggagg agtccggggg
tcgcctggtc acgcctggga cacccctgac actcacctgc 120acagtctctg
gcttctccct cagtagctat gcaatgagct gggtccgcca ggctccagga
180aaggggctgg agtggatcgg aatcattggt ggttttggta ccacatacta
cgcgacctgg 240gcgaaaggcc gattcaccat ctccaaaacc tcgaccacgg
tggatctgag aatcaccagt 300ccgacaaccg aggacacggc cacctatttc
tgtgccagag gtggtcctgg taatggtggt 360gacatc 366683122PRTOryctolagus
cuniculus 683Met Asp Thr Arg Ala Pro Thr Gln Leu Leu Gly Leu Leu
Leu Leu Trp 1 5 10 15 Leu Pro Gly Ala Thr Phe Ala Ala Val Leu Thr
Gln Thr Pro Ser Pro 20 25 30 Val Ser Val Pro Val Gly Gly Thr Val
Thr Ile Lys Cys Gln Ser Ser 35 40 45 Gln Ser Val Tyr Asn Asn Phe
Leu Ser Trp Tyr Gln Gln Lys Pro Gly 50 55 60 Gln Pro Pro Lys Leu
Leu Ile Tyr Gln Ala Ser Lys Leu Ala Ser Gly 65 70 75 80 Val Pro Asp
Arg Phe Ser Gly Ser Gly Ser Gly Thr Gln Phe Thr Leu 85 90 95 Thr
Ile Ser Gly Val Gln Cys Asp Asp Ala Ala Thr Tyr Tyr Cys Leu 100 105
110 Gly Gly Tyr Asp Asp Asp Ala Asp Asn Ala 115 120
684128PRTOryctolagus cuniculus 684Met Glu Thr Gly Leu Arg Trp Leu
Leu Leu Val Ala Val Leu Lys Gly 1 5 10 15 Val Gln Cys Gln Ser Val
Glu Glu Ser Gly Gly Arg Leu Val Thr Pro 20 25 30 Gly Thr Pro Leu
Thr Leu Thr Cys Thr Val Ser Gly Ile Asp Leu Ser 35 40 45 Asp Tyr
Ala Met Ser Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu 50 55 60
Trp Ile Gly Ile Ile Tyr Ala Gly Ser Gly Ser Thr Trp Tyr Ala Ser 65
70 75 80 Trp Ala Lys Gly Arg Phe Thr Ile Ser Lys Thr Ser Thr Thr
Val Asp 85 90 95 Leu Lys Ile Thr Ser Pro Thr Thr Glu Asp Thr Ala
Thr Tyr Phe Cys 100 105 110 Ala Arg Asp Gly Tyr Asp Asp Tyr Gly Asp
Phe Asp Arg Leu Asp Leu 115 120 125 685366DNAOryctolagus cuniculus
685atggacacga gggcccccac tcagctgctg gggctcctgc tgctctggct
cccaggtgcc 60acatttgcag ccgtgctgac ccagacacca tcgcccgtgt ctgtacctgt
gggaggcaca 120gtcaccatca agtgccagtc cagtcagagt gtttataata
atttcttatc gtggtatcag 180cagaaaccag ggcagcctcc caagctcctg
atctaccagg catccaaact ggcatctggg 240gtcccagata ggttcagcgg
cagtggatct gggacacagt tcactctcac catcagcggc 300gtgcagtgtg
acgatgctgc cacttactac tgtctaggcg gttatgatga tgatgctgat 360aatgct
366686384DNAOryctolagus cuniculus 686atggagactg ggctgcgctg
gcttctcctg gtcgctgtgc tcaaaggtgt ccagtgtcag 60tcggtggagg agtccggggg
tcgcctggtc acgcctggga cacccctgac gctcacctgc 120acagtctctg
gaatcgacct cagtgactat gcaatgagct gggtccgcca ggctccaggg
180aaggggctgg aatggatcgg aatcatttat gctggtagtg gtagcacatg
gtacgcgagc 240tgggcgaaag gccgattcac catctccaaa acctcgacca
cggtggatct gaaaatcacc 300agtccgacaa ccgaggacac ggccacctat
ttctgtgcca gagatggata cgatgactat 360ggtgatttcg atcgattgga tctc
384687122PRTOryctolagus cuniculus 687Met Asp Thr Arg Ala Pro Thr
Gln Leu Leu Gly Leu Leu Leu Leu Trp 1 5 10 15 Leu Pro Gly Ala Arg
Cys Ala Tyr Asp Met Thr Gln Thr Pro Ala Ser 20 25 30 Val Ser Ala
Ala Val Gly Gly Thr Val Thr Ile Lys Cys Gln Ala Ser 35 40 45 Gln
Ser Ile Asn Asn Glu Leu Ser Trp Tyr Gln Gln Lys Ser Gly Gln 50 55
60 Arg Pro Lys Leu Leu Ile Tyr Arg Ala Ser Thr Leu Ala Ser Gly Val
65 70 75 80 Ser Ser Arg Phe Lys Gly Ser Gly Ser Gly Thr Glu Phe Thr
Leu Thr 85 90 95 Ile Ser Asp Leu Glu Cys Ala Asp Ala Ala Thr Tyr
Tyr Cys Gln Gln 100 105 110 Gly Tyr Ser Leu Arg Asn Ile Asp Asn Ala
115 120 688125PRTOryctolagus cuniculus 688Met Glu Thr Gly Leu Arg
Trp Leu Leu Leu Val Ala Val Leu Ser Gly 1 5 10 15 Val Gln Cys Gln
Ser Leu Glu Glu Ser Gly Gly Arg Leu Val Thr Pro 20 25 30 Gly Thr
Pro Leu Thr Leu Thr Cys Thr Ala Ser Gly Phe Ser Leu Ser 35 40 45
Asn Tyr Tyr Met Thr Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu 50
55 60 Trp Ile Gly Met Ile Tyr Gly Ser Asp Glu Thr Ala Tyr Ala Asn
Trp 65 70 75 80 Ala Ile Gly Arg Phe Thr Ile Ser Lys Thr Ser Thr Thr
Val Asp Leu 85 90 95 Lys Met Thr Ser Leu Thr Ala Ala Asp Thr Ala
Thr Tyr Phe Cys Ala 100 105 110 Arg Asp Asp Ser Ser Asp Trp Asp Ala
Lys Phe Asn Leu 115 120 125 689366DNAOryctolagus cuniculus
689atggacacga gggcccccac tcagctgctg gggctcctgc tgctctggct
cccaggtgcc 60agatgtgcct atgatatgac ccagactcca gcctcggtgt ctgcagctgt
gggaggcaca 120gtcaccatca aatgccaggc cagtcagagc attaacaatg
aattatcctg gtatcagcag 180aaatcagggc agcgtcccaa gctcctgatc
tatagggcat ccactctggc atctggggtc 240tcatcgcggt tcaaaggcag
tggatctggg acagagttca ctctcaccat cagcgacctg 300gagtgtgccg
atgctgccac ttactactgt caacagggtt atagtctgag gaatattgat 360aatgct
366690375DNAOryctolagus cuniculus 690atggagactg ggctgcgctg
gcttctcctg gtcgctgtgc tctcaggtgt ccagtgtcag 60tcgctggagg agtccggggg
tcgcctggtc acgcctggga cacccctgac actcacctgc 120acagcctctg
gattctccct cagtaactac tacatgacct gggtccgcca ggctccaggg
180aaggggctgg aatggatcgg aatgatttat ggtagtgatg aaacagccta
cgcgaactgg 240gcgataggcc gattcaccat ctccaaaacc tcgaccacgg
tggatctgaa aatgaccagt 300ctgacagccg cggacacggc cacctatttc
tgtgccagag atgatagtag tgactgggat 360gcaaaattta acttg
375691450PRTOryctolagus cuniculus 691Glu Val Gln Leu Val Glu Ser
Gly Gly Gly Leu Val Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser
Cys Ala Ala Ser Gly Phe Ser Leu Ser Asn Tyr 20 25 30 Tyr Met Thr
Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45 Gly
Met Ile Tyr Gly Ser Asp Glu Thr Ala Tyr Ala Asn Trp Ala Ile 50 55
60 Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr Leu
65 70 75 80 Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr
Cys Ala 85 90 95 Arg Asp Asp Ser Ser Asp Trp Asp Ala Lys Phe Asn
Leu Trp Gly Gln 100 105 110 Gly Thr Leu Val Thr Val Ser Ser Ala Ser
Thr Lys Gly Pro Ser Val 115 120 125 Phe Pro Leu Ala Pro Ser Ser Lys
Ser Thr Ser Gly Gly Thr Ala Ala 130 135 140 Leu Gly Cys Leu Val Lys
Asp Tyr Phe Pro Glu Pro Val Thr Val Ser 145 150 155 160 Trp Asn Ser
Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala Val 165 170 175 Leu
Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro 180 185
190 Ser Ser Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn His Lys
195 200 205 Pro Ser Asn Thr Lys Val Asp Lys Arg Val Glu Pro Lys Ser
Cys Asp 210 215 220 Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu
Leu Leu Gly Gly 225 230 235 240 Pro Ser Val Phe Leu Phe Pro Pro Lys
Pro Lys Asp Thr Leu Met Ile 245 250 255 Ser Arg Thr Pro Glu Val Thr
Cys Val Val Val Asp Val Ser His Glu 260 265 270 Asp Pro Glu Val Lys
Phe Asn Trp Tyr Val Asp Gly Val Glu Val His 275 280 285 Asn Ala Lys
Thr Lys Pro Arg Glu Glu Gln Tyr Ala Ser Thr Tyr Arg 290 295 300 Val
Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys 305 310
315 320 Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile
Glu 325 330 335 Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro
Gln Val Tyr 340 345 350 Thr Leu Pro Pro Ser Arg Asp Glu Leu Thr Lys
Asn Gln Val Ser Leu 355 360 365 Thr Cys Leu Val Lys Gly Phe Tyr Pro
Ser Asp Ile Ala Val Glu Trp 370 375 380 Glu Ser Asn Gly Gln Pro Glu
Asn Asn Tyr Lys Thr Thr Pro Pro Val 385 390 395 400 Leu Asp Ser Asp
Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp 405 410 415 Lys Ser
Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met His 420 425 430
Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro 435
440 445 Gly Lys 450 692450PRTOryctolagus cuniculus 692Glu Val Gln
Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly 1 5 10 15 Ser
Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Ser Leu Ser Asn Tyr 20 25
30 Tyr Met Thr Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val
35 40 45 Gly Met Ile Tyr Gly Ser Asp Glu Thr Ala Tyr Ala Asn Ser
Ala Ile 50 55 60 Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn
Thr Leu Tyr Leu 65 70 75 80 Gln Met Asn Ser Leu Arg Ala Glu Asp Thr
Ala Val Tyr Tyr Cys Ala 85 90 95 Arg Asp Asp Ser Ser Asp Trp Asp
Ala Lys Phe Asn Leu Trp Gly Gln 100 105 110 Gly Thr Leu Val Thr Val
Ser Ser Ala Ser Thr Lys Gly Pro Ser Val 115 120 125 Phe Pro Leu Ala
Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala Ala 130 135 140 Leu Gly
Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser 145 150 155
160 Trp Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala Val
165 170 175 Leu Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr
Val Pro 180 185 190 Ser Ser Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn
Val Asn His Lys 195 200 205 Pro Ser Asn Thr Lys Val Asp Lys Arg Val
Glu Pro Lys Ser Cys Asp 210 215 220 Lys Thr His Thr Cys Pro Pro Cys
Pro Ala Pro Glu Leu Leu Gly Gly 225 230 235 240 Pro Ser Val Phe Leu
Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile 245 250 255 Ser Arg Thr
Pro Glu Val Thr Cys Val Val Val Asp Val Ser His Glu 260 265 270 Asp
Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His 275 280
285 Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Ala Ser Thr Tyr Arg
290 295 300 Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn
Gly Lys 305 310 315 320 Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu
Pro Ala Pro Ile Glu 325 330 335 Lys Thr Ile Ser Lys Ala Lys Gly Gln
Pro Arg Glu Pro Gln Val Tyr 340 345 350 Thr Leu Pro
Pro Ser Arg Asp Glu Leu Thr Lys Asn Gln Val Ser Leu 355 360 365 Thr
Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp 370 375
380 Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val
385 390 395 400 Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu
Thr Val Asp 405 410 415 Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser
Cys Ser Val Met His 420 425 430 Glu Ala Leu His Asn His Tyr Thr Gln
Lys Ser Leu Ser Leu Ser Pro 435 440 445 Gly Lys 450
693217PRTOryctolagus cuniculus 693Asp Ile Gln Met Thr Gln Ser Pro
Ser Thr Leu Ser Ala Ser Val Gly 1 5 10 15 Asp Arg Val Thr Ile Thr
Cys Gln Ala Ser Gln Ser Ile Asn Asn Glu 20 25 30 Leu Ser Trp Tyr
Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile 35 40 45 Tyr Arg
Ala Ser Thr Leu Ala Ser Gly Val Pro Ser Arg Phe Ser Gly 50 55 60
Ser Gly Ser Gly Thr Glu Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro 65
70 75 80 Asp Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Gly Tyr Ser Leu
Arg Asn 85 90 95 Ile Asp Asn Ala Phe Gly Gly Gly Thr Lys Val Glu
Ile Lys Arg Thr 100 105 110 Val Ala Ala Pro Ser Val Phe Ile Phe Pro
Pro Ser Asp Glu Gln Leu 115 120 125 Lys Ser Gly Thr Ala Ser Val Val
Cys Leu Leu Asn Asn Phe Tyr Pro 130 135 140 Arg Glu Ala Lys Val Gln
Trp Lys Val Asp Asn Ala Leu Gln Ser Gly 145 150 155 160 Asn Ser Gln
Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser Thr Tyr 165 170 175 Ser
Leu Ser Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu Lys His 180 185
190 Lys Val Tyr Ala Cys Glu Val Thr His Gln Gly Leu Ser Ser Pro Val
195 200 205 Thr Lys Ser Phe Asn Arg Gly Glu Cys 210 215
69433DNAOryctolagus cuniculus 694caggccagtc agagcattaa caatgagtta
tcc 3369536DNAOryctolagus cuniculus 695caacagggtt atagtctgag
gaacattgat aatgct 3669648DNAOryctolagus cuniculus 696atcatctatg
gtagtgatga aaccgcctac gctacctccg ctataggc 4869736DNAOryctolagus
cuniculus 697gatgatagta gtgactggga tgcaaagttc aacttg
36698336DNAOryctolagus cuniculus 698gctatccaga tgacccagtc
tccttcctcc ctgtctgcat ctgtaggaga cagagtcacc 60atcacttgcc aggccagtca
gagcattaac aatgagttat cctggtatca gcagaaacca 120gggaaagccc
ctaagctcct gatctatagg gcatccactc tggcatctgg ggtcccatca
180aggttcagcg gcagtggatc tgggacagac ttcactctca ccatcagcag
cctgcagcct 240gatgattttg caacttatta ctgccaacag ggttatagtc
tgaggaacat tgataatgct 300ttcggcggag ggaccaaggt ggaaatcaaa cgtacg
336699112PRTOryctolagus cuniculus 699Ala Ile Gln Met Thr Gln Ser
Pro Ser Ser Leu Ser Ala Ser Val Gly 1 5 10 15 Asp Arg Val Thr Ile
Thr Cys Gln Ala Ser Gln Ser Ile Asn Asn Glu 20 25 30 Leu Ser Trp
Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile 35 40 45 Tyr
Arg Ala Ser Thr Leu Ala Ser Gly Val Pro Ser Arg Phe Ser Gly 50 55
60 Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro
65 70 75 80 Asp Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Gly Tyr Ser Leu
Arg Asn 85 90 95 Ile Asp Asn Ala Phe Gly Gly Gly Thr Lys Val Glu
Ile Lys Arg Thr 100 105 110 700360DNAOryctolagus cuniculus
700gaggtgcagc tggtggagtc tgggggaggc ttggtccagc ctggggggtc
cctgagactc 60tcctgtgcag cctctggatt ctccctcagt aactactacg tgacctgggt
ccgtcaggct 120ccagggaagg ggctggagtg ggtcggcatc atctatggta
gtgatgaaac cgcctacgct 180acctccgcta taggccgatt caccatctcc
agagacaatt ccaagaacac cctgtatctt 240caaatgaaca gcctgagagc
tgaggacact gctgtgtatt actgtgctag agatgatagt 300agtgactggg
atgcaaagtt caacttgtgg ggccaaggga ccctcgtcac cgtctcgagc
360701651DNAOryctolagus cuniculus 701gctatccaga tgacccagtc
tccttcctcc ctgtctgcat ctgtaggaga cagagtcacc 60atcacttgcc aggccagtca
gagcattaac aatgagttat cctggtatca gcagaaacca 120gggaaagccc
ctaagctcct gatctatagg gcatccactc tggcatctgg ggtcccatca
180aggttcagcg gcagtggatc tgggacagac ttcactctca ccatcagcag
cctgcagcct 240gatgattttg caacttatta ctgccaacag ggttatagtc
tgaggaacat tgataatgct 300ttcggcggag ggaccaaggt ggaaatcaaa
cgtacggtgg ctgcaccatc tgtcttcatc 360ttcccgccat ctgatgagca
gttgaaatct ggaactgcct ctgttgtgtg cctgctgaat 420aacttctatc
ccagagaggc caaagtacag tggaaggtgg ataacgccct ccaatcgggt
480aactcccagg agagtgtcac agagcaggac agcaaggaca gcacctacag
cctcagcagc 540accctgacgc tgagcaaagc agactacgag aaacacaaag
tctacgcctg cgaagtcacc 600catcagggcc tgagctcgcc cgtcacaaag
agcttcaaca ggggagagtg t 651702217PRTOryctolagus cuniculus 702Ala
Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly 1 5 10
15 Asp Arg Val Thr Ile Thr Cys Gln Ala Ser Gln Ser Ile Asn Asn Glu
20 25 30 Leu Ser Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu
Leu Ile 35 40 45 Tyr Arg Ala Ser Thr Leu Ala Ser Gly Val Pro Ser
Arg Phe Ser Gly 50 55 60 Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr
Ile Ser Ser Leu Gln Pro 65 70 75 80 Asp Asp Phe Ala Thr Tyr Tyr Cys
Gln Gln Gly Tyr Ser Leu Arg Asn 85 90 95 Ile Asp Asn Ala Phe Gly
Gly Gly Thr Lys Val Glu Ile Lys Arg Thr 100 105 110 Val Ala Ala Pro
Ser Val Phe Ile Phe Pro Pro Ser Asp Glu Gln Leu 115 120 125 Lys Ser
Gly Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe Tyr Pro 130 135 140
Arg Glu Ala Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln Ser Gly 145
150 155 160 Asn Ser Gln Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser
Thr Tyr 165 170 175 Ser Leu Ser Ser Thr Leu Thr Leu Ser Lys Ala Asp
Tyr Glu Lys His 180 185 190 Lys Val Tyr Ala Cys Glu Val Thr His Gln
Gly Leu Ser Ser Pro Val 195 200 205 Thr Lys Ser Phe Asn Arg Gly Glu
Cys 210 215 7031350DNAOryctolagus cuniculus 703gaggtgcagc
tggtggagtc tgggggaggc ttggtccagc ctggggggtc cctgagactc 60tcctgtgcag
cctctggatt ctccctcagt aactactacg tgacctgggt ccgtcaggct
120ccagggaagg ggctggagtg ggtcggcatc atctatggta gtgatgaaac
cgcctacgct 180acctccgcta taggccgatt caccatctcc agagacaatt
ccaagaacac cctgtatctt 240caaatgaaca gcctgagagc tgaggacact
gctgtgtatt actgtgctag agatgatagt 300agtgactggg atgcaaagtt
caacttgtgg ggccaaggga ccctcgtcac cgtctcgagc 360gcctccacca
agggcccatc ggtcttcccc ctggcaccct cctccaagag cacctctggg
420ggcacagcgg ccctgggctg cctggtcaag gactacttcc ccgaaccggt
gacggtgtcg 480tggaactcag gcgccctgac cagcggcgtg cacaccttcc
cggctgtcct acagtcctca 540ggactctact ccctcagcag cgtggtgacc
gtgccctcca gcagcttggg cacccagacc 600tacatctgca acgtgaatca
caagcccagc aacaccaagg tggacaagag agttgagccc 660aaatcttgtg
acaaaactca cacatgccca ccgtgcccag cacctgaact cctgggggga
720ccgtcagtct tcctcttccc cccaaaaccc aaggacaccc tcatgatctc
ccggacccct 780gaggtcacat gcgtggtggt ggacgtgagc cacgaagacc
ctgaggtcaa gttcaactgg 840tacgtggacg gcgtggaggt gcataatgcc
aagacaaagc cgcgggagga gcagtacgcc 900agcacgtacc gtgtggtcag
cgtcctcacc gtcctgcacc aggactggct gaatggcaag 960gagtacaagt
gcaaggtctc caacaaagcc ctcccagccc ccatcgagaa aaccatctcc
1020aaagccaaag ggcagccccg agaaccacag gtgtacaccc tgcccccatc
ccgggaggag 1080atgaccaaga accaggtcag cctgacctgc ctggtcaaag
gcttctatcc cagcgacatc 1140gccgtggagt gggagagcaa tgggcagccg
gagaacaact acaagaccac gcctcccgtg 1200ctggactccg acggctcctt
cttcctctac agcaagctca ccgtggacaa gagcaggtgg 1260cagcagggga
acgtcttctc atgctccgtg atgcatgagg ctctgcacaa ccactacacg
1320cagaagagcc tctccctgtc tccgggtaaa 1350704450PRTOryctolagus
cuniculus 704Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln
Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe
Ser Leu Ser Asn Tyr 20 25 30 Tyr Val Thr Trp Val Arg Gln Ala Pro
Gly Lys Gly Leu Glu Trp Val 35 40 45 Gly Ile Ile Tyr Gly Ser Asp
Glu Thr Ala Tyr Ala Thr Ser Ala Ile 50 55 60 Gly Arg Phe Thr Ile
Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr Leu 65 70 75 80 Gln Met Asn
Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys Ala 85 90 95 Arg
Asp Asp Ser Ser Asp Trp Asp Ala Lys Phe Asn Leu Trp Gly Gln 100 105
110 Gly Thr Leu Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val
115 120 125 Phe Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr
Ala Ala 130 135 140 Leu Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro
Val Thr Val Ser 145 150 155 160 Trp Asn Ser Gly Ala Leu Thr Ser Gly
Val His Thr Phe Pro Ala Val 165 170 175 Leu Gln Ser Ser Gly Leu Tyr
Ser Leu Ser Ser Val Val Thr Val Pro 180 185 190 Ser Ser Ser Leu Gly
Thr Gln Thr Tyr Ile Cys Asn Val Asn His Lys 195 200 205 Pro Ser Asn
Thr Lys Val Asp Lys Arg Val Glu Pro Lys Ser Cys Asp 210 215 220 Lys
Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly Gly 225 230
235 240 Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met
Ile 245 250 255 Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val
Ser His Glu 260 265 270 Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp
Gly Val Glu Val His 275 280 285 Asn Ala Lys Thr Lys Pro Arg Glu Glu
Gln Tyr Ala Ser Thr Tyr Arg 290 295 300 Val Val Ser Val Leu Thr Val
Leu His Gln Asp Trp Leu Asn Gly Lys 305 310 315 320 Glu Tyr Lys Cys
Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu 325 330 335 Lys Thr
Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr 340 345 350
Thr Leu Pro Pro Ser Arg Glu Glu Met Thr Lys Asn Gln Val Ser Leu 355
360 365 Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu
Trp 370 375 380 Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr
Pro Pro Val 385 390 395 400 Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr
Ser Lys Leu Thr Val Asp 405 410 415 Lys Ser Arg Trp Gln Gln Gly Asn
Val Phe Ser Cys Ser Val Met His 420 425 430 Glu Ala Leu His Asn His
Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro 435 440 445 Gly Lys 450
705705DNAOryctolagus cuniculus 705atgaagtggg taacctttat ttcccttctg
tttctcttta gcagcgctta ttccgctatc 60cagatgaccc agtctccttc ctccctgtct
gcatctgtag gagacagagt caccatcact 120tgccaggcca gtcagagcat
taacaatgag ttatcctggt atcagcagaa accagggaaa 180gcccctaagc
tcctgatcta tagggcatcc actctggcat ctggggtccc atcaaggttc
240agcggcagtg gatctgggac agacttcact ctcaccatca gcagcctgca
gcctgatgat 300tttgcaactt attactgcca acagggttat agtctgagga
acattgataa tgctttcggc 360ggagggacca aggtggaaat caaacgtacg
gtggctgcac catctgtctt catcttcccg 420ccatctgatg agcagttgaa
atctggaact gcctctgttg tgtgcctgct gaataacttc 480tatcccagag
aggccaaagt acagtggaag gtggataacg ccctccaatc gggtaactcc
540caggagagtg tcacagagca ggacagcaag gacagcacct acagcctcag
cagcaccctg 600acgctgagca aagcagacta cgagaaacac aaagtctacg
cctgcgaagt cacccatcag 660ggcctgagct cgcccgtcac aaagagcttc
aacaggggag agtgt 705706235PRTOryctolagus cuniculus 706Met Lys Trp
Val Thr Phe Ile Ser Leu Leu Phe Leu Phe Ser Ser Ala 1 5 10 15 Tyr
Ser Ala Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser 20 25
30 Val Gly Asp Arg Val Thr Ile Thr Cys Gln Ala Ser Gln Ser Ile Asn
35 40 45 Asn Glu Leu Ser Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro
Lys Leu 50 55 60 Leu Ile Tyr Arg Ala Ser Thr Leu Ala Ser Gly Val
Pro Ser Arg Phe 65 70 75 80 Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr
Leu Thr Ile Ser Ser Leu 85 90 95 Gln Pro Asp Asp Phe Ala Thr Tyr
Tyr Cys Gln Gln Gly Tyr Ser Leu 100 105 110 Arg Asn Ile Asp Asn Ala
Phe Gly Gly Gly Thr Lys Val Glu Ile Lys 115 120 125 Arg Thr Val Ala
Ala Pro Ser Val Phe Ile Phe Pro Pro Ser Asp Glu 130 135 140 Gln Leu
Lys Ser Gly Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe 145 150 155
160 Tyr Pro Arg Glu Ala Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln
165 170 175 Ser Gly Asn Ser Gln Glu Ser Val Thr Glu Gln Asp Ser Lys
Asp Ser 180 185 190 Thr Tyr Ser Leu Ser Ser Thr Leu Thr Leu Ser Lys
Ala Asp Tyr Glu 195 200 205 Lys His Lys Val Tyr Ala Cys Glu Val Thr
His Gln Gly Leu Ser Ser 210 215 220 Pro Val Thr Lys Ser Phe Asn Arg
Gly Glu Cys 225 230 235 7071404DNAOryctolagus cuniculus
707atgaagtggg taacctttat ttcccttctg tttctcttta gcagcgctta
ttccgaggtg 60cagctggtgg agtctggggg aggcttggtc cagcctgggg ggtccctgag
actctcctgt 120gcagcctctg gattctccct cagtaactac tacgtgacct
gggtccgtca ggctccaggg 180aaggggctgg agtgggtcgg catcatctat
ggtagtgatg aaaccgccta cgctacctcc 240gctataggcc gattcaccat
ctccagagac aattccaaga acaccctgta tcttcaaatg 300aacagcctga
gagctgagga cactgctgtg tattactgtg ctagagatga tagtagtgac
360tgggatgcaa agttcaactt gtggggccaa gggaccctcg tcaccgtctc
gagcgcctcc 420accaagggcc catcggtctt ccccctggca ccctcctcca
agagcacctc tgggggcaca 480gcggccctgg gctgcctggt caaggactac
ttccccgaac cggtgacggt gtcgtggaac 540tcaggcgccc tgaccagcgg
cgtgcacacc ttcccggctg tcctacagtc ctcaggactc 600tactccctca
gcagcgtggt gaccgtgccc tccagcagct tgggcaccca gacctacatc
660tgcaacgtga atcacaagcc cagcaacacc aaggtggaca agagagttga
gcccaaatct 720tgtgacaaaa ctcacacatg cccaccgtgc ccagcacctg
aactcctggg gggaccgtca 780gtcttcctct tccccccaaa acccaaggac
accctcatga tctcccggac ccctgaggtc 840acatgcgtgg tggtggacgt
gagccacgaa gaccctgagg tcaagttcaa ctggtacgtg 900gacggcgtgg
aggtgcataa tgccaagaca aagccgcggg aggagcagta cgccagcacg
960taccgtgtgg tcagcgtcct caccgtcctg caccaggact ggctgaatgg
caaggagtac 1020aagtgcaagg tctccaacaa agccctccca gcccccatcg
agaaaaccat ctccaaagcc 1080aaagggcagc cccgagaacc acaggtgtac
accctgcccc catcccggga ggagatgacc 1140aagaaccagg tcagcctgac
ctgcctggtc aaaggcttct atcccagcga catcgccgtg 1200gagtgggaga
gcaatgggca gccggagaac aactacaaga ccacgcctcc cgtgctggac
1260tccgacggct ccttcttcct ctacagcaag ctcaccgtgg acaagagcag
gtggcagcag 1320gggaacgtct tctcatgctc cgtgatgcat gaggctctgc
acaaccacta cacgcagaag 1380agcctctccc tgtctccggg taaa
1404708468PRTOryctolagus cuniculus 708Met Lys Trp Val Thr Phe Ile
Ser Leu Leu Phe Leu Phe Ser Ser Ala 1 5 10 15 Tyr Ser Glu Val Gln
Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro 20 25 30 Gly Gly Ser
Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Ser Leu Ser 35 40 45 Asn
Tyr Tyr Val Thr Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu 50 55
60 Trp Val Gly Ile Ile Tyr Gly Ser Asp Glu Thr Ala Tyr Ala Thr Ser
65 70 75 80 Ala Ile Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn
Thr Leu 85 90 95 Tyr Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr
Ala Val Tyr Tyr 100 105
110 Cys Ala Arg Asp Asp Ser Ser Asp Trp Asp Ala Lys Phe Asn Leu Trp
115 120 125 Gly Gln Gly Thr Leu Val Thr Val Ser Ser Ala Ser Thr Lys
Gly Pro 130 135 140 Ser Val Phe Pro Leu Ala Pro Ser Ser Lys Ser Thr
Ser Gly Gly Thr 145 150 155 160 Ala Ala Leu Gly Cys Leu Val Lys Asp
Tyr Phe Pro Glu Pro Val Thr 165 170 175 Val Ser Trp Asn Ser Gly Ala
Leu Thr Ser Gly Val His Thr Phe Pro 180 185 190 Ala Val Leu Gln Ser
Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr 195 200 205 Val Pro Ser
Ser Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn 210 215 220 His
Lys Pro Ser Asn Thr Lys Val Asp Lys Arg Val Glu Pro Lys Ser 225 230
235 240 Cys Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu
Leu 245 250 255 Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys
Asp Thr Leu 260 265 270 Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val
Val Val Asp Val Ser 275 280 285 His Glu Asp Pro Glu Val Lys Phe Asn
Trp Tyr Val Asp Gly Val Glu 290 295 300 Val His Asn Ala Lys Thr Lys
Pro Arg Glu Glu Gln Tyr Ala Ser Thr 305 310 315 320 Tyr Arg Val Val
Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn 325 330 335 Gly Lys
Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro 340 345 350
Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln 355
360 365 Val Tyr Thr Leu Pro Pro Ser Arg Glu Glu Met Thr Lys Asn Gln
Val 370 375 380 Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp
Ile Ala Val 385 390 395 400 Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn
Asn Tyr Lys Thr Thr Pro 405 410 415 Pro Val Leu Asp Ser Asp Gly Ser
Phe Phe Leu Tyr Ser Lys Leu Thr 420 425 430 Val Asp Lys Ser Arg Trp
Gln Gln Gly Asn Val Phe Ser Cys Ser Val 435 440 445 Met His Glu Ala
Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu 450 455 460 Ser Pro
Gly Lys 465 709111PRTOryctolagus cuniculus 709Ala Ile Gln Met Thr
Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly 1 5 10 15 Asp Arg Val
Thr Ile Thr Cys Gln Ala Ser Gln Ser Ile Asn Asn Glu 20 25 30 Leu
Ser Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile 35 40
45 Tyr Arg Ala Ser Thr Leu Ala Ser Gly Val Pro Ser Arg Phe Ser Gly
50 55 60 Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu
Gln Pro 65 70 75 80 Asp Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Gly Tyr
Ser Leu Arg Asn 85 90 95 Ile Asp Asn Ala Phe Gly Gly Gly Thr Lys
Val Glu Ile Lys Arg 100 105 110 71011PRTOryctolagus cuniculus
710Arg Ala Ser Gln Gly Ile Arg Asn Asp Leu Gly 1 5 10
71111PRTOryctolagus cuniculus 711Arg Ala Ser Gln Gly Ile Ser Asn
Tyr Leu Ala 1 5 10 71211PRTOryctolagus cuniculus 712Arg Ala Ser Gln
Ser Ile Ser Ser Trp Leu Ala 1 5 10 7137PRTOryctolagus cuniculus
713Ala Ala Ser Ser Leu Gln Ser 1 5 7147PRTOryctolagus cuniculus
714Ala Ala Ser Thr Leu Gln Ser 1 5 7157PRTOryctolagus cuniculus
715Lys Ala Ser Ser Leu Glu Ser 1 5 7165PRTOryctolagus cuniculus
716Ser Asn Tyr Met Ser 1 5 71716PRTOryctolagus cuniculus 717Val Ile
Tyr Ser Gly Gly Ser Thr Tyr Tyr Ala Asp Ser Val Lys Gly 1 5 10 15
71817PRTOryctolagus cuniculus 718Val Ile Tyr Ser Gly Gly Ser Ser
Thr Tyr Tyr Ala Asp Ser Val Lys 1 5 10 15 Gly
719330PRTArtificialGamma-1 constant domain 719Ala Ser Thr Lys Gly
Pro Ser Val Phe Pro Leu Ala Pro Ser Ser Lys 1 5 10 15 Ser Thr Ser
Gly Gly Thr Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr 20 25 30 Phe
Pro Glu Pro Val Thr Val Ser Trp Asn Ser Gly Ala Leu Thr Ser 35 40
45 Gly Val His Thr Phe Pro Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser
50 55 60 Leu Ser Ser Val Val Thr Val Pro Ser Ser Ser Leu Gly Thr
Gln Thr 65 70 75 80 Tyr Ile Cys Asn Val Asn His Lys Pro Ser Asn Thr
Lys Val Asp Lys 85 90 95 Arg Val Glu Pro Lys Ser Cys Asp Lys Thr
His Thr Cys Pro Pro Cys 100 105 110 Pro Ala Pro Glu Leu Leu Gly Gly
Pro Ser Val Phe Leu Phe Pro Pro 115 120 125 Lys Pro Lys Asp Thr Leu
Met Ile Ser Arg Thr Pro Glu Val Thr Cys 130 135 140 Val Val Val Asp
Val Ser His Glu Asp Pro Glu Val Lys Phe Asn Trp 145 150 155 160 Tyr
Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu 165 170
175 Glu Gln Tyr Ala Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu
180 185 190 His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val
Ser Asn 195 200 205 Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser
Lys Ala Lys Gly 210 215 220 Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu
Pro Pro Ser Arg Asp Glu 225 230 235 240 Leu Thr Lys Asn Gln Val Ser
Leu Thr Cys Leu Val Lys Gly Phe Tyr 245 250 255 Pro Ser Asp Ile Ala
Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn 260 265 270 Asn Tyr Lys
Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe 275 280 285 Leu
Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn 290 295
300 Val Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn His Tyr Thr
305 310 315 320 Gln Lys Ser Leu Ser Leu Ser Pro Gly Lys 325 330
720297DNAOryctolagus cuniculus 720atccagatga cccagtctcc ttcctccctg
tctgcatctg taggagacag agtcaccatc 60acttgccagg ccagtcagag cattaacaat
gagttatcct ggtatcagca gaaaccaggg 120aaagccccta agctcctgat
ctatagggca tccactctgg catctggggt cccatcaagg 180ttcagcggca
gtggatctgg gacagacttc actctcacca tcagcagcct gcagcctgat
240gattttgcaa cttattactg ccaacagggt tatagtctga ggaacattga taatgct
297721333DNAOryctolagus cuniculus 721gcctatgata tgacccagac
tccagcctcg gtgtctgcag ctgtgggagg cacagtcacc 60atcaagtgcc aggccagtca
gagcattaac aatgaattat cctggtatca gcagaaacca 120gggcagcgtc
ccaagctcct gatctatagg gcatccactc tggcatctgg ggtctcatcg
180cggttcaaag gcagtggatc tgggacagag ttcactctca ccatcagcga
cctggagtgt 240gccgatgctg ccacttacta ctgtcaacag ggttatagtc
tgaggaatat tgataatgct 300ttcggcggag ggaccgaggt ggtggtcaaa cgt
333722648DNAOryctolagus cuniculus 722atccagatga cccagtctcc
ttcctccctg tctgcatctg taggagacag agtcaccatc 60acttgccagg ccagtcagag
cattaacaat gagttatcct ggtatcagca gaaaccaggg 120aaagccccta
agctcctgat ctatagggca tccactctgg catctggggt cccatcaagg
180ttcagcggca gtggatctgg gacagacttc actctcacca tcagcagcct
gcagcctgat 240gattttgcaa cttattactg ccaacagggt tatagtctga
ggaacattga taatgctttc 300ggcggaggga ccaaggtgga aatcaaacgt
acggtggctg caccatctgt cttcatcttc 360ccgccatctg atgagcagtt
gaaatctgga actgcctctg ttgtgtgcct gctgaataac 420ttctatccca
gagaggccaa agtacagtgg aaggtggata acgccctcca atcgggtaac
480tcccaggaga gtgtcacaga gcaggacagc aaggacagca cctacagcct
cagcagcacc 540ctgacgctga gcaaagcaga ctacgagaaa cacaaagtct
acgcctgcga agtcacccat 600cagggcctga gctcgcccgt cacaaagagc
ttcaacaggg gagagtgt 648723333DNAOryctolagus cuniculus 723gctatccaga
tgacccagtc tccttcctcc ctgtctgcat ctgtaggaga cagagtcacc 60atcacttgcc
aggccagtca gagcattaac aatgagttat cctggtatca gcagaaacca
120gggaaagccc ctaagctcct gatctatagg gcatccactc tggcatctgg
ggtcccatca 180aggttcagcg gcagtggatc tgggacagac ttcactctca
ccatcagcag cctgcagcct 240gatgattttg caacttatta ctgccaacag
ggttatagtc tgaggaacat tgataatgct 300ttcggcggag ggaccaaggt
ggaaatcaaa cgt 333724327DNAOryctolagus cuniculus 724gaggtgcagc
tggtggagtc tgggggaggc ttggtccagc ctggggggtc cctgagactc 60tcctgtgcag
cctctggatt ctccctcagt aactactacg tgacctgggt ccgtcaggct
120ccagggaagg ggctggagtg ggtcggcatc atctatggta gtgatgaaac
cgcctacgct 180acctccgcta taggccgatt caccatctcc agagacaatt
ccaagaacac cctgtatctt 240caaatgaaca gcctgagagc tgaggacact
gctgtgtatt actgtgctag agatgatagt 300agtgactggg atgcaaagtt caacttg
327725351DNAOryctolagus cuniculus 725cagtcgctgg aggagtccgg
gggtcgcctg gtcacgcctg ggacacccct gacactcacc 60tgcacagcct ctggattctc
cctcagtaac tactacgtga cctgggtccg ccaggctcca 120gggaaggggc
tggaatggat cggaatcatt tatggtagtg atgaaacggc ctacgcgacc
180tgggcgatag gccgattcac catctccaaa acctcgacca cggtggatct
gaaaatgacc 240agtctgacag ccgcggacac ggccacctat ttctgtgcca
gagatgatag tagtgactgg 300gatgcaaaat ttaacttgtg gggccaaggc
accctggtca ccgtctcgag c 351726224PRTHomo sapiens 726Met Glu Lys Leu
Leu Cys Phe Leu Val Leu Thr Ser Leu Ser His Ala 1 5 10 15 Phe Gly
Gln Thr Asp Met Ser Arg Lys Ala Phe Val Phe Pro Lys Glu 20 25 30
Ser Asp Thr Ser Tyr Val Ser Leu Lys Ala Pro Leu Thr Lys Pro Leu 35
40 45 Lys Ala Phe Thr Val Cys Leu His Phe Tyr Thr Glu Leu Ser Ser
Thr 50 55 60 Arg Gly Tyr Ser Ile Phe Ser Tyr Ala Thr Lys Arg Gln
Asp Asn Glu 65 70 75 80 Ile Leu Ile Phe Trp Ser Lys Asp Ile Gly Tyr
Ser Phe Thr Val Gly 85 90 95 Gly Ser Glu Ile Leu Phe Glu Val Pro
Glu Val Thr Val Ala Pro Val 100 105 110 His Ile Cys Thr Ser Trp Glu
Ser Ala Ser Gly Ile Val Glu Phe Trp 115 120 125 Val Asp Gly Lys Pro
Arg Val Arg Lys Ser Leu Lys Lys Gly Tyr Thr 130 135 140 Val Gly Ala
Glu Ala Ser Ile Ile Leu Gly Gln Glu Gln Asp Ser Phe 145 150 155 160
Gly Gly Asn Phe Glu Gly Ser Gln Ser Leu Val Gly Asp Ile Gly Asn 165
170 175 Val Asn Met Trp Asp Phe Val Leu Ser Pro Asp Glu Ile Asn Thr
Ile 180 185 190 Tyr Leu Gly Gly Pro Phe Ser Pro Asn Val Leu Asn Trp
Arg Ala Leu 195 200 205 Lys Tyr Glu Val Gln Gly Glu Val Phe Thr Lys
Pro Gln Leu Trp Pro 210 215 220 727468PRTHomo sapiens 727Met Leu
Ala Val Gly Cys Ala Leu Leu Ala Ala Leu Leu Ala Ala Pro 1 5 10 15
Gly Ala Ala Leu Ala Pro Arg Arg Cys Pro Ala Gln Glu Val Ala Arg 20
25 30 Gly Val Leu Thr Ser Leu Pro Gly Asp Ser Val Thr Leu Thr Cys
Pro 35 40 45 Gly Val Glu Pro Glu Asp Asn Ala Thr Val His Trp Val
Leu Arg Lys 50 55 60 Pro Ala Ala Gly Ser His Pro Ser Arg Trp Ala
Gly Met Gly Arg Arg 65 70 75 80 Leu Leu Leu Arg Ser Val Gln Leu His
Asp Ser Gly Asn Tyr Ser Cys 85 90 95 Tyr Arg Ala Gly Arg Pro Ala
Gly Thr Val His Leu Leu Val Asp Val 100 105 110 Pro Pro Glu Glu Pro
Gln Leu Ser Cys Phe Arg Lys Ser Pro Leu Ser 115 120 125 Asn Val Val
Cys Glu Trp Gly Pro Arg Ser Thr Pro Ser Leu Thr Thr 130 135 140 Lys
Ala Val Leu Leu Val Arg Lys Phe Gln Asn Ser Pro Ala Glu Asp 145 150
155 160 Phe Gln Glu Pro Cys Gln Tyr Ser Gln Glu Ser Gln Lys Phe Ser
Cys 165 170 175 Gln Leu Ala Val Pro Glu Gly Asp Ser Ser Phe Tyr Ile
Val Ser Met 180 185 190 Cys Val Ala Ser Ser Val Gly Ser Lys Phe Ser
Lys Thr Gln Thr Phe 195 200 205 Gln Gly Cys Gly Ile Leu Gln Pro Asp
Pro Pro Ala Asn Ile Thr Val 210 215 220 Thr Ala Val Ala Arg Asn Pro
Arg Trp Leu Ser Val Thr Trp Gln Asp 225 230 235 240 Pro His Ser Trp
Asn Ser Ser Phe Tyr Arg Leu Arg Phe Glu Leu Arg 245 250 255 Tyr Arg
Ala Glu Arg Ser Lys Thr Phe Thr Thr Trp Met Val Lys Asp 260 265 270
Leu Gln His His Cys Val Ile His Asp Ala Trp Ser Gly Leu Arg His 275
280 285 Val Val Gln Leu Arg Ala Gln Glu Glu Phe Gly Gln Gly Glu Trp
Ser 290 295 300 Glu Trp Ser Pro Glu Ala Met Gly Thr Pro Trp Thr Glu
Ser Arg Ser 305 310 315 320 Pro Pro Ala Glu Asn Glu Val Ser Thr Pro
Met Gln Ala Leu Thr Thr 325 330 335 Asn Lys Asp Asp Asp Asn Ile Leu
Phe Arg Asp Ser Ala Asn Ala Thr 340 345 350 Ser Leu Pro Val Gln Asp
Ser Ser Ser Val Pro Leu Pro Thr Phe Leu 355 360 365 Val Ala Gly Gly
Ser Leu Ala Phe Gly Thr Leu Leu Cys Ile Ala Ile 370 375 380 Val Leu
Arg Phe Lys Lys Thr Trp Lys Leu Arg Ala Leu Lys Glu Gly 385 390 395
400 Lys Thr Ser Met His Pro Pro Tyr Ser Leu Gly Gln Leu Val Pro Glu
405 410 415 Arg Pro Arg Pro Thr Pro Val Leu Val Pro Leu Ile Ser Pro
Pro Val 420 425 430 Ser Pro Ser Ser Leu Gly Ser Asp Asn Thr Ser Ser
His Asn Arg Pro 435 440 445 Asp Ala Arg Asp Pro Arg Ser Pro Tyr Asp
Ile Ser Asn Thr Asp Tyr 450 455 460 Phe Phe Pro Arg 465
728918PRTHomo sapiens 728Met Leu Thr Leu Gln Thr Trp Val Val Gln
Ala Leu Phe Ile Phe Leu 1 5 10 15 Thr Thr Glu Ser Thr Gly Glu Leu
Leu Asp Pro Cys Gly Tyr Ile Ser 20 25 30 Pro Glu Ser Pro Val Val
Gln Leu His Ser Asn Phe Thr Ala Val Cys 35 40 45 Val Leu Lys Glu
Lys Cys Met Asp Tyr Phe His Val Asn Ala Asn Tyr 50 55 60 Ile Val
Trp Lys Thr Asn His Phe Thr Ile Pro Lys Glu Gln Tyr Thr 65 70 75 80
Ile Ile Asn Arg Thr Ala Ser Ser Val Thr Phe Thr Asp Ile Ala Ser 85
90 95 Leu Asn Ile Gln Leu Thr Cys Asn Ile Leu Thr Phe Gly Gln Leu
Glu 100 105 110 Gln Asn Val Tyr Gly Ile Thr Ile Ile Ser Gly Leu Pro
Pro Glu Lys 115 120 125 Pro Lys Asn Leu Ser Cys Ile Val Asn Glu Gly
Lys Lys Met Arg Cys 130 135 140 Glu Trp Asp Gly Gly Arg Glu Thr His
Leu Glu Thr Asn Phe Thr Leu 145 150 155 160 Lys Ser Glu Trp Ala Thr
His Lys Phe Ala Asp Cys Lys Ala Lys Arg 165 170 175 Asp Thr Pro Thr
Ser Cys Thr Val Asp Tyr Ser Thr Val Tyr Phe Val 180 185 190 Asn Ile
Glu Val Trp Val Glu Ala Glu Asn Ala Leu Gly Lys Val Thr 195 200 205
Ser Asp His Ile Asn Phe Asp Pro Val Tyr Lys Val Lys Pro Asn Pro 210
215 220 Pro His Asn Leu Ser Val Ile Asn Ser Glu Glu Leu Ser Ser Ile
Leu 225 230 235 240 Lys Leu Thr Trp Thr Asn Pro Ser Ile Lys Ser Val
Ile Ile Leu Lys 245 250 255 Tyr Asn Ile Gln Tyr Arg Thr Lys Asp Ala
Ser Thr Trp Ser Gln Ile 260
265 270 Pro Pro Glu Asp Thr Ala Ser Thr Arg Ser Ser Phe Thr Val Gln
Asp 275 280 285 Leu Lys Pro Phe Thr Glu Tyr Val Phe Arg Ile Arg Cys
Met Lys Glu 290 295 300 Asp Gly Lys Gly Tyr Trp Ser Asp Trp Ser Glu
Glu Ala Ser Gly Ile 305 310 315 320 Thr Tyr Glu Asp Arg Pro Ser Lys
Ala Pro Ser Phe Trp Tyr Lys Ile 325 330 335 Asp Pro Ser His Thr Gln
Gly Tyr Arg Thr Val Gln Leu Val Trp Lys 340 345 350 Thr Leu Pro Pro
Phe Glu Ala Asn Gly Lys Ile Leu Asp Tyr Glu Val 355 360 365 Thr Leu
Thr Arg Trp Lys Ser His Leu Gln Asn Tyr Thr Val Asn Ala 370 375 380
Thr Lys Leu Thr Val Asn Leu Thr Asn Asp Arg Tyr Leu Ala Thr Leu 385
390 395 400 Thr Val Arg Asn Leu Val Gly Lys Ser Asp Ala Ala Val Leu
Thr Ile 405 410 415 Pro Ala Cys Asp Phe Gln Ala Thr His Pro Val Met
Asp Leu Lys Ala 420 425 430 Phe Pro Lys Asp Asn Met Leu Trp Val Glu
Trp Thr Thr Pro Arg Glu 435 440 445 Ser Val Lys Lys Tyr Ile Leu Glu
Trp Cys Val Leu Ser Asp Lys Ala 450 455 460 Pro Cys Ile Thr Asp Trp
Gln Gln Glu Asp Gly Thr Val His Arg Thr 465 470 475 480 Tyr Leu Arg
Gly Asn Leu Ala Glu Ser Lys Cys Tyr Leu Ile Thr Val 485 490 495 Thr
Pro Val Tyr Ala Asp Gly Pro Gly Ser Pro Glu Ser Ile Lys Ala 500 505
510 Tyr Leu Lys Gln Ala Pro Pro Ser Lys Gly Pro Thr Val Arg Thr Lys
515 520 525 Lys Val Gly Lys Asn Glu Ala Val Leu Glu Trp Asp Gln Leu
Pro Val 530 535 540 Asp Val Gln Asn Gly Phe Ile Arg Asn Tyr Thr Ile
Phe Tyr Arg Thr 545 550 555 560 Ile Ile Gly Asn Glu Thr Ala Val Asn
Val Asp Ser Ser His Thr Glu 565 570 575 Tyr Thr Leu Ser Ser Leu Thr
Ser Asp Thr Leu Tyr Met Val Arg Met 580 585 590 Ala Ala Tyr Thr Asp
Glu Gly Gly Lys Asp Gly Pro Glu Phe Thr Phe 595 600 605 Thr Thr Pro
Lys Phe Ala Gln Gly Glu Ile Glu Ala Ile Val Val Pro 610 615 620 Val
Cys Leu Ala Phe Leu Leu Thr Thr Leu Leu Gly Val Leu Phe Cys 625 630
635 640 Phe Asn Lys Arg Asp Leu Ile Lys Lys His Ile Trp Pro Asn Val
Pro 645 650 655 Asp Pro Ser Lys Ser His Ile Ala Gln Trp Ser Pro His
Thr Pro Pro 660 665 670 Arg His Asn Phe Asn Ser Lys Asp Gln Met Tyr
Ser Asp Gly Asn Phe 675 680 685 Thr Asp Val Ser Val Val Glu Ile Glu
Ala Asn Asp Lys Lys Pro Phe 690 695 700 Pro Glu Asp Leu Lys Ser Leu
Asp Leu Phe Lys Lys Glu Lys Ile Asn 705 710 715 720 Thr Glu Gly His
Ser Ser Gly Ile Gly Gly Ser Ser Cys Met Ser Ser 725 730 735 Ser Arg
Pro Ser Ile Ser Ser Ser Asp Glu Asn Glu Ser Ser Gln Asn 740 745 750
Thr Ser Ser Thr Val Gln Tyr Ser Thr Val Val His Ser Gly Tyr Arg 755
760 765 His Gln Val Pro Ser Val Gln Val Phe Ser Arg Ser Glu Ser Thr
Gln 770 775 780 Pro Leu Leu Asp Ser Glu Glu Arg Pro Glu Asp Leu Gln
Leu Val Asp 785 790 795 800 His Val Asp Gly Gly Asp Gly Ile Leu Pro
Arg Gln Gln Tyr Phe Lys 805 810 815 Gln Asn Cys Ser Gln His Glu Ser
Ser Pro Asp Ile Ser His Phe Glu 820 825 830 Arg Ser Lys Gln Val Ser
Ser Val Asn Glu Glu Asp Phe Val Arg Leu 835 840 845 Lys Gln Gln Ile
Ser Asp His Ile Ser Gln Ser Cys Gly Ser Gly Gln 850 855 860 Met Lys
Met Phe Gln Glu Val Ser Ala Ala Asp Ala Phe Gly Pro Gly 865 870 875
880 Thr Glu Gly Gln Val Glu Arg Phe Glu Thr Val Gly Met Glu Ala Ala
885 890 895 Thr Asp Glu Gly Met Pro Lys Ser Tyr Leu Pro Gln Thr Val
Arg Gln 900 905 910 Gly Gly Tyr Met Pro Gln 915
* * * * *