U.S. patent application number 11/554521 was filed with the patent office on 2007-03-22 for interferon-alpha polypeptides and conjugates.
This patent application is currently assigned to MAXYGEN, INC.. Invention is credited to Sridhar Govindarajan, Torben Lauesgaard Nissen, Phillip A. Patten, Sridhar Viswanathan.
Application Number | 20070065407 11/554521 |
Document ID | / |
Family ID | 36777639 |
Filed Date | 2007-03-22 |
United States Patent
Application |
20070065407 |
Kind Code |
A1 |
Patten; Phillip A. ; et
al. |
March 22, 2007 |
Interferon-Alpha Polypeptides and Conjugates
Abstract
The present invention provides interferon-alpha polypeptides and
conjugates, and nucleic acids encoding the polypeptides. The
invention also includes compositions comprising these polypeptides,
conjugates, and nucleic acids; cells containing or expressing the
polypeptides, conjugates, and nucleic acids; methods of making the
polypeptides, conjugates, and nucleic acids; and methods of using
the polypeptides, conjugates, and nucleic acids.
Inventors: |
Patten; Phillip A.; (Portola
Valley, CA) ; Govindarajan; Sridhar; (Redwood City,
CA) ; Viswanathan; Sridhar; (Menlo Park, CA) ;
Nissen; Torben Lauesgaard; (London, GB) |
Correspondence
Address: |
MAXYGEN, INC.;INTELLECTUAL PROPERTY DEPARTMENT
515 GALVESTON DRIVE
REDWOOD CITY
CA
94063
US
|
Assignee: |
MAXYGEN, INC.
|
Family ID: |
36777639 |
Appl. No.: |
11/554521 |
Filed: |
October 30, 2006 |
Related U.S. Patent Documents
|
|
|
|
|
|
Application
Number |
Filing Date |
Patent Number |
|
|
10848827 |
May 19, 2004 |
|
|
|
11554521 |
Oct 30, 2006 |
|
|
|
10714817 |
Nov 17, 2003 |
|
|
|
10848827 |
May 19, 2004 |
|
|
|
60502560 |
Sep 12, 2003 |
|
|
|
60427612 |
Nov 18, 2002 |
|
|
|
Current U.S.
Class: |
424/85.7 ;
435/320.1; 435/325; 435/69.51; 530/351; 536/23.5 |
Current CPC
Class: |
A61P 31/14 20180101;
A61P 1/16 20180101; A61P 31/18 20180101; A61K 47/60 20170801; A61P
37/02 20180101; A61K 38/212 20130101; A61P 43/00 20180101; C07K
14/56 20130101; A61K 9/0019 20130101; A61P 31/20 20180101; A61P
31/12 20180101 |
Class at
Publication: |
424/085.7 ;
435/069.51; 435/320.1; 435/325; 530/351; 536/023.5 |
International
Class: |
A61K 38/21 20060101
A61K038/21; C07K 14/565 20060101 C07K014/565; C07H 21/04 20060101
C07H021/04; C12P 21/04 20060101 C12P021/04 |
Claims
1.-34. (canceled)
35. A method for reducing the level of a virus in the serum of a
patient infected with the virus, comprising administering to the
patient a polypeptide in an amount effective to reduce the level of
virus in the serum compared to the level present prior to the start
of treatment, wherein the polypeptide comprises a sequence which
differs in 0 to 16 amino acid positions from the sequence of SEQ ID
NO:10 and wherein the polypeptide exhibits an interferon-alpha
activity, wherein the interferon-alpha activity is an anti-viral
activity.
36. The method of claim 35 wherein the virus is Hepatitis C
Virus.
37. The method of claim 35 wherein the virus is Hepatitis B
Virus.
38. The method of claim 35 wherein the virus is Human
Immunodeficiency Virus.
39. A method for reducing the level of a virus in the serum of a
patient infected with the virus, comprising administering to the
patient a conjugate in an amount effective to reduce the level of
virus in the serum compared to the level present prior to the start
of treatment, wherein the conjugate comprises: a) a polypeptide
comprising a sequence which differs in 0 to 16 amino acid positions
from the sequence of SEQ ID NO:10; and b) a non-polypeptide moiety
covalently attached to the polypeptide, and wherein the conjugate
exhibits an interferon-alpha activity, wherein the interferon-alpha
activity is an antiviral activity.
40. The method of claim 39 wherein the virus is Hepatitis C
Virus.
41. The method of claim 39 wherein the virus is Hepatitis B
Virus.
42. The method of claim 39 wherein the virus is Human
Immunodeficiency Virus.
43. The method of claim 40 wherein the non-polypeptide moiety is a
polymer.
44. The method of claim 43 wherein the polymer is polyethylene
glycol.
45. The method of claim 44 wherein a polyethylene glycol moiety is
covalently attached to a cysteine residue.
46. The method of claim 44 wherein a polyethylene glycol moiety is
covalently attached to a lysine residue or to the N-terminal amino
group.
47. The method of claim 44 wherein a polyethylene glycol moiety is
covalently attached to a lysine residue.
48. The method of claim 44 wherein a polyethylene glycol moiety is
covalently attached to the N-terminal amino group.
49. The method of claim 44, wherein at least two polyethylene
glycol moieties are attached to the polypeptide and wherein each
polyethylene glycol moiety is covalently attached to a different
amino acid residue of the polypeptide.
50. The method of claim 49, wherein the at least two polyethylene
glycol moieties are attached to different lysine residues.
51. The method of claim 49, wherein one of the at least two
polyethylene glycol moieties is attached to the N-terminal amino
group and one of the at least two polyethylene glycol moieties is
attached to a lysine residue.
52. A method of treating a patient infected with Hepatitis C Virus
comprising administering to the patient a pharmaceutically
acceptable excipient and a therapeutically effective amount of a
conjugate, wherein the conjugate comprises: a) a polypeptide
comprising a sequence which differs in 0 to 16 amino acid positions
from the sequence of SEQ ID NO:10; and b) a non-polypeptide moiety
covalently attached to the polypeptide, and wherein the conjugate
exhibits an interferon-alpha activity, wherein the interferon-alpha
activity is an antiviral activity.
53. The method of claim 52 wherein the conjugate is administered by
weekly subcutaneous injection.
54. The method of claim 52 wherein the conjugate is administered by
subcutaneous injection every two weeks.
55. The method of claim 52 wherein the non-polypeptide moiety is a
polymer.
56. The method of claim 55 wherein the polymer is polyethylene
glycol.
57. The method of claim 56 wherein a polyethylene glycol moiety is
covalently attached to a cysteine residue.
58. The method of claim 56 wherein a polyethylene glycol moiety is
covalently attached to a lysine residue or to the N-terminal amino
group.
59. The method of claim 56 wherein a polyethylene glycol moiety is
covalently attached to a lysine residue.
60. The method of claim 56 wherein a polyethylene glycol moiety is
covalently attached to the N-terminal amino group.
61. The method of claim 56, wherein at least two polyethylene
glycol moieties are attached to the polypeptide and wherein each
polyethylene glycol moiety is covalently attached to a different
amino acid residue of the polypeptide.
62. The method of claim 61, wherein the at least two polyethylene
glycol moieties are attached to different lysine residues.
63. The method of claim 61, wherein one of the at least two
polyethylene glycol moieties is attached to the N-terminal amino
group and one of the at least two polyethylene glycol moieties is
attached to a lysine residue.
Description
CROSS-REFERENCE TO RELATED APPLICATIONS
[0001] This application is a Continuation-in-part of U.S.
application Ser. No. 10/714,817 filed on Nov. 17, 2003, which
claims the benefit of U.S. Provisional Application Ser. No.
60/502,560 filed on Sep. 12, 2003 and U.S. Provisional Application
Ser. No. 60/427,612 filed on Nov. 18, 2002, the disclosures of each
of which are incorporated by reference herein in their entirety for
all purposes.
COPYRIGHT NOTIFICATION
[0002] Pursuant to 37 C.F.R. 1.71(e), Applicants note that a
portion of this disclosure contains material which is subject to
copyright protection. The copyright owner has no objection to the
facsimile reproduction by anyone of the patent document or patent
disclosure, as it appears in the Patent and Trademark Office patent
file or records, but otherwise reserves all copyright rights
whatsoever.
FIELD OF THE INVENTION
[0003] The present invention relates generally to polynucleotides
and polypeptides encoded therefrom, conjugates of the polypeptides,
as well as vectors, cells, antibodies, and methods for using and
producing the polynucleotides, polypeptides, and conjugates.
BACKGROUND OF THE INVENTION
[0004] Interferon-alphas are members of the diverse helical-bundle
superfamily of cytokine genes (Sprang, S. R. et al. (1993) Curr.
Opin. Struct. Biol. 3:815-827). The human interferon-alphas are
encoded by a family of over 20 tandemly duplicated nonallelic genes
and psuedogenes that share 85-98% sequence identity at the amino
acid level (Henco, K. et al. (1985) J. Mol. Biol. 185:227-260).
Genes which express active interferon-alpha proteins have been
grouped into 13 families according to genetic loci. Known expressed
human interferon-alpha proteins and their allelic variations are
tabulated in Allen G. and Diaz M. O. (1996) J. Interferon and
Cytokine Res. 16:181-184.
[0005] Interferon-alphas have been shown to inhibit various types
of cellular proliferation, and are especially useful for the
treatment of a variety of cellular proliferation disorders
frequently associated with cancer, particularly hematologic
malignancies such as leukemias. These proteins have shown
antiproliferative activity against multiple myeloma, chronic
lymphocytic leukemia, low-grade lymphoma, Kaposi's sarcoma, chronic
myelogenous leukemia, renal-cell carcinoma, urinary bladder tumors
and ovarian cancers (Bonnem, E. M. et al. (1984) J. Biol. Response
Modifiers 3:580; Oldham, R. K. (1985) Hospital Practice 20:71).
[0006] Interferon-alphas are also useful against various types of
viral infections (Finter, N. B. et al. (1991) Drugs 42(5):749).
Interferon-alphas have activity against human papillomavirus
infection, Hepatitis B, and Hepatitis C infections (Finter, N. B.
et al., 1991, supra; Kashima, H. et al. (1988) Laryngoscope 98:334;
Dusheiko, G. M. et al. (1986) J. Hematology 3 (Supple. 2):S199;
Davis, G L et al. (1989) N. England J. Med. 321:1501).
[0007] The role of interferons and interferon receptors in the
pathogenesis of certain autoimmune and inflammatory diseases has
also been investigated (Benoit, P. et al. (1993) J. Immunol.
150(3):707).
[0008] Although these proteins possess therapeutic value in the
treatment of a number of diseases, they have not been optimized for
use as pharmaceuticals. For example, dose-limiting toxicity,
receptor cross-reactivity, and short serum half-lives significantly
reduce the clinical utility of many of these cytokines (Dusheiko,
G. (1997) Hepatology 26:112S-121S; Vial, T. and Descotes, J. (1994)
Drug Experience 10:115-150; Funke, I. et al. (1994) Ann. Hematol.
68:49-52; Schomburg, A. et al. (1993) J. Cancer Res. Clin. Oncol.
119:745-755). Diverse and severe side effect profiles which
accompany interferon administration include flu-like symptoms,
fatigue, hallucination, fever, hepatic enzyme elevation, and
leukopenia (Pontzer, C. H. et al. (1991) Cancer Res. 51:5304;
Oldham, 1985, supra).
[0009] Hepatitis C virus (HCV) is a nonhost integrated RNA virus
with a very high rate of replication and is therefore associated
with a large degree of genetic diversity. At least six genotypes
and more than thirty subtypes of HCV RNA have been identified. HCV
genotype has been shown to be a predictor of response to IFN-alpha
therapy. Patients infected with HCV genotypes 2 and 3 have been
found to generally respond well to interferon therapy. Patients
infected with genotypes 4, 5 and 6 tend to respond less well.
Patients infected with HCV genotype 1 tend to respond very poorly
to interferon therapy, with about 50% of Genotype 1 patients
classified as "nonresponders" towards IFN-alpha therapy. Genotype 1
is currently the most prevalent form of Hepatitis C, infecting
approximately 70% of patients in the US and 50% of patients in
Europe. Clearly, there is a pressing need for more effective
therapies for HCV infection, particularly of the Genotype 1
variety.
[0010] There is genetic and biochemical evidence that Genotype 1
HCV (and other subtypes) actively attenuate the IFN-alpha signaling
pathway by inhibiting key IFN responsive proteins such as the
dsRNA-activated serine/threonine protein kinase PKR (Katze M.
(2002) Nat. Rev. Immunol. 2(9):675-687). As a likely consequence of
this genetic diversity and active inhibition of the antiviral
response, HCV (particularly Genotype 1) has the ability to escape
the host's immune surveillance, leading to a high rate of chronic
infection. The extensive genetic heterogeneity of HCV has important
diagnostic and clinical implications, potentially accounting for
variations in clinical course, difficulties in vaccine development,
and lack of response to therapy.
[0011] The present invention addresses the need for
interferon-alpha molecules which exhibit enhanced antiviral
efficacy and/or enhanced immunomodulatory efficacy compared to
interferon-alphas currently in clinical use. The invention provides
novel interferon-alpha polypeptides and polypeptide conjugates,
nucleic acids encoding the polypeptides, and methods of using such
molecules. Such molecules would be of beneficial use in a variety
of applications, including, e.g., therapeutic and prophylactic
treatments, particularly for viral infections and diseases and
conditions associated with viral infections. The present invention
fulfills these and other needs.
SUMMARY OF THE INVENTION
[0012] The present invention provides novel polypeptides, including
variants and fusion polypeptides. The invention also provides
conjugates comprising a polypeptide of the invention covalently
linked to one or more non-polypeptide moieties. The invention also
provides nucleic acids encoding any of the polypeptides of the
invention, and vectors and host cells comprising such nucleic
acids. In addition, the invention provides methods of making and
using such polypeptides, conjugates, and nucleic acids, and other
features apparent upon further review.
[0013] In one aspect, the invention provides an isolated or
recombinant polypeptide, the polypeptide comprising a sequence
identified as one of SEQ ID NOs:1-15 and SEQ ID NOs:44-104 (such as
one of SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID
NO:5, SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:9, SEQ ID
NO:10, SEQ ID NO:11, SEQ ID NO:12, SEQ ID NO:13, SEQ ID NO:14, SEQ
ID NO:15, SEQ ID NO:44, SEQ ID NO:45, SEQ ID NO:46, SEQ ID NO:47,
SEQ ID NO:48, SEQ ID NO:49, SEQ ID NO:50, SEQ ID NO:51, SEQ ID
NO:52, SEQ ID NO:53, SEQ ID NO:54, SEQ ID NO:55, SEQ ID NO:56, SEQ
ID NO:57, SEQ ID NO:58, SEQ ID NO:59, SEQ ID NO:60, SEQ ID NO:61,
SEQ ID NO:62, SEQ ID NO:63, SEQ ID NO:64, SEQ ID NO:65, SEQ ID
NO:66, SEQ ID NO:67, SEQ ID NO:68, SEQ ID NO:69, SEQ ID NO:70, SEQ
ID NO:71, SEQ ID NO:72, SEQ ID NO:73, SEQ ID NO:74, SEQ ID NO:75,
SEQ ID NO:76, SEQ ID NO:77, SEQ ID NO:78, SEQ ID NO:79, SEQ ID
NO:80, SEQ ID NO:81, SEQ ID NO:82, SEQ ID NO:83, SEQ ID NO:84, SEQ
ID NO:85, SEQ ID NO:86, SEQ ID NO:87, SEQ ID NO:88, SEQ ID NO:89,
SEQ ID NO:90, SEQ ID NO:91, SEQ ID NO:92, SEQ ID NO:93, SEQ ID
NO:94, SEQ ID NO:95, SEQ ID NO:96, SEQ ID NO:97, SEQ ID NO:98, SEQ
ID NO:99, SEQ ID NO:100, SEQ ID NO:101, SEQ ID NO:102, SEQ ID
NO:103, and SEQ ID NO:104).
[0014] The invention also provides isolated or recombinant
polypeptides which each comprise a sequence which differs in 0-16
amino acid positions (such as in 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10,
11, 12, 13, 14, 15, or 16 amino acid positions), e.g. in 0-14 amino
acid positions, in 0-12 amino acid positions, in 0-10 amino acid
positions, in 0-8 amino acid positions, in 0-6 amino acid
positions, in 0-5 amino acid positions, in 0-4 amino acid
positions, in 0-3 amino acid positions, in 0-2 amino acid
positions, or in 0-1 amino acid position, from one of SEQ ID
NOs:1-15 and SEQ ID NOs:44-104, such as, for example, one of SEQ ID
NOs:1-15, 47, or 53 (for example, SEQ ID NO:1, SEQ ID NO:3, SEQ ID
NO:8, SEQ ID NO:12, SEQ ID NO:47, or SEQ ID NO:53). In some
instances, the polypeptide exhibits an interferon-alpha activity
(such as, e.g., antiviral activity, T.sub.H1 differentiation
activity, and/or antiproliferative activity). In some instances,
the polypeptide sequence comprises a substitution at one or more of
positions 47, 51, 52, 53, 54, 55, 56, 57, 58, 60, 61, 64, 69, 71,
72, 75, 76, 77, 78, 79, 80, 83, 84, 85, 86, 87, 90, 93, 133, 140,
154, 160, 161, and 162, relative to one of SEQ ID NOs: 1-15, 47, or
53, such as, for example, one of SEQ ID NO:1, SEQ ID NO:3, SEQ ID
NO:8, SEQ ID NO:12, SEQ ID NO:47, or SEQ ID NO:53. In some
instances, the polypeptide sequence comprises one or more of: His
or Gln at position 47; Val, Ala or Thr at position 51; Gln, Pro or
Glu at position 52; Ala or Thr at position 53; Phe, Ser, or Pro at
position 55; Leu, Val or Ala at position 56; Phe or Leu at position
57; Tyr or His at position 58; Met, Leu or Val at position 60; Met
or Ile at position 61; Thr or Ile at position 64; Ser or Thr at
position 69; Lys or Glu at position 71; Asn or Asp at position 72;
Ala or Val at position 75; Ala or Thr at position 76; Trp or Leu at
position 77; Asp or Glu at position 78; Glu or Gln at position 79;
Thr, Asp, Ser, or Arg at position 80; Glu or Asp at position 83;
Lys or Glu at position 84; Phe or Leu at position 85; Tyr, Cys or
Ser at position 86; Ile or Thr at position 87; Phe, Tyr, Asp or Asn
at position 90; Met or Leu at position 93; Lys or Glu at position
133; Ser or Ala at position 140; Phe or Leu at position 154; Lys or
Glu at position 160; Arg or Ser at position 161; and Arg or Ser at
position 162; the position numbering relative to that of SEQ ID
NO:1. In some instances, the polypeptide sequence comprises one or
more of His47, Val51, Phe55, Leu56, Tyr58, Lys133, and Ser140, the
position numbering relative to that of SEQ ID NO:1. Some such
polypeptides include SEQ ID NOs:1-15 and SEQ ID NOs:44-104. The
invention also provides fusion proteins and conjugates comprising
any of these polypeptides, nucleic acids encoding such
polypeptides, and methods of making such polypeptides.
[0015] Some polypeptides of the invention comprise one or more
substitution, including but not limited to a substitution selected
from: D2C, L3C, P4C, Q5C, T6C, H.sub.7C, S8C, L9C, G10C, R12C,
R13C, M16C, A19C, Q20C, R22C, R23C, 124C, S25C, L26C, F27C, S28C,
L30C, K31C, R33C, H.sub.34C, D35C, R37C, Q40C, E41C, E42C, D44C,
N46C, H.sub.47C, Q49C, K50C, V51C, Q52C, E59C, Q62C, Q63C, N66C,
S69C, T70C, K71C, N72C, S74C, A75C, D78C, E79C, T80C, L81C, E83C,
K84C, 187C, F90C, Q91C, N94C, D95C, E97C, A98C, V100C, M101C,
Q102C, E103C, V104C, G105C, E107C, E108C, T109C, P10C, L10C, M112C,
N113C, V114C, D115C, L118C, R121C, K122C, Q125C, R126C, T128C,
L129C, T132C, K133C, K134C, K135C, Y136C, S137C, P138C, A146C,
M149C, R150C, S153C, F154C, N157C, Q159C, K160C, R161C, L162C,
R163C, R164C, K165C and E166C (or equivalent position relative to
SEQ ID NO:1), and combinations thereof.
[0016] Some polypeptides of the invention comprise one or more
substitution, including but not limited to a substitution selected
from: D2K, L3K, P4K, Q5K, T6K, H7K, S8K, L9K, G10K, R12K, R13K,
M16K, A19K, Q20K, R22K, R23K, 124K, S25K, L26K, F27K, S28K, L30K,
R33K, H34K, D35K, R37K, Q40K, E41K, E42K, D44K, N46K, H47K, Q49K,
V51K, Q52K, E59K, Q62K, Q63K, N66K, S69K, T70K, N72K, S74K, A75K,
D78K, E79K, T80K, L81K, E83K, 187K, F90K, Q91K, N94K, D95K, E97K,
A98K, V100K, M101K, Q102K, E103K, V104K, G105K, E107K, E108K,
T109K, P110K, L111K, M112K, N113K, V114K, D115K, L118K, R121K,
Q125K, R126K, T128K, L129K, T132K, Y136K, S137K, P138K, A146K,
M149K, R150K, S153K, F154K, N157K, Q159K, R161K, L162K, R163K,
R164K, and E166K (or equivalent position relative to SEQ ID NO:1),
and combinations thereof.
[0017] Some polypeptides of the invention comprise one or more
substitution of an amino acid residue for a different amino acid
residue, or one or more deletion of an amino acid residue, which
removes one or more lysines, e.g., K31, K50, K71, K84, K122, K133,
K134, K135, K160, and/or K165 (relative to SEQ ID NO:1) from any
polypeptide of the invention such as, for example, any one of SEQ
ID NOs:1-15 and SEQ ID NOs:44-104 (such as, e.g., SEQ ID NO:1, SEQ
ID NO:3, SEQ ID NO:8, SEQ ID NO:12, SEQ ID NO:47 or SEQ ID NO:53).
The one or more lysine residue(s) to be removed may be substituted
with any other amino acid, may be substituted with an Arg (R) or
Gln (Q), or may be deleted.
[0018] Some polypeptides of the invention comprise one or more
substitution of an amino acid residue for a different amino acid
residue, or one or more deletion of an amino acid residue, which
removes one or more histidines, e.g., H7, H11, H34, and/or H47
(relative to SEQ ID NO:1) from any polypeptide of the invention
such as, for example, one of SEQ ID NOs:1-15 and SEQ ID NOs:44-104
(such as, e.g., SEQ ID NO:1, SEQ ID NO:3, SEQ ID NO:8, SEQ ID
NO:12, SEQ ID NO:47, or SEQ ID NO:53). The one or more histidine
residue(s) to be removed may be substituted with any other amino
acid, may be substituted with an Arg (R) or Gln (Q), or may be
deleted.
[0019] Some polypeptides of the invention comprise one or more
substitution, including but not limited to substitutions selected
from: D2N+P4S/T, L3N+Q5S/T, P4Q, P4Q+T6S, Q5N+H7S/T, T6N, T6N+S8T,
H.sub.7N+L9S/T, S8N+G10S/T, L9N+H11S/T, G10N+R12S/T, R12N,
R12N+T14S, R13N+M15S/T, M16N+L18S/T, A19N+M21S/T, Q20N+R22S/T,
R22N+124S/T, R23N, R23N+S25T, 124N+L26S/T, S25N+F27S/T, L26N,
L26N+S28T, S28N+L30S/T, L30N+D32S/T, K31N+R33S/T, R33N+D35S/T,
H.sub.34N+F36S/T, D35N+R37S/T, R37N+P39S/T, Q40N+E42S/T,
E41N+F43S/T, E42N+D44S/T, D44N+N46S/T, F48S/T, H.sub.47N+Q49S/T,
Q49N+V51S/T, K50N+Q52S/T, V51N+A53S/T, Q52N+154S/T, E59N+M61S/T,
Q62N, Q62N+T64S, Q63N+F65S/T, F68S/T, S69N+K71S/T, T70N+N72S/T,
K71N, K71N+S73T, S74T, S74N+A76S/T, A75N+W77S/T, D78N, D78N+T80S,
E79N+L81S/T, T80N+L82S/T, L81N+E83S/T, E83N+F85S/T, K84N+Y86S/T,
187N+L89S/T, F90N+Q92S/T, Q91N+M93S/T, L96S/T, D95N+E97S/T,
E97N+C99S/T, A98N+V100S/T, V100N+Q102S/T, M101N+E103S/T,
Q102N+V104S/T, E103N+G105S/T, V104N+V106S/T, G105N+E107S/T, E107,
E107N+T109S, E108N+P110S/T, L111N+N113S/T, M112N+V114S/T,
N113N+D115S/T, V114N, V114N+S116T, D115N+1117S/T, L118N+V120S/T,
R121N+Y123S/T, K122N+F124S/T, Q125N+1127S/T, R126N, R126N+T128S,
T128N+Y130S/T, L129N+L131S/T, T132N+K134S/T, K133N+K135S/T,
K134N+Y136S/T, K135N, K135N+S137T, Y136N+P138S/T, P138N,
P138N+S140T, A146N+I148S/T, M149N, M149N+S151T, R150N+F152S/T,
S153N, S153N+S155T, F154N+F156S/T, Q159S/T, K160N+L162S/T,
R161N+R163S/T, L162N+R164S/T, R163N+K165S/T and R164N+E166S/T (or
equivalent positions relative to SEQ ID NO:1), and combinations
thereof.
[0020] The invention also provides fusion proteins and conjugates
comprising any of the above polypeptides, nucleic acids encoding
such polypeptides, and methods of making and using such
polypeptides.
[0021] In another aspect, the invention provides isolated or
recombinant polypeptides which each comprise a sequence having at
least about 90% amino acid sequence identity to one of SEQ ID
NOs:1-15 and SEQ ID NOs:44-104, such as, for example, one of SEQ ID
NOs:1-15, 47, and 53 (such as, for example, SEQ ID NO:1, SEQ ID NO:
3, SEQ ID NO:8, SEQ ID NO:12, SEQ ID NO:47, or SEQ ID NO:53). Some
such polypeptides exhibit an interferon-alpha activity (such as,
e.g., antiviral activity, T.sub.H1 differentiation activity, and/or
antiproliferative activity). In some instances, the sequence has at
least about 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity
to one of SEQ ID NOs:1-15 and SEQ ID NOs 44-104 (such as, e.g., SEQ
ID NO:1, SEQ ID NO:3, SEQ ID NO:8, SEQ ID NO:12, SEQ ID NO:47, or
SEQ ID NO:53). In some instances, the polypeptide sequence
comprises a substitution at one or more of positions 47, 51, 52,
53, 54, 55, 56, 57, 58, 60, 61, 64, 69, 71, 72, 75, 76, 77, 78, 79,
80, 83, 84, 85, 86, 87, 90, 93, 133, 140, 154, 160, 161, and 162,
relative to, e.g., one of SEQ ID NOs:1-15. In some instances, the
polypeptide sequence comprises one or more of: His or Gln at
position 47; Val, Ala or Thr at position 51; Gln, Pro or Glu at
position 52; Ala or Thr at position 53; Phe, Ser, or Pro at
position 55; Leu, Val or Ala at position 56; Phe or Leu at position
57; Tyr or His at position 58; Met, Leu or Val at position 60; Met
or Ile at position 61; Thr or Ile at position 64; Ser or Thr at
position 69; Lys or Glu at position 71; Asn or Asp at position 72;
Ala or Val at position 75; Ala or Thr at position 76; Trp or Leu at
position 77; Asp or Glu at position 78; Glu or Gln at position 79;
Thr, Asp, Ser, or Arg at position 80; Glu or Asp at position 83;
Lys or Glu at position 84; Phe or Leu at position 85; Tyr, Cys or
Ser at position 86; Ile or Thr at position 87; Phe, Tyr, Asp or Asn
at position 90; Met or Leu at position 93; Lys or Glu at position
133; Ser or Ala at position 140; Phe or Leu at position 154; Lys or
Glu at position 160; Arg or Ser at position 161; and Arg or Ser at
position 162 (the position numbering relative to that of SEQ ID
NO:1). In some instances, the polypeptide sequence comprises one or
more of His47, Val51, Phe55, Leu56, Tyr58, Lys133, and Ser140, the
position numbering relative to that of SEQ ID NO:1. Some such
polypeptides comprise a sequence selected from SEQ ID NOs:1-15 and
SEQ ID NOs:44-104. The invention also provides fusion proteins and
conjugates comprising any of the above polypeptides, nucleic acids
encoding such polypeptides, and methods of making such
polypeptides.
[0022] In another aspect, the invention provides isolated or
recombinant polypeptides which are variants of a parent
polypeptide, each variant comprising a variant sequence which
differs from the parent polypeptide sequence in least one amino
acid position relative to the parent polypeptide sequence, wherein
the parent polypeptide sequence is one of SEQ ID NOs:1-15 and SEQ
ID NOs:44-104, such as, for example, one of SEQ ID NOs:1-15, 47,
and 53, e.g., SEQ ID NO:1, SEQ ID NO:3, SEQ ID NO:8, SEQ ID NO:12,
SEQ ID NO:47, or SEQ ID NO:53. In some instances, the variant
exhibits an interferon-alpha activity (such as, antiviral activity,
T.sub.H1 differentiation activity, and/or antiproliferative
activity). In some instances, the variant sequence differs from the
parent polypeptide sequence in 1-16 amino acid positions (such as
in 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, or 16 amino
acid positions), e.g. in 1-14 amino acid positions, in 1-12 amino
acid positions, in 1-10 amino acid positions, in 1-8 amino acid
positions, in 1-6 amino acid positions, in 1-5 amino acid
positions, in 1-4 amino acid positions, in 1-3 amino acid
positions, or in 1-2 amino acid positions. In some instances, the
variant sequence comprises a substitution at one or more of
positions 47, 51, 52, 53, 54, 55, 56, 57, 58, 60, 61, 64, 69, 71,
72, 75, 76, 77, 78, 79, 80, 83, 84, 85, 86, 87, 90, 93, 133, 140,
154, 160, 161, and 162, relative to one of SEQ ID NOs:1-15. In some
instances, the variant sequence comprises one or more of: His or
Gln at position 47; Val, Ala or Thr at position 51; Gln, Pro or Glu
at position 52; Ala or Thr at position 53; Phe, Ser, or Pro at
position 55; Leu, Val or Ala at position 56; Phe or Leu at position
57; Tyr or His at position 58; Met, Leu or Val at position 60; Met
or Ile at position 61; Thr or Ile at position 64; Ser or Thr at
position 69; Lys or Glu at position 71; Asn or Asp at position 72;
Ala or Val at position 75; Ala or Thr at position 76; Trp or Leu at
position 77; Asp or Glu at position 78; Glu or Gln at position 79;
Thr, Asp, Ser, or Arg at position 80; Glu or Asp at position 83;
Lys or Glu at position 84; Phe or Leu at position 85; Tyr, Cys or
Ser at position 86; Ile or Thr at position 87; Phe, Tyr, Asp or Asn
at position 90; Met or Leu at position 93; Lys or Glu at position
133; Ser or Ala at position 140; Phe or Leu at position 154; Lys or
Glu at position 160; Arg or Ser at position 161; and Arg or Ser at
position 162; the position numbering relative to that of SEQ ID
NO:1. In some instances, the variant sequence comprises one or more
of His47, Val51, Phe55, Leu56, Tyr58, Lys133, and Ser140, the
position numbering relative to that of SEQ ID NO:1. Some such
variants comprise a sequence selected from SEQ ID NOs:1-15 and SEQ
ID NOs:44-104. The invention also provides fusion proteins and
conjugates comprising any of these variants, nucleic acids encoding
any of these variants, and methods of making such variants.
[0023] In another aspect, the invention provides isolated or
recombinant polypeptides which are variants of a parent
interferon-alpha polypeptide, each variant comprising a variant
sequence which differs from the parent interferon-alpha polypeptide
sequence in least one amino acid position, wherein the variant
sequence comprises one or more of His47, Val51, Phe55, Leu56,
Tyr58, Lys133, and Ser140, the position numbering relative to that
of SEQ ID NO:1. In some instances the parent interferon-alpha
polypeptide sequence is a sequence of a naturally-occurring human
interferon-alpha, such as one of SEQ ID NO:31-SEQ ID NO:42 or SEQ
ID NO:32+R23K, or a non-naturally occurring (i.e., synthetic)
interferon-alpha, such as SEQ ID NO:43. In some instances, the
variant sequence differs from the parent interferon-alpha
polypeptide sequence in 1-16 amino acid positions (such as in 1, 2,
3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, or 16 amino acid
positions), e.g. in 1-14 amino acid positions, in 1-12 amino acid
positions, in 1-10 amino acid positions, in 1-8 amino acid
positions, in 1-6 amino acid positions, in 1-5 amino acid
positions, in 1-4 amino acid positions, in 1-3 amino acid
positions, or in 1-2 amino acid positions. In some instances, the
variant exhibits an interferon-alpha activity (such as, e.g.,
antiviral activity, T.sub.H1 differentiation activity, and/or
antiproliferative activity). The invention also provides fusion
proteins and conjugates comprising any of these variants, nucleic
acids encoding any of these variants, and methods of making such
variants.
[0024] The invention also provides conjugates comprising a
polypeptide of the invention, such as any of the polypeptides of
the invention (including variants) described above, and at least
one non-polypeptide moiety attached to an attachment group of the
polypeptide, wherein the conjugate exhibits an interferon-alpha
activity. In some instances, the non-polypeptide moiety is a
polymer (such as, e.g., PEG or mPEG), or a sugar moiety. The at
least one non-polypeptide moiety may be attached to a cysteine, to
a lysine, to the N-terminal amino group of the polypeptide, to an
in vivo glycosylation site of the polypeptide. The invention also
provides methods of making and using such conjugates.
[0025] The invention also provides isolated or recombinant nucleic
acids encoding any of the polypeptides (including variants) of the
invention. The invention also provides vectors and host cells
comprising such nucleic acids, and methods of making polypeptides
of the invention, comprising culturing host cells comprising such
nucleic acids.
[0026] In another aspect, the invention provides a method of
inhibiting viral replication in virus-infected cells, the method
comprising contacting the virus-infected cells with a polypeptide
or a conjugate of the invention. The invention also provides a
method of reducing the number of copies of a virus in
virus-infected cells, comprising contacting the virus-infected
cells with a polypeptide or a conjugate of the invention.
[0027] In another aspect, the invention provides a method for
reducing the level of a virus in the serum of a patient infected
with the virus, comprising administering to the patient the
polypeptide or a conjugate of the invention in an amount effective
to reduce the level of the virus in the serum compared to the level
present prior to the start of treatment.
[0028] These and other objects and features of the invention will
become more fully apparent when the following detailed description
is read in conjunction with the accompanying figures.
BRIEF DESCRIPTION OF THE FIGURES
[0029] FIGS. 1A and 1B show biphasic timecourses for viral
clearance from HCV-infected cells following IFN-alpha treatment (A.
Nonresponder kinetics; B. Responder kinetics).
[0030] FIG. 2 shows an alignment of the sequence of a polypeptide
of the invention (SEQ ID NO:1) with the following human
interferon-alpha polypeptide sequences: huIFN-alpha 1a (SEQ ID
NO:31), huIFN-alpha 2b (SEQ ID NO:32), huIFN-alpha 4b (SEQ ID
NO:33), huIFN-alpha 5 (SEQ ID NO:34), huIFN-alpha 6 (SEQ ID NO:35),
huIFN-alpha 7 (SEQ ID NO:36), huIFN-alpha 8b (SEQ ID NO:37),
huIFN-alpha 10a (SEQ ID NO:38), huIFN-alpha 14a (SEQ ID NO:39),
huIFN-alpha 16 (SEQ ID NO:40), huIFN-alpha 17b (SEQ ID NO:41) and
huIFN-alpha 21b (SEQ ID NO:42). The naming conventions for the
huIFN-alpha sequences are according to Allen G. and Diaz M. O.
(1996) J. Interferon and Cytokine Res. 16:181-184. The arrows
indicate residues His47, Val51, Phe55, Leu56, Tyr58, Lys133, and
Ser140 of SEQ ID NO:1, which are not present in any of SEQ ID
NOs:31-SEQ ID NO:42. Amino acid residue positions in SEQ ID
NOs:31-42 which are identical to SEQ ID NO:1 are indicated with a
period (.), and gaps in the sequence are indicated with a dash
(-).
[0031] FIG. 3 shows an alignment of the sequence of a polypeptide
of the invention (SEQ ID NO:3) with huIFN-alpha 14a (SEQ ID NO:39)
(LeIF H; Goeddel et al. (1981) Nature 290:20-26) using the
following parameters: BLOSUM62 matrix, gap open penalty 11, gap
extension penalty 1. Amino acid positions in SEQ ID NO:39 which are
identical to SEQ ID NO:3 are indicated with a period (.).
[0032] FIG. 4 shows an alignment of the sequence of a polypeptide
of the invention (SEQ ID NO:8) with human interferon-alpha
polypeptide sequences SEQ ID NO:31-SEQ ID NO:42. Amino acid residue
positions in SEQ ID NOs:31-42 which are identical to SEQ ID NO:8
are indicated with a period (.), and gaps in the sequence are
indicated with a dash (-).
[0033] FIG. 5 shows an alignment of the sequence of a polypeptide
of the invention (SEQ ID NO:12) with huIFN-alpha 14a (SEQ ID NO:39)
(LeIF H; Goeddel et al. (1981) Nature 290:20-26) using the
following parameters: BLOSUM62 matrix, gap open penalty 11, gap
extension penalty 1. Amino acid positions in SEQ ID NO:39 which are
identical to SEQ ID NO:12 are indicated with a period (.).
[0034] FIG. 6 shows the BLOSUM62 substitution matrix.
[0035] FIGS. 7A, 7B and 7C show examples of calculations of
alignment scores used to determine optimal sequence alignments,
using the following parameters: BLOSUM62 matrix, gap open
penalty=11, and gap extension penalty=1.
DETAILED DESCRIPTION OF THE INVENTION
Definitions
[0036] Unless otherwise defined herein or in the remainder of the
specification, all technical and scientific terms used herein have
the same meaning as commonly understood by those of ordinary skill
in the art to which the invention belongs.
[0037] A "polypeptide sequence" (e.g., a protein, polypeptide,
peptide, etc.) is a polymer of amino acids comprising naturally
occurring amino acids or artificial amino acid analogues, or a
character string representing an amino acid polymer, depending on
context. Given the degeneracy of the genetic code, one or more
nucleic acids, or the complementary nucleic acids thereof, that
encode a specific polypeptide sequence can be determined from the
polypeptide sequence.
[0038] A "polynucleotide sequence" (e.g., a nucleic acid,
polynucleotide, oligonucleotide, etc.) is a polymer of nucleotides
comprising nucleotides A,C,T,U,G, or other naturally occurring
nucleotides or artificial nucleotide analogues, or a character
string representing a nucleic acid, depending on context. Either
the given nucleic acid or the complementary nucleic acid can be
determined from any specified polynucleotide sequence.
[0039] Numbering of a given amino acid polymer or nucleic acid
polymer "corresponds to" or is "relative to" the numbering of a
selected amino acid polymer or nucleic acid polymer when the
position of any given polymer component (e.g., amino acid,
nucleotide, also referred to generically as a "residue") is
designated by reference to the same or an equivalent position in
the selected amino acid or nucleic acid polymer, rather than by the
actual numerical position of the component in the given polymer.
Thus, for example, the numbering of a given amino acid position in
a given polypeptide sequence corresponds to the same or equivalent
amino acid position in a selected polypeptide sequence used as a
reference sequence.
[0040] An "equivalent position" (for example, an "equivalent amino
acid position" or "equivalent residue position") is defined herein
as a position (such as, an amino acid position or a residue
position) of a test polypeptide sequence which aligns with a
corresponding position of a reference polypeptide sequence, using
an alignment algorithm as described herein. The equivalent amino
acid position of the test polypeptide sequence need not have the
same numerical position number as the corresponding position of the
test polypeptide. As an example, FIG. 2 shows the sequence of a
polypeptide of the invention (SEQ ID NO:1) aligned with various
known human interferon-alpha polypeptide sequences. In this
example, amino acid position number 47 of SEQ ID NO:1 is considered
to be an equivalent amino acid position to (i.e. is "equivalent
to") that of amino acid position number 46 of SEQ ID NO:32
(huIFN-alpha 2b), since amino acid number 47 of SEQ ID NO:1 aligns
with amino acid number 46 of SEQ ID NO:32. In other words, amino
acid position 47 of SEQ ID NO:1 corresponds to amino acid position
46 of SEQ ID NO:32. Likewise, residue H47 in SEQ ID NO:1 is
understood to correspond to residue Q47 in SEQ ID NO:5, so that for
example the substitution H.sub.47C relative to SEQ ID NO:1 is
understood to correspond to the substitution Q47C in, e.g., SEQ ID
NO:5 (and so on).
[0041] Two polypeptide sequences are "optimally aligned" when they
are aligned using defined parameters, i.e., a defined amino acid
substitution matrix, gap existence penalty (also termed gap open
penalty), and gap extension penalty, so as to arrive at the highest
similarity score possible for that pair of sequences. The BLOSUM62
matrix (Henikoff and Henikoff (1992) Proc. Natl. Acad. Sci. USA
89(22):10915-10919) is often used as a default scoring substitution
matrix in polypeptide sequence alignment algorithms (such as
BLASTP). The gap existence penalty is imposed for the introduction
of a single amino acid gap in one of the aligned sequences, and the
gap extension penalty is imposed for each residue position in the
gap. Unless otherwise stated, alignment parameters employed herein
are: BLOSUM62 scoring matrix, gap existence penalty=11, and gap
extension penalty=1. The alignment score is defined by the amino
acid positions of each sequence at which the alignment begins and
ends (e.g. the alignment window), and optionally by the insertion
of a gap or multiple gaps into one or both sequences, so as to
arrive at the highest possible similarity score, as described in
more detail below in the section entitled "Percent Sequence
Identity".
[0042] The terminology used for identifying amino acid positions
and amino acid substitutions is illustrated as follows: H47
indicates position number 47 occupied by a histidine (His) residue
in a reference amino acid sequence, e.g. SEQ ID NO:1. H47Q
indicates that the histidine residue of position 47 has been
substituted with a glutamine (Gln) residue. Alternative
substitutions are indicated with a "/", e.g., H47S/T means an amino
acid sequence in which the histidine residue in position 47 is
substituted with a serine or a threonine residue. Multiple
substitutions may be indicated with a "+", e.g. H47Q+V51 S/T means
an amino acid sequence which comprises a substitution of the
histidine residue at position 47 with an glutamine residue and a
substitution of the valine residue at position 51 with a serine or
a threonine residue. Deletions are indicated by an asterix. For
example, H47* indicates that the histidine residue in position 47
has been deleted. Deletions of two or more continuous amino acids
may be indicated as follows, e.g., R161*-E166* indicates the
deletion of residues R161-E166 inclusive (that is, residues 161,
162, 163, 164, 164, and 166 are deleted). Insertions are indicated
the following way: Insertion of an additional serine residue after
the histidine residue located at position 47 is indicated as H47HS.
Combined substitutions and insertions are indicated in the
following way: Substitution of the histidine residue at position 47
with a serine residue and insertion of an alanine residue after the
position 47 amino acid residue is indicated as H47SA.
[0043] Unless otherwise indicated, the position numbering of amino
acid residues recited herein is relative to the amino acid sequence
SEQ ID NO:1. It is to be understood that while the examples and
modifications to the parent polypeptide are generally provided
herein relative to the sequence SEQ ID NO:1 (or relative to another
specified sequence), the examples pertain to other polypeptides of
the invention, and the modifications described herein may be made
in equivalent amino acid positions of any of the other polypeptides
described herein. Thus, as an example, the substitution H.sub.47C
relative to SEQ ID NO:1 is understood to correspond to the
substitution Q47C in SEQ ID NO:5, and so on.
[0044] The term "exhibiting (or exhibits, or having, or has) an
interferon-alpha activity" is intended to indicate that the
polypeptide or conjugate of the invention has at least one activity
exhibited by a reference interferon-alpha polypeptide (such as, for
example, a human interferon-alpha polypeptide, e.g., huIFN-alpha 2b
identified herein as SEQ ID NO:32, huIFN-alpha 2a identified herein
as SEQ ID NO:32+R23K, huIFN-alpha 8b identified herein as SEQ ID
NO:37, or any other human interferon alpha polypeptide known in the
art, such as, for example, those shown in FIGS. 2 and 4 herein
and/or listed in Allen G. and Diaz M. O. (1996), supra). Such
activity includes the ability to signal through an interferon-alpha
receptor, as evidenced by, for example, one or more of: inhibition
of viral replication in virus-infected cells ("antiviral
activity"); enhancement of differentiation of naive T-cells to a
T.sub.H1 phenotype and/or suppression of differentiation of naive
T-cells to a T.sub.H2 phenotype ("T.sub.H1 differentiation
activity"); or inhibition of cell proliferation ("antiproliferative
activity"). The one or more interferon-alpha activity is assayed
using assays known in the art and/or described in the Examples.
[0045] A polypeptide or a conjugate exhibiting an interferon-alpha
activity is considered to have such activity when it displays a
measurable activity, e.g., a measurable antiviral activity,
antiproliferative activity, or T.sub.H1 differentiation activity
(e.g., as determined by assays known in the art and/or described in
the Examples). One of skill in the art recognizes that what
constitutes a measurable activity depends in part on the nature of
the assay being undertaken, but as a general guideline a measurable
activity is one in which the assay signal generated in the presence
of the test compound (e.g., a polypeptide of the invention) is
quantifiably different than the assay signal generated in the
absence of the test compound. It is to be understood that the
polypeptide or conjugate of the invention need not exhibit all of
the known activities of a particular reference interferon-alpha, or
exhibit such activities to the same extent as the reference
interferon-alpha. In some instances the activity exhibited by a
polypeptide or conjugate of the invention (as evidenced, e.g., by
an EC.sub.50, specific activity, or other value related to
activity) may be about equal to, be less than, or be greater than
that of the particular activity exhibited by the reference
interferon-alpha.
[0046] A "variant" is a polypeptide comprising a sequence which
differs in one or more amino acid position(s) from that of a parent
polypeptide sequence. For example, a variant may comprise a
sequence which differs from the parent polypeptides sequence in up
to 10% of the total number of residues of the parent polypeptide
sequence, such as in up to 8% of the residues, e.g., in up to 5%,
4%, 3% 2% or 1% of the total number of residue of the parent
polypeptide sequence. For example, a variant of SEQ ID NO:1 may
comprise a sequence which differs from SEQ ID NO:1 in 1-16 amino
acid positions (such as in 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12,
13, 14, 15, or 16 amino acid positions), e.g. in 1-15 amino acid
positions, in 1-14 amino acid positions, in 1-13 amino acid
positions, in 1-12 amino acid positions, in 1-11 amino acid
positions, in 1-10 amino acid positions, in 1-9 amino acid
positions, in 1-8 amino acid positions, in 1-7 amino acid
positions, in 1-6 amino acid positions, in 1-5 amino acid
positions, in 1-4 amino acid positions, in 1-3 amino acid
positions, or in 1-2 amino acid positions.
[0047] The term "parent polypeptide" or "parent interferon-alpha"
is intended to indicate the polypeptide sequence to be modified in
accordance with the present invention. The parent polypeptide
sequence may be that of a naturally occurring IFN-alpha (such as a
mammalian IFN-alpha, e.g., a primate IFN-alpha, such as a human
IFN-alpha, such as a huIFN-alpha polypeptide identified herein as
SEQ ID NOs:31-42, SEQ ID NO:32+R23K, or other huIFN-alpha sequence
described herein and/or in Allen G. and Diaz M. O. (1996), supra).
The parent polypeptide sequence may be that of a non-naturally
occurring (i.e., "synthetic") interferon-alpha, such as IFN-alpha
Con1 (SEQ ID NO:43). In some instances, the parent polypeptide to
be modified may itself be a polypeptide of the invention, such as,
e.g. any one of SEQ ID NOs:1-15 and SEQ ID NOs:44-104.
[0048] "Naturally occurring" as applied to an object refers to the
fact that the object can be found in nature as distinct from being
artificially produced by man. For example, a polypeptide or
polynucleotide sequence that is present in an organism (including
viruses, bacteria, protozoa, insects, plants or mammalian tissue)
that can be isolated from a source in nature and which has not been
intentionally modified by man in the laboratory is naturally
occurring. "Non-naturally occurring" (also termed "synthetic" or
"artificial") as applied to an object means that the object is not
naturally-occurring--i.e., the object cannot be found in nature as
distinct from being artificially produced by man.
[0049] A "fragment" or "subsequence" is any portion of an entire
sequence, up to but not including the entire sequence. Thus, a
fragment or subsequence refers to a sequence of amino acids or
nucleic acids that comprises a part of a longer sequence of amino
acids (e.g., polypeptide) or nucleic acids (e.g.,
polynucleotide).
[0050] One type of fragment contemplated by the present invention
is a fragment in which amino acid residues are removed from the
N-terminus or the C-terminus of the parent polypeptide (or both);
such a polypeptide is considered to be "N-terminally truncated" or
"C-terminally truncated", respectively. It is known that deletion
of at least the first four amino acids from the N-terminus does not
significantly affect interferon-alpha activity (Lydon, N. B. et al.
(1985) Biochemistry 24: 4131-41). Furthermore, variants retaining
interferon-alpha activity have been described wherein between 7 and
11 amino acids have been deleted from the C-terminus (Cheetham B.
F. et al. (1991) Antiviral Res. 15(1):27-39; Chang N. T. et al.
(1983) Arch. Biochem Biophys. 221(2): 585-589; Franke A. E. et al.
(1982) DNA 1(3):223-230).
[0051] A "receptor" e.g., an "interferon-alpha receptor" (also
known as a "Type I interferon receptor") is a receptor which is
activated in cells by an interferon-alpha, e.g., binds an
interferon-alpha and initiates intracellular signaling, such as a
type I interferon receptor comprising receptor subunits IFNAR-2 and
IFNAR-1 (Domanski et al. (1998) J. Biol. Chem. 273(6):3144-3147;
Mogensen et al., (1999) Journal of Interferon and Cytokine
Research, 19:1069-1098). In the context of this invention, receptor
is also meant to include truncated forms of a full-length receptor
molecule, such as for example a receptor molecule which lacks a
membrane-binding portion, such as a soluble form of a receptor
molecule (also known as a "soluble receptor") which comprises an
extracelluar binding domain, which binds an interferon-alpha, but
may not necessarily bind to a membrane and/or initiate
intracellular signaling.
[0052] A "specific binding affinity" between two molecules, e.g., a
ligand and a receptor, means a preferential binding of one molecule
for another in a mixture of molecules. The binding of the molecules
is typically considered specific if the binding affinity is about
1.times.10.sup.4 M.sup.-1 to about 1.times.10.sup.9 M.sup.-1 or
greater (i.e., K.sub.D of about 10.sup.-4 to 10.sup.-9 M or less).
Binding affinity of a ligand and a receptor may be measured by
standard techniques known to those of skill in the art.
Non-limiting examples of well-known techniques for measuring
binding affinities include Biacore.RTM. technology (Biacore AB,
Sweden), isothermal titration microcalorimetry (MicroCal LLC,
Northampton, Mass. USA), ELISA, and FACS. For example, FACS or
other sorting methods may be used to select for populations of
molecules (such as for example, cell surface-displayed ligands)
which specifically bind to the associated binding pair member (such
as a receptor, e.g., a soluble receptor). Ligand-receptor complexes
may be detected and sorted e.g., by fluorescence (e.g., by reacting
the complex with a fluorescent antibody that recognizes the
complex). Molecules of interest which bind an associated binding
pair member (e.g., receptor) are pooled and re-sorted in the
presence of lower concentrations of receptor. By performing
multiple rounds sorting in the presence of decreasing
concentrations of receptor (an exemplary concentration range being
on the order of 10.sup.-6 M down to 10.sup.-9 M, i.e., 1 micromolar
(.mu.M) down to 1 nanomolar (nM), or less, depending on the nature
of the ligand-receptor interaction), populations of the molecule of
interest exhibiting specific binding affinity for the receptor may
be isolated.
[0053] A polypeptide, nucleic acid, or other component is
"isolated" when it is partially or completely separated from
components with which it is normally associated (other peptides,
polypeptides, proteins (including complexes, e.g., polymerases and
ribosomes which may accompany a native sequence), nucleic acids,
cells, synthetic reagents, cellular contaminants, cellular
components, etc.), e.g., such as from other components with which
it is normally associated in the cell from which it was originally
derived. A polypeptide, nucleic acid, or other component is
isolated when it is partially or completely recovered or separated
from other components of its natural environment such that it is
the predominant species present in a composition, mixture, or
collection of components (i.e., on a molar basis it is more
abundant than any other individual species in the composition). In
some instances, the preparation consists of more than about 60%,
70% or 75%, typically more than about 80%, or preferably more than
about 90% of the isolated species.
[0054] A "substantially pure" or "isolated" nucleic acid (e.g., RNA
or DNA), polypeptide, protein, or composition also means where the
object species (e.g., nucleic acid or polypeptide) comprises at
least about 50, 60, or 70 percent by weight (on a molar basis) of
all macromolecular species present. A substantially pure or
isolated composition can also comprise at least about 80, 90, or 95
percent by weight of all macromolecular species present in the
composition. An isolated object species can also be purified to
essential homogeneity (contaminant species cannot be detected in
the composition by conventional detection methods) wherein the
composition consists essentially of derivatives of a single
macromolecular species. The term "purified" generally denotes that
a nucleic acid, polypeptide, or protein gives rise to essentially
one band in an electrophoretic gel. It typically means that the
nucleic acid, polypeptide, or protein is at least about 50% pure,
60% pure, 70% pure, 75% pure, more preferably at least about 85%
pure, and most preferably at least about 99% pure.
[0055] The term "isolated nucleic acid" may refer to a nucleic acid
(e.g., DNA or RNA) that is not immediately contiguous with both of
the coding sequences with which it is immediately contiguous (i.e.,
one at the 5' and one at the 3' end) in the naturally occurring
genome of the organism from which the nucleic acid of the invention
is derived. Thus, this term includes, e.g., a cDNA or a genomic DNA
fragment produced by polymerase chain reaction (PCR) or restriction
endonuclease treatment, whether such cDNA or genomic DNA fragment
is incorporated into a vector, integrated into the genome of the
same or a different species than the organism, including, e.g., a
virus, from which it was originally derived, linked to an
additional coding sequence to form a hybrid gene encoding a
chimeric polypeptide, or independent of any other DNA sequences.
The DNA may be double-stranded or single-stranded, sense or
antisense.
[0056] A "recombinant polynucleotide" or a "recombinant
polypeptide" is a non-naturally occurring polynucleotide or
polypeptide which may include nucleic acid or amino acid sequences,
respectively, from more than one source nucleic acid or
polypeptide, which source nucleic acid or polypeptide can be a
naturally occurring nucleic acid or polypeptide, or can itself have
been subjected to mutagenesis or other type of modification. A
nucleic acid or polypeptide may be deemed "recombinant" when it is
synthetic or artificial or engineered, or derived from a synthetic
or artificial or engineered polypeptide or nucleic acid. A
recombinant nucleic acid (e.g., DNA or RNA) can be made by the
combination (e.g., artificial combination) of at least two segments
of sequence that are not typically included together, not typically
associated with one another, or are otherwise typically separated
from one another. A recombinant nucleic acid can comprise a nucleic
acid molecule formed by the joining together or combination of
nucleic acid segments from different sources and/or artificially
synthesized. A "recombinant polypeptide" often refers to a
polypeptide that results from a cloned or recombinant nucleic acid.
The source polynucleotides or polypeptides from which the different
nucleic acid or amino acid sequences are derived are sometimes
homologous (i.e., have, or encode a polypeptide that encodes, the
same or a similar structure and/or function), and are often from
different isolates, serotypes, strains, species, of organism or
from different disease states, for example.
[0057] The term "recombinant" when used with reference, e.g., to a
cell, polynucleotide, vector, protein, or polypeptide typically
indicates that the cell, polynucleotide, or vector has been
modified by the introduction of a heterologous (or foreign) nucleic
acid or the alteration of a native nucleic acid, or that the
protein or polypeptide has been modified by the introduction of a
heterologous amino acid, or that the cell is derived from a cell so
modified. Recombinant cells express nucleic acid sequences that are
not found in the native (non-recombinant) form of the cell or
express native nucleic acid sequences that would otherwise be
abnormally expressed, under-expressed, or not expressed at all. The
term "recombinant" when used with reference to a cell indicates
that the cell replicates a heterologous nucleic acid, or expresses
a polypeptide encoded by a heterologous nucleic acid. Recombinant
cells can contain coding sequences that are not found within the
native (non-recombinant) form of the cell. Recombinant cells can
also contain coding sequences found in the native form of the cell
wherein the coding sequences are modified and re-introduced into
the cell by artificial means. The term also encompasses cells that
contain a nucleic acid endogenous to the cell that has been
modified without removing the nucleic acid from the cell; such
modifications include those obtained by gene replacement,
site-specific mutation, recombination, and related techniques.
[0058] The term "recombinantly produced" refers to an artificial
combination usually accomplished by either chemical synthesis
means, recursive sequence recombination of nucleic acid segments or
other diversity generation methods (such as, e.g., shuffling) of
nucleotides, or manipulation of isolated segments of nucleic acids,
e.g., by genetic engineering techniques known to those of ordinary
skill in the art. "Recombinantly expressed" typically refers to
techniques for the production of a recombinant nucleic acid in
vitro and transfer of the recombinant nucleic acid into cells in
vivo, in vitro, or ex vivo where it may be expressed or
propagated.
[0059] A "recombinant expression cassette" or simply an "expression
cassette" is a nucleic acid construct, generated recombinantly or
synthetically, with nucleic acid elements that are capable of
effecting expression of a structural gene in hosts compatible with
such sequences. Expression cassettes include at least promoters and
optionally, transcription termination signals. Typically, the
recombinant expression cassette includes a nucleic acid to be
transcribed (e.g., a nucleic acid encoding a desired polypeptide),
and a promoter. Additional factors necessary or helpful in
effecting expression may also be used as described herein. For
example, an expression cassette can also include nucleotide
sequences that encode a signal sequence that directs secretion of
an expressed protein from the host cell. Transcription termination
signals, enhancers, and other nucleic acid sequences that influence
gene expression, can also be included in an expression
cassette.
[0060] An "immunogen" refers to a substance capable of provoking an
immune response, and includes, e.g., antigens, autoantigens that
play a role in induction of autoimmune diseases, and
tumor-associated antigens expressed on cancer cells. An immune
response generally refers to the development of a cellular or
antibody-mediated response to an agent, such as an antigen or
fragment thereof or nucleic acid encoding such agent. In some
instances, such a response comprises a production of at least one
or a combination of CTLs, B cells, or various classes of T cells
that are directed specifically to antigen-presenting cells
expressing the antigen of interest.
[0061] An "antigen" refers to a substance that is capable of
eliciting the formation of antibodies in a host or generating a
specific population of lymphocytes reactive with that substance.
Antigens are typically macromolecules (e.g., proteins and
polysaccharides) that are foreign to the host.
[0062] An "adjuvant" refers to a substance that enhances an
antigen's immune-stimulating properties or the pharmacological
effect(s) of a drug. An adjuvant may non-specifically enhance the
immune response to an antigen. "Freund's Complete Adjuvant," for
example, is an emulsion of oil and water containing an immunogen,
an emulsifying agent and mycobacteria. Another example, "Freund's
incomplete adjuvant," is the same, but without mycobacteria.
[0063] A vector is a component or composition for facilitating cell
transduction or transfection by a selected nucleic acid, or
expression of the nucleic acid in the cell.
[0064] Vectors include, e.g., plasmids, cosmids, viruses, YACs,
bacteria, poly-lysine, etc. An "expression vector" is a nucleic
acid construct or sequence, generated recombinantly or
synthetically, with a series of specific nucleic acid elements that
permit transcription of a particular nucleic acid in a host cell.
The expression vector can be part of a plasmid, virus, or nucleic
acid fragment. The expression vector typically includes a nucleic
acid to be transcribed operably linked to a promoter. The nucleic
acid to be transcribed is typically under the direction or control
of the promoter.
[0065] "Substantially the entire length of a polynucleotide
sequence" or "substantially the entire length of a polypeptide
sequence" refers to at least 50%, generally at least about 60%,
70%, or 75%, usually at least about 80%, or typically at least
about 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more
of a length of a polynucleotide sequence or polypeptide
sequence.
[0066] The term "immunoassay" includes an assay that uses an
antibody or immunogen to bind or specifically bind an antigen. The
immunoassay is typically characterized by the use of specific
binding properties of a particular antibody to isolate, target,
and/or quantify the antigen.
[0067] The term "subject" as used herein includes, but is not
limited to, an organism; a mammal, including, e.g., a human,
non-human primate (e.g., baboon, orangutan, monkey), mouse, pig,
cow, goat, cat, rabbit, rat, guinea pig, hamster, horse, monkey,
sheep, or other non-human mammal; a non-mammal, including, e.g., a
non-mammalian vertebrate, such as a bird (e.g., a chicken or duck)
or a fish, and a non-mammalian invertebrate.
[0068] The term "pharmaceutical composition" means a composition
suitable for pharmaceutical use in a subject, including an animal
or human. A pharmaceutical composition generally comprises an
effective amount of an active agent and a carrier, including, e.g.,
a pharmaceutically acceptable carrier.
[0069] The term "effective amount" means a dosage or amount
sufficient to produce a desired result. The desired result may
comprise an objective or subjective improvement in the recipient of
the dosage or amount.
[0070] A "prophylactic treatment" is a treatment administered to a
subject who does not display signs or symptoms of a disease,
pathology, or medical disorder, or displays only early signs or
symptoms of a disease, pathology, or disorder, such that treatment
is administered for the purpose of diminishing, preventing, or
decreasing the risk of developing the disease, pathology, or
medical disorder. A prophylactic treatment functions as a
preventative treatment against a disease or disorder. A
"prophylactic activity" is an activity of an agent, such as a
nucleic acid, vector, gene, polypeptide, protein, substance, or
composition thereof that, when administered to a subject who does
not display signs or symptoms of pathology, disease or disorder, or
who displays only early signs or symptoms of pathology, disease, or
disorder, diminishes, prevents, or decreases the risk of the
subject developing a pathology, disease, or disorder. A
"prophylactically useful" agent or compound (e.g., nucleic acid or
polypeptide) refers to an agent or compound that is useful in
diminishing, preventing, treating, or decreasing development of
pathology, disease or disorder.
[0071] A "therapeutic treatment" is a treatment administered to a
subject who displays symptoms or signs of pathology, disease, or
disorder, in which treatment is administered to the subject for the
purpose of diminishing or eliminating those signs or symptoms of
pathology, disease, or disorder. A "therapeutic activity" is an
activity of an agent, such as a nucleic acid, vector, gene,
polypeptide, protein, substance, or composition thereof, that
eliminates or diminishes signs or symptoms of pathology, disease or
disorder, when administered to a subject suffering from such signs
or symptoms. A "therapeutically useful" agent or compound (e.g.,
nucleic acid or polypeptide) indicates that an agent or compound is
useful in diminishing, treating, or eliminating such signs or
symptoms of a pathology, disease or disorder.
[0072] The term "gene" broadly refers to any segment of DNA
associated with a biological function. Genes include coding
sequences and/or regulatory sequences required for their
expression. Genes also include non-expressed DNA nucleic acid
segments that, e.g., form recognition sequences for other proteins
(e.g., promoter, enhancer, or other regulatory regions). Genes can
be obtained from a variety of sources, including cloning from a
source of interest or synthesizing from known or predicted sequence
information, and may include sequences designed to have desired
parameters.
[0073] Generally, the nomenclature used hereafter and the
laboratory procedures in cell culture, molecular genetics,
molecular biology, nucleic acid chemistry, and protein chemistry
described below are those well known and commonly employed by those
of ordinary skill in the art. Standard techniques, such as
described in Sambrook et al., Molecular Cloning--A Laboratory
Manual (2nd Ed.), Vols. 1-3, Cold Spring Harbor Laboratory, Cold
Spring Harbor, N.Y., 1989 (hereinafter "Sambrook") and Current
Protocols in Molecular Biology, F. M. Ausubel et al., eds., Current
Protocols, a joint venture between Greene Publishing Associates,
Inc. and John Wiley & Sons, Inc. (1994, supplemented through
1999) (hereinafter "Ausubel"), are used for recombinant nucleic
acid methods, nucleic acid synthesis, cell culture methods, and
transgene incorporation, e.g., electroporation, injection, gene
gun, impressing through the skin, and lipofection.
[0074] Generally, oligonucleotide synthesis and purification steps
are performed according to specifications. The techniques and
procedures are generally performed according to conventional
methods in the art and various general references which are
provided throughout this document. The procedures therein are
believed to be well known to those of ordinary skill in the art and
are provided for the convenience of the reader.
[0075] As used herein, an "antibody" refers to a protein comprising
one or more polypeptides substantially or partially encoded by
immunoglobulin genes or fragments of immunoglobulin genes. The term
antibody is used to mean whole antibodies and binding fragments
thereof. The recognized immunoglobulin genes include the kappa,
lambda, alpha, gamma, delta, epsilon and mu constant region genes,
as well as myriad immunoglobulin variable region genes. Light
chains are classified as either kappa or lambda. Heavy chains are
classified as gamma, mu, alpha, delta, or epsilon, which in turn
define the immunoglobulin classes, IgG, IgM, IgA, IgD and IgE,
respectively. A typical immunoglobulin (e.g., antibody) structural
unit comprises a tetramer. Each tetramer is composed of two
identical pairs of polypeptide chains, each pair having one "light"
(about 25 KDa) and one "heavy" chain (about 50-70 KDa). The
N-terminus of each chain defines a variable region of about 100 to
110 or more amino acids primarily responsible for antigen
recognition. The terms variable light chain (VL) and variable heavy
chain (VH) refer to these light and heavy chains, respectively.
[0076] Antibodies also include single-armed composite monoclonal
antibodies, single chain antibodies, including single chain Fv
(sFv) antibodies in which a variable heavy and a variable light
chain are joined together (directly or through a peptide linker) to
form a continuous polypeptide, as well as diabodies, tribodies, and
tetrabodies (Pack et al. (1995) J Mol Biol 246:28; Biotechnol
11:1271; and Biochemistry 31:1579). The antibodies are, e.g.,
polyclonal, monoclonal, chimeric, humanized, single chain, Fab
fragments, fragments produced by an Fab expression library, or the
like.
[0077] The term "epitope" means a protein determinant capable of
specific binding to an antibody. Epitopes usually consist of
chemically active surface groupings of molecules such as amino
acids or sugar side chains and usually have specific three
dimensional structural characteristics, as well as specific charge
characteristics. Conformational and nonconformational epitopes are
distinguished in that the binding to the former but not the latter
is lost in the presence of denaturing solvents.
[0078] An "antigen-binding fragment" of an antibody is a peptide or
polypeptide fragment of the antibody that binds an antigen. An
antigen-binding site is formed by those amino acids of the antibody
that contribute to, are involved in, or affect the binding of the
antigen. See Scott, T. A. and Mercer, E. I., Concise Encyclopedia:
Biochemistry and Molecular Biology (de Gruyter, 3d ed. 1997), and
Watson, J. D. et al., Recombinant DNA (2d ed. 1992) [hereinafter
"Watson, Recombinant DNA"], each of which is incorporated herein by
reference in its entirety for all purposes.
[0079] The term "screening" describes, in general, a process that
identifies optimal molecules of the present invention, such as,
e.g., polypeptides of the invention, and related fusion
polypeptides including the same, and nucleic acids encoding all
such molecules. Several properties of these respective molecules
can be used in selection and screening, for example: an ability of
a respective molecule to bind a ligand or to a receptor, to inhibit
cell proliferation, to inhibit viral replication in virus-infected
cells, to induce or inhibit cellular cytokine production, to alter
an immune response, e.g., induce or inhibit a desired immune
response, in a test system or an in vitro, ex vivo or in vivo
application. In the case of antigens, several properties of the
antigen can be used in selection and screening including antigen
expression, folding, stability, immunogenicity and presence of
epitopes from several related antigens.
[0080] "Selection" is a form of screening in which identification
and physical separation are achieved simultaneously by, e.g.,
expression of a selection marker, which, in some genetic
circumstances, allows cells expressing the marker to survive while
other cells die (or vice versa). Screening markers include, for
example, luciferase, beta-galactosidase and green fluorescent
protein, and the like. Selection markers include drug and toxin
resistance genes, and the like. Another mode of selection involves
physical sorting based on a detectable event, such as binding of a
ligand to a receptor, reaction of a substrate with an enzyme, or
any other physical process which can generate a detectable signal
either directly (e.g., by utilizing a chromogenic substrate or
ligand) or indirectly (e.g., by reacting with a chromogenic
secondary antibody). Selection by physical sorting can by
accomplished by a variety of methods, such as by FACS in whole cell
or microdroplet formats.
[0081] An "exogenous" nucleic acid," "exogenous DNA segment,"
"heterologous sequence," or "heterologous nucleic acid," as used
herein, is one that originates from a source foreign to the
particular host cell, or, if from the same source, is modified from
its original form. Thus, a heterologous gene in a host cell
includes a gene that is endogenous to the particular host cell, but
has been modified. Modification of a heterologous sequence in the
applications described herein typically occurs through the use of
recursive sequence recombination. The terms refer to a DNA segment
which is foreign or heterologous to the cell, or homologous to the
cell but in a position within the host cell nucleic acid in which
the element is not ordinarily found. Exogenous DNA segments are
expressed to yield exogenous polypeptides.
[0082] The term "nucleic acid" refers to deoxyribonucleotides or
ribonucleotides and polymers thereof in either single- or
double-stranded form. Unless specifically limited, the term
encompasses nucleic acids containing known analogues of natural
nucleotides which have similar binding properties as the reference
nucleic acid and are metabolized in a manner similar to naturally
occurring nucleotides. Unless otherwise indicated, a particular
nucleic acid sequence also implicitly encompasses conservatively
modified variants thereof (e.g., degenerate codon substitutions)
and complementary sequences and as well as the sequence explicitly
indicated. Specifically, degenerate codon substitutions may be
achieved by generating sequences in which the third position of one
or more selected (or all) codons is substituted with mixed-base
and/or deoxyinosine residues (Batzer et al. (1991) Nucleic Acid Res
19:5081; Ohtsuka et al. (1985) J Biol Chem 260:2605-2608; Cassol et
al. (1992); Rossolini et al. (1994) Mol Cell Probes 8:91-98). The
term nucleic acid is used interchangeably with gene, cDNA, and mRNA
encoded by a gene.
[0083] "Nucleic acid derived from a gene" refers to a nucleic acid
for whose synthesis the gene, or a subsequence thereof, has
ultimately served as a template. Thus, an mRNA, a cDNA reverse
transcribed from an mRNA, an RNA transcribed from that cDNA, a DNA
amplified from the cDNA, an RNA transcribed from the amplified DNA,
etc., are all derived from the gene and detection of such derived
products is indicative of the presence and/or abundance of the
original gene and/or gene transcript in a sample. A nucleic acid is
"operably linked" when it is placed into a functional relationship
with another nucleic acid sequence. For instance, a promoter or
enhancer is operably linked to a coding sequence if it increases
the transcription of the coding sequence. Operably linked means
that the DNA sequences being linked are typically contiguous and,
where necessary to join two protein coding regions, contiguous and
in reading frame. However, since enhancers generally function when
separated from the promoter by several kilobases and intronic
sequences may be of variable lengths, some polynucleotide elements
may be operably linked but not contiguous.
[0084] The term "cytokine" includes, for example, interleukins,
interferons, chemokines, hematopoietic growth factors, tumor
necrosis factors and transforming growth factors. In general these
are low molecular weight proteins that regulate maturation,
activation, proliferation, and differentiation of cells of the
immune system.
[0085] In the present description and claims, any reference to "a"
component, e.g. in the context of a non-polypeptide moiety, an
amino acid residue, a substitution, a buffer, a cation, etc., is
intended to refer to one or more of such components, unless stated
otherwise or unless it is clear from the particular context that
this is not the case. For example, the expression "a component
selected from the group consisting of A, B and C" is intended to
include all combinations of A, B and C, e.g., A, B, C, A+B, A+C,
B+C or A+B+C. Various additional terms are defined or otherwise
characterized herein.
Molecules and Methods of the Invention
[0086] Molecules of the invention (e.g., polypeptides of the
invention, conjugates of the invention, and nucleic acids encoding
said polypeptides) are useful for the treatment of diseases and
conditions which are responsive to treatment by interferon-alpha,
particularly diseases and conditions associated with viral
infection, such as, for example, infection by HCV.
[0087] Patients with chronic HCV infection have viral loads
typically in the range of 10.sup.4-10.sup.7 copies of HCV RNA/ml of
serum prior to treatment. Upon treatment with IFN-alpha, viral load
in these patients characteristically undergoes two distinct
log-linear phases of decline (FIG. 1B; Neumann A. (1998) Science
282:103-107). The initial rapid drop in viral load that occurs
within the first two days of IFN-alpha therapy is believed to be
due to interferon-alpha mediated reduction in virus production in
the infected liver cells and concomitant protection of naive cells
against infection. The rate of viral production reaches a new
steady state at about two days, at which time a second less rapid
log-linear phase of viral clearance is observed. This second phase
of viral clearance is generally believed to be due in part to
T-cell mediated killing of infected liver cells (Neumann, et al.,
supra). IFN-alpha is believed to play a key role in this biological
response through the stimulation of antigen specific T cells to
differentiate into T.sub.H1 cells. Furthermore, the mode of action
of Ribavirin is believed to be due to augmentation of the T.sub.H1
response, and is thought to be the mechanistic basis of its
efficacy in combination therapy with IFN-alpha. HCV-infected
patients who are non-responsive to interferon-alpha therapies
currently in use (generally termed "non-responders") exhibit much
shallower viral load clearance profiles (FIG. 1A).
[0088] Although the present invention is not intended to be limited
by a particular theory of underlying mechanism, it is proposed that
antiviral activity in surrogate assay systems (such as those
described in more detail herein) may be predictive of
interferon-alpha efficacy, for example in the first phase of viral
clearance. An exemplary antiviral assay, described in the Examples
section, monitors the effectiveness of IFN-alpha in protecting
against the cytopathic effect of Encephalomyocarditis Virus (EMCV)
in HuH7 human liver-derived cells, as a surrogate system for
effectiveness against HCV in human liver cells. Example 2 shows
antiviral activities of representative polypeptides of the
invention in the EMCV/HuH7 antiviral activity assay. Preliminary
experiments (data not shown) indicates that polypeptides of the
invention exhibit antiviral activity in other virus/cell systems,
including EMCV in WISH human amniotic tissue-derived cells, EMCV in
HeLa human cervical carcinoma cells, Vesicular Stomatitis Virus
(VSV) in HuH7 cells, Vaccinia Virus (VV) in HeLa cells, Yellow
Fever Virus (YFV) in HepG2 human hepatocarcinoma cells, as well as
Human Immunodeficiency Virus (HIV) in human primary CD4+ T-cells.
This suggests that polypeptides of the invention exhibit antiviral
activity against a broad spectrum of viruses and cell types.
[0089] Other surrogate assay system for HCV replication in infected
hepatocytes include HCV replicon systems, as described, for
example, by Lohmann V., et al., (1999) Science 285(5424):285-3;
Randall G. and Rice C. M. (2001) Curr Opin Infect Dis 14(6):743-7;
and Bartenschlager, R. (2002) Nature Reviews/Drug Discovery 1:911.
An example of a useful in vivo system for monitoring HCV antiviral
efficacy is a chimeric human liver SCID mouse, as described by
Mercer, et al. (2001) Nature Medicine 7(8):927-933.
[0090] It is furthermore proposed, without being limited by theory,
that enhancement of T.sub.H1 differentiation and/or suppression of
T.sub.H2 differentiation by IFN-alpha may be a contributing factor
to interferon-alpha efficacy, for example, in the second phase of
viral clearance. According to this theory, evolved IFN-alphas with
increased potency in these biological activities (i.e., enhancement
of T.sub.H1 differentiation and/or suppression of T.sub.H2
differentiation) would be predicted to have increased efficacy
relative to, for example, currently approved therapeutic
interferon-alpha molecules administered at the same dosage. An
exemplary assay, described in the Examples section herein, monitors
the enhancement of T.sub.H1 differentiation and/or suppression of
T.sub.H2 differentiation by IFN-alpha on naive T.sub.H0 cells, by
measuring production of cytokines associated with the
T.sub.H1-phenotype (e.g., IFN-gamma) and/or the T.sub.H2-phenotype
(e.g., IL-5, IL4) via ELISA or via intracellular staining and FACS
sorting.
[0091] The therapeutic efficacy of IFN-alpha molecules tends to be
diminished in part due to dose-limiting toxicities, e.g.
thrombocytopenia and neutropenia. Although the present invention is
not intended to be limited by a particular theory of underlying
mechanism, it is proposed that such toxicity may be associated with
anti-proliferative effects of IFN-alpha on platelet and neutrophil
precursors, and that antiproliferative activity in surrogate assay
systems (such as those described herein) may be predictive of the
relative toxicity of an interferon-alpha molecule. Thus,
dose-limiting toxicities associated with IFN-alpha therapy may be
diminished in IFN-alpha molecules that exhibit reduced
antiproliferative activity relative to, for example, currently
approved therapeutic interferon-alpha molecules, such as
ROFERON.RTM.-A (Interferon alfa-2a, recombinant; Hoffmann-La Roche
Inc.), INTRON.RTM. A (Interferon alfa-2b, recombinant; Schering
Corporation), and INFERGEN.RTM. (interferon alfacon-1; InterMune,
Inc.). An exemplary antiproliferative activity assay, described in
the Examples section herein, monitors the effect of IFN-alpha on
the proliferation of human Daudi lymphoid cells. Alternatively, or
in addition, dose-limiting toxicities may be reduced as a result of
administering more therapeutically active molecules, which would
permit dosing in lower concentrations or at lower frequency than
currently approved molecules.
[0092] It is an object of the invention to provide novel
interferon-alpha polypeptides, and nucleic acids which encode the
polypeptides. Polypeptides of the invention are useful for the
treatment of diseases and disorders which are responsive to
treatment by interferon-alpha, particularly diseases associated
with viral infection, such as, for example, infection by HCV. Some
polypeptides of the invention exhibit an interferon-alpha activity,
such as, for example, antiviral activity, antiproliferative
activity, and/or T.sub.H1 differentiation activity. Some
polypeptides of the invention exhibit one or more of the following
properties: increased or decreased antiviral activity compared to a
reference IFN-alpha polypeptide; increased or decreased T.sub.H1
differentiation activity compared to a reference IFN-alpha
polypeptide; increased or decreased antiproliferative activity
compared to a reference IFN-alpha polypeptide. The reference
IFN-alpha polypeptide may comprise a sequence of a non-naturally
occurring interferon-alpha, such as IFN-alpha Con1 (SEQ ID NO:43),
or may comprise a sequence of a naturally-occurring (i.e.,
wild-type) interferon-alpha polypeptide. Examples of sequences of
naturally occurring interferon-alpha polypeptides include sequences
of human IFN-alpha polypeptides, such as, for example, huIFN-alpha
2b (SEQ ID NO:32), huIFN-alpha 2a (SEQ ID NO:32 with position
23=Lys), huIFN-alpha 2c (SEQ ID NO:32 with position 34=Arg),
huIFN-alpha 8b (SEQ ID NO:33), huIFN-alpha 8a (SEQ ID NO:33 with
positions 98=Val, 99=Leu, 100=Cys, and 101=Asp), huIFN-alpha 8c
(SEQ ID NO:33 with position 161=Asp and amino acids at positions
162-166 deleted), huIFN-alpha 14a (SEQ ID NO:39), huIFN-alpha 14c
(SEQ ID NO:39 with position 152=Leu), or a sequence of any other
naturally occurring human interferon alpha polypeptide, such as
those shown in FIGS. 2 and 4 herein (SEQ ID NOs:31-42) and/or
listed in Allen G. and Diaz M. O. (1996), supra.
[0093] In another aspect, the invention provides interferon-alpha
polypeptides which exhibit enhanced efficacy in clearing a virus
from virus-infected cells, compared to a reference interferon-alpha
molecule, such as one currently employed as a therapeutic (such as,
for example, ROFERON-A, INTRON A, or INFERGEN). Exemplary viruses
include, but are not limited to, viruses of the Flaviviridae
family, such as, for example, Hepatitis C Virus, Yellow Fever
Virus, West Nile Virus, Japanese Encephalitis Virus, Dengue Virus,
and Bovine Viral Diarrhea Virus; viruses of the Hepadnaviridae
family, such as, for example, Hepatitis B Virus; viruses of the
Picornaviridae family, such as, for example, Encephalomyocarditis
Virus, Human Rhinovirus, and Hepatitis A Virus; viruses of the
Retroviridae family, such as, for example, Human Immunodeficiency
Virus, Simian Immunodeficiency Virus, Human T-Lymphotropic Virus,
and Rous Sarcoma Virus; viruses of the Coronaviridae family, such
as, for example, SARS coronavirus; viruses of the Rhabdoviridae
family, such as, for example, Rabies Virus and Vesicular Stomatitis
Virus, viruses of the Paramyxoviridae family, such as, for example,
Respiratory Syncytial Virus and Parainfluenza Virus, viruses of the
Papillomaviridae family, such as, for example, Human
Papillomavirus, and viruses of the Herpesviridae family, such as,
for example, Herpes Simplex Virus. Such enhanced efficacy may arise
from enhanced antiviral activity, enhanced T.sub.H1-differentiation
activity, or both, relative to the reference molecule. For example,
some interferon-alpha polypeptides of the invention may be
particularly useful in clearing viruses or viral strains that show
poor response to treatment with interferon-alpha molecules
currently in use, such as, for example, Genotype 1 of HCV.
[0094] Some polypeptides of the invention exhibit an increased
ratio of (antiviral activity/antiproliferative activity) compared
to the reference IFN-alpha molecule, and/or an increased ratio of
(T.sub.H1 differentiation activity/antiproliferative activity)
compared to the reference IFN-alpha molecule. Polypeptides
exhibiting such properties may be particularly effective in
treatment of viral infections, such as, for example, infection by a
virus listed above. Some such polypeptides may, for example,
provide enhanced therapeutic efficacy over currently-approved
interferon-alpha molecules in the treatment of HCV, in one or both
phases of the biphasic viral clearance profile, and/or may exhibit
reduced toxicity. Some such polypeptides may provide enhanced
therapeutic efficacy over currently-approved interferon-alpha
molecules in the treatment of Genotype 1 HCV.
[0095] It is another object of the invention to provide conjugates,
such conjugates comprising one or more non-polypeptide moiety
linked to a polypeptide of the invention, which conjugate exhibits
an interferon-alpha activity (such as one or more of the activities
listed above), and which optionally exhibits other desirable
properties, such as increased serum half-life and/or functional in
vivo half-life, and/or decreased antigenicity, compared to the
non-conjugated polypeptide. Some such conjugates may exhibit
enhanced efficacy in clearing a virus from cells infected with the
virus, compared to a reference interferon-alpha molecule, such as
an interferon-alpha conjugate currently employed as a therapeutic
(such as, for example, PEGASYS.RTM. (Peginterferon alfa-2a;
Hoffmann-La Roche, Inc.) or PEG-INTRON.RTM. (peginterferon alfa-2b;
Schering Corporation). Exemplary viruses include, but are not
limited to, viruses of the Flaviviridae family, such as, for
example, Hepatitis C Virus, Yellow Fever Virus, West Nile Virus,
Japanese Encephalitis Virus, Dengue Virus, and Bovine Viral
Diarrhea Virus; viruses of the Hepadnaviridae family, such as, for
example, Hepatitis B Virus; viruses of the Picornaviridae family,
such as, for example, Encephalomyocarditis Virus, Human Rhinovirus,
and Hepatitis A Virus; viruses of the Retroviridae family, such as,
for example, Human Immunodeficiency Virus, Simian Immunodeficiency
Virus, Human T-Lymphotropic Virus, and Rous Sarcoma Virus; viruses
of the Coronaviridae family, such as, for example, SARS
coronavirus; viruses of the Rhabdoviridae family, such as, for
example, Rabies Virus and Vesicular Stomatitis Virus, viruses of
the Paramyxoviridae family, such as, for example, Respiratory
Syncytial Virus and Parainfluenza Virus, viruses of the
Papillomaviridae family, such as, for example, Human
Papillomavirus, and viruses of the Herpesviridae family, such as,
for example, Herpes Simplex Virus. Such enhanced efficacy may arise
from enhanced antiviral activity, enhanced T.sub.H1-differentiation
activity, or both, relative to the reference molecule. For example,
some interferon-alpha conjugates of the invention may be
particularly useful in clearing viruses or viral strains that show
poor response to treatment with interferon-alpha molecules
currently in use, such as, for example, Genotype 1 of HCV.
[0096] Some conjugates of the invention exhibit an increased ratio
of (antiviral activity/antiproliferative activity) compared to the
reference IFN-alpha molecule, and/or an increased ratio of
(T.sub.H1 differentiation activity/antiproliferative activity)
compared to the reference IFN-alpha molecule. Conjugates exhibiting
such properties may be particularly effective in treatment of viral
infections, such as infection by a virus listed above, such as, for
example, HCV. Some such conjugates may, for example, provide
enhanced therapeutic efficacy over currently-approved
interferon-alpha molecules in the treatment of HCV, in one or both
phases of the biphasic viral clearance profile, and/or may exhibit
reduced toxicity. Some such conjugates may provide enhanced
therapeutic efficacy over currently-approved interferon-alpha
molecules in the treatment of Genotype 1 HCV.
[0097] It is another object of the invention to provide a method of
inhibiting viral replication in virus-infected cells, the method
comprising administering to the virus-infected cells a polypeptide
or conjugate of the invention in an amount effective to inhibit
viral replication in said cells. The invention also provides a
method of reducing the number of copies of a virus in
virus-infected cells, comprising administering to the
virus-infected cells a polypeptide or conjugate of the invention in
an amount effective to reduce the number of copies of the virus in
said cells. The virus may, for example, be a virus of the
Flaviviridae family, such as, for example, Hepatitis C Virus,
Yellow Fever Virus, West Nile Virus, Japanese Encephalitis Virus,
Dengue Virus, or Bovine Viral Diarrhea Virus; a virus of the
Hepadnaviridae family, such as, for example, Hepatitis B Virus; a
virus of the Picornaviridae family, such as, for example,
Encephalomyocarditis Virus, Human Rhinovirus, or Hepatitis A Virus;
a virus of the Retroviridae family, such as, for example, Human
Immunodeficiency Virus, Simian Immunodeficiency Virus, Human
T-Lymphotropic Virus, or Rous Sarcoma Virus; a virus of the
Coronaviridae family, such as, for example, SARS coronavirus; a
virus of the Rhabdoviridae family, such as, for example, Rabies
Virus or Vesicular Stomatitis Virus, a virus of the Paramyxoviridae
family, such as, for example, Respiratory Syncytial Virus or
Parainfluenza Virus, a virus of the Papillomaviridae family, such
as, for example, Human Papillomavirus, or a virus of the
Herpesviridae family, such as, for example, Herpes Simplex Virus.
The virus may for example be an RNA virus, such as HCV, a DNA
virus, such as HBV, or a retrovirus, such as HIV. The cells may be
in culture or otherwise isolated from a mammal (i.e., in vitro or
ex vivo), or may be in vivo, e.g., in a mammal (e.g. such as a SCID
mouse model as described by Mercer, et al. (2001) Nature Medicine.
7(8): 927-933), in a primate, or in man.
[0098] The invention also provides a method of enhancing T.sub.H1
differentiation of T.sub.H0 cells, comprising administering to a
population comprising T.sub.H0 cells a polypeptide or conjugate of
the invention in an amount effective to increase the production of
a cytokine associated with the T.sub.H1-phenotype (e.g., IFN-gamma)
and/or decrease the production of a cytokine associated with the
T.sub.H2-phenotype (e.g., IL-4 or IL-5) in said population. The
population may be in culture or otherwise isolated from a mammal
(i.e., in vitro or ex vivo), or may be in vivo, e.g., in a mammal,
in a primate, or in man.
[0099] The invention also provides a method of inhibiting
proliferation of a cell population, comprising contacting the cell
population with a polypeptide or conjugate of the invention in an
amount effective to decrease proliferation of the cell population.
The cell population may be in culture or otherwise isolated from a
mammal (i.e., in vitro or ex vivo), or may be in vivo, e.g., in a
mammal, a primate, or man.
[0100] These and other objects of the invention are discussed in
more detail below.
Polypeptides of the Invention
[0101] The invention provides novel interferon-alpha polypeptides,
collectively referred to herein as "polypeptides of the invention".
The term "polypeptide(s) of the invention" is intended throughout
to include variants of the polypeptide sequences disclosed herein.
Also included in this invention are fusion proteins comprising
polypeptides of the invention, and conjugates comprising
polypeptides of the invention.
[0102] Fragments of various interferon-alpha coding sequences were
recursively recombined to form libraries comprising recombinant
polynucleotides, from which some polypeptides of the invention were
discovered. Methods for obtaining libraries of recombinant
polynucleotides and/or for obtaining diversity in nucleic acids
used as the substrates for recursive sequence recombination are
also described infra.
[0103] Exemplary polypeptides of the invention include polypeptides
comprising sequences identified herein as SEQ ID NOs:1-15, such as
SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5,
SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:9, SEQ ID NO:10,
SEQ ID NO:11, SEQ ID NO:12, SEQ ID NO:13, SEQ ID NO:14 and SEQ ID
NO:15, encoded by nucleic acids identified herein as SEQ ID
NOs:16-30, such as SEQ ID NO:16, SEQ ID NO:17, SEQ ID NO:18, SEQ ID
NO:19, SEQ ID NO:20, SEQ ID NO:21, SEQ ID NO:22, SEQ ID NO:23, SEQ
ID NO:24, SEQ ID NO:25, SEQ ID NO:26, SEQ ID NO:27, SEQ ID NO:28,
SEQ ID NO:29 and SEQ ID NO:30. Polypeptides of the invention also
include those comprising sequences identified herein as SEQ ID
NOs:44-104, such as SEQ ID NO:44, SEQ ID NO:45, SEQ ID NO:46, SEQ
ID NO:47, SEQ ID NO:48, SEQ ID NO:49, SEQ ID NO:50, SEQ ID NO:51,
SEQ ID NO:52, SEQ ID NO:53, SEQ ID NO:54, SEQ ID NO:55, SEQ ID
NO:56, SEQ ID NO:57, SEQ ID NO:58, SEQ ID NO:59, SEQ ID NO:60, SEQ
ID NO:61, SEQ ID NO:62, SEQ ID NO:63, SEQ ID NO:64, SEQ ID NO:65,
SEQ ID NO:66, SEQ ID NO:67, SEQ ID NO:68, SEQ ID NO:69, SEQ ID
NO:70, SEQ ID NO:71, SEQ ID NO:72, SEQ ID NO:73, SEQ ID NO:74, SEQ
ID NO:75, SEQ ID NO:76, SEQ ID NO:77, SEQ ID NO:78, SEQ ID NO:79,
SEQ ID NO:80, SEQ ID NO:81, SEQ ID NO:82, SEQ ID NO:83, SEQ ID
NO:84, SEQ ID NO:85, SEQ ID NO:86, SEQ ID NO:87, SEQ ID NO:88, SEQ
ID NO:89, SEQ ID NO:90, SEQ ID NO:91, SEQ ID NO:92, SEQ ID NO:93,
SEQ ID NO:94, SEQ ID NO:95, SEQ ID NO:96, SEQ ID NO:97, SEQ ID
NO:98, SEQ ID NO:99, SEQ ID NO:100, SEQ ID NO:101, SEQ ID NO:102,
SEQ ID NO:103, and SEQ ID NO:104. Some such polypeptides further
comprise an additional amino acid, such as a methionine, added to
the N-terminus. The invention also provides fusion proteins and
conjugates comprising these polypeptides, and isolated or
recombinant nucleic acids encoding these polypeptides.
[0104] The invention also includes polypeptides comprising
sequences which differ in 0-16 amino acid positions (such as in 0,
1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, or 16 amino acid
positions), e.g. in 0-16 positions, 0-15 positions, 0-14 positions,
0-13 positions, 0-12 positions, 0-11 positions, 0-10 positions, 0-9
positions, 0-8 positions, 0-7 positions, 0-6 positions, 0-5
positions, 0-4 positions, 0-3 positions, 0-2 positions, or 0-1
positions, from any one of SEQ ID NOs:1-15 and SEQ ID NOs:44-104,
such as, one of SEQ ID NOs:1-15, 47, and 53 (for example, SEQ ID
NO:1, SEQ ID NO:3, SEQ ID NO:8, SEQ ID NO:12, SEQ ID NO:47, or SEQ
ID NO:53). In some instances, the polypeptide exhibits an
interferon-alpha activity (e.g., antiviral activity, T.sub.H1
differentiation activity, and/or antiproliferative activity). Some
such polypeptides further comprise an additional amino acid, such
as a methionine, added to the N-terminus. The invention also
provides fusion proteins and conjugates comprising these
polypeptides, and isolated or recombinant nucleic acids encoding
these polypeptides.
[0105] In some instances, the sequence of the polypeptide of the
invention comprises a substitution of an amino acid for a different
amino acid at one or more positions, including, but not limited to,
positions 47, 51, 52, 53, 54, 55, 56, 57, 58, 60, 61, 64, 69, 71,
72, 75, 76, 77, 78, 79, 80, 83, 84, 85, 86, 87, 90, 93, 133, 140,
154, 160, 161, and 162, relative to any one of SEQ ID NOs:1-15, 47,
and 53, such as, for example, SEQ ID NO:1, SEQ ID NO:3, SEQ ID
NO:8, SEQ ID NO:12, SEQ ID NO:47, or SEQ ID NO:53. In some
instances, the polypeptide sequence comprises one or more of: His
or Gln at position 47; Val, Ala or Thr at position 51; Gln, Pro or
Glu at position 52; Ala or Thr at position 53; Phe, Ser, or Pro at
position 55; Leu, Val or Ala at position 56; Phe or Leu at position
57; Tyr or His at position 58; Met, Leu or Val at position 60; Met
or Ile at position 61; Thr or Ile at position 64; Ser or Thr at
position 69; Lys or Glu at position 71; Asn or Asp at position 72;
Ala or Val at position 75; Ala or Thr at position 76; Trp or Leu at
position 77; Asp or Glu at position 78; Glu or Gln at position 79;
Thr, Asp, Ser, or Arg at position 80; Glu or Asp at position 83;
Lys or Glu at position 84; Phe or Leu at position 85; Tyr, Cys or
Ser at position 86; Ile or Thr at position 87; Phe, Tyr, Asp or Asn
at position 90; Met or Leu at position 93; Lys or Glu at position
133; Ser or Ala at position 140; Phe or Leu at position 154; Lys or
Glu at position 160; Arg or Ser at position 161; and Arg or Ser at
position 162; the position numbering relative to that of SEQ ID
NO:1. The invention also provides fusion proteins and conjugates
comprising these polypeptides, and isolated or recombinant nucleic
acids encoding these polypeptides.
[0106] Some polypeptides of the invention comprise a substitution
at a position which in a parent molecule is predicted to contain an
amino acid residue that is exposed to the surface of the molecule,
e.g., that is calculated to have at least 25%, such as at least 50%
of its side chain exposed to the surface. Some such polypeptides of
the invention comprise a substitution of an amino acid for a
different amino acid at one or more positions including, but not
limited to, the following positions which contain amino acid
residues having more than 25% fractional Accessible Surface Area
(ASA): positions 2, 3, 4, 5, 6, 7, 8, 9, 10, 12, 13, 16, 19, 20,
22, 23, 24, 25, 26, 27, 28, 30, 31, 33, 34, 35, 37, 40, 41, 42, 44,
46, 47, 49, 50, 51, 52, 59, 62, 63, 66, 69, 70, 71, 72, 74, 75, 78,
79, 80, 81, 83, 84, 87, 90, 91, 94, 95, 97, 98, 100, 101, 102, 103,
104, 105, 107, 108, 109, 110, 111, 112, 113, 114, 115, 118, 121,
122, 125, 126, 128, 129, 132, 133, 134, 135, 136, 137, 138, 139,
146, 149, 150, 153, 154, 157, 159, 160, 161, 162, 163, 164, 165,
and 166, relative to any one of SEQ ID NOs:1-15, 47, and 53 (for
example, SEQ ID NO:1, SEQ ID NO:3, SEQ ID NO:8, SEQ ID NO:12, SEQ
ID NO:47, or SEQ ID NO:53). Some such polypeptides of the invention
comprise a substitution of an amino acid for a different amino acid
at one or more positions including, but not limited to, the
following positions which contain amino acid residues having more
than 50% fractional ASA: 2, 3, 4, 5, 6, 7, 8, 9, 12, 13, 16, 19,
25, 27, 28, 31, 33, 34, 35, 37, 41, 44, 46, 47, 49, 50, 66, 71, 75,
78, 79, 80, 83, 84, 87, 90, 91, 94, 95, 101, 102, 103, 105, 107,
108, 109, 110, 111, 114, 115, 118, 121, 122, 125, 126, 129, 132,
133, 135, 138, 150, 160, 162, 163, 164, 165, and 166, relative to
any one of SEQ ID NOs:1-15, 47, and 53 (for example, SEQ ID NO:1,
SEQ ID NO:3, SEQ ID NO:8, SEQ ID NO:12, SEQ ID NO:47, or SEQ ID
NO:53).
[0107] Some polypeptides of the invention comprise one or more of
the following substitutions which introduce a cysteine residue into
a position which has more than 25% fractional ASA: D2C, L3C, P4C,
Q5C, T6C, H.sub.7C, S8C, L9C, G10C, R12C, R13C, M16C, A19C, Q20C,
R22C, R23C, 124C, S25C, L26C, F27C, S28C, L30C, K31C, R33C,
H.sub.34C, D35C, R37C, Q40C, E41C, E42C, D44C, N46C, H.sub.47C,
Q49C, K50C, V51C, Q52C, E59C, Q62C, Q63C, N66C, S69C, T70C, K71C,
N72C, S74C, A75C, D78C, E79C, T80C, L81C, E83C, K84C, 187C, F90C,
Q91C, N94C, D95C, E97C, A98C, V100C, M101C, Q102C, E103C, V104C,
G105C, E107C, E108C, T109C, P101C, L111C, M112C, N113C, V114C,
D115C, L118C, R121C, K122C, Q125C, R126C, T128C, L129C, T132C,
K133C, K134C, K135C, Y136C, S137C, P138C, A146C, M149C, R150C,
S153C, F154C, N157C, Q159C, K160C, R161C, L162C, R163C, R164C,
K165C and E166C (or equivalent position relative to SEQ ID NO:1),
and combinations thereof.
[0108] Some polypeptides of the invention comprise one or more of
the following substitutions which introduce a lysine residue into a
position which has more than 25% fractional ASA: D2K, L3K, P4K,
Q5K, T6K, H7K, S8K, L9K, G10K, R12K, R13K, M16K, A19K, Q20K, R22K,
R23K, 124K, S25K, L26K, F27K, S28K, L30K, R33K, H34K, D35K, R37K,
Q40K, E41K, E42K, D44K, N46K, H47K, Q49K, V51K, Q52K, E59K, Q62K,
Q63K, N66K, S69K, T70K, N72K, S74K, A75K, D78K, E79K, T80K, L81K,
E83K, 187K, F90K, Q91K, N94K, D95K, E97K, A98K, V100K, M101K,
Q102K, E103K, V104K, G105K, E107K, E108K, T109K, P110K, L111K,
M112K, N113K, V114K, D115K, L118K, R121K, Q125K, R126K, T128K,
L129K, T132K, Y136K, S137K, P138K, A146K, M149K, R150K, S153K,
F154K, N157K, Q159K, R161K, L162K, R163K, R164K, and E166K (or
equivalent position relative to SEQ ID NO:1), and combinations
thereof.
[0109] Some polypeptides of the invention comprise a substitution
of an amino acid residue for a different amino acid residue, or a
deletion of an amino acid residue, which removes one or more
lysines, e.g., K31, K50, K71, K84, K122, K133, K134, K135, K160,
and/or K165 (relative to SEQ ID NO:1) from any polypeptide of the
invention, such as one of SEQ ID NOs:1-15 and SEQ ID NOs:44-104,
such as, for example, one of SEQ ID NOs:1-15, 47, and 53 (for
example, SEQ ID NO:1, SEQ ID NO:3, SEQ ID NO:8, SEQ ID NO:12, SEQ
ID NO:47, or SEQ ID NO:53). The one or more lysine residue(s) to be
removed may be substituted with any other amino acid, may be
substituted with an Arg (R) or Gln (Q), or may be deleted. Some
such polypeptides comprise the substitutions K31R+K122R;
K31R+K133R; K122R+K133R; or K31R+K122R+K133R. Other exemplary
substitutions include K71E; K84E; K133E/G; and K160E.
[0110] Some polypeptides of the invention comprise a substitution
or a deletion which removes one or more histidines, e.g., H7, H11,
H34, and/or H47 (relative to SEQ ID NO:1) from any polypeptide of
the invention, such as one of SEQ ID NOs:1-15 and SEQ ID
NOs:44-104, such as, for example, one of SEQ ID NOs:1-15, 47, and
53 (for example, SEQ ID NO:1, SEQ ID NO:3, SEQ ID NO:8, SEQ ID
NO:12, SEQ ID NO:47, or SEQ ID NO:53). The one or more histidine
residue(s) to be removed may be substituted with any other amino
acid, may be substituted with an Arg (R) or Gln (Q), or may be
deleted. Some such polypeptides comprise the substitutions H34Q;
H47Q; or H34Q+H47Q.
[0111] Some polypeptides of the invention comprise a substitution
of an amino acid for a different amino acid, a deletion of an amino
acid, or an insertion of an amino acid, which removes or otherwise
disrupts the spatial arrangement of the N-linked glycosylation site
N72 S73 S74 (relative to SEQ ID NO:1). Removal of this site may be
accomplished in a number of ways, for example by deletion of N72 or
substitution of N72 for a different amino acid, substitution of
Ser73 with Pro, substitution of Ser74 for an amino acid other than
Ser, Thr, or Cys, or insertion of an amino acid residue other than
Ser, Thr, or Cys between positions 73 and 74. For example, some
such polypeptides comprise the substitution N72D.
[0112] Some polypeptides of the invention comprise one or more
amino acid substitution, deletion or insertion which removes one or
more basic residues or one or more pairs of basic residues (such
as, Arg-Arg, Arg-Lys, Lys-Arg, Lys-Lys) in order to, for example,
minimize the presence of potential protease-sensitive sites, or in
some instances to remove sites potentially reactive towards
amine-reactive conjugation (e.g. PEGylation) reagents. For example,
removal of dibasic sequences near the C-terminus may be
accomplished by removal of one or more of Lys160, Arg161, Arg163,
Arg164, and Lys165 (relative to SEQ ID NO:1). The one or more Lys
or Arg to be removed may for example be deleted, or substituted
with any amino acid other than Lys or Arg. Some such polypeptides
of the invention comprise a substitution of one or more of Lys160,
Arg161, and Arg164 for an amino acid other than Lys or Arg, such
as, for example, one or more of the substitutions Lys160Glu;
Arg161Ser/Cys; and Arg164Ser/Cys. Some such polypeptides
alternatively or in addition comprise a deletion of one or more of
Lys 160, Arg161, Arg163, Arg164, and Lys165, which may be via
individual deletions (e.g., K165*) or in groups of more than one,
including via C-terminal truncation (e.g., K165*-E166*).
[0113] Other modifications contemplated for polypeptides of the
invention include those described below and in the section entitled
"INTERFERON-ALPHA CONJUGATES".
[0114] It is to be understood that while the examples and
modifications to the parent polypeptide are generally provided
herein relative to the sequence SEQ ID NO:1 (or relative to some
other specified sequence), the disclosed modifications may also be
made in equivalent amino acid positions of any of the other
polypeptides of the invention (including SEQ ID NOs:2-15 and SEQ ID
NOs:44-104 and variants thereof) described herein. Thus, as an
example, the substitution H.sub.47C relative to SEQ ID NO:1 is
understood to correspond to Q47C in SEQ ID NO:5, and so on.
[0115] The following tables provide sequences of some
interferon-alpha polypeptides of the invention. For clarity, the
sequences are shown relative to SEQ ID NO:3 (Table 1) or SEQ ID
NO:12 (Table 2). Some such polypeptides exhibit an interferon-alpha
activity, such as antiviral activity, T.sub.H1 differentiation
activity, and/or antiproliferative activity. TABLE-US-00001 TABLE 1
Polypeptide Sequence (relative to Clone SEQ ID NO:3) name(s) SEQ ID
SEQ ID NO:3 B9x11 SEQ ID NO:1 + E133K, A140S SEQ ID NO:3 B9x12 SEQ
ID NO:2 + H47Q, E133K, A140S SEQ ID NO:3 B9x14, SEQ ID NO:3
B9x14CHO2 SEQ ID NO:3 B9x15 SEQ ID NO:4 + H47Q, V51T, F55S, L56V,
Y58H, E133K, A140S SEQ ID NO:3 B9x16 SEQ ID NO:5 + H47Q SEQ ID NO:3
B9x17 SEQ ID NO:6 + V51T, F555, L56V, Y58H SEQ ID NO:3 B9x18 SEQ ID
NO:7 + H47Q, V51T, F555, L56V, Y58H SEQ ID NO:3 + F154L, K160E,
R161S, B9x14C2a SEQ ID NO:44 R164S SEQ ID NO:3 B9x14CHO1 SEQ ID
NO:45 + E166EC SEQ ID NO:3 B9x14CHO3 SEQ ID NO:46 + N72D SEQ ID
NO:3 B9x14CHO4, SEQ ID NO:47 + N72D, K160E, R161S, B9x14EC4 R164S
SEQ ID NO:3 B9x14CHO5, SEQ ID NO:48 + N72D, K160E, R161C, B9x14EC5
R164S SEQ ID NO:3 B9x14CHO6, SEQ ID NO:49 + N72D, K160E, R161S,
B9x14EC3 R164C SEQ ID NO:3 14Ep01 SEQ ID NO:50 + H47Q, V51A, F555,
L56V, F57L, Y58H, N72D, F154L, K160E, R161S, R164S SEQ ID NO:3
14Ep02 SEQ ID NO:51 + M61I, N72D, E83D, M93L, M101T, T109I, P110A,
V114E, F154L, K160E, R161S, R164S SEQ ID NO:3 14Ep03 SEQ ID NO:52 +
N72D, M101I, F154L, K160E, R161S, R164S SEQ ID NO:3 14Ep04 SEQ ID
NO:53 + N72D, F154L, K160E, R161S, R164S SEQ ID NO:3 14Ep05 SEQ ID
NO:54 + H47Q, V51A, F55S, L56V, F57L, Y58H, N72D, M101I, F154L,
K160E, R161S, R164S SEQ ID NO:3 14EF SEQ ID NO:55 + H47Q, V51A,
F55S, L56V, F57L, Y58H, M61I, N72D, E83D, M93L, M101T, T109I,
P110A, V114E, F154L, K160E, R161S, R164S SEQ ID NO:3 B9x14Ep04C31
SEQ ID NO:56 + K31C, N72D, F154L, K160E, R161S, R164S SEQ ID NO:3
B9x14CHO4C31 SEQ ID NO:57 + K31C, N72D, K160E, R161S, R164S SEQ ID
NO:3 B9x14HO4C46 SEQ ID NO:58 + N46C, N72D, K160E, R161S, R164S SEQ
ID NO:3 B9x14CHO4C71 SEQ ID NO:59 + K71C, N72D, K160E, R161S, R164S
SEQ ID NO:3 B9x14CHO4C75 SEQ ID NO:60 + N72D, A75C, K160E, R161S,
R164S SEQ ID NO:3 B9x14CHO4C79 SEQ ID NO:61 + N72D, E79C, K160E,
R161S, R164S SEQ ID NO:3 B9x14CHO4C107 SEQ ID NO:62 + N72D, E107C,
K160E, R161S, R164S SEQ ID NO:3 B9x14CHO4C122 SEQ ID NO:63 + N72D,
K122C, K160E, R161S, R164S SEQ ID NO:3 B9x14CHO4C134 SEQ ID NO:64 +
N72D, K134C, K160E, R161S, R164S SEQ ID NO:3 B9x14Ep04 SEQ ID NO:65
+ N72D, F154L, K160E, .DELTA.161-166 R161*-E166* SEQ ID NO:3
B9x14Ep04 SEQ ID NO:66 + N72D, F154L, K160E, .DELTA.165-166 R161S,
R164S, K165*-E166* SEQ ID NO:3 B9x14Ep04.DELTA.1-4 SEQ ID NO:67 +
C1*-P4*, D44*, N72D, D44*.DELTA.165-166 F154L, K160E, R161S, R164S,
K165*-E166* SEQ ID NO:3 B9x14CHO4NP1 SEQ ID NO:68 + H34Q, N72D,
K160E, R161S, R164S SEQ ID NO:3 B9x14CHO4NP2 SEQ ID NO:69 + H34Q,
H47Q, N72D, K160E, R161S, R164S SEQ ID NO:3 B9x14CHO8 SEQ ID NO:70
+ K31R, N72D, K160E, R161S, R164S SEQ ID NO:3 B9x14CHO9 SEQ ID
NO:71 + K50R, N72D, K160E, R161S, R164S SEQ ID NO:3 B9x14CHO10 SEQ
ID NO:72 + K71R, N72D, K160E, R161S, R164S SEQ ID NO:3 B9x14CHO11
SEQ ID NO:73 + N72D, K84R, K160E, R161S, R164S SEQ ID NO:3
B9x14CHO12 SEQ ID NO:74 + N72D, K122R, K160E, R161S, R164S SEQ ID
NO:3 B9x14CHO13 SEQ ID NO:75 + N72D, K134R, K160E, R161S, R164S SEQ
ID NO:3 B9x14CHO14 SEQ ID NO:76 + N72D, K135R, K160E, R161S, R164S
SEQ ID NO:3 B9x14CHO15 SEQ ID NO:77 + N72D, K160E, R161S, R164S,
K165R SEQ ID NO:3 B9x14CHO16 SEQ ID NO:78 + N72D, K122R, K135R,
K160E, R161S, R164S SEQ ID NO:3 B9x14CHO17 SEQ ID NO:79 + K31R,
N72D, K135R, K160E, R161S, R164S SEQ ID NO:3 B9x14CHO18 SEQ ID
NO:80 + K31R, N72D, K122R, K160E, R161S, R164S SEQ ID NO:3
B9x14CHO18NP2 SEQ ID NO:81 + K31R, H34Q, H47Q, N72D, K122R, K160E,
R161S, R164S SEQ ID NO:3 B9x14CHO18NP2 SEQ ID NO:82 + K31R, H34Q,
H47Q, .DELTA.165-166 N72D, K122R, K160E, R161S, R164S,
K165*-E166*
[0116] TABLE-US-00002 TABLE 2 Polypeptide Sequence (relative to
Clone SEQ ID NO:12) name(s) SEQ ID SEQ ID NO:12 B9X21 SEQ ID NO:8 +
H47Q, V51T, F55S, L56V, Y58H SEQ ID NO:12 B9X22 SEQ ID NO:9 + V51T,
F55S, L56V, Y58H SEQ ID NO:12 B9X23 SEQIDNO:1O + H47Q SEQ ID NO:12
+ H47Q, V51T, F55S, L56V, B9X24 SEQ ID NO:11 Y58H, E133K, A140S SEQ
ID NO:12 B9X25 SEQ ID NO:12 SEQ ID NO:12 B9X26 SEQ ID NO:13 + V51T,
F55S, L56V, Y58H, E133K, A140S SEQ ID NO:12 B9X27 SEQ ID NO:14 +
H47Q, E133K, A140S SEQ ID NO:12 B9X28 SEQ ID NO:15 + E133K, A140S
SEQ ID NO:12 B9x25CHO1 SEQ ID NO:83 + N72D SEQ ID NO:12 B9x25CHO2,
SEQ ID NO:84 + N72D, F154L, K160E, 25Ep09, R161S, R164S B9x25EC1
SEQ ID NO:12 B9x25CHO3, SEQ ID NO:85 + N72D, F154L, K160E, B9x25EC2
R161C, R164S SEQ ID NO:12 B9x25CHO4, SEQ ID NO:86 + N72D, F154L,
K160E, B9x25EC3 R161S, R164C SEQ ID NO:12 25Ep01 SEQ ID NO:87 +
H47Q, V51T, F55S, L56V, Y58H, N72D SEQ ID NO:12 25Ep02 SEQ ID NO:88
+ L17I, R22G, H47Q, V51T, F55S, L56V, Y58H, N72D, F154L, K160E,
R161S, R164S SEQ ID NO:12 25Ep03 SEQ ID NO:89 + D2N, P45, S10N,
H47Q, V51T, F55S, L56V, Y58H, N72D, F154L, K160E, R161S, R164S SEQ
ID NO:12 25Ep04 SEQ ID NO:90 + H47Q, V51T, F55S, L56V, Y58H, L60M,
N72D, F154L, K160E, R161S, R164S SEQ ID NO:12 25Ep05 SEQ ID NO:91 +
H47Q, V51T, F55S, L56V, Y58H, N72D, N95D, F154L, K160E, R161S,
R164S SEQ ID NO:12 25Ep06 SEQ ID NO:92 + H47Q, V51T, F55S, L56V,
Y58H, N72D, E83D, M93L, N95D, I101T, V114E, F154L, K160E, R161S,
R164S SEQ ID NO:12 25Ep07 SEQ ID NO:93 + H47Q, V51T, F55S, L56V,
Y58H, N72D, R125Q, F154L, K160E, R161S, R164S SEQ ID NO:12 25Ep08
SEQ ID NO:94 + H47Q, V51T, F55S, L56V, Y58H, N72D, F154L, K160E,
R161S, R164S SEQ ID NO:12 25Ep10 SEQ ID NO:95 + L171, R22G, N72D,
F154L, K160E, R161S, R164S SEQ ID NO:12 25Ep11 SEQ ID NO:96 + D2N,
P45, S10N, N72D, F154L, K160E, R161S, R164S SEQ ID NO:12 25Ep12 SEQ
ID NO:97 + N72D, R125Q, F154L, K160E, R161S, R164S SEQ ID NO:12
25Ep13 SEQ ID NO:98 + L171, R22G, N72D, R125Q, F154L, K160E, R161S,
R164S SEQ ID NO:12 25Ep14 SEQ ID NO:99 + D2N, P45, S10N, N72D,
R125Q, F154L, K160E, R161S, R164S SEQ ID NO:12 25Ep15 SEQ ID NO:100
+ H47Q, V51T, F55S, L56V, Y58H, N72D, F154L, K160E, R161S, R164S
SEQ ID NO:12 25Ep16 SEQ ID NO:101 + L17I, R22G, H47Q, V51T, F55S,
L56V, Y58H, N72D, R125Q, F154L, K160E, R161S, R164S SEQ ID NO:12
25Ep17 SEQ ID NO:102 + D2N, P45, S10N, H47Q, V51T, F55S, L56V,
Y58H, N72D, R125Q, F154L, K160E, R161S, R164S SEQ ID NO:12 25EF1
SEQ ID NO:103 + L17I, R22G, H47Q, V51T, F55S, L56V, Y58H, L60M,
N72D, E83D, M93L, N95D, I101T, V114E, R125Q, F154L, K160E, R161S,
R164S SEQ ID NO:12 25EF2 SEQ ID NO:104 + D2N, P4S, S10N, H47Q,
V51T, F55S, L56V, Y58H, L60M, N72D, E83D, M93L, N95D, I101T, V114E,
R125Q, F154L, K160E, R161S, R164S
Variants
[0117] In another aspect, the invention provides an isolated or
recombinant polypeptide which is a variant of a parent
interferon-alpha polypeptide, the variant comprising a sequence
which differs from the parent polypeptide sequence in least one
amino acid position, wherein the variant sequence comprises one or
more of His at position 47, Val at position 51, Phe at position 55,
Leu at position 56, Tyr at position 58, Lys at position 133, and at
position Ser140, the position numbering relative to that of SEQ ID
NO:1. In some instances the parent interferon-alpha polypeptide
sequence is a sequence of a naturally-occurring human
interferon-alpha (such as, for example, huIFN-alpha 2b (SEQ ID
NO:32), huIFN-alpha 2a (SEQ ID NO:32 with position 23=Lys),
huIFN-alpha 2c (SEQ ID NO:32 with position 34=Arg), huIFN-alpha 8b
(SEQ ID NO:33), huIFN-alpha 8a (SEQ ID NO:33 with positions 98=Val,
99=Leu, 100=Cys, and 101=Asp), huIFN-alpha 8c (SEQ ID NO:33 with
position 161=Asp and amino acids at positions 162-166 deleted),
huIFN-alpha 14a (SEQ ID NO:39), huIFN-alpha 14c (SEQ ID NO:39 with
position 152=Leu), or a sequence of any other naturally occurring
human interferon alpha polypeptide, such as those shown in FIGS. 2
and 4 herein (SEQ ID NOs:31-42) and/or listed in Allen G. and Diaz
M. O. (1996), supra). In some instances the parent interferon-alpha
polypeptide sequence is a sequence of a non-naturally occurring
(i.e., synthetic) interferon-alpha, such as IFN-alphaCon1 (SEQ ID
NO:43) In some instances, the parent polypeptide to be modified may
itself be a polypeptide of the invention, such as, e.g. any one of
SEQ ID NOs:1-15 and SEQ ID NOs:44-104. In some instances, the
variant sequence differs from the parent polypeptide sequence in
1-16 amino acid positions (such as in 1, 2, 3, 4, 5, 6, 7, 8, 9,
10, 11, 12, 13, 14, 15, or 16 amino acid positions), e.g. in 1-14
amino acid positions, in 1-12 amino acid positions, in 1-10 amino
acid positions, in 1-8 amino acid positions, in 1-6 amino acid
positions, in 1-5 amino acid positions, in 1-4 amino acid
positions, in 1-3 amino acid positions, or in 1-2 amino acid
positions. Some such variants exhibit an interferon-alpha activity.
The invention also provides fusion proteins and conjugates
comprising these variants, and isolated or recombinant nucleic
acids encoding these variants.
Sequence Variations
[0118] As noted above, polypeptides of the present invention
include polypeptides comprising sequences which differ from any one
of SEQ ID NOs:1-15 and SEQ ID NOs:44-104, such as one of SEQ ID
NOs:1-15, 47, and 53 (for example, SEQ ID NO:1, SEQ ID NO:3, SEQ ID
NO:8, SEQ ID NO:12, SEQ ID NO:47, or SEQ ID NO:53), in 0-16 amino
acid positions (such as in 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11,
12, 13, 14, 15, or 16 amino acid positions), e.g. in 0-16
positions, 0-15 positions, 0-14 positions, 0-13 positions, 0-12
positions, 0-11 positions, 0-10 positions, 0-9 positions, 0-8
positions, 0-7 positions, 0-6 positions, 0-5 positions, 0-4
positions, 0-3 positions, 0-2 positions, or 0-1 positions. Some
such polypeptides exhibit an interferon-alpha activity.
[0119] For example, some such polypeptides of the invention
comprise a sequence having a length of about 150 amino acids, such
as about 151, 152, 153, 154, 155, 156, 157, 158, 159, 160, 161,
162, 163, 164, or 165 amino acids, corresponding to a deletion of
between 1 and 16 amino acids relative to a parent polypeptide
sequence (such as, for example, one of SEQ ID NOs:1-15). In some
instances, between 1 and 11, e.g., between 1 and 10, such as
between 1 and 7, e.g. between 1 and 5, such as between 1 and 3
amino acids are deleted from the C-terminus, i.e. the polypeptide
is C-terminally truncated compared to the parent polypeptide
sequence (such as, for example, one of SEQ ID NOs: 1-15, 47, or 53)
by 1-11 amino acid residues (e.g. by 1, 2, 3, 4, 5, 6, 7, 8, 9, 10,
or 11 amino acid residues), such as by 1-10, 1-7, e.g., by 1-5 or
by 1-3 amino acid residues. Alternatively, or in addition, some
such polypeptides are N-terminally truncated compared to the parent
polypeptide sequence (such as, one of SEQ ID NOs:1-15, 47, or 53)
by 1-4 amino acid residues (e.g. by 1, 2, 3, or 4 amino acid
residues), e.g., 1-4, 1-3, 1-2 or 1 amino acid residue(s) are
removed from the N-terminus. Some such polypeptides further
comprise a methionine at the N-terminus. Some such polypeptides
exhibit an interferon-alpha activity.
[0120] As another example, some such polypeptides of the invention
comprise a sequence containing between 0 and 16 amino acid
substitutions relative to one of SEQ ID NOs:1-15, 47, or 53 (e.g.
0, 1, 2, 3, 4, 5, 6, 7, 8,9, 10, 11, 12, 13, 14, 15, or 16 amino
acid substitutions), such as 0-14 or 0-12 or 0-10 or 0-8 or 0-6 or
0-5 or 0-4 or 0-3 or 0-2 or 0-1 amino acid substitutions. In some
instances, one or more of the amino acid substitutions are made
according to, for example, a substitution group (such as, a
conservative substitution group), such as one set forth below. Some
such polypeptides exhibit an interferon-alpha activity.
[0121] Some polypeptides of the invention comprise a sequence
comprising between 0 and 16 amino acid substitutions relative to
one of SEQ ID NOs:1-15 and SEQ ID NOs:44-104 (for example, SEQ ID
NO:1, SEQ ID NO:3, SEQ ID NO:8, SEQ ID NO:12, SEQ ID NO:47, or SEQ
ID NO:53), e.g. 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14,
15, or 16 amino acid substitutions, such as 0-14 or 0-12 or 0-10 or
0-8 or 0-6 or 0-5 or 0-4 or 0-3 or 0-2 or 0-1 amino acid
substitutions, where at least one of said substitution(s)
introduces an amino acid residue comprising an attachment group for
a non-polypeptide moiety. Examples include introduction of one or
more N-glycosylation site(s), or introduction of one or more
cysteine residue(s) or lysine residue(s), as described above and in
the section entitled "INTERFERON-ALPHA CONJUGATES". Some such
polypeptides exhibit an interferon-alpha activity.
[0122] Some polypeptides of the invention comprise a sequence
containing between 0 and 16 amino acid substitutions or deletions
or insertions relative to one of SEQ ID NOs:1-15 and SEQ ID
NOs:44-104 (for example, SEQ ID NO:1, SEQ ID NO:3, SEQ ID NO:8, SEQ
ID NO:12, SEQ ID NO:47, or SEQ ID NO:53), such as 0-14 or 0-12 or
0-10 or 0-8 or 0-6 or 0-5 or 0-4 or 0-3 or 0-2 or 0-1 amino acid
substitutions deletions or insertions (or a combination thereof),
where at least one of said substitution or deletion removes an
amino acid residue from a parent polypeptide sequence which
comprises an attachment group for a non-polypeptide moiety or, in
the case of an amino acid insertion, disrupts the spatial
arrangement of residues required for such attachment group (e.g.,
an insertion of an amino acid to disrupt an N-glycosylation N-X-S/T
motif). Examples include removal from the parent polypeptide
sequence of an N-glycosylation site, or removal of a lysine,
histidine, or cysteine residue, as described above and in the
section entitled "INTERFERON-ALPHA CONJUGATES". Some such
polypeptides exhibit an interferon-alpha activity.
[0123] As a non-limiting example, a polypeptide of the invention
may have the sequence SEQ ID NO:3 or a sequence which differs from
SEQ ID NO:3 in a total of up to 16 positions (which may be a
combination of amino acid substitutions, deletions, and/or
insertions, including those described above). In some instances,
none, some, or all of the substitutions are substitutions according
to a substitution group defined below.
[0124] Amino acid substitutions in accordance with the invention
may include, but are not limited to, one or more conservative amino
acid substitutions. Conservative substitution tables providing
functionally similar amino acids are well known in the art. One
example is provided in the table below (Table 3), which sets forth
six exemplary groups that contain amino acids which may be
considered "conservative substitutions" for one another.
TABLE-US-00003 TABLE 3 Conservative Substitution Groups 1 Alanine
(A) Glycine (G) Serine (S) Threonine (T) 2 Aspartic acid (D)
Glutamic acid (E) 3 Asparagine (N) Glutamine (Q) 4 Arginine (R)
Lysine (K) Histidine (H) 5 Isoleucine (I) Leucine (L) Methionine
(M) Valine (V) 6 Phenylalanine (F) Tyrosine (Y) Tryptophan (W)
[0125] Other substitution groups of amino acids can be envisioned.
For example, amino acids can be grouped by similar function or
chemical structure or composition (e.g., acidic, basic, aliphatic,
aromatic, sulfur-containing). For example, an Aliphatic grouping
may comprise: Glycine (G), Alanine (A), Valine (V), Leucine (L),
Isoleucine (I). Other groups containing amino acids that are
considered conservative substitutions for one another include:
Aromatic: Phenylalanine (F), Tyrosine (Y), Tryptophan (W);
Sulfur-containing: Methionine (M), Cysteine (C); Basic: Arginine
(R), Lysine (K), Histidine (H); Acidic: Aspartic acid (D), Glutamic
acid (E), Asparagine (N), Glutamine (Q). See also Creighton (1984)
Proteins, W.H. Freeman and Company, for additional groupings of
amino acids. Listing of a polypeptide sequence herein, in
conjunction with the above substitution groups, provides an express
listing of all conservatively substituted polypeptide
sequences.
Percent Sequence Identity
[0126] In one aspect, the invention provides an isolated or
recombinant polypeptides each comprising a sequence having at least
90% sequence identity (e.g., at least about 91%, at least about
92%, at least about 93%, at least about 94%, at least about 95%, at
least about 96%, at least about 97%, at least about 98%, or at
least about 99% amino acid sequence identity) to any one of SEQ ID
NOs: 1-15 and SEQ ID NOs:44-104, such as, for example, to one of
SEQ ID NOs:1-15, 47, and 53 (for example, SEQ ID NO:1, SEQ ID NO:3,
SEQ ID NO:8, SEQ ID NO:12, SEQ ID NO:47, or SEQ ID NO:53). In some
instances the polypeptide exhibits an interferon-alpha activity. In
some instances, the polypeptide sequence differs at one or more
amino acid positions, e.g., in up to 16 positions (such as, 1-16
positions, 1-15 positions, 1-14 positions, 1-13 positions, 1-12
positions, 1-11 positions, 1-10 positions, 1-9 positions, 1-8
positions, 1-7 positions, 1-6 positions, 1-5 positions, 1-4
positions, 1-3 positions, or 1-2 positions) from any one of SEQ ID
NO:1-15 and 44-104, such as, for example, SEQ ID NO:1, SEQ ID NO:3,
SEQ ID NO:8, SEQ ID NO:12, SEQ ID NO:47, or SEQ ID NO:53. As an
example, some positions which may be substituted for another amino
acid in accordance with the invention include, but are not limited
to, one or more of positions 47, 51, 52, 53, 54, 55, 56, 57, 58,
60, 61, 64, 69, 71, 72, 75, 76, 77, 78, 79, 80, 83, 84, 85, 86, 87,
90, 93, 133, 140, 154, 160, 161, and 162, relative to one of SEQ ID
NOs:1-15 and 44-104. In some instances, the sequence comprises one
or more of: His or Gln at position 47; Val, Ala or Thr at position
51; Gln, Pro or Glu at position 52; Ala or Thr at position 53; Phe,
Ser, or Pro at position 55; Leu, Val or Ala at position 56; Phe or
Leu at position 57; Tyr or His at position 58; Met, Leu or Val at
position 60; Met or Ile at position 61; Thr or Ile at position 64;
Ser or Thr at position 69; Lys or Glu at position 71; Asn or Asp at
position 72; Ala or Val at position 75; Ala or Thr at position 76;
Trp or Leu at position 77; Asp or Glu at position 78; Glu or Gln at
position 79; Thr, Asp, Ser, or Arg at position 80; Glu or Asp at
position 83; Lys or Glu at position 84; Phe or Leu at position 85;
Tyr, Cys or Ser at position 86; Ile or Thr at position 87; Phe,
Tyr, Asp or Asn at position 90; Met or Leu at position 93; Lys or
Glu at position 133; Ser or Ala at position 140; Phe or Leu at
position 154; Lys or Glu at position 160; Arg or Ser at position
161; and Arg or Ser at position 162; the position numbering
relative to that of SEQ ID NO:1. Other substitutions contemplated
in sequences of the invention are described above and in the
section entitled "INTERFERON-ALPHA CONJUGATES". The invention also
provides fusion proteins comprising such polypeptides, conjugates
comprising such polypeptides, and isolated or recombinant nucleic
acids encoding such polypeptides.
[0127] In another aspect, the present invention provides nucleic
acids having at least about 60%, 70%, 75%, 80%, 85%, 90%, 91%, 92%,
93%, 94%, 95%, 96%, 97%, 98%, 99% or more percent sequence identity
to one or more of SEQ ID NOS:16-30. Some such nucleic acids encode
polypeptides exhibiting an interferon-alpha activity as described
herein.
[0128] The degree to which a sequence (polypeptide or nucleic acid)
is similar to another provides an indication of similar structural
and functional properties for the two sequences. Accordingly, in
the context of the present invention, sequences which have a
similar sequence to any given exemplar sequence are a feature of
the present invention. In particular, sequences that have percent
sequence identities as defined below are a feature of the
invention.
[0129] A variety of methods of determining sequence relationships
can be used, including manual alignment and computer assisted
sequence alignment and analysis. A variety of computer programs for
performing sequence alignments are available, or an alignment can
be prepared manually by one of skill, as described below.
[0130] As noted above, the sequences of the nucleic acids and
polypeptides employed in the subject invention need not be
identical, but can be substantially identical to the corresponding
sequence of a polypeptide of the invention or nucleic acid of the
invention. For example, polypeptides of the invention can be
subject to various changes, such as one or more amino acid
insertions, deletions, and/or substitutions, either conservative or
non-conservative, including where, e.g., such changes might provide
for certain advantages in their use, such as, in their therapeutic
or prophylactic use or administration or diagnostic application.
The nucleic acids of the invention can also be subject to various
changes, such as one or more substitutions of one or more nucleic
acids in one or more codons such that a particular codon encodes
the same or a different amino acid, resulting in either a silent
variation (as defined herein) or non-silent variation, or one or
more deletions of one or more nucleic acids (or codons) in the
sequence. The nucleic acids can also be modified to include one or
more codons that provide for optimum expression in an expression
system (e.g., bacterial or mammalian), while, if desired, said one
or more codons still encode the same amino acid(s). Such nucleic
acid changes might provide for certain advantages in their
therapeutic or prophylactic use or administration, or diagnostic
application. The nucleic acids and polypeptides can be modified in
a number of ways so long as they comprise a sequence substantially
identical (as defined below) to a sequence in a respective nucleic
acid or polypeptide of the invention.
[0131] The term "identical" or "identity," in the context of two or
more nucleic acid or polypeptide sequences, refers to two or more
sequences that are the same or have a specified percentage of amino
acid residues or nucleotides that are the same, when compared and
aligned for maximum similarity, as determined using the sequence
comparison algorithm described below or by visual inspection.
[0132] The "percent sequence identity" ("% identity") of a subject
sequence to a reference (i.e. query) sequence means that the
subject sequence is identical (i.e., on an amino acid-by-amino acid
basis for a polypeptide sequence, or a nucleotide-by-nucleotide
basis for a polynucleotide sequence) by a specified percentage to
the query sequence over a comparison length.
[0133] The percent sequence identity of a subject sequence to a
query sequence is calculated as follows. First, the optimal
alignment of the two sequences is determined using a sequence
comparison algorithm with specific alignment parameters. This
determination of the optimal alignment may be performed using a
computer, or may be manually calculated, as described below. Then,
the two optimally aligned sequences are compared over the
comparison length, and the number of positions in the optimal
alignment at which identical residues occur in both sequences are
determined, which provides the number of matched positions. The
number of matched positions is then divided by the total number of
positions of the comparison length (which, unless otherwise
specified, is the length of the query sequence), and then the
result is multiplied by 100, to yield the percent sequence identity
of the subject sequence to the query sequence.
[0134] With regard to polypeptide sequences, typically one sequence
is regarded as a "query sequence" (for example, a polypeptide
sequence of the invention) to which one or more other sequences,
i.e., "subject sequence(s)" (for example, sequences present in a
sequence database) are compared. The sequence comparison algorithm
uses the designated alignment parameters to determine the optimal
alignment between the query sequence and the subject sequence(s).
When comparing a query sequence against a sequence database, such
as, e.g., GENBANK.RTM. (Genetic Sequence Data Bank; U.S. Department
of Health and Human Services) or GENESEQ.RTM. (Thomson Derwent;
also available as DGENE.RTM. on STN), usually only the query
sequence and the alignment parameters are input into the computer;
optimal alignments between the input query sequence and each
subject sequence present in the database are returned, generally
for up to a desired number of subject sequences.
[0135] Two polypeptide sequences are "optimally aligned" when they
are aligned using defined parameters, i.e., a defined amino acid
substitution matrix, gap existence penalty (also termed gap open
penalty), and gap extension penalty, so as to arrive at the highest
similarity score possible for that pair of sequences. The BLOSUM62
matrix (Henikoff and Henikoff (1992) Proc. Natl. Acad. Sci. USA
89(22):10915-10919) is often used as a default scoring substitution
matrix in polypeptide sequence alignment algorithms (such as
BLASTP, described below). The gap existence penalty is imposed for
the introduction of a single amino acid gap in one of the aligned
sequences, and the gap extension penalty is imposed for each
residue position in the gap. Unless otherwise stated, alignment
parameters employed herein are: BLOSUM62 scoring matrix, gap
existence penalty=11, and gap extension penalty=1. The alignment
score is defined by the amino acid positions of each sequence at
which the alignment begins and ends (e.g. the alignment window),
and optionally by the insertion of a gap or multiple gaps into one
or both sequences, so as to arrive at the highest possible
similarity score.
[0136] While optimal alignment between two or more sequences can be
determined manually (as described below), the process is
facilitated by the use of a computer-implemented alignment
algorithm such as BLAST.RTM. (National Library of Medicine), e.g.,
BLASTP for polypeptide sequences and BLASTN for nucleic acid
sequences, described in Altschul et al. (1997) Nucleic Acids Res.
25:3389-3402, and made available to the public through various
sources, such as the National Center for Biotechnology Information
(NCBI) Website. When using a computerized BLAST interface, if the
option exists to use a "low complexity filter", this option should
be turned off (i.e., no filter).
[0137] FIG. 3 shows an alignment of a polypeptide of the invention
(B9.times.14, SEQ ID NO:3) with human IFN-alpha 14a (also known as
LeIF H, Goeddel et al. (1981) Nature 290:20-26; SEQ ID NO:39),
which was the most closely-related sequence retrieved in a BLASTP
search of query sequence SEQ ID NO:3 against the GENBANK and
GENESEQ databases using the BLOSUM62 matrix, gap open penalty 11,
gap extension penalty 1. These two sequences differ in 18 amino
acid positions over a length of 166 amino acids (i.e., SEQ ID NO:39
differs from SEQ ID NO:3 in 18 amino acid positions); furthermore,
SEQ ID NO:39 is 89% identical to SEQ ID NO:3, since
((166-18)/166).times.100=89.
[0138] FIG. 5 shows an alignment of another polypeptide of the
invention (B9.times.25, SEQ ID NO:12) with human IFN-alpha 14a (SEQ
ID NO:39) which was the most closely-related sequence retrieved in
a BLASTP search of query sequence SEQ ID NO:12 against the GENBANK
and GENESEQ databases using the parameters specified above. SEQ ID
NO:39 differs from SEQ ID NO:12 in 20 amino acid positions over a
length of 166 amino acids; furthermore, SEQ ID NO:39 is 88%
identical to SEQ ID NO:3, since ((166-20)/166).times.100=88.
[0139] The optimal alignment between two polypeptide sequences can
also be determined by a manual calculation of the BLASTP algorithm
(i.e., without aid of a computer) using the same alignment
parameters specified above (matrix=BLOSUM62, gap open penalty=11,
and gap extension penalty=1). To begin, the two sequences are
initially aligned by visual inspection. An initial alignment score
is then calculated as follows: for each individual position of the
alignment (i.e., for each pair of aligned residues), a numerical
value is assigned according to the BLOSUM62 matrix (FIG. 6). The
sum of the values assigned to each pair of residues in the
alignment is the initial alignment score. If the two sequences
being aligned are highly similar, often this initial alignment
provides the highest possible alignment score. The alignment with
the highest possible alignment score is the optimal alignment based
on the alignment parameters employed. FIG. 7A shows an example
calculation of an alignment score for two sequences, a "query"
sequence, identified herein as residues 29-50 of SEQ ID NO:3
(upper), and a "subject" sequence, identified herein as residues
30-52 of SEQ ID NO:5 (lower). The sequences were aligned by visual
inspection, and the numerical value assigned by the BLOSUM62 matrix
for each aligned pair of amino acids is shown beneath each position
in the alignment (to aid in visualization, each identical pair of
amino acids in the alignment is shown in boldface). In this
example, this initial alignment provided the highest possible
alignment score (the sum of the values shown beneath each aligned
position); any other alignment of these two sequences, with or
without gaps, would result in a lower alignment score.
[0140] In some instances, a higher alignment score might be
obtained by introducing one or more gaps into the alignment.
Whenever a gap is introduced into an alignment, a gap open penalty
is assigned, and in addition a gap extension penalty is assessed
for each residue position within that gap. Therefore, using the
alignment parameters described above (including gap open penalty=11
and gap extension penalty=1), a gap of one residue in the alignment
would correspond to a value of -(11+(1.times.1))=-12 assigned to
the gap; a gap of three residues would correspond to a value of
-(11+(3.times.1))=-14 assigned to the gap, and so on. This
calculation is repeated for each new gap introduced into the
alignment. FIGS. 7B and 7C show an example which demonstrates how
introduction of a gap into an alignment can result in a higher
alignment score, despite the gap penalty. FIG. 7B shows an initial
alignment of residues 29-50 of SEQ ID NO:3 (upper, query) and
residues 30-50 of SEQ ID NO:32 (lower, subject) made by visual
inspection, which results in an initial alignment score of 67. FIG.
7C shows the effect of a one-residue gap in SEQ ID NO:32 on the
alignment score; despite the gap penalty of -12, the overall
alignment score of the two sequences increases to 88. In this
example, the alignment shown in FIG. 7C provides the highest
possible alignment score, and is thus the optimal alignment of
these two sequences; any other alignment of these two sequences
(with or without gaps) would result in a lower alignment score.
[0141] It is to be understood that the examples of sequence
alignment calculations described above, which use relatively short
sequences, are provided for illustrative purposes only; in
practice, the alignment parameters employed (BLOSUM62 matrix, gap
open penalty=11, and gap extension penalty=1) are generally
intended for polypeptide sequences 85 amino acids in length or
longer. The NCBI website provides the following alignment
parameters for sequences of other lengths (which are suitable for
computer-aided as well as manual alignment calculation, using the
same procedure as described above). For sequences of 50-85 amino
acids in length, optimal parameters are BLOSUM80 matrix (Henikoff
and Henikoff, supra), gap open penalty=10, and gap extension
penalty=1. For sequences of 35-50 amino acids in length, optimal
parameters are PAM70 matrix (Dayhoff, M. O., Schwartz, R. M. &
Orcutt, B. C. (1978) "A model of evolutionary change in proteins."
In Atlas of Protein Sequence and Structure, vol. 5, suppl. 3, M. O.
Dayhoff (ed.), pp. 345-352, Natl. Biomed. Res. Found., Washington,
D.C.), gap open penalty=10, and gap extension penalty=1. For
sequences of less than 35 amino acids in length, optimal parameters
are PAM30 matrix (Dayhoff, M. O., supra), gap open penalty=9, and
gap extension penalty=1.
[0142] Once the sequences are optimally aligned, the percent
identity of the subject sequence relative to the query sequence is
calculated by counting the number of positions in the optimal
alignment which contain identical residue pairs, divide that by the
number of residues in the comparison length (which, unless
otherwise specified, is the number of residues in the query
sequence), and multiplying the resulting number by 100. Referring
back to the examples shown in FIG. 7, in each example the sequence
designated as the query sequence is 22 amino acids in length.
Referring to the alignment of FIG. 7A, 20 pairs of aligned amino
acid residues (shown in boldface) are identical in the optimal
alignment of the query sequence (upper) with the subject sequence
(lower). Thus, this particular subject sequence has
(20/22).times.100=91.1% identity to the query sequence; in other
words, the subject sequence in the alignment of FIG. 7A has at
least 91% amino acid sequence identity to the query sequence. In
the alignment shown in FIG. 7C, 18 pairs of amino acid residues
(shown in boldface) in the optimal alignment are identical; thus
this particular subject sequence has (18/22).times.100=81.8%
identity to the query sequence; in other words, the subject
sequence in the alignment of FIG. 7C has at least 81% amino acid
sequence identity to the query sequence.
[0143] As applied to polypeptides, the term "substantial identity"
(or "substantially identical") typically means that when two amino
acid sequences (i.e. a query sequence and a subject sequence) are
optimally aligned using the BLASTP algorithm (manually or via
computer) using appropriate parameters described above, the subject
sequence has at least about 60%, 70%, 75%, 80%, 85%, 90%, 91%, 92%,
93%, 94%, 95%, 96%, 97%, 98%, 99%, or more percent amino acid
sequence identity to the query sequence. In some instances, the
substantial identity exists over a comparison length of at least
about 100 amino acid residues, such as, at least about 110, 120,
125, 130, 135, 140, 145, 150, 155, 156, 157, 158, 159, 160, 161,
162, 163, 164, 165, or 166 amino acid residues.
[0144] Similarly, as applied in the context of two nucleic acid
sequences, the term substantial identity (or substantially
identical) means that when two nucleic acid sequences (i.e. a query
and a subject sequence) are optimally aligned using the BLASTN
algorithm (manually or via computer) using appropriate parameters
described below, the subject sequence has at least about 60%, 70%,
75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or
more percent nucleic acid sequence identity to the query sequence.
Parameters used for nucleic acid sequence alignments are: match
reward 1, mismatch penalty -3, gap existence penalty 5, gap
extension penalty 2 (substitution matrices are not used in the
BLASTN algorithm). In some instances, the substantial identity
exists over a comparison length of at least about 300 nucleotide
residues, such as at least about 330, 360, 375, 390, 405, 420, 435,
450, 465, 480, 483, 486, 489, 492, 495, or 498 nucleotides.
Additional Aspects
[0145] Any polypeptide of the invention may be present as part of a
larger polypeptide sequence, e.g. a fusion protein, such as occurs
upon the addition of one or more domains or subsequences for
stabilization or detection or purification of the polypeptide. A
polypeptide purification subsequence may include, e.g., an epitope
tag, a FLAG tag, a polyhistidine sequence, a GST fusion, or any
other detection/purification subsequence or "tag" known in the art.
These additional domains or subsequences either have little or no
effect on the activity of the polypeptide of the invention, or can
be removed by post synthesis processing steps such as by treatment
with a protease, inclusion of an intein, or the like.
[0146] Any polypeptide of the invention may also comprise one or
more modified amino acid. The modified amino acid may be, e.g., a
glycosylated amino acid, a PEGylated amino acid, a farnesylated
amino acid, an acetylated amino acid, a biotinylated amino acid, an
amino acid conjugated to a lipid moiety, or an amino acid
conjugated to an organic derivatizing agent. The presence of
modified amino acids may be advantageous in, for example, (a)
increasing polypeptide serum half-life and/or functional in vivo
half-life, (b) reducing polypeptide antigenicity, (c) increasing
polypeptide storage stability, or (d) increasing bioavailability,
e.g. increasing the AUC.sub.sc. Amino acid(s) are modified, for
example, co-translationally or post-translationally during
recombinant production (e.g., N-linked glycosylation at N-X-S/T
motifs during expression in mammalian cells) or modified by
synthetic means. This aspect is described in more detail in the
section herein entitled "INTERFERON-ALPHA CONJUGATES".
[0147] The invention also provides a composition comprising at
least one polypeptide of the invention, and an excipient or
carrier. In one aspect, the composition comprises an isolated or
recombinant polypeptide comprising an amino acid sequence which
differs in 0-16 amino acid positions (such as in 0, 1, 2, 3, 4, 5,
6, 7, 8, 9, 10, 11, 12, 13, 14, 15, or 16 amino acid positions),
from one of SEQ ID NOs:1-15 and SEQ ID NOs:44-104 (such as, for
example, one of SEQ ID NO:1, SEQ ID NO:3, SEQ ID NO:8, SEQ ID
NO:12, SEQ ID NO:47, or SEQ ID NO:53), plus a carrier or excipient.
The composition may be a composition comprising a pharmaceutically
acceptable excipient or carrier. Exemplary compositions and
excipients and carriers are described below.
Making Polypeptides
[0148] Recombinant methods for producing and isolating polypeptides
of the invention are described herein. In addition to recombinant
production, the polypeptides may be produced by direct peptide
synthesis using solid-phase techniques (see, e.g., Stewart et al.
(1969) Solid-Phase Peptide Synthesis, WH Freeman Co, San Francisco;
Merrifield J. (1963) J Am Chem Soc 85:2149-2154). Peptide synthesis
may be performed using manual techniques or by automation.
Automated synthesis may be achieved, for example, using Applied
Biosystems 431A Peptide Synthesizer (Perkin Elmer, Foster City,
Calif.) in accordance with the instructions provided by the
manufacturer. For example, subsequences may be chemically
synthesized separately and combined using chemical methods to
provide full-length polypeptides or fragments thereof.
Alternatively, such sequences may be ordered from any number of
companies which specialize in production of polypeptides. Most
commonly, polypeptides of the invention may be produced by
expressing coding nucleic acids and recovering polypeptides, e.g.,
as described below.
[0149] Methods for producing the polypeptides of the invention are
also included. One such method comprises introducing into a
population of cells any nucleic acid of the invention described
herein, which is operatively linked to a regulatory sequence
effective to produce the encoded polypeptide, culturing the cells
in a culture medium to express the polypeptide, and isolating the
polypeptide from the cells or from the culture medium. An amount of
nucleic acid sufficient to facilitate uptake by the cells
(transfection) and/or expression of the polypeptide is utilized.
The nucleic acid is introduced into such cells by any delivery
method described herein, including, e.g., injection, gene gun,
passive uptake, etc. The nucleic acid may be part of a vector, such
as a recombinant expression vector, including a DNA plasmid vector,
or any vector described herein. The nucleic acid or vector
comprising a nucleic acid of the invention may be prepared and
formulated as described herein, above. Such a nucleic acid or
expression vector may be introduced into a population of cells of a
mammal in vivo, or selected cells of the mammal (e.g., tumor cells)
may be removed from the mammal and the nucleic acid expression
vector introduced ex vivo into the population of such cells in an
amount sufficient such that uptake and expression of the encoded
polypeptide results. Or, a nucleic acid or vector comprising a
nucleic acid of the invention is produced using cultured cells in
vitro. In one aspect, the method of producing a polypeptide of the
invention comprises introducing into a population of cells a
recombinant expression vector comprising any nucleic acid of the
invention described herein in an amount and formula such that
uptake of the vector and expression of the encoded polypeptide will
result; administering the expression vector into a mammal by any
introduction/delivery format described herein; and isolating the
polypeptide from the mammal or from a byproduct of the mammal.
Antibodies
[0150] In another aspect of the invention, a polypeptide of the
invention (or an antigenic fragment thereof) is used to produce
antibodies which have, e.g., diagnostic, therapeutic, or
prophylactic uses, e.g., related to the activity, distribution, and
expression of polypeptides and fragments thereof. Antibodies to
polypeptides of the invention may be generated by methods well
known in the art. Such antibodies may include, but are not limited
to, polyclonal, monoclonal, chimeric, humanized, single chain, Fab
fragments and fragments produced by a Fab expression library.
Antibodies, e.g., those that block receptor binding, are especially
preferred for therapeutic and/or prophylactic use.
[0151] Polypeptides for antibody induction do not require
biological activity; however, the polypeptides or peptides should
be antigenic. Peptides used to induce specific antibodies may have
an amino acid sequence consisting of at least about 10 amino acids,
preferably at least about 15 or 20 amino acids or at least about 25
or 30 amino acids. Short stretches of a polypeptide may be fused
with another protein, such as keyhole limpet hemocyanin, and
antibody produced against the chimeric molecule.
[0152] Methods of producing polyclonal and monoclonal antibodies
are known to those of skill in the art, and many antibodies are
available. See, e.g., Current Protocols in Immunology, John
Colligan et al., eds., Vols. I-IV (John Wiley & Sons, Inc., NY,
1991 and 2001 Supplement); and Harlow and Lane (1989) Antibodies: A
Laboratory Manual Cold Spring Harbor Press, NY; Stites et al.
(eds.) Basic and Clinical Immunology (4th ed.) Lange Medical
Publications, Los Altos, Calif., and references cited therein; and
Goding (1986) Monoclonal Antibodies: Principles and Practice (2d
ed.) Academic Press, New York, N.Y.; and Kohler and Milstein (1975)
Nature 256:495-497. Other suitable techniques for antibody
preparation include selection of libraries of recombinant
antibodies in phage or similar vectors. See, Huse et al. (1989)
Science 246:1275-1281; and Ward et al. (1989) Nature 341:544-546.
Specific monoclonal and polyclonal antibodies and antisera will
usually bind with a K.sub.D of at least about 0.1 .mu.M, preferably
at least about 0.01 .mu.M or better, and most typically and
preferably, 0.001 .mu.M or better.
[0153] Detailed methods for preparation of chimeric (humanized)
antibodies can be found in U.S. Pat. No. 5,482,856. Additional
details on humanization and other antibody production and
engineering techniques can be found in Borrebaeck (ed.) (1995)
Antibody Engineering, 2.sup.nd Edition Freeman and Company, NY
(Borrebaeck); McCafferty et al. (1996) Antibody Engineering, A
Practical Approach IRL at Oxford Press, Oxford, England
(McCafferty), and Paul (1995) Antibody Engineering Protocols Humana
Press, Towata, N.J. (Paul).
[0154] In one aspect, this invention provides for fully humanized
antibodies against the polypeptides of the invention or fragments
thereof. Humanized antibodies are especially desirable in
applications where the antibodies are used as therapeutics and/or
prophylactics in vivo in human patients. Human antibodies consist
of characteristically human immunoglobulin sequences. The human
antibodies of this invention can be produced in using a wide
variety of methods (see, e.g., Larrick et al., U.S. Pat. No.
5,001,065, and Borrebaeck McCafferty and Paul, supra, for a
review). In one aspect, the human antibodies of the present
invention are produced initially in trioma cells. Genes encoding
the antibodies are then cloned and expressed in other cells, such
as nonhuman mammalian cells. The general approach for producing
human antibodies by trioma technology is described by Ostberg et
al. (1983), Hybridoma 2:361-367, Ostberg, U.S. Pat. No. 4,634,664,
and Engelman et al., U.S. Pat. No. 4,634,666. The
antibody-producing cell lines obtained by this method are called
triomas because they are descended from three cells--two human and
one mouse. Triomas have been found to produce antibody more stably
than ordinary hybridomas made from human cells.
[0155] Other uses contemplated for polypeptides of the invention
are provided throughout the specification.
Interferon-Alpha Conjugates
[0156] In another aspect, the invention relates to a conjugate
comprising a polypeptide exhibiting an interferon-alpha activity
which comprises an amino acid sequence of any one of SEQ ID NOs:
1-15 and SEQ ID NOs:44-104, and at least one non-polypeptide moiety
attached to the polypeptide, such as e.g., 1-6, 1-5, 1-4, 1-3, e.g.
1 or 2 non-polypeptide moieties attached to the polypeptide. It
will be understood that the conjugate also exhibits an
interferon-alpha activity (such as, antiviral activity, T.sub.H1
differentiation activity, and/or antiproliferative activity).
[0157] In another aspect, the invention relates to a conjugate
comprising a polypeptide exhibiting an interferon-alpha activity,
which polypeptide comprises an amino acid sequence that differs
from the amino acid sequence of any one of SEQ ID NOs:1-15 and SEQ
ID NOs:44-104 (such as, one of SEQ ID NOs:1-15, 47, or 53), in at
least one amino acid residue selected from an introduced or removed
amino acid residue comprising an attachment group for a
non-polypeptide moiety. Examples of amino acid residues to be
introduced and/or removed according to this aspect are described in
further detail in the following sections. It will be understood
that the conjugate itself also exhibits an interferon-alpha
activity.
[0158] In another aspect the conjugate comprises an amino acid
sequence which differs from the amino acid sequence of any of SEQ
ID NOs:1-15 and SEQ ID NOs:44-104 (such as, one of SEQ ID NOs:1-15,
47, or 53) in 0-16 amino acid positions (such as in 0, 1, 2, 3, 4,
5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, or 16 amino acid positions),
e.g. in 0-14 amino acid positions, in 0-12 amino acid positions, in
0-10 amino acid positions, in 0-8 amino acid positions, in 0-6
amino acid positions, in 0-5 amino acid positions, in 0-4 amino
acid positions, in 0-3 amino acid positions, in 0-2 amino acid
positions, or in 0-1 amino acid positions. In one aspect of the
invention, the amino acid residue comprising an attachment group
for the non-polypeptide moiety is introduced (e.g., by substitution
of an amino acid residue for a different residue which comprises an
attachment group for the non-polypeptide moiety, or by insertion of
an additional amino acid residue which comprises an attachment
group for the non-polypeptide moiety).
[0159] The term "conjugate" (or interchangeably "polypeptide
conjugate" or "conjugated polypeptide") is intended to indicate a
heterogeneous (in the sense of composite) molecule formed by the
covalent attachment of one or more polypeptides of the invention to
one or more non-polypeptide moieties. The term "covalent
attachment" means that the polypeptide and the non-polypeptide
moiety are either directly covalently joined to one another, or
else are indirectly covalently joined to one another through an
intervening moiety or moieties, such as a bridge, spacer, or
linkage moiety or moieties. Preferably, a conjugated polypeptide is
soluble at relevant concentrations and conditions, i.e. soluble in
physiological fluids such as blood. Examples of conjugated
polypeptides of the invention include glycosylated and/or PEGylated
polypeptides. The term "non-conjugated polypeptide" may be used to
refer to the polypeptide part of the conjugated polypeptide.
[0160] The term "non-polypeptide moiety" is intended to mean a
molecule that is capable of conjugating to an attachment group of
the polypeptide. Preferred examples of non-polypeptide moieties
include polymer molecules, sugar moieties, lipophilic compounds, or
organic derivatizing agents, in particular polymer molecules or
sugar moieties. It will be understood that the non-polypeptide
moiety is linked to the polypeptide through an attachment group of
the polypeptide. Except where the number of non-polypeptide
moieties, such as polymer molecule(s), attached to the polypeptide
is expressly indicated, every reference to "a non-polypeptide
moiety" attached to the polypeptide or otherwise used in the
present invention shall be a reference to one or more
non-polypeptide moieties attached to the polypeptide.
[0161] The term "polymer molecule" is defined as a molecule formed
by covalent linkage of two or more monomers, wherein none of the
monomers is an amino acid residue. The term "polymer" may be used
interchangeably with the term "polymer molecule".
[0162] The term "sugar moiety" is intended to indicate a
carbohydrate molecule attached by in vivo or in vitro
glycosylation, such as N- or O-glycosylation.
[0163] An "N-glycosylation site" has the sequence N-X-S/T/C,
wherein X is any amino acid residue except proline, N is asparagine
and S/T/C is either serine, threonine or cysteine, preferably
serine or threonine, and most preferably threonine.
[0164] An "O-glycosylation site" comprises the OH-group of a serine
or threonine residue.
[0165] The term "attachment group" is intended to indicate an amino
acid residue group capable of coupling to the relevant
non-polypeptide moiety such as a polymer molecule or a sugar
moiety. Non-limiting examples of useful attachment groups and some
corresponding non-polypeptide moieties are provided in Table 4
below. TABLE-US-00004 TABLE 4 Useful attachment groups and examples
of corresponding non-polypeptide moieties Conjugation Attachment
Examples of non- method/activated group Amino acid polypeptide
moiety PEG Reference --NH.sub.2 N-terminus, Lys Polymer, e.g. PEG
mPEG-SPA Nektar Inc. 2003 mPEG2-NHS Catalog mPEG2- butryALD --COOH
C-terminus, Asp, Polymer, e.g. PEG mPEG-Hz Nektar Inc. 2003 Glu
Catalog Sugar moiety In vitro coupling --SH Cys Polymer, e.g. PEG,
mPEG-VS Nektar Inc. 2003 mPEG2-MAL Catalog; Delgado et al, Sugar
moiety In vitro coupling Critical Reviews in Therapeutic Drug
Carrier Systems 9(3, 4): 249-304 (1992) --OH Ser, Thr, OH--, Lys
Sugar moiety In vivo O-linked glycosylation --CONH.sub.2 Asn as
part of an Sugar moiety In vivo N-glycosylation glycosylation site
Aromatic Phe, Tyr, Trp Sugar moiety In vitro coupling residue
--CONH.sub.2 Gln Sugar moiety In vitro coupling Yan and Wold,
Biochemistry, 1984, Jul 31; 23(16): 3759-65 Aldehyde Oxidized
Polymer, e.g. PEG, PEGylation Andresz et al., Ketone carbohydrate
PEG-hydrazide 1978, Makromol. Chem. 179: 301; WO 92/16555, WO
00/23114 Guanidino Arg Sugar moiety In vitro coupling Lundblad and
Noyes, Chemical Reagents for Protein Modification, CRC Press Inc.
Boca Raton, FI Imidazole ring His Sugar moiety In vitro coupling As
for guanidine
[0166] For in vivo N-glycosylation, the term "attachment group" is
used in an unconventional way to indicate the amino acid residues
constituting an N-glycosylation site (with the sequence N-X-S/T/C,
wherein X is any amino acid residue except proline, N is asparagine
and S/T/C is either serine, threonine or cysteine, preferably
serine or threonine, and most preferably threonine). Although the
asparagine residue of the N-glycosylation site is the one to which
the sugar moiety is attached during glycosylation, such attachment
cannot be achieved unless the other amino acid residues of the
N-glycosylation site is present. Accordingly, when the
non-polypeptide moiety is a sugar moiety and the conjugation is to
be achieved by N-glycosylation, the term "amino acid residue
comprising an attachment group for the non-polypeptide moiety" as
used in connection with alterations of the amino acid sequence of
the polypeptide of the invention is to be understood as one, two or
all of the amino acid residues constituting an N-glycosylation site
is/are to be altered in such a manner that either a functional
N-glycosylation site is introduced into the amino acid sequence,
removed from said sequence or a functional N-glycosylation site is
retained in the amino acid sequence (e.g. by substituting a serine
residue, which already constitutes part of an N-glycosylation site,
with a threonine residue and vice versa).
[0167] The term "introduce" (i.e., an "introduced" amino acid
residue, "introduction" of an amino acid residue) is primarily
intended to mean substitution of an existing amino acid residue for
another amino acid residue, but may also mean insertion of an
additional amino acid residue.
[0168] The term "remove" (i.e., a "removed" amino acid residue,
"removal" of an amino acid residue) is primarily intended to mean
substitution of the amino acid residue to be removed for another
amino acid residue, but may also mean deletion (without
substitution) of the amino acid residue to be removed.
[0169] The term "amino acid residue comprising an attachment group
for the non-polypeptide moiety" is intended to indicate that the
amino acid residue is one to which the non-polypeptide moiety binds
(in the case of an introduced amino acid residue) or would have
bound (in the case of a removed amino acid residue).
[0170] The term "functional in vivo half-life" is used in its
normal meaning, i.e. the time at which 50% of the biological
activity of the polypeptide is still present in the body/target
organ, or the time at which the activity of the polypeptide is 50%
of the initial value. The functional in vivo half-life may be
determined in an experimental animal, such as rat, mice, rabbit,
dog or monkey. Preferably, the functional in vivo half half-life is
determined in a non-human primate, such as a monkey. Furthermore,
the functional in vivo half-life may be determined for a sample
that has been administered intravenously or subcutaneously.
[0171] As an alternative to determining functional in vivo
half-life, "serum half-life" may be determined, i.e. the time at
which 50% of the polypeptide circulates in the plasma or
bloodstream prior to being cleared. Determination of serum
half-life is often more simple than determining the functional in
vivo half-life and the magnitude of serum half-life is usually a
good indication of the magnitude of functional in vivo half-life.
Alternatively terms to serum half-life include "plasma half-life",
"circulating half-life", "serum clearance", "plasma clearance" and
"clearance half-life". The serum half-life may be determined as
described above in connection with determination of functional in
vivo half-life.
[0172] The term "serum" is used in its normal meaning, i.e. as
blood plasma without fibrinogen and other clotting factors.
[0173] The term "increased" as used about the functional in vivo
half-life or serum half-life is used to indicate that the relevant
half-life of the conjugate of the invention is statistically
significantly increased relative to that of a reference molecule,
such as a wild-type interferon-alpha, e.g., a human
interferon-alpha, such as one of SEQ ID NO:31-SEQ ID NO:42 (or
other huIFN-alpha sequences as described herein and/or in Allen G.
and Diaz M. O. (1996), supra), or the corresponding non-conjugated
polypeptide. Thus, interesting conjugates of the invention include
those which have an increased functional in vivo half-life or an
increased serum half-life as compared to a reference molecule
mentioned above.
[0174] The term "AUC.sub.sc" or "Area Under the Curve when
administered subcutaneously" is used in its normal meaning, i.e. as
the area under the interferon-alpha-activity-in-serum vs. time
curve, where the conjugated molecule has been administered
subcutaneously to an experimental animal. Once the experimental
interferon-alpha activity time points have been determined, the
AUC.sub.sc may conveniently be calculated by a computer program,
such as GraphPad Prism 3.01.
[0175] The term "increased" as used about the AUC.sub.sc is used to
indicate that the Area Under the Curve for a conjugate of the
invention, when administered subcutaneously, is statistically
significantly increased relative to that of a reference molecule,
such as wild-type interferon-alpha, e.g., a human interferon-alpha,
such as one of SEQ ID NO:31-SEQ ID NO:42 (or other huIFN-alpha
sequences as described herein and/or in Allen G. and Diaz M. O.
(1996), supra), or the corresponding non-conjugated polypeptide,
when determined under comparable conditions. Evidently, the same
amount of interferon-alpha activity should be administered for the
conjugate of the invention and the reference molecule.
Consequently, in order to make direct comparisons between different
interferon-alpha molecules, the AUC.sub.sc values should typically
be normalized, i.e. be expressed as AUC.sub.sc dose
administered.
[0176] The term "T.sub.max,sc" is used about the time point in the
interferon-alpha-activity-in-serum vs. time curve where the highest
interferon-alpha activity in serum is observed.
[0177] It will be understood that while the examples and
modifications to the parent polypeptide are generally provided
herein in regards to the sequence SEQ ID NO:1, the disclosed
modifications may also be made in equivalent amino acid positions
of any of the other polypeptides of the invention (including SEQ ID
NOs:2-15 and 44-104 and variants thereof) described above.
[0178] By removing and/or introducing amino acid residues
comprising an attachment group for the non-polypeptide moiety it is
possible to specifically adapt the polypeptide so as to make the
molecule more susceptible to conjugation to the non-polypeptide
moiety of choice, to optimize the conjugation pattern (e.g. to
ensure an optimal distribution of non-polypeptide moieties on the
surface of the interferon-alpha molecule and thereby, e.g.,
effectively shield epitopes and other surface parts of the
polypeptide without significantly impairing the function thereof).
For instance, by introduction of attachment groups, the
interferon-alpha polypeptide is altered in the content of the
specific amino acid residues to which the relevant non-polypeptide
moiety binds, whereby a more efficient, specific and/or extensive
conjugation is achieved. By removal of one or more attachment
groups it is possible to avoid conjugation to the non-polypeptide
moiety in parts of the polypeptide in which such conjugation is
disadvantageous, e.g. to an amino acid residue located at or near a
functional site of the polypeptide (since conjugation at such a
site may result in inactivation or reduced interferon- alpha
activity of the resulting conjugate due to impaired receptor
recognition). Further, it may be advantageous to remove an
attachment group located close to another attachment group.
[0179] It will be understood that the amino acid residue comprising
an attachment group for a non-polypeptide moiety, whether it be
removed or introduced, is selected on the basis of the nature of
the non-polypeptide moiety and, in some instances, on the basis of
the conjugation method to be used. For instance, when the
non-polypeptide moiety is a polymer molecule, such as a
polyethylene glycol or polyalkylene oxide derived molecule, amino
acid residues capable of functioning as an attachment group may be
selected from the group consisting of cysteine, lysine (and/or the
N-terminal amino group of the polypeptide), aspartic acid, glutamic
acid, histidine and arginine. When the non-polypeptide moiety is a
sugar moiety, the attachment group is an in vivo or in vitro N- or
O-glycosylation site, preferably an N-glycosylation site.
[0180] In some instances, when an attachment group for a
non-polypeptide moiety is to be introduced into or removed from the
interferon-alpha polypeptide, the position of the interferon-alpha
polypeptide to be modified may be conveniently selected as
follows:
[0181] The position to be modified may be located at the surface of
the interferon-alpha polypeptide, such as a position occupied by an
amino acid residue which has more than 25% of its side chain
exposed to the solvent, such as more than 50% of its side chain
exposed to the solvent. Such positions have been identified on the
basis of an analysis of a 3D structure of the human
interferon-alpha 2a molecule as described in the "Materials and
Methods" section herein.
[0182] Alternatively or additionally, the position to be modified
may be identified on the basis of an analysis of an
interferon-alpha protein sequence family (such as shown in the
alignments depicted in FIGS. 2 and 4). For the purposes of the
following example, SEQ ID NO:1 as shown in the top line of the
alignment of FIG. 2 may be considered the parent interferon-alpha
to be modified, and the human interferon-alpha sequences in the
rest of the alignment are considered the other members of the
family. For example, the position to be modified in the parent
sequence may be one which, in one or more members of the family
other than the parent interferon-alpha, is (a) occupied by an amino
acid residue comprising the relevant attachment group (when such
amino acid residue is to be introduced into the parent sequence) or
(b) which in the parent interferon- alpha, but not in one or more
other members of the family, is occupied by an amino acid residue
comprising the relevant attachment group (when such amino acid
residue is to be removed from the parent sequence).
[0183] In order to determine an optimal distribution of attachment
groups, the distance between amino acid residues located at the
surface of the interferon-alpha molecule was calculated on the
basis of a 3D structure of an interferon-alpha polypeptide. More
specifically, the distance between the CB's of the amino acid
residues comprising such attachment groups, or the distance between
the functional group (NZ for lysine, CG for aspartic acid, CD for
glutamic acid, SG for cysteine) of one and the CB of another amino
acid residue comprising an attachment group were determined. In
case of glycine, CA was used instead of CB. In the
interferon-.alpha. polypeptide part of a conjugate of the
invention, any of said distances may be more than 8 .ANG., such as
more than 10 .ANG. in order to avoid or reduce heterogeneous
conjugation and to provide a uniform distribution of attachment
groups, e.g. with the aim of epitope shielding. Furthermore, in the
interferon-alpha polypeptide part of a conjugate of the invention,
in some instances attachment groups located at or near the receptor
binding sites of interferon-alpha are removed, such as by
substitution of the amino acid residue comprising such group. In
some instances, amino acid residues comprising an attachment group
for a non-polypeptide moiety, such as cysteine or lysine, are often
not introduced at or near the receptor binding site of the
interferon alpha molecule.
[0184] Another approach for modifying an interferon-alpha
polypeptide is to shield and thereby modify or destroy or otherwise
inactivate an epitope present in the parent interferon-alpha, by
conjugation to a non-polypeptide moiety. Epitopes of
interferon-alpha polypeptides may be identified by use of methods
known in the art, also known as epitope mapping, see e.g. Romagnoli
et al., J. Biol. Chem., 1999, 380(5):553-9, DeLisser H M, Methods
Mol Biol, 1999, 96:11-20, Van de Water et al., Clin Immunol
Immunopathol, 1997, 85(3):229-35, Saint-Remy J M, Toxicology, 1997,
119(1):77-81, and Lane D P and Stephen C W, Curr Opin Immunol,
1993, 5(2):268-71. One method is to establish a phage display
library expressing random oligopeptides of, e.g., 9 amino acid
residues. IgGl antibodies from specific antisera towards human
interferon-alpha are purified by immunoprecipitation and the
reactive phages are identified by immunoblotting. By sequencing the
DNA of the purified reactive phages, the sequence of the
oligopeptide can be determined followed by localization of the
sequence on the 3D-structure of the interferon-alpha.
Alternatively, epitopes can be identified according to the method
described in U.S. Pat. No. 5,041,376. The thereby identified region
on the structure constitutes an epitope that then can be selected
as a target region for introduction of an attachment group for the
non-polypeptide moiety. Preferably, at least one epitope, such as
two, three or four epitopes of interferon-alpha are shielded by a
non-polypeptide moiety according to the present invention.
Accordingly, in one aspect, the conjugate of the invention has at
least one shielded epitope as compared to a wild type human
interferon-alpha, including any commercially available interferon-
alpha. This may be done by introduction of an attachment group for
a non-polypeptide moiety into a position located in the vicinity of
(i.e. within 4 amino acid residues in the primary sequence or
within about 10 .ANG. in the tertiary sequence) of a given epitope.
The 10 .ANG. distance is measured between CB's (CA's in case of
glycine). Such specific introductions are described in the
following sections.
[0185] In case of removal of an attachment group, the relevant
amino acid residue comprising such group and occupying a position
as defined above may be substituted with a different amino acid
residue that does not comprise an attachment group for the
non-polypeptide moiety in question, or may be deleted. Removal of
an N-glycosylation group, may also be accomplished by insertion or
removal of an amino acid reside within the motif N-X-S/T/C.
[0186] In case of introduction of an attachment group, an amino
acid residue comprising such group is introduced into the position,
such as by substitution of the amino acid residue occupying such
position.
[0187] The exact number of attachment groups available for
conjugation and present in the interferon-alpha polypeptide is
dependent on the effect desired to be achieved by conjugation. The
effect to be obtained is, e.g., dependent on the nature and degree
of conjugation (e.g. the identity of the non-polypeptide moiety,
the number of non-polypeptide moieties desirable or possible to
conjugate to the polypeptide, where they should be conjugated or
where conjugation should be avoided, etc.). For instance, if
reduced immunogenicity is desired, the number (and location of)
attachment groups should be sufficient to shield most or all
epitopes. This is normally obtained when a greater proportion of
the interferon-alpha polypeptide is shielded. Effective shielding
of epitopes is normally achieved when the total number of
attachment groups available for conjugation is in the range of 1-6
attachment groups, e.g., 1-5, such as in the range of 1-3, such as
1, 2, or 3 attachment groups.
[0188] Functional in vivo half-life is i.a. dependent on the
molecular weight of the conjugate, and the number of attachment
groups needed for providing increased half-life thus depends on the
molecular weight of the non-polypeptide moiety in question. Some
such conjugates comprise 1-6, e.g., 1-5, such as 1-3, e.g. 1, 2, or
3 non-polypeptide moieties each having a MW of about 2-40 kDa, such
as about 2 kDa, about 5 kDa, about 12 kDa, about 15 kDa, about 20
kDa, about 30 kDa, or about 40 kDa.
[0189] In the conjugate of the invention, some, most, or
substantially all conjugatable attachment groups are occupied by
the relevant non-polypeptide moiety.
[0190] The conjugate of the invention may exhibit one or more of
the following improved properties:
[0191] For example, the conjugate may exhibit a reduced
immunogenicity as compared to a human interferon-alpha (such as any
of the polypeptides defined herein as SEQ ID NO:31-42, SEQ ID
NO:32+R23K, or any other huIFN-alpha described herein and/or in
Allen G. and Diaz M. O. (1996), supra) or as compared to the
corresponding non-conjugated polypeptide, e.g. a reduction of at
least 10%, such as a reduction of at least of 25%, such as a
reduction of at least of 50%, e.g. a reduction of at least 75%
compared to the non-conjugated polypeptide or compared to a human
interferon-alpha.
[0192] In another aspect the conjugate may exhibit a reduced
reaction or no reaction with neutralizing antibodies from patients
treated with a human interferon-alpha (such as any of the
polypeptides defined herein as SEQ ID NO:31-42, SEQ ID NO:32+R23K,
or any other huIFN-alpha described herein and/or in Allen G. and
Diaz M. O. (1996), supra) or as compared to the corresponding
non-conjugated polypeptide, e.g. a reduction of neutralisation of
at least 10%, such as at least of 25%, such as of at least 50%,
e.g., at least 75%.
[0193] In another aspect of the invention the conjugate may exhibit
an increased functional in vivo half-life and/or increased serum
half-life as compared to a reference molecule such as a human
interferon-alpha (e.g. any of the polypeptides defined herein as
SEQ ID NO:31-42, SEQ ID NO:32+R23K, or any other huIFN-alpha
described herein and/or in Allen G. and Diaz M. O. (1996), supra)
or as compared to the corresponding non-conjugated polypeptide.
Particular preferred conjugates are such conjugates where the ratio
between the functional in vivo half-life (or serum half-life) of
said conjugate and the functional in vivo half-life (or serum
half-life) of said reference molecule is at least 1.25, such as at
least 1.50, such as at least 1.75, such as at least 2, such as at
least 3, such as at least 4, such as at least 5, such as at least
6, such as at least 7, such as at least 8. As mentioned above, the
half-life is conveniently determined in an experimental animal,
such as rat or monkey, and may be based on intravenously or
subcutaneously administration.
[0194] In a further aspect the conjugate may exhibit an increased
bioavailability as compared to a reference molecule such as a human
interferon-alpha (e.g. any of the polypeptides defined herein as
SEQ ID NO:31-42, SEQ ID NO:32+R23K, or any other huIFN-alpha
described herein and/or in Allen G. and Diaz M. O. (1996), supra)
or the corresponding non-conjugated polypeptide. For example, the
conjugate may exhibit an increased AUC.sub.sc as compared to a
reference molecule such as a human interferon-alpha or the
corresponding non-conjugated polypeptide. Thus, exemplary
conjugates are such conjugates where the ratio between the
AUC.sub.sc of said conjugate and the AUC.sub.sc of said reference
molecule is at least 1.25, such as at least 1.5, such as at least
2, such as at least 3, such as at least 4, such as at least 5 or at
least 6, such as at least 7, such as at least 8, such as at least 9
or at least 10, such as at least 12, such as at least 14, e.g. at
least 16, at least 18 or at least 20 when administered
subcutaneously, in particular when administered subcutaneously in
an experimental animal such as rat or monkey. Analogously, some
conjugates of the invention are such conjugates wherein the ratio
between T.sub.max for said conjugate and T.sub.max for said
reference molecule, such as a human interferon-alpha or the
corresponding non-conjugated polypeptide, is at least 1.2, such as
at least 1.4, e.g. at least 1.6, such as at least 1.8, such as at
least 2, e.g. at least 2.5, such as at least 3, such as at least 4,
e.g. at least 5, such as at least 6, such as at least 7, e.g. at
least 8, such as at least 9, such as at least 10, when administered
subcutaneously, in particular when administered subcutaneously in
an experimental animal such as rat or monkey.
[0195] In some instances, the magnitude of the antiviral activity
of a conjugate of the invention may be reduced (e.g. by at least
about 75%, at least about 50%, at least about 25%, at least about
10%) or increased (e.g. by at least about 10%) or is about equal
(e.g. within about +/-10% or about +/-5%) to that of a human
interferon-alpha (e.g. any of the polypeptides identified herein as
SEQ ID NO:31-42, SEQ ID NO:32+R23K, or any other huIFN-alpha
described herein and/or in Allen G. and Diaz M. O. (1996), supra)
or to that of the corresponding non-conjugated polypeptide. In some
instances the degree of antiviral activity as compared to
antiproliferative activity of a conjugate of the invention may
vary, and thus be higher, lower or about equal to that of a human
interferon-alpha or to that of the corresponding non-conjugated
polypeptide.
Conjugate of the Invention where the Non-Polypeptide Moiety Binds
to a Cysteine Residue
[0196] In another aspect, the invention relates to a conjugate
exhibiting an interferon-alpha activity and comprising at least one
non-polypeptide moiety conjugated to at least one cysteine residue
of an interferon-alpha, the amino acid sequence of which differs in
0-16 amino acid positions from that of a parent interferon-alpha
polypeptide, such as an interferon-alpha polypeptide comprising the
amino acid sequence of any of SEQ ID NOs:1-15 and 44-104 (such as,
one of SEQ ID NO:1, SEQ ID NO:3, SEQ ID NO:8, SEQ ID NO:12, SEQ ID
NO:47 or SEQ ID NO:53), in that at least one cysteine residue has
been introduced, such as by substitution or insertion, into a
position that is occupied in the parent interferon-alpha by an
amino acid residue that is exposed to the surface of the molecule,
preferably one that has at least 25%, such as at least 50% of its
side chain exposed to the surface. Typically, the conjugate
comprises an amino acid sequence which differs from the amino acid
sequence of any one of, e.g., SEQ ID NOs:1-15, 47 or 53, in 1-16
amino acid positions (such as in 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11,
12, 13, 14, 15, or 16 amino acid positions), e.g. in 1-14 amino
acid positions, in 1-12 amino acid positions, in 1-10 amino acid
positions, in 1-8 amino acid positions, in 1-6 amino acid
positions, in 1-5 amino acid positions, in 1-4 amino acid
positions, in 1-3 amino acid positions or in 1-2 amino acid
positions.
[0197] Some conjugates of the invention comprise a polypeptide
sequence comprising one or more of the following substitutions,
relative to SEQ ID NO:1, which introduces a cysteine residue into a
position which is predicted to be exposed at the surface of the
molecule with more than a 25% fractional ASA: D2C, L3C, P4C, Q5C,
T6C, H.sub.7C, S8C, L9C, G1.degree. C., R12C, R13C, M16C, A19C,
Q20C, R22C, R23C, 124C, S25C, L26C, F27C, S28C, L30C, K31C, R33C,
H.sub.34C, D35C, R37C, Q40C, E41C, E42C, D44C, N46C, H.sub.47C,
Q49C, K50C, V51C, Q52C, E59C, Q62C, Q63C, N66C, S69C, T70C, K71C,
N72C, S74C, A75C, D78C, E79C, T80C, L81C, E83C, K84C, 187C, F90C,
Q91C, N94C, D95C, E97C, A98C, V100C, M101C, Q102C, E103C, V104C,
G105C, E107C, E108C, T109C, P10C, L10C, M112C, N113C, V114C, D115C,
L118C, R121C, K122C, Q125C, R126C, T128C, L129C, T132C, K133C,
K134C, K135C, Y136C, S137C, P138C, A146C, M149C, R150C, S153C,
F154C, N157C, Q159C, K160C, R161C, L162C, R163C, R164C, K165C and
E166C, said amino acid residue positions relative to SEQ ID NO:1.
In some instances, among the above-mentioned positions, one or more
of the amino acid residues at positions 47, 51 and 133 are not
substituted with cysteine.
[0198] For example, some such conjugates of the invention comprise
a polypeptide sequence comprising one or more of the following
substitutions, relative to SEQ ID NO:1, which introduces a cysteine
residue into a position which is predicted to be exposed at the
surface of the molecule with more than a 50% fractional ASA: D2C,
L3C, P4C, Q5C, T6C, H.sub.7C, S8C, L9C, R12C, R13C, M16C, A19C,
S25C, F27C, S28C, K31C, R33C, H.sub.34C, D35C, R37C, E41C, D44C,
N46C, H.sub.47C, Q49C, K50C, N66C, K71C, A75C, D78C, E79C, T80C,
E83C, K84C, 187C, F90C, Q91C, N94C, D95C, M101C, Q102C, E103C,
G105C, E107C, E108C, T109C, P10C, L10C, V114C, D115C, L118C, R121C,
K122C, Q125C, R126C, L129C, T132C, K133C, K135C, P138C, R150C,
K160C, L162C, R163C, R164C, K165C and E166C, said amino acid
residue positions relative to SEQ ID NO:1. In some instances, one
or both of the amino acid residues at positions 47 and 133 are not
are not substituted with cysteine.
[0199] As indicated above, in some instances it may be preferable
to introduce cysteine residues outside of potential receptor
binding sites of interferon-alpha, i.e., outside of about positions
29-40, 79-96, and 124-141, position numbering relative to SEQ ID
NO:1. Thus, in some instances the one or more cysteine
substitutions are selected from the group consisting of D2C, L3C,
P4C, Q5C, T6C, H.sub.7C, S8C, L9C, G10C, R12C, R13C, M16C, A19C,
Q20C, R22C, R23C, 124C, S25C, L26C, F27C, S28C, E41C, E42C, D44C,
N46C, H.sub.47C, Q49C, K50C, V51C, Q52C, E59C, Q62C, Q63C, N66C,
S69C, T70C, K71C, N72C, S74C, A75C, E97C, A98C, V100C, M101C,
Q102C, E103C, V104C, G105C, E107C, E108C, T109C, P101C, L111C,
M112C, N113C, V114C, D115C, L118C, R121C, K122C, A146C, M149C,
R150C, S153C, F154C, N157C, Q159C, K160C, R161C, L162C, R163C,
R164C, K165C and E166C (positions which are predicted to be exposed
at the surface of the molecule with more than a 25% fractional ASA
and are not part of the putative receptor binding sites), relative
to SEQ ID NO:1. In some instances, one or both of positions 47 and
51 are not substituted with cysteine.
[0200] In other aspects the cysteine substitution is selected from
the group consisting of: D2C, L3C, P4C, Q5C, T6C, H.sub.7C, S8C,
L9C, R12C, R13C, M16C, A19C, S25C, F27C, S28C, E41C, D44C, N46C,
H.sub.47C, Q49C, K50C, N66C, K71C, A75C, M101C, Q102C, E103C,
G105C, E107C, E108C, T109C, P110C, L111C, V114C, D115C, L118C,
R121C, K122C, R150C, K160C, L162C, R163C, R164C, K165C and E166C
(positions which are predicted to be exposed at the surface of the
molecule with more than a 50% fractional ASA and are not part of
the putative receptor binding sites), relative to SEQ ID NO:1. In
some instances, position 47 is not substituted with a cysteine.
[0201] Some such conjugates of the invention comprise a polypeptide
sequence containing one or more of the substitutions S25C, S28C,
L30C, K31C, N46C, K71C, S74C, A75C, E79C, E107C, E108C, T132C,
K133C, P138C, and K135C, relative to SEQ ID NO:1.
[0202] In another aspect, the one or more cysteine residue is
introduced at or near the C-terminus either by substitution (for
example, Q159C, K160C, R161C, L162C, R163C, R164C, K165C or E166C,
relative to SEQ ID NO:1) or by insertion (for example, E166EC, also
referred to herein as 167C). It will be understood that cysteine
residues may also be introduced, either by substitution or by
insertion, in C-terminally truncated fragments of the
interferon-alpha molecules described herein.
[0203] In some instances, only a single cysteine residue is
introduced in order to avoid formation of disulfide bridges between
two or more introduced cysteine residues.
[0204] In interferon alphas, disulfide bonds are formed between
cysteines at positions 1/99 and 29/139. The disulfide bond 29/139
is essential for biological activity, while the 1/99 bond can be
reduced without significantly affecting biological activity
(Beilharz M. W. et al. (1986) J. Interferon Res. 6(6):677-685).
Thus, in another aspect of the invention one of C1 or C99 is
removed, preferably by substitution, e.g. C1S or C99S, thereby
leaving the other cysteine residue available for conjugation to a
non-polypeptide moiety.
[0205] Non-polypeptide moieties contemplated in this aspect of the
invention include polymer molecules, such as any of the molecules
mentioned in the section entitled "Conjugation to a polymer
molecule", such as PEG or mPEG. The conjugation between the
cysteine-containing polypeptide and the polymer molecule may be
achieved in any suitable manner, e.g. as described in the section
entitled "Conjugation to a polymer molecule", e.g. in using a one
step method or in the stepwise manner referred to in said section.
An exemplary method for PEGylating the interferon-alpha polypeptide
is to covalently attach PEG to cysteine residues using
cysteine-reactive PEGs. A number of highly specific,
cysteine-reactive PEGs with different groups (e.g.
orthopyridyl-disulfide (OPSS), maleimide (MAL) and vinylsulfone
(VS)) and different size linear or branched PEGs (e.g., 2-40 kDa,
such as 2 kDa, 5 kDa, 12 kDa, 15 kDa, 20 kDa, 30 kDa, or 40 kDa)
are commercially available, e.g. from Nektar Therapeutics Inc.,
Huntsville, Ala., USA, or SunBio, Anyang City, South Korea.
[0206] It is to be understood that while the examples of
modifications to the parent polypeptide are generally provided
herein relative to the sequence SEQ ID NO:1 (or relative to some
other specified sequence), the disclosed modifications may also be
made in equivalent amino acid positions of any of the other
polypeptides of the invention (including SEQ ID NOs:2-15 and SEQ ID
NOs:44-104 and variants thereof) described herein. Thus, as an
example, the substitution H.sub.47C relative to SEQ ID NO:1 is
understood to correspond to Q47C in SEQ ID NO:5, and so on.
Conjugate of the Invention where the Non-Polypeptide Moiety Binds
to a Lysine Residue
[0207] In another aspect, the invention relates to a conjugate
exhibiting an interferon-alpha activity and comprising at least one
non-polypeptide moiety conjugated to at least one lysine residue,
and/or to the N-terminal amino group, of an interferon-alpha
polypeptide comprising a sequence selected from SEQ ID NOs:1-15 and
44-104, or comprising a sequence which differs from any of SEQ ID
NOs: 1-15 and 44-104 (such as, one of SEQ ID NO:1, SEQ ID NO:3, SEQ
ID NO:8, SEQ ID NO:12, SEQ ID NO:47 or SEQ ID NO:53), in 0-16 amino
acid positions (such as in 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11,
12, 13, 14, 15, or 16 amino acid positions), e.g. in 0-14 amino
acid positions, in 0-12 amino acid positions, in 0-10 amino acid
positions, in 0-8 amino acid positions, in 0-6 amino acid
positions, in 0-5 amino acid positions, in 0-4 amino acid
positions, in 0-3 amino acid positions, in 0-2 amino acid positions
or in 0-1 amino acid position. Some conjugates according to this
aspect comprise at least one removed lysine residue and/or at least
one removed histidine residue, and/or at least one introduced
lysine residue.
[0208] Some conjugates of the invention comprise a polypeptide
sequence comprising a substitution of an amino acid residue for a
different amino acid residue, or a deletion of an amino acid
residue, which removes one or more lysines, e.g., K31, K50, K71,
K84, K122, K133, K134, K135, K160, and/or K165 (relative to SEQ ID
NO:1) from any polypeptide of the invention such as, for example,
one of SEQ ID NO:1, SEQ ID NO:3, SEQ ID NO:8, SEQ ID NO:12, SEQ ID
NO:47 or SEQ ID NO:53. The one or more lysine residue(s) to be
removed may be substituted with any other amino acid, may be
substituted with an Arg (R) or Gln (Q), or may be deleted. Some
such conjugates comprise the substitutions K31R+K122R; K31R+K133R;
K122R+K133R; or K31R+K122R+K133R. Other exemplary substitutions
include K71E; K84E; K133E/G; and K160E.
[0209] In instances where amine-reactive conjugation chemistries
are employed, it may be advantageous to avoid or to minimize the
potential for conjugation to histidine residues. Therefore, some
conjugates of the invention comprise a polypeptide sequence
comprising a substitution or a deletion which removes one or more
histidines, e.g., H7, H11, H34, and/or H47 (relative to SEQ ID
NO:1) from any polypeptide sequence of the invention such as, for
example, one of SEQ ID NO:1, SEQ ID NO:3, SEQ ID NO:8, SEQ ID
NO:12, SEQ ID NO:47 or SEQ ID NO:53. The one or more histidine
residue(s) to be removed may be substituted with any other amino
acid, may be substituted with an Arg (R) or Gln (Q), or may be
deleted. Some such conjugates comprise the substitutions H34Q;
H47Q; or H34Q+H47Q.
[0210] Alternatively, or in addition, some conjugates of the
invention comprise a polypeptide sequence comprising a modification
which introduces a lysine into a position that is occupied in the
parent sequence (e.g., one of SEQ ID NOs:1-15, 47, or 53) by an
amino acid residue that is exposed to the surface of the molecule,
e.g., one that has at least 25%, such as at least 50% of its side
chain exposed to the surface. Some such conjugates comprise a
polypeptide sequence comprising one or more of the following
substitutions, relative to SEQ ID NO:1, which introduces a lysine
residue into a position which is predicted to be exposed at the
surface of the molecule with more than a 25% fractional ASA: D2K,
L3K, P4K, Q5K, T6K, H7K, S8K, L9K, G10K, R12K, R13K, M16K, A19K,
Q20K, R22K, R23K, 124K, S25K, L26K, F27K, S28K, L30K, R33K, H34K,
D35K, R37K, Q40K, E41K, E42K, D44K, N46K, H47K, Q49K, V51K, Q52K,
E59K, Q62K, Q63K, N66K, S69K, T70K, N72K, S74K, A75K, D78K, E79K,
T80K, L81K, E83K, 187K, F90K, Q91K, N94K, D95K, E97K, A98K, V100K,
M101K, Q102K, E103K, V104K, G105K, E107K, E108K, T109K, P110K,
L111K, M112K, N113K, V114K, D115K, L118K, R121K, Q125K, R126K,
T128K, L129K, T132K, Y136K, S137K, P138K, A146K, M149K, R150K,
S153K, F154K, N157K, Q159K, R161K, L162K, R163K, R164K, and E166K,
said amino acid residue positions relative to SEQ ID NO:1. In some
instances, among the above-mentioned positions, one or more of the
amino acid residues at positions 47, 51, 52, and 154 are not
substituted with lysine.
[0211] Some such conjugates of the invention comprise a polypeptide
sequence comprising one or more of the following substitutions,
relative to SEQ ID NO:1, which introduces a lysine residue into a
position which is predicted to be exposed at the surface of the
molecule with more than a 50% fractional ASA: D2K, L3K, P4K, Q5K,
T6K, H7K, S8K, L9K, R12K, R13K, M16K, A19K, S25K, F27K, S28K, R33K,
H34K, D35K, R37K, E41K, D44K, N46K, H47K, Q49K, N66K, A75K, D78K,
E79K, T80K, E83K, 187K, F90K, Q91K, N94K, D95K, M101K, Q102K,
E103K, G105K, E107K, E108K, T109K, P110K, L111K, V114K, D115K,
L118K, R121K, Q125K, R126K, L129K, T132K, P138K, R150K, L162K,
R163K, R164K and E166K, said amino acid residue positions relative
to SEQ ID NO:1. In some instances, among the above-mentioned
positions, positions 47 is not substituted with lysine.
[0212] As indicated above, in some instances it may be preferable
to introduce lysine residues outside of potential receptor binding
sites of interferon-alpha, i.e., outside of about positions 29-40,
79-96, and 124-141, position numbering relative to SEQ ID NO:1.
Thus, in some instances the one or more lysine substitutions are
selected from the group consisting of D2K, L3K, P4K, Q5K, T6K, H7K,
S8K, L9K, G10K, R12K, R13K, M16K, A19K, Q20K, R22K, R23K, 124K,
S25K, L26K, F27K, S28K, E41K, E42K, D44K, N46K, H47K, Q49K, V51K,
Q52K, E59K, Q62K, Q63K, N66K, S69K, T70K, N72K, S74K, A75K, E97K,
A98K, V100K, M101K, Q102K, E103K, V104K, G105K, E107K, E108K,
T109K, P110K, L111K, M112K, N113K, V114K, D115K, L118K, R121K,
A146K, M149K, R150K, S153K, F154K, N157K, Q159K, R161K, L162K,
R163K, R164K, and E166K (residues having more than 25% of the side
chain exposed to the surface and not forming part of the putative
receptor binding sites), relative to SEQ ID NO:1. In some
instances, one or more of positions 47, 51, and 154 are not
substituted with lysine.
[0213] In some instances the one or more lysine substitutions are
selected from the group consisting of: D2K, L3K, P4K, Q5K, T6K,
H7K, S8K, L9K, R12K, R13K, M16K, A19K, S25K, F27K, S28K, E41K,
D44K, N46K, H47K, Q49K, N66K, A75K, M101K, Q102K, E103K, G105K,
E107K, E108K, T109K, P110K, L111K, Vi 14K, D115K, L118K, R121K,
R150K, L162K, R163K, R164K and E166K (residues having more than 50%
of the side chain exposed to the surface an not forming part of the
putative receptor binding sites), relative to SEQ ID NO:1. In some
instances, position 47 is not substituted with a lysine.
[0214] Non-polypeptide moieties contemplated for this aspect of the
invention include polymer molecules, such as any of the molecules
mentioned in the section entitled "Conjugation to a polymer
molecule", such as PEG or mPEG. The conjugation between the
lysine-containing polypeptide and the polymer molecule may be
achieved in any suitable manner, e.g. as described in the section
entitled "Conjugation to a polymer molecule", e.g. in using a one
step method or in the stepwise manner referred to in said section.
An exemplary method for PEGylating the interferon-alpha polypeptide
is to covalently attach PEG to lysine residues using
lysine-reactive PEGs. A number of highly specific, lysine-reactive
PEGs (such as for example, succinimidyl propionate (SPA),
succinimidyl butanoate (SBA), N-hydroxylsuccinimide (NHS), and
aldehyde (e.g., ButyrALD)) and different size linear or branched
PEGs (e.g., 2-40 kDa, such as 2 kDa, 5 kDa, 12 kDa, 15 kDa, 20 kDa,
30 kDa, or 40 kDa,) are commercially available, e.g. from Nektar
Therapeutics Inc., Huntsville, Ala., USA, or SunBio, Anyang City,
South Korea.
[0215] It is to be understood that while the examples of
modifications to the parent polypeptide are generally provided
herein relative to the sequence SEQ ID NO:1 (or relative to some
other specified sequence), the disclosed modifications may also be
made in equivalent amino acid positions of any of the other
polypeptides of the invention (including SEQ ID NOs:2-15 and SEQ ID
NOs:44-104 and variants thereof) described herein. Thus, as an
example, the substitution H47K relative to SEQ ID NO:1 is
understood to correspond to Q47K in SEQ ID NO:5, and so on.
Conjugate of the Invention where the Non-Polypeptide Moiety is a
Sugar Moiety
[0216] In another aspect, the invention relates to a conjugate
exhibiting interferon-alpha activity and comprising at least one
sugar moiety conjugated to an interferon-alpha polypeptide, the
amino acid sequence of which differs from that of a parent
interferon-alpha polypeptide, such as any one of SEQ ID NOs:1-15
and SEQ ID NOs:44-104 (such as one of SEQ ID NO:1, SEQ ID NO:3, SEQ
ID NO:8, SEQ ID NO:12, SEQ ID NO:47 or SEQ ID NO:53), 1-16 amino
acid positions, in that at least one glycosylation site, preferably
an in vivo N-glycosylation site, has been introduced, preferably by
substitution, into a position that in the parent interferon-alpha
polypeptide is occupied by an amino acid residue that is exposed to
the surface of the molecule, e.g. one that has at least 25%, such
as at least 50% of its side chain exposed to the surface.
Typically, the conjugate comprises an amino acid sequence which
differs from the amino acid sequence of any of, for example, SEQ ID
NOs:1-15, 47, or 53, in 1-16 amino acid positions (such as in 1, 2,
3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, or 16 amino acid
positions), e.g. in 1-14 amino acid positions, in 1-12 amino acid
positions, in 1-10 amino acid positions, in 1-8 amino acid
positions, in 1-6 amino acid positions, in 1-5 amino acid
positions, in 1-4 amino acid positions, in 1-3 amino acid positions
or in 1-2 amino acid positions.
[0217] The N-glycosylation site is introduced in such a way that
the N-residue (Asn) of said site is located in the designated
position. Analogously, an O-glycosylation site is introduced so
that the S (Ser) or T (Thr) residue making up such site is located
in said position. It should be understood that when the term "at
least 25% (or 50%) of its side chain exposed to the surface" is
used in connection with introduction of an in vivo N-glycosylation
site this term refers to the surface accessibility of the amino
acid side chain in the position where the sugar moiety is actually
attached. In many cases it will be necessary to introduce a serine
or a threonine residue in position +2 relative to the asparagine
residue to which the sugar moiety is actually attached and these
positions, where the serine or threonine residues are introduced,
are allowed to be buried, i.e. to have less than 25% (or 50%) of
their side chains exposed to the surface of the molecule.
[0218] Some conjugates of the invention comprise a polypeptide
sequence comprising one or more of the following substitutions,
relative to SEQ ID NO:1, which introduces an N-glycosylation site
into a position which is predicted to be exposed at the surface of
the molecule with more than a 25% fractional ASA: D2N+P4S/T,
L3N+Q5S/T, P4Q, P4Q+T6S, Q5N+H7S/T, T6N, T6N+S8T, H.sub.7N+L9S/T,
S8N+G10S/T, L9N+H11S/T, G10N+R12S/T, R12N, R12N+T14S, R13N+M15S/T,
M16N+L18S/T, A19N+M21S/T, Q20N+R22S/T, R22N+124S/T, R23N,
R23N+S25T, 124N+L26S/T, S25N+F27S/T, L26N, L26N+S28T, S28N+L30S/T,
L30N+D32S/T, K31N+R33S/T, R33N+D35S/T, H.sub.34N+F36S/T,
D35N+R37S/T, R37N+P39S/T, Q40N+E42S/T, E41N+F43S/T, E42N+D44S/T,
D44N+N46S/T, F48S/T, H.sub.47N+Q49S/T, Q49N+V51S/T, K50N+Q52S/T,
V51N+A53S/T, Q52N+154S/T, E59N+M61S/T, Q62N, Q62N+T64S,
Q63N+F65S/T, F68S/T, S69N+K71S/T, T70N+N72S/T, K71N, K71N+S73T,
S74T, S74N+A76S/T, A75N+W77S/T, D78N, D78N+T80S, E79N+L81S/T,
T80N+L82S/T, L81N+E83S/T, E83N+F85S/T, K84N+Y86S/T, 187N+L89S/T,
F90N+Q92S/T, Q91N+M93S/T, L96S/T, D95N+E97S/T, E97N+C99S/T,
A98N+V100S/T, V100N+Q102S/T, M101N+E103S/T, Q102N+V104S/T,
E103N+G105S/T, V104N+V106S/T, G105N+E107S/T, E107, E107N+T109S,
E108N+P110S/T, L111N+N113S/T, M112N+V114S/T, N113N+D115S/T, V114N,
V114N+S116T, D115N+1117S/T, L118N+V120S/T, R121N+Y123S/T,
K122N+F124S/T, Q125N+1127S/T, R126N, R126N+T128S, T128N+Y130S/T,
L129N+L131S/T, T132N+K134S/T, K133N+K135S/T, K134N+Y136S/T, K135N,
K135N+S137T, Y136N+P138S/T, P138N, P138N+S140T, A146N+I148S/T,
M149N, M149N+S151T, R150N+F152S/T, S153N, S153N+S155T,
F154N+F156S/T, Q159S/T, K160N+L162S/T, R161N+R163S/T,
L162N+R164S/T, R163N+K165S/T and R164N+E166S/T, relative to SEQ ID
NO:1. In some instances, among the above-mentioned positions, the
amino acid residues at one or more of positions 47, 51, 133 and 140
are not modified as shown above. S/T indicates a substitution to a
serine or threonine residue, preferably a threonine residue.
[0219] Some conjugates of the invention comprise a polypeptide
sequence comprising one or more of the following substitutions,
relative to SEQ ID NO:1, which introduces an N-glycosylation site
into a position which is predicted to be exposed at the surface of
the molecule with more than a 50% fractional ASA: D2N+P4S/T,
L3N+Q5S/T, P4Q, P4Q+T6S, Q5N+H7S/T, T6N, T6N+S8T, H.sub.7N+L9S/T,
S8N+G10S/T, L9N+H11S/T, R12N, R12N+T14S, R13N+M15S/T, M16N+L18S/T,
A19N+M21S/T, S25N+F27S/T, S28N+L30S/T, R33N+D35S/T,
H.sub.34N+F36S/T, D35N+R37S/T, R37N+P39S/T, E41N+F43S/T,
D44N+N46S/T, F48S/T, H.sub.47N+Q49S/T, Q49N+V51S/T, K50N+Q52S/T,
F68S/T, K71N, K71N+S73T, A75N+W77S/T, D78N, D78N+T80S, E79N+L81S/T,
T80N+L82S/T, E83N+F85S/T, K84N+Y86S/T, 187N+L89S/T, F90N+Q92S/T,
Q91N+M93S/T, L96S/T, D95N+E97S/T, M101N+E103S/T, Q102N+V104S/T,
E103N+G105S/T, G105N+E107S/T, E107, E107N+T109S, E108N+P110S/T,
L111N+N113S/T, V114N, V114N+S116T, D115N+117S/T, L118N+V120S/T,
R121N+Y123S/T, K122N+F124S/T, Q125N+1127S/T, R126N, R126N+T128S,
L129N+L131S/T, T132N+K134S/T, K133N+K135S/T, K135N, K135N+S137T,
P138N, P138N+S140T, R150N+F152S/T, K160N+L162S/T, L162N+R164S/T,
R163N+K165S/T and R164N+E166S/T, relative to SEQ ID NO:1. In some
instances, among the above-mentioned positions, the amino acid
residues at one or more of positions 47, 51, 133 and 140 are not
modified as described above. S/T indicates a substitution to a
serine or threonine residue, preferably a threonine residue.
[0220] In some instances it may be preferable to introduce
N-glycosylation site(s) outside of potential receptor binding sites
of interferon-alpha, i.e., outside of about positions 29-40, 79-96,
and 124-141, position numbering relative to SEQ ID NO:1. Thus, the
substitution(s) leading to introduction of one or more
N-glycosylation site may include one or more of D2N+P4S/T,
L3N+Q5S/T, P4Q, P4Q+T6S, Q5N+H7S/T, T6N, T6N+S8T, H.sub.7N+L9S/T,
S8N+G10S/T, L9N+H11S/T, G10N+R12S/T, R12N, R12N+T14S, R13N+M15S/T,
M16N+L18S/T, A19N+M21S/T, Q20N+R22S/T, R22N+124S/T, R23N,
R23N+S25T, 124N+L26S/T, S25N+F27S/T, L26N, L26N+S28T, S28N+L30S/T,
E41N+F43S/T, E42N+D44S/T, D44N+N46S/T, F48S/T, H.sub.47N+Q49S/T,
Q49N+V51S/T, K50N+Q52S/T, V51N+A53S/T, Q52N+154S/T, E59N+M61S/T,
Q62N, Q62N+T64S, Q63N+F65S/T, F68S/T, S69N+K71S/T, T70N+N72S/T,
K71N, K71N+S73T, S74T, S74N+A76S/T, A75N+W77S/T, E97N+C99S/T,
A98N+V100S/T, V100N+Q102S/T, M101N+E103S/T, Q102N+V104S/T,
E103N+G105S/T, V104N+V106S/T, G105N+E107S/T, E107, E107N+T109S,
E108N+P110S/T, L111+N113S/T, M112N+V114S/T, N113N+D115S/T, V114N,
V114N+S116T, D115N+I117S/T, L118N+V120S/T, R121N+Y123S/T,
K122N+F124S/T, A146N+I148S/T, M149N, M149N+S151T, R150N+F152S/T,
S153N, S153N+S155T, F154N+F156S/T, Q159S/T, K160N+L162S/T,
R161N+R163S/T, L162N+R164S/T, R163N+K165S/T and R164N+E166S/T
(residues having more than 25% of the side chain exposed to the
surface an not forming part of the putative binding sites). In some
instances, among the above-mentioned positions, the amino acid
residues at one or both of positions 47 and 51 are not modified as
shown above. S/T indicates a substitution to a serine or threonine
residue, preferably a threonine residue.
[0221] In some instances the substitution(s) are selected from the
group consisting of: D2N+P4S/T, L3N+Q5S/T, P4Q, P4Q+T6S, Q5N+H7S/T,
T6N, T6N+S8T, H.sub.7N+L9S/T, S8N+G10S/T, L9N+H11S/T, R12N,
R12N+T14S, R13N+M15S/T, M16N+L18S/T, A19N+M21S/T, S25N+F27S/T,
S28N+L30S/T, E41N+F43S/T, D44N+N46S/T, F48S/T, H.sub.47N+Q49S/T,
Q49N+V51S/T, K50N+Q52S/T, F68S/T, K71N, K71N+S73T, A75N+W77S/T,
M101N+E103S/T, Q102N+V104S/T, E103N+G105S/T, G105N+E107S/T, E107,
E107N+T109S, E108N+P110S/T, L111N+N113S/T, V114N, V114N+S116T,
D115N+1117S/T, L118N+V120S/T, R121N+Y123S/T, K122N+F124S/T,
R150N+F152S/T, K160N+L162S/T, L162N+R164S/T, R163N+K165S/T and
R164N+E166S/T (residues having more than 50% of the side chain
exposed to the surface an not forming part of the putative binding
sites). In some instances, among the above-mentioned positions, the
amino acid residues at one or both of positions 47 and 51 are not
modified as shown above. S/T indicates a substitution to a serine
or threonine residue, preferably a threonine residue.
[0222] In order to obtain efficient utilization of the introduced
N-glycosylation site it is desirable to select any of the
above-mentioned substitutions within about the 125 N-terminal amino
acid residues, such as within about the 100 N-terminal amino acid
residues, e.g. within the 75 N-terminal amino acid residues or
within the 50 N-terminal amino acid residues.
[0223] When the interferon-alpha polypeptide part of a conjugate of
the invention is glycosylated, it may contain a single introduced
in vivo glycosylation site, such as a single introduced in vivo
N-glycosylation site. However, in order to obtain efficient
shielding of epitopes present on the surface of the parent
polypeptide it may be desirable that the polypeptide comprises more
than one in vivo glycosylation site, such as 2-5 in vivo
glycosylation sites, e.g. 2, 3, 4, or 5 in vivo glycosylation
sites.
[0224] It is to be understood that while the examples of
modifications to the parent polypeptide are generally provided
herein relative to the sequence SEQ ID NO:1 (or relative to some
other specified sequence), the disclosed modifications may also be
made in equivalent amino acid positions of any of the other
polypeptides of the invention (including SEQ ID NOs:2-15 and SEQ ID
NOs:44-104 and variants thereof) described herein. Thus, as an
example, the substitution H.sub.47N+Q49S/T relative to SEQ ID NO:1
is understood to correspond to Q47N+Q49S/T in SEQ ID NO:5, and so
on.
Non-Polypeptide Moiety of the Conjugate of the Invention
[0225] As indicated above, the non-polypeptide moiety of the
conjugate of the invention is generally selected from the group
consisting of a polymer molecule, a lipophilic compound, a sugar
moiety (e.g., by way of in vivo N-glycosylation) and an organic
derivatizing agent. All of these agents may confer desirable
properties to the polypeptide part of the conjugate, such as
reduced immunogenicity, increased functional in vivo half-life,
increased serum half-life, increased bioavailability and/or
increased AUC.sub.sc. The polypeptide part of the conjugate is
often conjugated to only one type of non-polypeptide moiety, but
may also be conjugated to two or more different types of
non-polypeptide moieties, e.g. to a polymer molecule and a sugar
moiety, etc. The conjugation to two or more different
non-polypeptide moieties may be done simultaneously or
sequentially. The choice of non-polypeptide moiety/moieties,
depends especially on the effect desired to be achieved by the
conjugation. For instance, sugar moieties have been found
particularly useful for reducing immunogenicity, whereas polymer
molecules such as PEG are of particular use for increasing
functional in vivo half-life and/or serum half-life. Using a
combination of a polymer molecule and a sugar moiety may enhance
the reduction in immunogenicity and the increase in functional in
vivo or serum half-life.
[0226] In the following sections "Conjugation to a lipophilic
compound", "Conjugation to a polymer molecule", "Conjugation to a
sugar moiety" and "Conjugation to an organic derivatizing agent"
conjugation to specific types of non-polypeptide moieties is
described.
Conjugation to a Lipophilic Compound
[0227] For conjugation to a lipophilic compound the following
polypeptide groups may function as attachment groups: the
N-terminus or C-terminus of the polypeptide, the hydroxy groups of
the amino acid residues Ser, Thr or Tyr, the .epsilon.-amino group
of Lys, the SH group of Cys or the carboxyl group of Asp and Glu.
The polypeptide and the lipophilic compound may be conjugated to
each other either directly or by use of a linker. The lipophilic
compound may be a natural compound such as a saturated or
unsaturated fatty acid, a fatty acid diketone, a terpene, a
prostaglandin, a vitamin, a carotenoid or steroid, or a synthetic
compound such as a carbon acid, an alcohol, an amine and sulphonic
acid with one or more alkyl, aryl, alkenyl or other multiple
unsaturated compounds. The conjugation between the polypeptide and
the lipophilic compound, optionally through a linker may be done
according to methods known in the art, e.g. as described by
Bodanszky in Peptide Synthesis, John Wiley, New York, 1976 and in
WO 96/12505.
Conjugation to a Polymer Molecule
[0228] The polymer molecule to be coupled to the polypeptide may be
any suitable polymer molecule, such as a natural or synthetic
homo-polymer or heteropolymer, typically with a molecular weight in
the range of about 300-100,000 Da, such as about 1000-50,000 Da,
e.g. in the range of about 1000-40,000 Da. More particularly, the
polymer molecule, such as PEG, in particular mPEG, will typically
have a molecular weight of about 2, 5, 10, 12, 15, 20, 30, 40 or 50
kDa, in particular a molecular weight of about 5 kDa, about 10 kDa,
about 12 kDa, about 15 kDa, about 20 kDa, about 30 kDa or about 40
kDa. The PEG molecule may be branched (e.g., mPEG2), or may be
unbranched (i.e., linear).
[0229] When used about polymer molecules herein, the word "about"
indicates an approximate average molecular weight and reflects the
fact that there will normally be a certain molecular weight
distribution in a given polymer preparation.
[0230] Examples of homo-polymers include a polyol (i.e. poly-OH), a
polyamine (i.e. poly-NH.sub.2) and a polycarboxylic acid (i.e.
poly-COOH). A hetero- polymer is a polymer which comprises one or
more different coupling groups, such as a hydroxyl group and an
amine group.
[0231] Examples of suitable polymer molecules include polymer
molecules selected from the group consisting of polyalkylene oxide
(PAO), including polyalkylene glycol (PAG), such as polyethylene
glycol (PEG) and polypropylene glycol (PPG), branched PEGs (PEG2),
poly-vinyl alcohol (PVA), poly-carboxylate, poly-(vinylpyrolidone),
polyethylene-co-maleic acid anhydride, polystyrene-co-malic acid
anhydride, dextran including carboxymethyl-dextran, or any other
biopolymer suitable for reducing immunogenicity and/or increasing
functional in vivo half-life and/or serum half-life.
[0232] Generally, polyalkylene glycol-derived polymers are
biocompatible, non-toxic, non-antigenic, non-immunogenic, have
various water solubility properties, and are easily excreted from
living organisms.
[0233] PEG is the preferred polymer molecule to be used, since it
has only few reactive groups capable of cross-linking compared to
e.g. polysaccharides such as dextran. In particular, monofunctional
PEG, e.g. monomethoxypolyethylene glycol (mPEG), is of interest
since its coupling chemistry is relatively simple (only one
reactive group is available for conjugating with attachment groups
on the polypeptide). Consequently, the risk of cross-linking is
eliminated, the resulting polypeptide conjugates are more
homogeneous and the reaction of the polymer molecules with the
polypeptide is easier to control.
[0234] To effect covalent attachment of the polymer molecule(s) to
the polypeptide, the hydroxyl end groups of the polymer molecule
must be provided in activated form, i.e. with reactive functional
groups (examples of which include primary amino groups, hydrazide
(HZ), thiol, succinate (SUC), succinimidyl succinate (SS),
succinimidyl succinamide (SSA), succinimidyl propionate (SPA),
succinimidyl butanoate (SBA), succinimidyl carboxymethylate (SCM),
benzotriazole carbonate (BTC), N-hydroxysuccinimide (NHS),
aldehyde, nitrophenylcarbonate (NPC), and tresylate (TRES)).
Suitably activated polymer molecules are commercially available,
e.g. from Nektar Therapeutics, Inc., Huntsville, Ala., USA;
PolyMASC Pharmaceuticals plc, UK; or SunBio Corporation, Anyang
City, South Korea. Alternatively, the polymer molecules can be
activated by conventional methods known in the art, e.g. as
disclosed in WO 90/13540. Specific examples of activated linear or
branched polymer molecules suitable for use in the present
invention are described in the Nektar Therapeutics, Inc. 2003
Catalog ("Nektar Molecule Engineering: Polyethylene Glycol and
Derivatives for Advanced Pegylation, Catalog 2003"), incorporated
by reference herein. Specific examples of activated PEG polymers
include the following linear PEGs: NHS-PEG, SPA-PEG, SSPA-PEG,
SBA-PEG, SS-PEG, SSA-PEG, SC-PEG, SG-PEG, SCM-PEG, NOR-PEG,
BTC-PEG, EPOX-PEG, NCO-PEG, NPC-PEG, CDI-PEG, ALD-PEG, TRES-PEG,
VS-PEG, OPSS-PEG, IODO-PEG, and MAL-PEG, and branched PEGs, such as
PEG2-NHS, PEG2-MAL, and those disclosed in U.S. Pat. No. 5,932,462
and U.S. Pat. No. 5,643,575, both of which are incorporated herein
by reference. Furthermore, the following publications, incorporated
herein by reference, disclose useful polymer molecules and/or
PEGylation chemistries: U.S. Pat. No. 5,824,778, U.S. Pat. No.
5,476,653, WO 97/32607, EP 229,108, EP 402,378, U.S. Pat. No.
4,902,502, U.S. Pat. No. 5,281,698, U.S. Pat. No. 5,122,614, U.S.
Pat. No. 5,219,564, WO 92/16555, WO 94/04193, WO 94/14758, WO
94/17039, WO 94/18247, WO 94/28024, WO 95/00162, WO 95/11924,
WO95/13090, WO 95/33490, WO 96/00080, WO 97/18832, WO 98/41562, WO
98/48837, WO 99/32134, WO 99/32139, WO 99/32140, WO 96/40791, WO
98/32466, WO 95/06058, EP 439 508, WO 97/03106, WO 96/21469, WO
95/13312, EP 921131, U.S. Pat. No. 5,736,625, WO 98/05363, EP 809
996, U.S. Pat. No. 5,629,384, WO 96/41813, WO 96/07670, U.S. Pat.
No. 5,473,034, U.S. Pat. No. 5,516,673, EP 605 963, U.S. Pat. No.
5,382,657, EP 510 356, EP 400 472, EP 183 503 and EP 154 316.
[0235] The conjugation of the polypeptide and the activated polymer
molecules is conducted by use of any conventional method, e.g. as
described in the following references (which also describe suitable
methods for activation of polymer molecules): Harris and Zalipsky,
eds., Poly(ethylene glycol) Chemistry and Biological Applications,
AZC, Washington; R. F. Taylor, (1991), "Protein immobilisation.
Fundamental and applications", Marcel Dekker, N.Y.; S. S. Wong,
(1992), "Chemistry of Protein Conjugation and Crosslinking", CRC
Press, Boca Raton; G. T. Hermanson et al., (1993), "Immobilized
Affinity Ligand Techniques", Academic Press, N.Y.
[0236] For PEGylation of cysteine residues the polypeptide is
usually treated with a reducing agent, such as dithiothreitol (DDT)
prior to PEGylation. The reducing agent is subsequently removed by
any conventional method, such as by desalting. Conjugation of PEG
to a cysteine residue typically takes place in a suitable buffer at
pH 6-9 at temperatures varying from 4.degree. C. to 25.degree. C.
for periods up to about 16 hours. Examples of activated PEG
polymers for coupling to cysteine residues include the following
linear and branched PEGs: vinylsulfone-PEG (PEG-VS), such as
vinylsulfone-mPEG (mPEG-VS); orthopyridyl-disulfide-PEG (PEG-OPSS),
such as orthopyridyl-disulfide-mPEG (mPEG-OPSS); and maleimide-PEG
(PEG-MAL), such as maleimide-mPEG (mPEG-MAL) and branched
maleimide-mPEG2 (mPEG2-MAL).
[0237] Pegylation of lysines often employs
PEG-N-hydroxylsuccinimide (e.g., mPEG-NHS or mPEG2-NHS), or esters
such as PEG succinimidyl propionate (e.g., mPEG-SPA) or PEG
succinimidyl butanoate (e.g., mPEG-SBA). One or more PEGs can be
attached to a protein within 30 minutes at pH 8-9.5 at room
temperature if about equimolar amounts of PEG and protein are
mixed. A molar ratio of PEG to protein amino groups of 1-5 to 1
will usually suffice. Increasing pH increases the rate of reaction,
while lowering pH reduces the rate of reaction. These highly
reactive active esters can couple at physiological pH, but less
reactive derivatives typically require higher pH. Low temperatures
may also be employed if a labile protein is being used. Under low
temperature conditions, a longer reaction time may be used.
[0238] N-terminal PEGylation is facilitated by the difference
between the pKa values of the .alpha.-amino group of the N-terminal
amino acid (.about.7.6 to 8.0) and the .epsilon.-amino group of
lysine (.about.10). PEGylation of the N-terminal amino group often
employs PEG-aldehydes (such as mPEG-propionaldehyde or
mPEG-butylaldehyde), which are more selective for amines and thus
are less likely to react with the imidazole group of histidine; in
addition, PEG reagents used for lysine conjugation (such as
mPEG-SPA or mPEG-SBA) may also be used for conjugation of the
N-terminal amine. Conjugation of a PEG-aldehyde to the N-terminal
amino group typically takes place in a suitable buffer (such as,
100 mM sodium acetate or 100 mM sodium bisphosphate buffer with 20
mM sodium cyanoborohydride) at pH.about.5.0 overnight at
temperatures varying from about 4.degree. C. to 25.degree. C.
Useful N-terminal PEGylation methods and chemistries are also
described in U.S. Pat. No. 5,985,265 and U.S. Pat. No. 6,077,939,
both incorporated herein by reference.
[0239] Typically, linear PEG or mPEG polymers will have a molecular
weight of about 5 kDa, about 10 kDa, about 12 kDa, about 15 kDa,
about 20 kDa, or about 30 kDa. Branched PEG (PEG2 or mPEG2)
polymers will typically have a molecular weight of about 10kDa,
about 20 kDa, or about 40 kDa. In some instances, the
higher-molecular weight branched PEG2 reagents, such as 20 kDa or
40 kDa PEG2, including e.g. mPEG2-NHS for lysine PEGylation,
mPEG2-MAL for cysteine PEGylation, or MPEG2-aldehyde for N-terminal
PEGylation (all available from Nektar Therapeutics, Inc, Huntsville
Ala.), may be used. The branched structure of the PEG2 compound
results in a relatively large molecular volume, so fewer attached
molecules (or, one attached molecule) may impart the desired
characteristics of the PEGylated molecule.
[0240] The skilled person will be aware that the activation method
and/or conjugation chemistry to be used depends on the attachment
group(s) of the interferon-alpha polypeptide as well as the
functional groups of the polymer (e.g., being amino, hydroxyl,
carboxyl, aldehyde or sulfhydryl). The PEGylation may be directed
towards conjugation to all available attachment groups on the
polypeptide (i.e. such attachment groups that are exposed at the
surface of the polypeptide) or may be directed towards specific
attachment groups, e.g. cysteine residues, lysine residues, or the
N-terminal amino group. Furthermore, the conjugation may be
achieved in one step or in a stepwise manner (e.g. as described in
WO 99/55377).
[0241] In some instances, the polymer conjugation is performed
under conditions aiming at reacting as many of the available
polymer attachment groups as possible with polymer molecules. This
is achieved by means of a suitable molar excess of the polymer in
relation to the polypeptide. Typical molar ratios of activated
polymer molecules to polypeptide are up to about 1000-1, such as up
to about 200-1 or up to about 100-1. In some cases, the ratio may
be somewhat lower, however, such as up to about 50-1, 10-1 or 5-1.
Also equimolar ratios may be used.
[0242] It is also contemplated according to the invention to couple
the polymer molecules to the polypeptide through a linker. Suitable
linkers are well known to the skilled person. A preferred example
is cyanuric chloride (Abuchowski et al., (1977), J. Biol. Chem.,
252, 3578-3581; U.S. Pat. No. 4,179,337; Shafer et al., (1986), J.
Polym. Sci. Polym. Chem. Ed., 24, 375-378).
[0243] Subsequent to the conjugation residual activated polymer
molecules are blocked according to methods known in the art, e.g.
by addition of primary amine to the reaction mixture, and the
resulting inactivated polymer molecules removed by a suitable
method.
[0244] Covalent in vitro coupling of a sugar moiety to amino acid
residues of interferon-alpha may be used to modify or increase the
number or profile of sugar substituents. Depending on the coupling
mode used, the carbohydrate(s) may be attached to a) arginine and
histidine (Lundblad and Noyes, Chemical Reagents for Protein
Modification, CRC Press Inc. Boca Raton, FI), b) free carboxyl
groups (e.g. of the C-terminal amino acid residue, asparagine or
glutamine), c) free sulfhydryl groups such as that of cysteine, d)
free hydroxyl groups such as those of serine, threonine, tyrosine
or hydroxyproline, e) aromatic residues such as those of
phenylalanine or tryptophan or f) the amide group of glutamine.
These amino acid residues constitute examples of attachment groups
for a sugar moiety, which may be introduced and/or removed in the
interferon-alpha polypeptide. Suitable methods of in vitro coupling
are described in WO 87/05330 and in Aplin et al., CRC Crit Rev.
Biochem., pp. 259-306, 1981. The in vitro coupling of sugar
moieties or PEG to protein- and peptide-bound Gln-residues can also
be carried out by transglutaminases (TGases), e.g. as described by
Sato et al., 1996 Biochemistry 35, 13072-13080 or in EP 725145.
Coupling to a Sugar Moiety
[0245] In order to achieve in vivo glycosylation of an
interferon-alpha polypeptide that has been modified by introduction
of one or more glycosylation sites (see the section "Conjugates of
the invention wherein the non-polypeptide moiety is a sugar
moiety"), the nucleotide sequence encoding the polypeptide part of
the conjugate is inserted in a glycosylating, eukaryotic expression
host. The expression host cell may be selected from fungal
(filamentous fungal or yeast), insect, mammalian animal cells, from
transgenic plant cells or from transgenic animals. Furthermore, the
glycosylation may be achieved in the human body when using a
nucleotide sequence encoding the polypeptide part of a conjugate of
the invention or a polypeptide of the invention in gene therapy. In
one aspect the host cell is a mammalian cell, such as a CHO cell, a
COS cell, a BHK or HEK cell, e.g. HEK293, or an insect cell, such
as an SF9 cell, or a yeast cell, e.g. Saccharomyces cerevisiae,
Pichia pastoris or any other suitable glycosylating host, e.g. as
described further below. Optionally, sugar moieties attached to the
interferon-.alpha. polypeptide by in vivo glycosylation are further
modified by use of glycosyltransferases, e.g. using the
GlycoAdvance.TM. technology marketed by Neose, Horsham, Pa., USA.
Thereby, it is possible to, e.g., increase the sialyation of the
glycosylated interferon-alpha polypeptide following expression and
in vivo glycosylation by CHO cells.
Coupling to an Organic Derivatizing Agent
[0246] Covalent modification of the interferon-alpha polypeptide
may be performed by reacting (an) attachment group(s) of the
polypeptide with an organic derivatizing agent.
[0247] Suitable derivatizing agents and methods are well known in
the art. For example, cysteinyl residues most commonly are reacted
with .alpha.-haloacetates (and corresponding amines), such as
chloroacetic acid or chloroacetamide, to give carboxymethyl or
carboxyamidomethyl derivatives. Cysteinyl residues also are
derivatized by reaction with bromotrifluoroacetone,
.alpha.-bromo-.beta.-(4-imidozoyl)propionic acid, chloroacetyl
phosphate, N-alkylmaleimides, 3-nitro-2-pyridyl disulfide, methyl
2-pyridyl disulfide, p-chloromercuribenzoate,
2-chloromercuri-4-nitrophenol, or
chloro-7-nitrobenzo-2-oxa-1,3-diazole. Histidyl residues are
derivatized by reaction with diethylpyrocarbonate at pH 5.5-7.0
because this agent is relatively specific for the histidyl side
chain. Para-bromophenacyl bromide is also useful; the reaction is
preferably performed in 0.1 M sodium cacodylate at pH 6.0. Lysinyl
and amino terminal residues are reacted with succinic or other
carboxylic acid anhydrides. Derivatization with these agents has
the effect of reversing the charge of the lysinyl residues. Other
suitable reagents for derivatizing .alpha.-amino-containing
residues include imidoesters such as methyl picolinimidate;
pyridoxal phosphate; pyridoxal; chloroborohydride;
trinitrobenzenesulfonic acid; O-methylisourea; 2,4-pentanedione;
and transaminase-catalyzed reaction with glyoxylate. Arginyl
residues are modified by reaction with one or several conventional
reagents, among them phenylglyoxal, 2,3-butanedione,
1,2-cyclohexanedione, and ninhydrin. Derivatization of arginine
residues requires that the reaction be performed in alkaline
conditions because of the high pKa of the guanidine functional
group. Furthermore, these reagents may react with the groups of
lysine as well as the arginine guanidino group. Carboxyl side
groups (aspartyl or glutamyl or C-terminal amino acid residue) are
selectively modified by reaction with carbodiimides
(R--N.dbd.C.dbd.N--R'), where R and R' are different alkyl groups,
such as 1-cyclohexyl-3-(2-morpholinyl-4-ethyl)carbodiimide or
1-ethyl-3-(4-azonia-4,4-dimethylpentyl)carbodiimide. Furthermore,
aspartyl and glutamyl residues are converted to asparaginyl and
glutaminyl residues by reaction with ammonium ions.
Blocking of a Functional Site
[0248] Since excessive polymer conjugation may lead to a loss of
activity of the interferon-.alpha. polypeptide to which the polymer
is conjugated, it may be advantageous to remove attachment groups
located at the functional site or to block the functional site
prior to conjugation. These latter strategies constitute further
aspects of the invention (the first strategy being exemplified
further above, e.g. by removal of lysine residues which may be
located close to a functional site). More specifically, according
to the second strategy the conjugation between the interferon-alpha
polypeptide and the non-polypeptide moiety is conducted under
conditions where the functional site of the polypeptide is blocked
by a helper molecule capable of binding to the functional site of
the polypeptide. Preferably, the helper molecule is one which
specifically recognizes a functional site of the polypeptide, such
as a receptor, in particular the type I interferon receptor.
Alternatively, the helper molecule may be an antibody, in
particular a monoclonal antibody recognizing the interferon-alpha
polypeptide. In particular, the helper molecule may be a
neutralizing monoclonal antibody.
[0249] The polypeptide is allowed to interact with the helper
molecule before effecting conjugation. This ensures that the
functional site of the polypeptide is shielded or protected and
consequently unavailable for derivatization by the non-polypeptide
moiety such as a polymer. Following its elution from the helper
molecule, the conjugate between the non-polypeptide moiety and the
polypeptide can be recovered with at least a partially preserved
functional site.
[0250] The subsequent conjugation of the polypeptide having a
blocked functional site to a polymer, a lipophilic compound, an
organic derivatizing agent or any other compound is conducted in
the normal way, e.g. as described in the sections above entitled
"Conjugation to . . . ".
[0251] Irrespective of the nature of the helper molecule to be used
to shield the functional site of the polypeptide from conjugation,
it is desirable that the helper molecule is free from or comprises
only a few attachment groups for the non-polypeptide moiety of
choice in parts of the molecule where the conjugation to such
groups would hamper the desorption of the conjugated polypeptide
from the helper molecule. Hereby, selective conjugation to
attachment groups present in non-shielded parts of the polypeptide
can be obtained and it is possible to reuse the helper molecule for
repeated cycles of conjugation. For instance, if the
non-polypeptide moiety is a polymer molecule such as PEG, which has
the epsilon amino group of a lysine or N-terminal amino acid
residue as an attachment group, it is desirable that the helper
molecule is substantially free from conjugatable epsilon amino
groups, preferably free from any epsilon amino groups. Accordingly,
in some instances the helper molecule is a protein or peptide
capable of binding to the functional site of the polypeptide, which
protein or peptide is free from any conjugatable attachment groups
for the non-polypeptide moiety of choice.
[0252] In a further aspect the helper molecule is first covalently
linked to a solid phase such as column packing materials, for
instance Sephadex or agarose beads, or a surface, e.g. reaction
vessel. Subsequently, the polypeptide is loaded onto the column
material carrying the helper molecule and conjugation carried out
according to methods known in the art, e.g. as described in the
sections above entitled "Conjugation to . . . ". This procedure
allows the polypeptide conjugate to be separated from the helper
molecule by elution. The polypeptide conjugate is eluted by
conventional techniques under physico-chemical conditions that do
not lead to a substantive degradation of the polypeptide conjugate.
The fluid phase containing the polypeptide conjugate is separated
from the solid phase to which the helper molecule remains
covalently linked. The separation can be achieved in other ways:
For instance, the helper molecule may be derivatized with a second
molecule (e.g. biotin) that can be recognized by a specific binder
(e.g. streptavidin). The specific binder may be linked to a solid
phase thereby allowing the separation of the polypeptide conjugate
from the helper molecule-second molecule complex through passage
over a second helper-solid phase column which will retain, upon
subsequent elution, the helper molecule-second molecule complex,
but not the polypeptide conjugate. The polypeptide conjugate may be
released from the helper molecule in any appropriate fashion.
De-protection may be achieved by providing conditions in which the
helper molecule dissociates from the functional site of the
interferon-.alpha. to which it is bound. For instance, a complex
between an antibody to which a polymer is conjugated and an
anti-idiotypic antibody can be dissociated by adjusting the pH to
an acid or alkaline pH.
Conjugation of a Tagged Interferon-Alpha Polypeptide
[0253] In another aspect the interferon-alpha polypeptide is
expressed as a fusion protein with a tag, i.e. an amino acid
sequence or peptide made up of typically 1-30, such as 1-20 or 1-15
or 1-10 or 1-5 amino acid residues, e.g. added to the N-terminus or
to the C-terminus of the polypeptide. Besides allowing for fast and
easy purification, the tag is a convenient tool for achieving
conjugation between the tagged polypeptide and the non-polypeptide
moiety. In particular, the tag may be used for achieving
conjugation in microtiter plates or other carriers, such as
paramagnetic beads, to which the tagged polypeptide can be
immobilised via the tag. The conjugation to the tagged polypeptide
in, e.g., microtiter plates has the advantage that the tagged
polypeptide can be immobilised in the microtiter plates directly
from the culture broth (in principle without any purification) and
subjected to conjugation. Thereby, the total number of process
steps (from expression to conjugation) can be reduced. Furthermore,
the tag may function as a spacer molecule ensuring an improved
accessibility to the immobilised polypeptide to be conjugated. The
conjugation using a tagged polypeptide may be to any of the
non-polypeptide moieties disclosed herein, e.g. to a polymer
molecule such as PEG.
[0254] The identity of the specific tag to be used is not critical
as long as the tag is capable of being expressed with the
polypeptide and is capable of being immobilised on a suitable
surface or carrier material. A number of suitable tags are
commercially available, e.g. from Unizyme Laboratories, Denmark.
Antibodies against such tags are commercially available, e.g. from
ADI, Aves Lab and Research Diagnostics.
Polynucleotides of the Invention
[0255] The invention provides isolated or recombinant nucleic acids
(also referred to herein as polynucleotides), collectively referred
to as "nucleic acids (or polynucleotides) of the invention", which
encode polypeptides of the invention. The polynucleotides of the
invention are useful in a variety of applications. As discussed
above, the polynucleotides are useful in producing polypeptides of
the invention. In addition, polynucleotides of the invention can be
incorporated into expression vectors useful for gene therapy, DNA
vaccination, and immunotherapy, as described in more detail
below.
[0256] In one aspect, the invention provides isolated or
recombinant nucleic acids that each comprise a polynucleotide
sequence selected from: (a) a polynucleotide sequence selected from
SEQ ID NOS:16-30, or a complementary polynucleotide sequence
thereof; (b) a polynucleotide sequence which encodes a polypeptide
selected from SEQ ID NOS:1-15 and 44-104, or a complementary
polynucleotide sequence thereof.
[0257] The invention also provides isolated or recombinant nucleic
acids that each comprise a polynucleotide sequence which encodes a
polypeptide comprising a sequence which differs in 0-16 amino acid
positions (such as in 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13,
14, 15, or 16 amino acid positions), e.g. in 0-16 positions, 0-15
positions, 0-14 positions, 0-13 positions, 0-12 positions, 0-11
positions, 0-10 positions, 0-9 positions, 0-8 positions, 0-7
positions, 0-6 positions, 0-5 positions, 0-4 positions, 0-3
positions, 0-2 positions, or 0-1 positions, from any one of SEQ ID
NOs:1-15 and SEQ ID NOs:44-104 (such as one of SEQ ID NO:1, SEQ ID
NO:3, SEQ ID NO:8, SEQ ID NO:12, SEQ ID NO:47 or SEQ ID NO:53). In
some instances the encoded polypeptide exhibits an interferon-alpha
activity.
[0258] The invention also provides isolated or recombinant nucleic
acids that each comprise a polynucleotide sequence which encodes a
polypeptide comprising a sequence having at least about 90%, 91%,
92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% or more amino acid
sequence identity to any one of SEQ ID NOs:1-15 and SEQ ID
NOs:44-104 (such as one of SEQ ID NO:1, SEQ ID NO:3, SEQ ID NO:8,
SEQ ID NO:12, SEQ ID NO:47 or SEQ ID NO:53). In some instances the
encoded polypeptide exhibits an interferon-alpha activity.
[0259] The invention also provides isolated or recombinant nucleic
acids that each comprise a polynucleotide sequence which encodes a
polypeptide which is a variant of a parent interferon-alpha
polypeptide, the encoded variant comprising a sequence which
differs from the parent interferon-alpha polypeptide sequence in
least one amino acid position, wherein the variant sequence
comprises one or more of His at position 47, Val at position 51,
Phe at position 55, Leu at position 56, Tyr at position 58, Lys at
position 133, and at position Ser140, the position numbering
relative to that of SEQ ID NO:1. In some instances the parent
interferon-alpha polypeptide sequence is a sequence of a
naturally-occurring human interferon-alpha (such as any one of SEQ
ID NO:31-SEQ ID NO:42, or SEQ ID NO:32+R23K, or other huIFN-alpha
sequence as described herein and/or in Allen G. and Diaz M. O.
(1996), supra), or is a sequence of a non-naturally occurring
(i.e., synthetic) interferon-alpha, such as IFN-alpha Con1 (SEQ ID
NO:43). In some instances, the variant sequence differs from the
parent polypeptide sequence in 1-16 amino acid positions (such as
in 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, or 16 amino
acid positions), e.g. in 1-10 amino acid positions, in 1-5 amino
acid positions, or in 1-3 amino acid positions. In some instances,
the variant exhibits an interferon-alpha activity.
[0260] In another aspect, the invention provides isolated or
recombinant nucleic acids that each comprise a polynucleotide
sequence which hybridizes under highly stringent conditions over
substantially the entire length of one of SEQ ID NOs:16-30, which
polynucleotide sequence encodes a polypeptide exhibiting an
interferon alpha activity.
Additional Aspects
[0261] Any of the nucleic acids of the invention (which includes
those described above) may encode a fusion protein comprising at
least one additional amino acid sequence, such as, for example, a
secretion/localization sequence, a sequence useful for
solubilization or immobilization (e.g., for cell surface display)
of the polypeptide, a sequence useful for detection and/or
purification of the polypeptide (e.g., a polypeptide purification
subsequence, such as an epitope tag, a polyhistidine sequence, and
the like). In another aspect, the invention provides cells
comprising one or more of the nucleic acids of the invention. Such
cells may express one or more polypeptides encoded by the nucleic
acids of the invention.
[0262] The invention also provides vectors comprising any of the
nucleic acids of the invention. Such vectors may comprise a
plasmid, a cosmid, a phage, a virus, or a fragment of a virus. Such
vectors may comprise an expression vector, and, if desired, the
nucleic acid is operably linked to a promoter, including those
discussed herein and below. Furthermore, in another aspect, the
invention provides compositions comprising an excipient or carrier
and at least one of any of the nucleic acids of the invention, or
vectors, cells, or host comprising such nucleic acids. Such
composition may be pharmaceutical compositions, and the excipient
or carrier may be a pharmaceutically acceptable excipient or
carrier.
[0263] The invention also includes compositions comprising two or
more nucleic acids of the invention, or fragments thereof (e.g., as
substrates for recombination). The composition can comprise a
library of recombinant nucleic acids, where the library contains at
least 2, at least 3, at least 5, at least 10, at least 20, at least
50, or at least 100 or more nucleic acids described above. The
nucleic acids are optionally cloned into expression vectors,
providing expression libraries.
[0264] The nucleic acids of the invention and fragments thereof, as
well as vectors comprising such polynucleotides, may be employed
for therapeutic or prophylactic uses in combination with a suitable
carrier, such as a pharmaceutical carrier. Such compositions
comprise a therapeutically and/or prophylactically effective amount
of the compound, and a pharmaceutically acceptable carrier or
excipient. Such a carrier or excipient includes, but is not limited
to, saline, buffered saline, dextrose, water, glycerol, ethanol,
and combinations thereof. The formulation should suit the mode of
administration. Methods of administering nucleic acids,
polypeptides, and proteins are well known in the art, and are
further discussed below.
[0265] The invention also includes compositions produced by
digesting one or more of any of the nucleic acids of the invention
with a restriction endonuclease, an RNAse, or a DNAse (e.g., as is
performed in certain of the recombination formats noted above); and
compositions produced by fragmenting or shearing one or more
nucleic acids of the invention by mechanical means (e.g.,
sonication, vortexing, and the like), which can also be used to
provide substrates for recombination in the methods described
herein. The invention also provides compositions produced by
cleaving at least one of any of the nucleic acids of the invention.
The cleaving may comprise mechanical, chemical, or enzymatic
cleavage, and the enzymatic cleavage may comprise cleavage with a
restriction endonuclease, an RNAse, or a DNAse.
[0266] Also included in the invention are compositions produced by
a process comprising incubating one or more of the fragmented
nucleic acids of the invention in the presence of ribonucleotide or
deoxyribonucleotide triphosphates and a nucleic acid polymerase.
This resulting composition forms a recombination mixture for many
of the recombination formats noted above. The nucleic acid
polymerase may be an RNA polymerase, a DNA polymerase, or an
RNA-directed DNA polymerase (e.g., a "reverse transcriptase"); the
polymerase can be, e.g., a thermostable DNA polymerase (e.g., VENT,
TAQ, or the like).
[0267] Similarly, compositions comprising sets of oligonucleotides
corresponding to more than one nucleic acids of the invention are
useful as recombination substrates and are a feature of the
invention. For convenience, these fragmented, sheared, or
oligonucleotide synthesized mixtures are referred to as fragmented
nucleic acid sets.
[0268] The invention also provides an isolated or recombinant
nucleic acid encoding a polypeptide that exhibits an
interferon-alpha activity, produced by mutating or recombining at
least one nucleic acid of the invention.
Making Polynucleotides
[0269] Polynucleotides, oligonucleotides, and nucleic acid
fragments of the invention can be prepared by standard solid-phase
methods, according to known synthetic methods. Typically, fragments
of up to about 100 bases are individually synthesized, then joined
(e.g., by enzymatic or chemical ligation methods, or polymerase
mediated recombination methods) to form essentially any desired
continuous sequence. For example, the polynucleotides and
oligonucleotides of the invention can be prepared by chemical
synthesis using, e.g., classical phosphoramidite method described
by, e.g., Beaucage et al. (1981) Tetrahedron Letters 22:1859-69, or
the method described by Matthes et al. (1984) EMBO J 3:801-05,
e.g., as is typically practiced in automated synthetic methods.
According to the phosphoramidite method, oligonucleotides are
synthesized, e.g., in an automatic DNA synthesizer, purified,
annealed, ligated and cloned into appropriate vectors.
[0270] In addition, essentially any polynucleotide can be custom
ordered from any of a variety of commercial sources, such as Operon
Technologies Inc. (Alameda, Calif.) and many others. Similarly,
peptides and antibodies can be custom ordered from any of a variety
of sources, e.g., Celtek Peptides (Nashville, Tenn.); Washington
Biotechnology, Inc. (Baltimore Md.); Global Peptide Services (Ft.
Collin Colo.), and many others.
[0271] Certain polynucleotides of the invention may also be
obtained by screening cDNA libraries (e.g., libraries generated by
recombining homologous nucleic acids as in typical recursive
sequence recombination methods) using oligonucleotide probes that
can hybridize to or PCR-amplify polynucleotides which encode
interferon-alpha polypeptides and fragments of those polypeptides.
Procedures for screening and isolating cDNA clones are well-known
to those of skill in the art. Such techniques are described in,
e.g., Berger and Kimmel, Guide to Molecular Cloning Techniques,
Methods in Enzymol. Vol. 152, Acad. Press, Inc., San Diego, Calif.
("Berger"); Sambrook, supra, and Current Protocols in Molecular
Biology, Ausubel, supra. Some polynucleotides of the invention can
be obtained by altering a naturally occurring sequence, e.g., by
mutagenesis, recursive sequence recombination (e.g., shuffling), or
oligonucleotide recombination. In other cases, such polynucleotides
can be made in silico or through oligonucleotide recombination
methods as described in the references cited herein.
[0272] As described in more detail herein, the polynucleotides of
the invention include polynucleotides that encode polypeptides of
the invention, polynucleotide sequences complementary to these
polynucleotide sequences, and polynucleotides that hybridize under
at least stringent conditions to the sequences defined herein. A
coding sequence refers to a polynucleotide sequence encoding a
particular polypeptide or domain, region, or fragment of said
polypeptide. A coding sequence may encode (code for) a polypeptide
of the invention exhibiting an interferon alpha activity as
described above. The polynucleotides of the invention may be in the
form of RNA or in the form of DNA, and include mRNA, cRNA,
synthetic RNA and DNA, and cDNA. The polynucleotides may be
double-stranded or single-stranded, and if single-stranded, can be
the coding strand or the non-coding (anti-sense, complementary)
strand. The polynucleotides of the invention include the coding
sequence of a polypeptide of the invention (i) in isolation, (ii)
in combination with one or more additional coding sequences, so as
to encode, e.g., a fusion protein, a pre-protein, a prepro-protein,
or the like, (iii) in combination with non-coding sequences, such
as introns, control elements, such as a promoter (e.g., naturally
occurring or recombinant or shuffled promoter), a terminator
element, or 5' and/or 3' untranslated regions effective for
expression of the coding sequence in a suitable host, and/or (iv)
in a vector, cell, or host environment in which the coding sequence
is a heterologous gene.
[0273] Polynucleotides of the invention can also be found in
combination with typical compositional formulations of nucleic
acids, including in the presence of carriers, buffers, adjuvants,
excipients, and the like, as are known to those of ordinary skill
in the art. Polynucleotide fragments typically comprise at least
about 200 nucleotide bases, such as at least about 250, 300, 350,
400, 450, 460, 470, or more bases. The nucleotide fragments of
polynucleotides of the invention may hybridize under highly
stringent conditions to a polynucleotide sequence described herein
and/or encode amino acid sequences having at least one of the
properties of polypeptides of the invention described herein.
Modified Coding Sequences
[0274] As will be understood by those of ordinary skill in the art,
it can be advantageous to modify a coding sequence to enhance its
expression in a particular host. The genetic code is redundant with
64 possible codons, but most organisms preferentially use a subset
of these codons. The codons that are utilized most often in a
species are considered optimal codons, and those not utilized very
often are classified as rare or low-usage codons (see, e.g., Zhang,
S. P. et al. (1991) Gene 105:61-72). Codons can be substituted to
reflect the preferred codon usage of the host, a process sometimes
termed "codon optimization" or "controlling for species codon
bias."
[0275] Modified coding sequence containing codons preferred by a
particular prokaryotic or eukaryotic host (see, e.g., Murray, E. et
al. (1989) Nuc Acids Res 17:477-508) can be prepared, for example,
to increase the rate of translation or to produce recombinant RNA
transcripts having desirable properties, such as a longer
half-life, as compared with transcripts produced from a
non-optimized sequence. Translation stop codons can also be
modified to reflect host preference. For example, preferred stop
codons for S. cerevisiaeand mammals are UAA and UGA respectively.
The preferred stop codon for monocotyledonous plants is UGA,
whereas insects and E. coli prefer to use UAA as the stop codon
(Dalphin, M. E. et al. (1996) Nucl. Acids Res. 24:216-218).
[0276] The polynucleotide sequences of the present invention can be
engineered in order to alter a coding sequence of the invention for
a variety of reasons, including but not limited to, alterations
which modify the cloning, processing and/or expression of the gene
product. For example, alterations may be introduced using
techniques which are well known in the art, e.g., site-directed
mutagenesis, to insert new restriction sites, to alter
glycosylation patterns, to introduce or remove attachment groups
(e.g., for pegylation or other conjugation), to change codon
preference, to introduce splice sites, etc.
[0277] Silent Variations
[0278] Because of the degeneracy of the genetic code, a large
number of functionally identical nucleic acids encode any given
polypeptide. For instance, inspection of the codon table below
(Table 5) shows that codons AGA, AGG, CGA, CGC, CGG, and CGU all
encode the amino acid arginine. Thus, at every position in a
nucleic acid sequence where an arginine is specified by a codon,
the codon can be altered to any of the corresponding codons
described above without altering the encoded polypeptide. Such
nucleic acid variations are "silent variations". It is to be
understood that U in an RNA sequence corresponds to T in a DNA
sequence. TABLE-US-00005 TABLE 5 Codon Table Amino acid Codon(s)
Alanine Ala A GCA GCC GCG GCU Cysteine Cys C UGC UGU Aspartic acid
Asp D GAC GAU Glutamic acid Glu E GAA GAG Phenylalanine Phe F UUC
UUU Glycine Gly G GGA GGC GGG GGU Histidine His H CAC CAU
Isoleucine Ile I AUA AUC AUU Lysine Lys K AAA AAG Leucine Leu L UUA
UUG CUA CUC CUG CUU Methionine Met M AUG Asparagine Asn N AAC AAU
Proline Pro P CCA CCC CCG CCU Glutamine Gln Q CAA CAG Arginine Arg
R AGA AGG CGA CGC CGG CGU Serine Ser S AGC AGU UCA UCC UCG UCU
Threonine Thr T ACA ACC ACG ACU Valine Val V GUA GUC GUG GUU
Tryptophan Trp W UGG Tyrosine Tyr Y UAC UAU
[0279] It will thus be appreciated by those skilled in the art that
due to the degeneracy of the genetic code, a multitude of nucleic
acids sequences encoding polypeptides of the invention may be
produced, some of which may bear minimal sequence identity to the
nucleic acid sequences explicitly disclosed herein. One of ordinary
skill in the art will recognize that each codon in a nucleic acid
(except AUG and UGC, which are ordinarily the only codon for
methionine and tryptophan, respectively) can be modified by
standard techniques to encode a functionally identical polypeptide.
Accordingly, each silent variation of a nucleic acid which encodes
a polypeptide is implicit in any described sequence. The invention
also provides each and every possible variation of a nucleic acid
sequence encoding a polypeptide of the invention that can be made
by selecting combinations based on possible codon choices. These
combinations are made in accordance with the standard triplet
(codon) genetic code (e.g., as set forth in Table 5), as applied to
the nucleic acid sequence encoding a polypeptide of the invention.
All such variations of every nucleic acid herein are specifically
provided and described by consideration of the sequence in
combination with the genetic code. One of skill is fully able to
generate any silent substitution of the sequences listed
herein.
Using Polynucleotides
[0280] The polynucleotides of the invention have a variety of uses
in, for example, recombinant production (i.e., expression) of the
polypeptides of the invention typically through expression of a
plasmid expression vector comprising a sequence encoding the
polypeptide or fragment thereof; as therapeutics; as prophylactics;
as diagnostic tools; as immunogens; as adjuvants; as diagnostic
probes for the presence of complementary or partially complementary
nucleic acids (including for detection of a wild-type
interferon-alpha nucleic acid), as substrates for further
reactions, e.g., recursive sequence recombination reactions or
mutation reactions to produce new and/or improved variants, and the
like.
Vectors, Promoters, and Expression Systems
[0281] The present invention also includes recombinant constructs
comprising one or more of the nucleic acid sequences as broadly
described above. The constructs comprise a vector, such as, a
plasmid, a cosmid, a phage, a virus, a bacterial artificial
chromosome (BAC), a yeast artificial chromosome (YAC), and the
like, into which a nucleic acid sequence of the invention has been
inserted, in a forward or reverse orientation. In some instances,
the construct further comprises regulatory sequences, including,
for example, a promoter, operably linked to the nucleic acid
sequence. Large numbers of suitable vectors and promoters are known
to those of skill in the art, and are commercially available.
[0282] General texts that describe molecular biological techniques
useful herein, including the use of vectors, promoters and many
other relevant topics, include Berger, supra; Sambrook (1989),
supra, and Ausubel, supra. Examples of techniques sufficient to
direct persons of skill through in vitro amplification methods,
including the polymerase chain reaction (PCR) the ligase chain
reaction (LCR), Q.beta.-replicase amplification and other RNA
polymerase mediated techniques (e.g., NASBA), e.g., for the
production of the homologous nucleic acids of the invention are
found in Berger, Sambrook, and Ausubel, all supra, as well as
Mullis et al. (1987) U.S. Pat. No. 4,683,202; PCR Protocols: A
Guide to Methods and Applications (Innis et al., eds.) Academic
Press Inc. San Diego, Calif. (1990) ("Innis"); Arnheim &
Levinson (Oct. 1, 1990) C&EN 36-47; The Journal Of NIH Research
(1991) 3:81-94; (Kwoh et al. (1989) Proc Natl Acad Sci USA
86:1173-1177; Guatelli et al. (1990) Proc Natl Acad Sci USA
87:1874-1878; Lomeli et al. (1989) J Clin Chem 35:1826-1831;
Landegren et al. (1988) Science 241:1077-1080; Van Brunt (1990)
Biotechnology 8:291-294; Wu and Wallace (1989) Gene 4:560-569;
Barringer et al. (1990) Gene 89:117-122, and Sooknanan and Malek
(1995) Biotechnology 13:563-564. Improved methods of cloning in
vitro amplified nucleic acids are described in Wallace et al., U.S.
Pat. No. 5,426,039. Improved methods of amplifying large nucleic
acids by PCR are summarized in Cheng et al. (1994) Nature
369:684-685 and the references therein, in which PCR amplicons of
up to 40 kilobases (kb) are generated. One of skill will appreciate
that essentially any RNA can be converted into a double stranded
DNA suitable for restriction digestion, PCR expansion and
sequencing using reverse transcriptase and a polymerase. See
Ausubel, Sambrook and Berger, all supra.
[0283] The present invention also provides host cells that are
transduced with vectors of the invention, and the production of
polypeptides of the invention by recombinant techniques. Host cells
are genetically engineered (e.g., transduced, transformed or
transfected) with the vectors of this invention, which may be, for
example, a cloning vector or an expression vector. The vector may
be, for example, in the form of a plasmid, a viral particle, a
phage, etc. The engineered host cells can be cultured in
conventional nutrient media modified as appropriate for activating
promoters, selecting transformants, or amplifying genes. 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 those skilled in the art and in the
references cited herein, including, e.g., Freshney (1994) Culture
of Animal Cells, a Manual of Basic Technique, third edition,
Wiley-Liss, New York and the references cited therein.
[0284] The polypeptides of the invention can also be produced in
non-animal cells such as plants, yeast, fungi, bacteria and the
like. In addition to Sambrook, Berger and Ausubel, details
regarding cell culture are found in, e.g., Payne et al. (1992)
Plant Cell and Tissue Culture in Liquid Systems John Wiley &
Sons, Inc. New York, N.Y.; Gamborg and Phillips (eds.) (1995) Plant
Cell, Tissue and Organ Culture; Fundamental Methods Springer Lab
Manual, Springer-Verlag (Berlin Heidelberg N.Y.); Atlas & Parks
(eds.) The Handbook of Microbiological Media (1993) CRC Press, Boca
Raton, Fla.
[0285] The polynucleotides of the present invention and fragments
thereof may be included in any one of a variety of expression
vectors for expressing a polypeptide. Such vectors include
chromosomal, nonchromosomal and synthetic DNA sequences, e.g.,
derivatives of SV40, bacterial plasmids, phage DNA, baculovirus,
yeast plasmids, vectors derived from combinations of plasmids and
phage DNA, viral DNA such as vaccinia, adenovirus, fowl pox virus,
pseudorabies, adeno-associated virus, retroviruses and many others.
Any vector that transduces genetic material into a cell, and, if
replication is desired, which is replicable and viable in the
relevant host can be used.
[0286] The nucleic acid sequence in the expression vector is
operatively linked to an appropriate transcription control sequence
(promoter) to direct mRNA synthesis. Examples of such promoters
include: LTR or SV40 promoter, E. coli lac or trp promoter, phage
lambda P.sub.L promoter, CMV promoter, and other promoters known to
control expression of genes in prokaryotic or eukaryotic cells or
their viruses. The expression vector also contains a ribosome
binding site for translation initiation, and a transcription
terminator. The vector optionally includes appropriate sequences
for amplifying expression, e.g., an enhancer. In addition, the
expression vectors optionally comprise one or more selectable
marker genes to provide a phenotypic trait for selection of
transformed host cells, such as dihydrofolate reductase or neomycin
resistance for eukaryotic cell culture, or such as tetracycline or
ampicillin resistance in E. coli.
[0287] The vector containing the appropriate DNA sequence encoding
a polypeptide of the invention, as well as an appropriate promoter
or control sequence, may be employed to transform an appropriate
host to permit the host to express the polypeptide. Examples of
appropriate expression hosts include: bacterial cells, such as E.
coli, Streptomyces, and Salmonella typhimurium; fungal cells, such
as Saccharomyces cerevisiae, Pichia pastoris, and Neurospora
crassa; insect cells such as Drosophila and Spodoptera frugiperda;
mammalian cells such as CHO, COS, BHK, HEK 293 or Bowes melanoma;
plant cells, etc. It is understood that not all cells or cell lines
need to be capable of producing fully functional polypeptides of
the invention or fragments thereof; for example, antigenic
fragments of the polypeptide may be produced in a bacterial or
other expression system. The invention is not limited by the host
cells employed.
[0288] In bacterial systems, a number of expression vectors may be
selected depending upon the use intended for the polypeptide or
fragment thereof. For example, when large quantities of a
polypeptide or fragments thereof are needed for the induction of
antibodies, vectors which direct high level expression of fusion
proteins that are readily purified may be desirable. Such vectors
include, but are not limited to, multifunctional E. coli cloning
and expression vectors such as BLUESCRIPT (Stratagene), in which
the nucleotide coding sequence may be ligated into the vector
in-frame with sequences for the amino-terminal Met and the
subsequent 7 residues of beta-galactosidase so that a hybrid
protein is produced; pIN vectors (Van Heeke & Schuster (1989) J
Biol Chem 264:5503-5509); pET vectors (Novagen, Madison Wis.); and
the like.
[0289] Similarly, in the yeast Saccharomyces cerevisiae a number of
vectors containing constitutive or inducible promoters such as
alpha factor, alcohol oxidase and PGH may be used for production of
the polypeptides of the invention. For reviews, see Ausubel, supra,
Berger, supra, and Grant et al. (1987) Methods in Enzymology
153:516-544.
[0290] In mammalian host cells, a number of expression systems,
such as viral-based systems, may be utilized. In cases where an
adenovirus is used as an expression vector, a coding sequence is
optionally ligated into an adenovirus transcription/translation
complex consisting of the late promoter and tripartite leader
sequence. Insertion in a nonessential E1 or E3 region of the viral
genome results in a viable virus capable of expressing a
polypeptide of the invention in infected host cells (Logan and
Shenk (1984) Proc Natl Acad Sci USA 81:3655-3659). In addition,
transcription enhancers, such as the rous sarcoma virus (RSV)
enhancer, are used to increase expression in mammalian host cells.
Host cells, media, expression systems, and methods of production
include those known for cloning and expression of various mammalian
interferon-alphas (e.g., human interferon-alphas).
Additional Expression Elements
[0291] Specific initiation signals can aid in efficient translation
of a polynucleotide coding sequence of the invention and/or
fragments thereof. These signals can include, e.g., the ATG
initiation codon and adjacent sequences. In cases where an coding
sequence, its initiation codon and upstream sequences are inserted
into the appropriate expression vector, no additional translational
control signals may be needed. However, in cases where only coding
sequence (e.g., a mature protein coding sequence), or a portion
thereof, is inserted, exogenous nucleic acid transcriptional
control signals including the ATG initiation codon must be
provided. Furthermore, the initiation codon must be in the correct
reading frame to ensure transcription of the entire insert.
Exogenous transcriptional elements and initiation codons can be of
various origins, both natural and synthetic. The efficiency of
expression can enhanced by the inclusion of enhancers appropriate
to the cell system in use (see, e.g., Scharf D. et al. (1994)
Results Probl Cell Differ 20:125-62; and Bittner et al. (1987)
Methods in Enzymol 153:516-544).
Secretion/Localization Sequences
[0292] Polynucleotides encoding polypeptides of the invention can
also be fused, for example, in-frame to nucleic acid encoding a
secretion/localization sequence, to target polypeptide expression
to a desired cellular compartment, membrane, or organelle, or to
direct polypeptide secretion to the periplasmic space or into the
cell culture media. Such sequences are known to those of skill, and
include secretion leader or signal peptides, organelle targeting
sequences (e.g., nuclear localization sequences, ER retention
signals, mitochondrial transit sequences, chloroplast transit
sequences), membrane localization/anchor sequences (e.g., stop
transfer sequences, GPI anchor sequences), and the like.
Expression Hosts
[0293] In a further aspect, the present invention relates to host
cells containing any of the above-described nucleic acids, vectors,
or other constructs of the invention. The host cell can be a
eukaryotic cell, such as a mammalian cell, a yeast cell, or a plant
cell, or the host cell can be a prokaryotic cell, such as a
bacterial cell. Introduction of the construct into the host cell
can be effected by calcium phosphate transfection, DEAE-Dextran
mediated transfection, electroporation, gene or vaccine gun,
injection, or other common techniques (see, e.g., Davis, L.,
Dibner, M., and Battey, I. (1986) Basic Methods in Molecular
Biology) for in vivo, ex vivo or in vitro methods.
[0294] A host cell strain is optionally chosen for its ability to
modulate the expression of the inserted sequences or to process the
expressed protein in the desired fashion. Such modifications of the
protein include, but are not limited to, acetylation,
carboxylation, glycosylation, phosphorylation, lipidation and
acylation. Post-translational processing which cleaves a "pre" or a
"prepro" form of the protein may also be important for correct
insertion, folding and/or function. Different host cells such as E.
coli, Bacillus sp., yeast or mammalian cells such as CHO, HeLa,
BHK, MDCK, HEK 293, W138, etc. have specific cellular machinery and
characteristic mechanisms for such post-translational activities
and may be chosen to ensure the correct modification and processing
of the introduced foreign protein.
[0295] Stable expression can be used for long-term, high-yield
production of recombinant proteins. For example, cell lines which
stably express a polypeptide of the invention are transduced using
expression vectors which contain viral origins of replication or
endogenous expression elements and a selectable marker gene.
Following the introduction of the vector, cells may be allowed to
grow for 1-2 days in an enriched media before they are switched to
selective media. The purpose of the selectable marker is to confer
resistance to selection, and its presence allows growth and
recovery of cells which successfully express the introduced
sequences. For example, resistant clumps of stably transformed
cells can be proliferated using tissue culture techniques
appropriate to the cell type.
[0296] Host cells transformed with a nucleotide sequence encoding a
polypeptide of the invention are optionally cultured under
conditions suitable for the expression and recovery of the encoded
protein from cell culture. The polypeptide produced by a
recombinant cell may be secreted, membrane-bound, or contained
intracellularly, depending on the sequence and/or the vector used.
As will be understood by those of skill in the art, expression
vectors containing polynucleotides encoding polypeptides of the
invention can be designed with signal sequences which direct
secretion of the mature polypeptides through a prokaryotic or
eukaryotic cell membrane.
Additional Sequences
[0297] The polynucleotides of the present invention optionally
comprise a coding sequence fused in-frame to a marker sequence
which, e.g., facilitates purification and/or detection of the
encoded polypeptide. Such purification subsequences include, but
are not limited to, metal chelating peptides such as
histidine-tryptophan modules that allow purification on immobilized
metals, a sequence which binds glutathione (e.g., GST), a
hemagglutinin (HA) tag (corresponding to an epitope derived from
the influenza hemagglutinin protein; Wilson, I. et al. (1984) Cell
37:767), maltose binding protein sequences, the FLAG epitope
utilized in the FLAGS extension/affinity purification system, and
the like. The inclusion of a protease-cleavable polypeptide linker
sequence between the purification domain and the polypeptide
sequence is useful to facilitate purification.
[0298] For example, one expression vector possible to use in the
compositions and methods described herein provides for expression
of a fusion protein comprising a polypeptide of the invention fused
to a polyhistidine region separated by an enterokinase cleavage
site. The histidine residues facilitate purification on IMIAC
(immobilized metal ion affinity chromatography, as described in
Porath et al. (1992) Protein Expression and Purification 3:263-281)
while the enterokinase cleavage site provides a method for
separating the desired polypeptide from the polyhistidine region.
pGEX vectors (Promega; Madison, Wis.) are optionally used to
express foreign polypeptides as fusion proteins with glutathione
S-transferase (GST). In general, such fusion proteins are soluble
and can easily be purified from lysed cells by adsorption to
ligand-agarose beads (e.g., glutathione-agarose in the case of
GST-fusions) followed by elution in the presence of free
ligand.
[0299] An additional construction in the compositions and methods
described herein provides for proteins, and their encoding nucleic
acids, comprising polypeptides of the invention (or one or more
fragments thereof), e.g., as described herein, fused to an Ig
molecule, e.g., human IgG Fc ("fragment crystallizable," or
fragment complement binding) hinge, CH2 domain and CH3 domain (and
nucleotide sequences encoding them). Fc is the portion of the
antibody responsible for binding to antibody receptors on cells and
the C1q component of complement. These fusion proteins or fragments
thereof and their encoding nucleic acids are optionally useful as
prophylactic and/or therapeutic drugs or as diagnostic tools (see
also, e.g., Challita-Eid, P. et al. (1998) J Immunol 160:3419-3426;
Sturmhoefel, K. et al. (1999) Cancer Res 59:4964-4972).
Polypeptide Production and Recovery
[0300] Following transduction of a suitable host strain and growth
of the host strain to an appropriate cell density, the selected
promoter is induced by appropriate means (e.g., temperature shift
or chemical induction) and cells are cultured for an additional
period. Cells are typically harvested by centrifugation, disrupted
by physical or chemical means, and the resulting crude extract
retained for further purification. Eukaryotic or microbial cells
employed in expression of the proteins can be disrupted by any
convenient method, including freeze-thaw cycling, sonication,
mechanical disruption, or use of cell lysing agents, or other
methods, which are well know to those skilled in the art.
[0301] As noted, many references are available for the culture and
production of many cells, including cells of bacterial, plant,
animal (especially mammalian) and archebacterial origin. See, e.g.,
Sambrook, Ausubel, and Berger (all supra), as well as Freshney
(1994) Culture of Animal Cells, a Manual of Basic Technique, third
edition, Wiley-Liss, New York and the references cited therein;
Doyle and Griffiths (1997) Mammalian Cell Culture: Essential
Techniques John Wiley and Sons, NY; Humason (1979) Animal Tissue
Techniques, fourth edition W.H. Freeman and Company; and
Ricciardelli et al. (1989) In vitro Cell Dev Biol 25:1016-1024. For
plant cell culture and regeneration see, e.g., Payne et al. (1992)
Plant Cell and Tissue Culture in Liquid Systems John Wiley &
Sons, Inc. New York, N.Y.; Gamborg and Phillips (eds.) (1995) Plant
Cell, Tissue and Organ Culture; Fundamental Methods Springer Lab
Manual, Springer-Verlag (Berlin Heidelberg N.Y.) and Plant
Molecular Biology (1993) R. R. D. Croy (ed.) Bios Scientific
Publishers, Oxford, U.K. ISBN 0 12 198370 6. Cell culture media in
general are set forth in Atlas and Parks (eds.) The Handbook of
Microbiological Media (1993) CRC Press, Boca Raton, Fla. Additional
information for cell culture is found in available commercial
literature such as the Life Science Research Cell Culture Catalogue
from Sigma-Aldrich, Inc (St Louis, Mo.) ("Sigma-LSRCCC") and, e.g.,
the Plant Culture Catalogue and supplement also from Sigma-Aldrich,
Inc (St Louis, Mo.) ("Sigma-PCCS").
[0302] Polypeptides of the invention can be recovered and purified
from recombinant cell cultures by any of a number of methods well
known in the art, including ammonium sulfate or ethanol
precipitation, acid extraction, anion or cation exchange
chromatography, phosphocellulose chromatography, hydrophobic
interaction chromatography, affinity chromatography (e.g., using
any of the tagging systems noted herein), hydroxylapatite
chromatography, and lectin chromatography. Protein refolding steps
can be used, as desired, in completing configuration of the mature
protein or fragments thereof. Finally, high performance liquid
chromatography (HPLC) can be employed in the final purification
steps. In addition to the references noted, supra, a variety of
purification methods are well known in the art, including, e.g.,
those set forth in Sandana (1997) Bioseparation of Proteins,
Academic Press, Inc.; Bollag et al. (1996) Protein Methods,
2.sup.nd Edition Wiley-Liss, NY; Walker (1996) The Protein
Protocols Handbook Humana Press, NJ; Harris and Angal (1990)
Protein Purification Applications: A Practical Approach IRL Press
at Oxford, Oxford, England; Harris and Angal Protein Purification
Methods: A Practical Approach IRL Press at Oxford, Oxford, England;
Scopes (1993) Protein Purification: Principles and Practice
3.sup.rd Edition Springer Verlag, NY; Janson and Ryden (1998)
Protein Purification: Principles, High Resolution Methods and
Applications, Second Edition Wiley-VCH, NY; and Walker (1998)
Protein Protocols on CD-ROM Humana Press, NJ.
In vitro Expression Systems
[0303] Cell-free transcription/translation systems can also be
employed to produce polypeptides of the invention using
polynucleotides of the present invention. Several such systems are
commercially available. A general guide to in vitro transcription
and translation protocols is found in Tymms (1995) In vitro
Transcription and Translation Protocols: Methods in Molecular
Biology Volume 37, Garland Publishing, NY.
In vivo Uses and Applications
[0304] Polynucleotides that encode a polypeptide of the invention,
or complements of the polynucleotides (including e.g., antisense or
ribozyme molecules), are optionally administered to a cell to
accomplish a therapeutically useful process or to express a
therapeutically useful product. These in vivo applications,
including gene therapy, include a multitude of techniques by which
gene expression may be altered in cells. Such methods include, for
instance, the introduction of genes for expression of, e.g.,
therapeutically and/or prophylactically useful polypeptides, such
as the polypeptides of the present invention.
In vivo Polypeptide Expression
[0305] Polynucleotides encoding polypeptides of the invention are
particularly useful for in vivo therapeutic applications, using
techniques well known to those skilled in the art. For example,
cultured cells are engineered ex vivo with at least one
polynucleotide (DNA or RNA) of the invention and/or other
polynucleotide sequences encoding, e.g., at least one of an
antigen, cytokine, other co-stimulatory molecule, adjuvant, etc.,
and the like, with the engineered cells then being returned to the
patient. Cells may also be engineered in vivo for expression of one
or more polypeptides in vivo. including polypeptides and/or
antigenic peptides of the invention.
[0306] A number of viral vectors suitable for organismal in vivo
transduction and expression are known. Such vectors include
retroviral vectors (see, e.g., Miller, Curr Top Microbiol Immunol
(1992) 158:1-24; Salmons and Gunzburg (1993) Human Gene Therapy
4:129-141; Miller et al. (1994) Methods in Enzymology 217:581-599)
and adeno-associated vectors (reviewed in Carter (1992) Curr
Opinion Biotech 3:533-539; Muzcyzka (1992) Curr Top Microbiol
Immunol. 158:97-129). Other viral vectors that are used include
adenoviral vectors, herpes viral vectors and Sindbis viral vectors,
as generally described in, e.g., Jolly (1994) Cancer Gene Therapy
1:51-64; Latchman (1994) Molec Biotechnol 2:179-195; and Johanning
et al. (1995) Nucl Acids Res 23:1495-1501.
[0307] In one aspect, a pox virus vector can be used. The pox viral
vector is transfected with a polynucleotide sequence encoding a
polypeptide of the invention, and is useful in prophylactic,
therapeutic and diagnostic applications where enhancement of an
immune response, such as e.g., increased or improved T cell
proliferation is desired. See viral vectors discussed in, e.g.,
Berencsi et al., J Infect Dis (2001) 183(8):1171-9; Rosenwirth et
al., Vaccine 2001 Feb. 8; 19 (13-14):1661-70; Kittlesen et al., J
Immunol (2000) 164(8):4204-11; Brown et al. Gene Ther 2000
7(19):1680-9; Kanesa-thasan et al., Vaccine (2000) 19 (4-5):483-91;
Sten (2000) Drug 60(2):249-71. Compositions comprising such vectors
and an acceptable excipient are also a feature of the
invention.
[0308] Gene therapy and genetic vaccines provide methods for
combating chronic infectious diseases (e.g., HIV infection, viral
hepatitis), as well as non-infectious diseases including cancer and
some forms of congenital defects such as enzyme deficiencies, and
such methods can be employed with polynucleotides of the invention,
including, e.g., vectors and cells comprising such polynucleotides.
Several approaches for introducing nucleic acids and vectors into
cells in vivo, ex vivo and in vitro have been used and can be
employed with polynucleotides of the invention, and vectors
comprising such polynucleotides. These approaches include liposome
based gene delivery (Debs and Zhu (1993) WO 93/24640 and U.S. Pat.
No. 5,641,662; Mannino and Gould-Fogerite (1988) BioTechniques
6(7):682-691; Rose, U.S. Pat. No. 5,279,833; Brigham (1991) WO
91/06309; and Felgner et al. (1987) Proc Natl Acad Sci USA
84:7413-7414; Brigham et al. (1989) Am J Med Sci 298:278-281; Nabel
et al. (1990) Science 249:1285-1288; Hazinski et al. (1991) Am J
Resp Cell Molec Biol 4:206-209; and Wang and Huang (1987) Proc Natl
Acad Sci USA 84:7851-7855); adenoviral vector mediated gene
delivery, e.g., to treat cancer (see, e.g., Chen et al. (1994) Proc
Natl Acad Sci USA 91:3054-3057; Tong et al. (1996) Gynecol Oncol
61:175-179; Clayman et al. (1995) Cancer Res. 5:1-6; O'Malley et
al. (1995) Cancer Res 55:1080-1085; Hwang et al. (1995) Am J Respir
Cell Mol Biol 13:7-16; Haddada et al. (1995) Curr Top Microbiol
Immunol. 1995 (Pt. 3):297-306; Addison et al. (1995) Proc Natl Acad
Sci USA 92:8522-8526; Colak et al. (1995) Brain Res 691:76-82;
Crystal (1995) Science 270:404-410; Elshami et al. (1996) Human
Gene Ther 7:141-148; Vincent et al. (1996) J Neurosurg 85:648-654),
and many others. Replication-defective retroviral vectors harboring
therapeutic polynucleotide sequence as part of the retroviral
genome have also been used, particularly with regard to simple MuLV
vectors. See, e.g., Miller et al. (1990) Mol Cell Biol 10:4239
(1990); Kolberg (1992) J NIH Res 4:43, and Cornetta et al. (1991)
Hum Gene Ther 2:215). Nucleic acid transport coupled to
ligand-specific, cation-based transport systems (Wu and Wu (1988) J
Biol Chem, 263:14621-14624) has also been used. Naked DNA
expression vectors have also been described (Nabel et al. (1990),
supra); Wolff et al. (1990) Science, 247:1465-1468). In general,
these approaches can be adapted to the invention by incorporating
nucleic acids encoding the polypeptides of the invention into the
appropriate vectors.
[0309] General texts which describe gene therapy protocols, which
can be adapted to the present invention by introducing the nucleic
acids of the invention into patients, include, e.g., Robbins (1996)
Gene Therapy Protocols, Humana Press, NJ, and Joyner (1993) Gene
Targeting: A Practical Approach, IRL Press, Oxford, England.
Antisense Technology
[0310] In addition to expression of the nucleic acids of the
invention as gene replacement nucleic acids, the nucleic acids are
also useful for sense and anti-sense suppression of expression,
e.g., to down-regulate expression of a nucleic acid of the
invention, once, or when, expression of the nucleic acid is
no-longer desired in the cell. Similarly, the nucleic acids of the
invention, or subsequences or anti-sense sequences thereof, can
also be used to block expression of naturally occurring homologous
nucleic acids. A variety of sense and anti-sense technologies are
known in the art, e.g., as set forth in Lichtenstein and Nellen
(1997) Antisense Technology: A Practical Approach IRL Press at
Oxford University, Oxford, England, and in Agrawal (1996) Antisense
Therapeutics Humana Press, NJ, and the references cited
therein.
Use as Probes
[0311] Also contemplated are uses of polynucleotides, also referred
to herein as oligonucleotides, typically having at least 12 bases,
preferably at least 15, more preferably at least 20, at least 30,
or at least 50 or more bases, which hybridize under highly
stringent conditions to a polynucleotide of the invention, or
fragments thereof. The polynucleotides may be used as probes,
primers, sense and antisense agents, and the like, according to
methods as noted supra.
Nucleic Acid Hybridization
[0312] Nucleic acids "hybridize" when they associate, typically in
solution. Nucleic acids hybridize due to a variety of well
characterized physico-chemical forces, such as hydrogen bonding,
solvent exclusion, base stacking and the like. An extensive guide
to the hybridization of nucleic acids is found in Tijssen (1993)
Laboratory Techniques in Biochemistry and Molecular
Biology--Hybridization with Nucleic Acid Probes, part I, chapter 2,
"Overview of principles of hybridization and the strategy of
nucleic acid probe assays," (Elsevier, N.Y.) (hereinafter
"Tjissen"), as well as in Ausubel, supra, Hames and Higgins (1995)
Gene Probes 1, IRL Press at Oxford University Press, Oxford,
England (Hames and Higgins 1) and Hames and Higgins (1995) Gene
Probes 2, IRL Press at Oxford University Press, Oxford, England
(Hames and Higgins 2) provide details on the synthesis, labeling,
detection and quantification of DNA and RNA, including
oligonucleotides.
[0313] An indication that two nucleic acid sequences are
substantially identical is that the two molecules hybridize to each
other under at least stringent conditions. The phrase "hybridizing
specifically to," refers to the binding, duplexing, or hybridizing
of a molecule only to a particular nucleotide sequence under
stringent conditions when that sequence is present in a complex
mixture (e.g., total cellular) DNA or RNA. "Bind(s) substantially"
refers to complementary hybridization between a probe nucleic acid
and a target nucleic acid and embraces minor mismatches that can be
accommodated by reducing the stringency of the hybridization media
to achieve the desired detection of the target polynucleotide
sequence.
[0314] "Stringent hybridization wash conditions" and "stringent
hybridization conditions" in the context of nucleic acid
hybridization experiments, such as Southern and northern
hybridizations, are sequence dependent, and are different under
different environmental parameters. An extensive guide to
hybridization of nucleic acids is found in Tijssen (1993), supra,
and in Hames and Higgins 1 and Hames and Higgins 2, supra.
[0315] For purposes of the present invention, generally, "highly
stringent" hybridization and wash conditions are selected to be
about 5.degree. C. or less lower than the thermal melting point
(T.sub.m) for the specific sequence at a defined ionic strength and
pH (as noted below, highly stringent conditions can also be
referred to in comparative terms). The T.sub.m is the temperature
(under defined ionic strength and pH) at which 50% of the test
sequence hybridizes to a perfectly matched probe. In other words,
the T.sub.m indicates the temperature at which the nucleic acid
duplex is 50% denatured under the given conditions and its
represents a direct measure of the stability of the nucleic acid
hybrid. Thus, the T.sub.m corresponds to the temperature
corresponding to the midpoint in transition from helix to random
coil; it depends on length, nucleotide composition, and ionic
strength for long stretches of nucleotides. Typically, under
"stringent conditions," a probe will hybridize to its target
subsequence, but to no other sequences. "Very stringent conditions"
are selected to be equal to the T.sub.m for a particular probe.
[0316] After hybridization, unhybridized nucleic acid material can
be removed by a series of washes, the stringency of which can be
adjusted depending upon the desired results. Low stringency washing
conditions (e.g., using higher salt and lower temperature) increase
sensitivity, but can product nonspecific hybridization signals and
high background signals. Higher stringency conditions (e.g., using
lower salt and higher temperature that is closer to the
hybridization temperature) lowers the background signal, typically
with only the specific signal remaining. See, Rapley, R. and
Walker, J. M. eds., Molecular Biomethods Handbook (Humana Press,
Inc. 1998) (hereinafter "Rapley and Walker"), which is incorporated
herein by reference in its entirety for all purposes.
[0317] The T.sub.m of a DNA-DNA duplex can be estimated using
equation (1): T.sub.m(.degree. C.)=81.5.degree.
C.+16.6(log.sub.10M)+0.41(% G+C)-0.72(% f)-500/n, where M is the
molarity of the monovalent cations (usually Na+), (% G+C) is the
percentage of guanosine (G) and cystosine (C) nucleotides, (% f) is
the percentage of formalize and n is the number of nucleotide bases
(i.e., length) of the hybrid. See, Rapley and Walker, supra.
[0318] The T.sub.m of an RNA-DNA duplex can be estimated using
equation (2): T.sub.m(.degree. C.)=79.8.degree. C.+18.5
(log.sub.10M)+0.58(% G+C)-11.8(% G+C)-0.56(% f)-820/n, where M is
the molarity of the monovalent cations (usually Na+), (% G+C) is
the percentage of guanosine (G) and cystosine (C) nucleotides, (%
f) is the percentage of formamide and n is the number of nucleotide
bases (i.e., length) of the hybrid. Id. Equations 1 and 2 above are
typically accurate only for hybrid duplexes longer than about
100-200 nucleotides. Id.
[0319] The T.sub.m of nucleic acid sequences shorter than 50
nucleotides can be calculated as follows: T.sub.m(.degree.
C.)=4(G+C)+2(A+T), where A (adenine), C, T (thymine), and G are the
numbers of the corresponding nucleotides.
[0320] An example of stringent hybridization conditions for
hybridization of complementary nucleic acids which have more than
100 complementary residues on a filter in a Southern or northern
blot is 50% formalin (or formamide) with 1 mg of heparin at
42.degree. C., with the hybridization being carried out overnight.
An example of stringent wash conditions is a 0.2.times.SSC wash at
65.degree. C. for 15 minutes (see Sambrook, supra, for a
description of SSC buffer). Often, the high stringency wash is
preceded by a low stringency wash to remove background probe
signal. An example low stringency wash is 2.times.SSC at 40.degree.
C. for 15 minutes. An example of highly stringent wash conditions
is 0.15M NaCl at 72.degree. C. for about 15 minutes. An example
medium stringency wash for a duplex of, e.g., more than 100
nucleotides, is 1.times.SSC at 45.degree. C. for 15 minutes. An
example low stringency wash for a duplex of, e.g., more than 100
nucleotides, is 4-6.times.SSC at 40.degree. C. for 15 minutes. For
short probes (e.g., about 10 to 50 nucleotides), stringent
conditions typically involve salt concentrations of less than about
1.0 M Na.sup.+ ion, typically about 0.01 to 1.0 M Na.sup.+ ion
concentration (or other salts) at pH 7.0 to 8.3, and the
temperature is typically at least about 30.degree. C. Stringent
conditions can also be achieved with the addition of destabilizing
agents such as formamide.
[0321] In general, a signal to noise ratio of 2.times. or
2.5.times.-5.times. (or higher) than that observed for an unrelated
probe in the particular hybridization assay indicates detection of
a specific hybridization. Detection of at least stringent
hybridization between two sequences in the context of the present
invention indicates relatively strong structural similarity or
homology to, e.g., the nucleic acids of the present invention
provided in the sequence listings herein.
[0322] As noted, "highly stringent" conditions are selected to be
about 5.degree. C. or less lower than the thermal melting point
(T.sub.m) for the specific sequence at a defined ionic strength and
pH. Target sequences that are closely related or identical to the
nucleotide sequence of interest (e.g., "probe") can be identified
under highly stringency conditions. Lower stringency conditions are
appropriate for sequences that are less complementary. See, e.g.,
Rapley and Walker; Sambrook, all supra.
[0323] Comparative hybridization can be used to identify nucleic
acids of the invention, and this comparative hybridization method
is a preferred method of distinguishing nucleic acids of the
invention. Detection of highly stringent hybridization between two
nucleotide sequences in the context of the present invention
indicates relatively strong structural similarity/homology to,
e.g., the nucleic acids provided in the sequence listing herein.
Highly stringent hybridization between two nucleotide sequences
demonstrates a degree of similarity or homology of structure,
nucleotide base composition, arrangement or order that is greater
than that detected by stringent hybridization conditions. In
particular, detection of highly stringent hybridization in the
context of the present invention indicates strong structural
similarity or structural homology (e.g., nucleotide structure, base
composition, arrangement or order) to, e.g., the nucleic acids
provided in the sequence listings herein. For example, it is
desirable to identify test nucleic acids which hybridize to the
exemplar nucleic acids herein under stringent conditions.
[0324] Thus, one measure of stringent hybridization is the ability
to hybridize to one of the listed nucleic acids of the invention
(e.g., nucleic acid sequences SEQ ID NOS:16-30, and complementary
polynucleotide sequences thereof) under highly stringent conditions
(or very stringent conditions, or ultra-high stringency
hybridization conditions, or ultra-ultra high stringency
hybridization conditions). Stringent hybridization (including,
e.g., highly stringent, ultra-high stringency, or ultra-ultra high
stringency hybridization conditions) and wash conditions can easily
be determined empirically for any test nucleic acid.
[0325] For example, in determining highly stringent hybridization
and wash conditions, the hybridization and wash conditions are
gradually increased (e.g., by increasing temperature, decreasing
salt concentration, increasing detergent concentration and/or
increasing the concentration of organic solvents, such as formalin,
in the hybridization or wash), until a selected set of criteria are
met. For example, the hybridization and wash conditions are
gradually increased until a probe comprising one or more nucleic
acid sequences selected from SEQ ID NOS:16-30, and complementary
polynucleotide sequences thereof, binds to a perfectly matched
complementary target (again, a nucleic acid comprising one or more
nucleic acid sequences selected from SEQ ID NOS:16-30, and
complementary polynucleotide sequences thereof), with a signal to
noise ratio that is at least 2.5.times., and optionally 5.times. or
more as high as that observed for hybridization of the probe to an
unmatched target. In this case, the unmatched target is a nucleic
acid corresponding to, e.g., a known interferon-alpha nucleic acid
sequence (e.g., an interferon-alpha nucleic acid sequence present
in a public database such as GenBank or GENESEQ at the time of
filing of the subject application).
[0326] A test nucleic acid is said to specifically hybridize to a
probe nucleic acid when it hybridizes at least 1/2 as well to the
probe as to the perfectly matched complementary target, i.e., with
a signal to noise ratio at least 1/2 as high as hybridization of
the probe to the target under conditions in which the perfectly
matched probe binds to the perfectly matched complementary target
with a signal to noise ratio that is at least about
2.5.times.-10.times., typically 5.times.-10.times. as high as that
observed for hybridization to any of the unmatched target nucleic
acids such as, e.g., a known interferon-alpha nucleic acid sequence
as set forth above. For some such nucleic acids, the stringent
conditions are selected such that a perfectly complementary
oligonucleotide to the coding oligonucleotide hybridizes to the
coding oligonucleotide with at least about a 5.times. higher signal
to noise ratio than for hybridization of the perfectly
complementary oligonucleotide to a control nucleic acid
corresponding to a known interferon-alpha sequence as set forth
above.
[0327] Ultra high-stringency hybridization and wash conditions are
those in which the stringency of hybridization and wash conditions
are increased until the signal to noise ratio for binding of the
probe to the perfectly matched complementary target nucleic acid is
at least 10.times. as high as that observed for hybridization to
any of the unmatched target nucleic acids, such as, e.g., a known
interferon-alpha nucleic acid sequence as set forth above. A target
nucleic acid which hybridizes to a probe under such conditions,
with a signal to noise ratio of at least 1/2 that of the perfectly
matched complementary target nucleic acid is said to bind to the
probe under ultra-high stringency conditions.
[0328] Similarly, even higher levels of stringency can be
determined by gradually increasing the hybridization and/or wash
conditions of the relevant hybridization assay. For example, those
in which the stringency of hybridization and wash conditions are
increased until the signal to noise ratio for binding of the probe
to the perfectly matched complementary target nucleic acid is at
least 10.times., 20.times., 50.times., 100.times., or 500.times. or
more as high as that observed for hybridization to any of the
unmatched target nucleic acids, such as, e.g., a known
interferon-alpha nucleic acid sequence as set forth above. A target
nucleic acid which hybridizes to a probe under such conditions,
with a signal to noise ratio of at least 1/2 that of the perfectly
matched complementary target nucleic acid is said to bind to the
probe under ultra-ultra-high stringency conditions.
[0329] Target nucleic acids which hybridize to the nucleic acids
represented by SEQ ID NOS:16-30 under high, ultra-high and
ultra-ultra high stringency conditions are a feature of the
invention. Examples of such nucleic acids include those with one or
a few silent or conservative nucleic acid substitutions as compared
to a given nucleic acid sequence.
Substrates and Formats for Sequence Recombination
[0330] The polynucleotides of the invention and fragments thereof
are optionally used as substrates for any of a variety of
recombination and recursive sequence recombination reactions, in
addition to their use in standard cloning methods as set forth in,
e.g., Ausubel, Berger and Sambrook, e.g., to produce additional
polynucleotides that encode polypeptides having desired properties.
A variety of such reactions are known, including those developed by
the inventors and their co-workers.
[0331] A variety of diversity generating protocols for generating
and identifying molecules having one of more of the properties
described herein are available and described in the art. The
procedures can be used separately, and/or in combination to produce
one or more variants of a nucleic acid or set of nucleic acids, as
well variants of encoded proteins. Individually and collectively,
these procedures provide robust, widely applicable ways of
generating diversified nucleic acids and sets of nucleic acids
(including, e.g., nucleic acid libraries) useful, e.g., for the
engineering or rapid evolution of nucleic acids, proteins,
pathways, cells and/or organisms with new and/or improved
characteristics. While distinctions and classifications are made in
the course of the ensuing discussion for clarity, it will be
appreciated that the techniques are often not mutually exclusive.
Indeed, the various methods can be used singly or in combination,
in parallel or in series, to access diverse sequence variants.
[0332] The result of any of the diversity generating procedures
described herein can be the generation of one or more nucleic
acids, which can be selected or screened for nucleic acids with or
which confer desirable properties, or that encode proteins with or
which confer desirable properties. Following diversification by one
or more of the methods herein, or otherwise available to one of
skill, any nucleic acids that are produced can be selected for a
desired activity or property, e.g., altered binding affinity for an
interferon-alpha receptor, altered antiviral or antiproliferative
activity, altered capacities to induce T.sub.H1 differentiation,
altered abilities to induce or inhibit cytokine production. This
can include identifying any activity that can be detected, for
example, in an automated or automatable format, by any of the
assays in the art and the assays of the invention discussed here
and in the Example section below. A variety of related (or even
unrelated) properties can be evaluated, in serial or in parallel,
at the discretion of the practitioner.
[0333] Descriptions of a variety of diversity generating procedures
for generating modified nucleic acid sequences that encode
polypeptides as described herein are found in the following
publications and the references cited therein: Soong, N. et al.
(2000) "Molecular breeding of viruses" Nat Genet 25(4):436-439;
Stemmer, et al. (1999) "Molecular breeding of viruses for targeting
and other clinical properties" Tumor Targeting 4:1-4; Ness et al.
(1999) "DNA Shuffling of subgenomic sequences of subtilisin" Nature
Biotechnology 17:893-896; Chang et al. (1999) "Evolution of a
cytokine using DNA family shuffling" Nature Biotechnology
17:793-797; Minshull and Stemmer (1999) "Protein evolution by
molecular breeding" Current Opinion in Chemical Biology 3:284-290;
Christians et al. (1999) "Directed evolution of thymidine kinase
for AZT phosphorylation using DNA family shuffling" Nature
Biotechnology 17:259-264; Crameri et al. (1998) "DNA shuffling of a
family of genes from diverse species accelerates directed
evolution" Nature 391:288-291; Crameri et al. (1997) "Molecular
evolution of an arsenate detoxification pathway by DNA shuffling,"
Nature Biotechnology 15:436-438; Zhang et al. (1997) "Directed
evolution of an effective fucosidase from a galactosidase by DNA
shuffling and screening" Proc. Natl. Acad. Sci. USA 94:4504-4509;
Patten et al. (1997) "Applications of DNA Shuffling to
Pharmaceuticals and Vaccines" Current Opinion in Biotechnology
8:724-733; Crameri et al. (1996) "Construction and evolution of
antibody-phage libraries by DNA shuffling" Nature Medicine
2:100-103; Crameri et al. (1996) "Improved green fluorescent
protein by molecular evolution using DNA shuffling" Nature
Biotechnology 14:315-319; Gates et al. (1996) "Affinity selective
isolation of ligands from peptide libraries through display on a
lac repressor `headpiece dimer`" Journal of Molecular Biology
255:373-386; Stemmer (1996) "Sexual PCR and Assembly PCR" In: The
Encyclopedia of Molecular Biology. VCH Publishers, New York. pp.
447-457; Crameri and Stemmer (1995) "Combinatorial multiple
cassette mutagenesis creates all the permutations of mutant and
wildtype cassettes" BioTechniques 18:194-195; Stemmer et al.,
(1995) "Single-step assembly of a gene and entire plasmid form
large numbers of oligodeoxy-ribonucleotides" Gene, 164:49-53;
Stemmer (1995) "The Evolution of Molecular Computation" Science
270: 1510; Stemmer (1995) "Searching Sequence Space" Bio/Technology
13:549-553; Stemmer (1994) "Rapid evolution of a protein in vitro
by DNA shuffling" Nature 370:389-391; and Stemmer (1994) "DNA
shuffling by random fragmentation and reassembly: In vitro
recombination for molecular evolution." Proc. Natl. Acad. Sci. USA
91:10747-10751.
[0334] The term "shuffling" is used herein to indicate
recombination between non-identical sequences, in some instances
shuffling may include crossover via homologous recombination or via
non-homologous recombination, such as via cre/lox and/or flp/frt
systems. Shuffling can be carried out by employing a variety of
different formats, including for example, in vitro and in vivo
shuffling formats, in silico shuffling formats, shuffling formats
that utilize either double-stranded or single-stranded templates,
primer based shuffling formats, nucleic acid fragmentation-based
shuffling formats, and oligonucleotide-mediated shuffling formats,
all of which are based on recombination events between
non-identical sequences and are described in more detail or
referenced herein below, as well as other similar
recombination-based formats.
[0335] Mutational methods of generating diversity include, for
example, site-directed mutagenesis (Ling et al. (1997) "Approaches
to DNA mutagenesis: an overview" Anal Biochem. 254(2): 157-178;
Dale et al. (1996) "Oligonucleotide-directed random mutagenesis
using the phosphorothioate method" Methods Mol. Biol. 57:369-374;
Smith (1985) "In vitro mutagenesis" Ann. Rev. Genet. 19:423-462;
Botstein & Shortle (1985) "Strategies and applications of in
vitro mutagenesis" Science 229:1193-1201; Carter (1986)
"Site-directed mutagenesis" Biochem. J. 237:1-7; and Kunkel (1987)
"The efficiency of oligonucleotide directed mutagenesis" in Nucleic
Acids & Molecular Biology (Eckstein, F. and Lilley, D. M. J.
eds., Springer Verlag, Berlin)); mutagenesis using uracil
containing templates (Kunkel (1985) "Rapid and efficient
site-specific mutagenesis without phenotypic selection" Proc. Natl.
Acad. Sci. USA 82:488-492; Kunkel et al. (1987) "Rapid and
efficient site-specific mutagenesis without phenotypic selection"
Methods in Enzymol. 154, 367-382; and Bass et al. (1988) "Mutant
Trp repressors with new DNA-binding specificities" Science
242:240-245); oligonucleotide-directed mutagenesis (Methods in
Enzymol. 100: 468-500 (1983); Methods in Enzymol. 154: 329-350
(1987); Zoller & Smith (1982) "Oligonucleotide-directed
mutagenesis using M13-derived vectors: an efficient and general
procedure for the production of point mutations in any DNA
fragment" Nucleic Acids Res. 10:6487-6500; Zoller & Smith
(1983) "Oligonucleotide-directed mutagenesis of DNA fragments
cloned into M13 vectors" Methods in Enzymol. 100:468-500; and
Zoller & Smith (1987) "Oligonucleotide-directed mutagenesis: a
simple method using two oligonucleotide primers and a
single-stranded DNA template" Methods in Enzymol. 154:329-350);
phosphorothioate-modified DNA mutagenesis (Taylor et al. (1985)
"The use of phosphorothioate-modified DNA in restriction enzyme
reactions to prepare nicked DNA" Nucl. Acids Res. 13: 8749-8764;
Taylor et al. (1985) "The rapid generation of
oligonucleotide-directed mutations at high frequency using
phosphorothioate-modified DNA" Nucl. Acids Res. 13: 8765-8787
(1985); Nakamaye & Eckstein (1986) "Inhibition of restriction
endonuclease Nci I cleavage by phosphorothioate groups and its
application to oligonucleotide-directed mutagenesis" Nucl. Acids
Res. 14: 9679-9698; Sayers et al. (1988) "Y-T Exonucleases in
phosphorothioate-based oligonucleotide-directed mutagenesis" Nucl.
Acids Res. 16:791-802; and Sayers et al. (1988) "Strand specific
cleavage of phosphorothioate-containing DNA by reaction with
restriction endonucleases in the presence of ethidium bromide"
Nucl. Acids Res. 16: 803-814); mutagenesis using gapped duplex DNA
(Kramer et al. (1984) "The gapped duplex DNA approach to
oligonucleotide-directed mutation construction" Nucl. Acids Res.
12: 9441-9456; Kramer & Fritz (1987) Methods in Enzymol.
"Oligonucleotide-directed construction of mutations via gapped
duplex DNA" 154:350-367; Kramer et al. (1988) "Improved enzymatic
in vitro reactions in the gapped duplex DNA approach to
oligonucleotide-directed construction of mutations" Nucl. Acids
Res. 16: 7207; and Fritz et al. (1988) "Oligonucleotide-directed
construction of mutations: a gapped duplex DNA procedure without
enzymatic reactions in vitro" Nucl. Acids Res. 16: 6987-6999).
[0336] Additional suitable methods include point mismatch repair
(Kramer et al. (1984) "Point Mismatch Repair" Cell 38:879-887),
mutagenesis using repair-deficient host strains (Carter et al.
(1985) "Improved oligonucleotide site-directed mutagenesis using
M13 vectors" Nucl. Acids Res. 13: 4431-4443; and Carter (1987)
"Improved oligonucleotide-directed mutagenesis using M13 vectors"
Methods in Enzymol. 154: 382-403), deletion mutagenesis
(Eghtedarzadeh & Henikoff (1986) "Use of oligonucleotides to
generate large deletions" Nucl. Acids Res. 14: 5115),
restriction-selection and restriction-purification (Wells et al.
(1986) "Importance of hydrogen-bond formation in stabilizing the
transition state of subtilisin" Phil. Trans. R. Soc. Lond. A 317:
415-423), mutagenesis by total gene synthesis (Nambiar et al.
(1984) "Total synthesis and cloning of a gene coding for the
ribonuclease S protein" Science 223: 1299-1301; Sakamar and Khorana
(1988) "Total synthesis and expression of a gene for the a-subunit
of bovine rod outer segment guanine nucleotide-binding protein
(transducin)" Nucl. Acids Res. 14: 6361-6372; Wells et al. (1985)
"Cassette mutagenesis: an efficient method for generation of
multiple mutations at defined sites" Gene 34:315-323; and
Grundstrom et al. (1985) "Oligonucleotide-directed mutagenesis by
microscale `shot-gun` gene synthesis" Nucl. Acids Res. 13:
3305-3316), double-strand break repair (Mandecki (1986)
"Oligonucleotide-directed double-strand break repair in plasmids of
Escherichia coli: a method for site-specific mutagenesis" Proc.
Natl. Acad. Sci. USA, 83:7177-7181; and Arnold (1993) "Protein
engineering for unusual environments" Current Opinion in
Biotechnology 4:450-455). Additional details on many of the above
methods can be found in Methods in Enzymology Volume 154, which
also describes useful controls for trouble-shooting problems with
various mutagenesis methods.
[0337] Additional details regarding various diversity generating
methods can be found in the following U.S. patents, PCT
publications and applications, and EPO publications: U.S. Pat. No.
5,605,793 to Stemmer (Feb. 25, 1997), "Methods for In vitro
Recombination;" U.S. Pat. No. 5,811,238 to Stemmer et al. (Sep. 22,
1998) "Methods for Generating Polynucleotides having Desired
Characteristics by Iterative Selection and Recombination;" U.S.
Pat. No. 5,830,721 to Stemmer et al. (Nov. 3, 1998), "DNA
Mutagenesis by Random Fragmentation and Reassembly;" U.S. Pat. No.
5,834,252 to Stemmer, et al. (Nov. 10, 1998) "End-Complementary
Polymerase Reaction;" U.S. Pat. No. 5,837,458 to Minshull, et al.
(Nov. 17, 1998), "Methods and Compositions for Cellular and
Metabolic Engineering;" WO 95/22625, Stemmer and Crameri,
"Mutagenesis by Random Fragmentation and Reassembly;" WO 96/33207
by Stemmer and Lipschutz "End Complementary Polymerase Chain
Reaction;" WO 97/20078 by Stemmer and Crameri "Methods for
Generating Polynucleotides having Desired Characteristics by
Iterative Selection and Recombination;" WO 97/35966 by Minshull and
Stemmer, "Methods and Compositions for Cellular and Metabolic
Engineering;" WO 99/41402 by Punnonen et al. "Targeting of Genetic
Vaccine Vectors;" WO 99/41383 by Punnonen et al. "Antigen Library
Immunization;" WO 99/41369 by Punnonen et al. "Genetic Vaccine
Vector Engineering;" WO 99/41368 by Punnonen et al. "Optimization
of Immunomodulatory Properties of Genetic Vaccines;" EP 752008 by
Stemmer and Crameri, "DNA Mutagenesis by Random Fragmentation and
Reassembly;" EP 0932670 by Stemmer "Evolving Cellular DNA Uptake by
Recursive Sequence Recombination;" WO 99/23107 by Stemmer et al.,
"Modification of Virus Tropism and Host Range by Viral Genome
Shuffling;" WO 99/21979 by Apt et al., "Human Papillomavirus
Vectors;" WO 98/31837 by del Cardayre et al. "Evolution of Whole
Cells and Organisms by Recursive Sequence Recombination;" WO
98/27230 by Patten and Stemmer, "Methods and Compositions for
Polypeptide Engineering;" WO 98/27230 by Stemmer et al., "Methods
for Optimization of Gene Therapy by Recursive Sequence Shuffling
and Selection," WO 00/00632, "Methods for Generating Highly Diverse
Libraries," WO 00/09679, "Methods for Obtaining in vitro Recombined
Polynucleotide Sequence Banks and Resulting Sequences," WO 98/42832
by Arnold et al., "Recombination of Polynucleotide Sequences Using
Random or Defined Primers," WO 99/29902 by Arnold et al., "Method
for Creating Polynucleotide and Polypeptide Sequences," WO 98/41653
by Vind, "An in vitro Method for Construction of a DNA Library," WO
98/41622 by Borchert et al., "Method for Constructing a Library
Using DNA Shuffling," and WO 98/42727 by Pati and Zarling,
"Sequence Alterations using Homologous Recombination;" WO 00/18906
by Patten et al., "Shuffling of Codon-Altered Genes;" WO 00/04190
by del Cardayre et al. "Evolution of Whole Cells and Organisms by
Recursive Recombination;" WO 00/42561 by Crameri et al.,
"Oligonucleotide Mediated Nucleic Acid Recombination;" WO 00/42559
by Selifonov and Stemmer "Methods of Populating Data Structures for
Use in Evolutionary Simulations;" WO 00/42560 by Selifonov et al.,
"Methods for Making Character Strings, Polynucleotides &
Polypeptides Having Desired Characteristics;" PCT/US00/26708 by
Welch et al., "Use of Codon-Varied Oligonucleotide Synthesis for
Synthetic Shuffling;" and PCT/US01/06775 "Single-Stranded Nucleic
Acid Template-Mediated Recombination and Nucleic Acid Fragment
Isolation" by Affholter.
[0338] Several different general classes of sequence modification
methods, such as mutation, recombination, etc. are applicable to
the present invention and set forth, e.g., in the references above
and below. The following exemplify some of the different types of
preferred formats for diversity generation in the context of the
present invention, including, e.g., certain recombination based
diversity generation formats.
[0339] Nucleic acids can be recombined in vitro by any of a variety
of techniques discussed in the references above, including e.g.,
DNAse digestion of nucleic acids to be recombined followed by
ligation and/or PCR reassembly of the nucleic acids. For example,
sexual PCR mutagenesis can be used in which random (or pseudo
random, or even non-random) fragmentation of the DNA molecule is
followed by recombination, based on sequence similarity, between
DNA molecules with different but related DNA sequences, in vitro,
followed by fixation of the crossover by extension in a polymerase
chain reaction. This process and many process variants is described
in several of the references above, e.g., in Stemmer (1994) Proc.
Natl. Acad. Sci. USA 91:10747-10751.
[0340] Similarly, nucleic acids can be recursively recombined in
vivo, e.g., by allowing recombination to occur between nucleic
acids in cells. Many such in vivo recombination formats are set
forth in the references noted above. Such formats optionally
provide direct recombination between nucleic acids of interest, or
provide recombination between vectors, viruses, plasmids, etc.,
comprising the nucleic acids of interest, as well as other formats.
Details regarding such procedures are found in the references noted
above.
[0341] Whole genome recombination methods can also be used in which
whole genomes of cells or other organisms are recombined,
optionally including spiking of the genomic recombination mixtures
with desired library components (e.g., genes corresponding to the
pathways of the present invention). These methods have many
applications, including those in which the identity of a target
gene is not known. Details on such methods are found, e.g., in WO
98/31837 by del Cardayre et al. "Evolution of Whole Cells and
Organisms by Recursive Sequence Recombination;" and in, e.g.,
PCT/US99/15972 by del Cardayre et al., also entitled "Evolution of
Whole Cells and Organisms by Recursive Sequence Recombination."
[0342] Synthetic recombination methods can also be used, in which
oligonucleotides corresponding to targets of interest are
synthesized and reassembled in PCR or ligation reactions which
include oligonucleotides which correspond to more than one parental
nucleic acid, thereby generating new recombined nucleic acids.
Oligonucleotides can be made by standard nucleotide addition
methods, or can be made, e.g., by tri-nucleotide synthetic
approaches. Details regarding such approaches are found in the
references noted above, including, e.g., WO 00/42561 by Crameri et
al., "Olgonucleotide Mediated Nucleic Acid Recombination;"
PCT/US00/26708 by Welch et al., "Use of Codon-Varied
Oligonucleotide Synthesis for Synthetic Shuffling;" WO 00/42560 by
Selifonov et al., "Methods for Making Character Strings,
Polynucleotides and Polypeptides Having Desired Characteristics;"
and WO 00/42559 by Selifonov and Stemmer "Methods of Populating
Data Structures for Use in Evolutionary Simulations."
[0343] In silico methods of recombination can be effected in which
genetic algorithms are used in a computer to recombine sequence
strings which correspond to homologous (or even non-homologous)
nucleic acids. The resulting recombined sequence strings are
optionally converted into nucleic acids by synthesis of nucleic
acids which correspond to the recombined sequences, e.g., in
concert with oligonucleotide synthesis/ gene reassembly techniques.
This approach can generate random, partially random or designed
variants. Many details regarding in silico recombination, including
the use of genetic algorithms, genetic operators and the like in
computer systems, combined with generation of corresponding nucleic
acids (and/or proteins), as well as combinations of designed
nucleic acids and/or proteins (e.g., based on cross-over site
selection) as well as designed, pseudo-random or random
recombination methods are described in WO 00/42560 by Selifonov et
al., "Methods for Making Character Strings, Polynucleotides and
Polypeptides Having Desired Characteristics" and WO 00/42559 by
Selifonov and Stemmer "Methods of Populating Data Structures for
Use in Evolutionary Simulations." Extensive details regarding in
silico recombination methods are found in these applications. This
methodology is generally applicable to the present invention in
providing for recombination of the molecules in silico and/or the
generation of corresponding nucleic acids or proteins.
[0344] Many methods of accessing natural diversity, e.g., by
hybridization of diverse nucleic acids or nucleic acid fragments to
single-stranded templates, followed by polymerization and/or
ligation to regenerate full-length sequences, optionally followed
by degradation of the templates and recovery of the resulting
modified nucleic acids can be similarly used. In one method
employing a single-stranded template, the fragment population
derived from the genomic library(ies) is annealed with partial, or,
often approximately full length ssDNA or RNA corresponding to the
opposite strand. Assembly of complex chimeric genes from this
population is then mediated by nuclease-base removal of
non-hybridizing fragment ends, polymerization to fill gaps between
such fragments and subsequent single stranded ligation. The
parental polynucleotide strand can be removed by digestion (e.g.,
if RNA or uracil-containing), magnetic separation under denaturing
conditions (if labeled in a manner conducive to such separation)
and other available separation/purification methods. Alternatively,
the parental strand is optionally co-purified with the chimeric
strands and removed during subsequent screening and processing
steps. Additional details regarding this approach are found, e.g.,
in "Single-Stranded Nucleic Acid Template-Mediated Recombination
and Nucleic Acid Fragment Isolation" by Affholter,
PCT/US01/06775.
[0345] In another approach, single-stranded molecules are converted
to double-stranded DNA (dsDNA) and the dsDNA molecules are bound to
a solid support by ligand-mediated binding. After separation of
unbound DNA, the selected DNA molecules are released from the
support and introduced into a suitable host cell to generate a
library enriched sequences which hybridize to the probe. A library
produced in this manner provides a desirable substrate for further
diversification using any of the procedures described herein.
[0346] Any of the preceding general recombination formats can be
practiced in a reiterative fashion (e.g., one or more cycles of
mutation/recombination or other diversity generation methods,
optionally followed by one or more selection methods) to generate a
more diverse set of recombinant nucleic acids.
[0347] Mutagenesis employing polynucleotide chain termination
methods have also been proposed (see e.g., U.S. Pat. No. 5,965,408,
"Method of DNA reassembly by interrupting synthesis" to Short, and
the references above), and can be applied to the present invention.
In this approach, double stranded DNAs corresponding to one or more
genes sharing regions of sequence similarity are combined and
denatured, in the presence or absence of primers specific for the
gene. The single stranded polynucleotides are then annealed and
incubated in the presence of a polymerase and a chain terminating
reagent (e.g., ultraviolet, gamma or X-ray irradiation; ethidium
bromide or other intercalators; DNA binding proteins, such as
single strand binding proteins, transcription activating factors,
or histones; polycyclic aromatic hydrocarbons; trivalent chromium
or a trivalent chromium salt; or abbreviated polymerization
mediated by rapid thermocycling; and the like), resulting in the
production of partial duplex molecules. The partial duplex
molecules, e.g., containing partially extended chains, are then
denatured and reannealed in subsequent rounds of replication or
partial replication resulting in polynucleotides which share
varying degrees of sequence similarity and which are diversified
with respect to the starting population of DNA molecules.
Optionally, the products, or partial pools of the products, can be
amplified at one or more stages in the process. Polynucleotides
produced by a chain termination method, such as described above,
are suitable substrates for any other described recombination
format.
[0348] Diversity also can be generated in nucleic acids or
populations of nucleic acids using a recombinational procedure
termed "incremental truncation for the creation of hybrid enzymes"
("ITCHY") described in Ostermeier et al. (1999) "A combinatorial
approach to hybrid enzymes independent of DNA homology" Nature
Biotech 17:1205. This approach can be used to generate an initial a
library of variants which can optionally serve as a substrate for
one or more in vitro or in vivo recombination methods. See, also,
Ostermeier et al. (1999) "Combinatorial Protein Engineering by
Incremental Truncation," Proc. Natl. Acad. Sci. USA, 96: 3562-67;
Ostermeier et al. (1999), "Incremental Truncation as a Strategy in
the Engineering of Novel Biocatalysts," Biological and Medicinal
Chemistry, 7: 2139-44.
[0349] Mutational methods which result in the alteration of
individual nucleotides or groups of contiguous or non-contiguous
nucleotides can be favorably employed to introduce nucleotide
diversity. Many mutagenesis methods are found in the above-cited
references; additional details regarding mutagenesis methods can be
found in following, which can also be applied to the present
invention. For example, error-prone PCR can be used to generate
nucleic acid variants. Using this technique, PCR is performed under
conditions where the copying fidelity of the DNA polymerase is low,
such that a high rate of point mutations is obtained along the
entire length of the PCR product. Examples of such techniques are
found in the references above and, e.g., in Leung et al. (1989)
Technique 1:11-15 and Caldwell et al. (1992) PCR Methods Applic.
2:28-33. Similarly, assembly PCR can be used, in a process which
involves the assembly of a PCR product from a mixture of small DNA
fragments. A large number of different PCR reactions can occur in
parallel in the same reaction mixture, with the products of one
reaction priming the products of another reaction.
[0350] Oligonucleotide directed mutagenesis can be used to
introduce site-specific mutations in a nucleic acid sequence of
interest. Examples of such techniques are found in the references
above and, e.g., in Reidhaar-Olson et al. (1988) Science,
241:53-57. Similarly, cassette mutagenesis can be used in a process
that replaces a small region of a double stranded DNA molecule with
a synthetic oligonucleotide cassette that differs from the native
sequence. The oligonucleotide can contain, e.g., completely and/or
partially randomized native sequence(s).
[0351] Recursive ensemble mutagenesis is a process in which an
algorithm for protein mutagenesis is used to produce diverse
populations of phenotypically related mutants, members of which
differ in amino acid sequence. This method uses a feedback
mechanism to monitor successive rounds of combinatorial cassette
mutagenesis. Examples of this approach are found in Arkin &
Youvan (1992) Proc. Natl. Acad. Sci. USA 89:7811-7815. Exponential
ensemble mutagenesis can be used for generating combinatorial
libraries with a high percentage of unique and functional mutants.
Small groups of residues in a sequence of interest are randomized
in parallel to identify, at each altered position, amino acids
which lead to functional proteins. Examples of such procedures are
in Delegrave & Youvan (1993) Biotechnology Research
11:1548-1552.
[0352] In vivo mutagenesis can be used to generate random mutations
in any cloned DNA of interest by propagating the DNA, e.g., in a
strain of E. coli that carries mutations in one or more of the DNA
repair pathways. These "mutator" strains have a higher random
mutation rate than that of a wild-type parent. Propagating the DNA
in one of these strains will eventually generate random mutations
within the DNA. Such procedures are described in the references
noted above.
[0353] Other procedures for introducing diversity into a genome,
e.g. a bacterial, fungal, animal or plant genome can be used in
conjunction with the above described and/or referenced methods. For
example, in addition to the methods above, techniques have been
proposed which produce nucleic acid multimers suitable for
transformation into a variety of species (see, e.g., Schellenberger
U.S. Pat. No. 5,756,316 and the references above). Transformation
of a suitable host with such multimers, consisting of genes that
are divergent with respect to one another, (e.g., derived from
natural diversity or through application of site directed
mutagenesis, error prone PCR, passage through mutagenic bacterial
strains, and the like), provides a source of nucleic acid diversity
for DNA diversification, e.g., by an in vivo recombination process
as indicated above.
[0354] Alternatively, a multiplicity of monomeric polynucleotides
sharing regions of partial sequence similarity can be transformed
into a host species and recombined in vivo by the host cell.
Subsequent rounds of cell division can be used to generate
libraries, members of which, include a single, homogenous
population, or pool of monomeric polynucleotides. Alternatively,
the monomeric nucleic acid can be recovered by standard techniques,
e.g., PCR and/or cloning, and recombined in any of the
recombination formats, including recursive recombination formats,
described above.
[0355] Methods for generating multispecies expression libraries
have been described (in addition to the reference noted above, see,
e.g., Peterson et al. (1998) U.S. Pat. No. 5,783,431 "METHODS FOR
GENERATING AND SCREENING NOVEL METABOLIC PATHWAYS," and Thompson,
et al. (1998) U.S. Pat. No. 5,824,485 METHODS FOR GENERATING AND
SCREENING NOVEL METABOLIC PATHWAYS) and their use to identify
protein activities of interest has been proposed (In addition to
the references noted above, see Short (1999) U.S. Pat. No.
5,958,672 "PROTEIN ACTIVITY SCREENING OF CLONES HAVING DNA FROM
UNCULTIVATED MICROORGANISMS"). Multispecies expression libraries
include, in general, libraries comprising cDNA or genomic sequences
from a plurality of species or strains, operably linked to
appropriate regulatory sequences, in an expression cassette. The
cDNA and/or genomic sequences are optionally randomly ligated to
further enhance diversity. The vector can be a shuttle vector
suitable for transformation and expression in more than one species
of host organism, e.g., bacterial species, eukaryotic cells. In
some cases, the library is biased by preselecting sequences which
encode a protein of interest, or which hybridize to a nucleic acid
of interest. Any such libraries can be provided as substrates for
any of the methods herein described.
[0356] The above-described procedures have been largely directed to
increasing nucleic acid and/or encoded protein diversity. However,
in many cases, not all of the diversity is useful, e.g.,
functional, and contributes merely to increasing the background of
variants that must be screened or selected to identify the few
favorable variants. In some applications, it is desirable to
preselect or prescreen libraries (e.g., an amplified library, a
genomic library, a cDNA library, a normalized library, etc.) or
other substrate nucleic acids prior to diversification, e.g., by
recombination-based mutagenesis procedures, or to otherwise bias
the substrates towards nucleic acids that encode functional
products. For example, in the case of antibody engineering, it is
possible to bias the diversity generating process toward antibodies
with functional antigen binding sites by taking advantage of in
vivo recombination events prior to manipulation by any of the
described methods. For example, recombined CDRs derived from B cell
cDNA libraries can be amplified and assembled into framework
regions (e.g., Jirholt et al. (1998) "Exploiting sequence space:
shuffling in vivo formed complementarity determining regions into a
master framework" Gene 215: 471) prior to diversifying according to
any of the methods described herein.
[0357] Libraries can be biased towards nucleic acids which encode
proteins with desirable enzyme activities. For example, after
identifying a clone from a library which exhibits a specified
activity, the clone can be mutagenized using any known method for
introducing DNA alterations. A library comprising the mutagenized
homologues is then screened for a desired activity, which can be
the same as or different from the initially specified activity. An
example of such a procedure is proposed in Short (1999) U.S. Pat.
No. 5,939,250 for "PRODUCTION OF ENZYMES HAVING DESIRED ACTIVITIES
BY MUTAGENESIS." Desired activities can be identified by any method
known in the art. For example, WO 99/10539 proposes that gene
libraries can be screened by combining extracts from the gene
library with components obtained from metabolically rich cells and
identifying combinations which exhibit the desired activity. It has
also been proposed (e.g., WO 98/58085) that clones with desired
activities can be identified by inserting bioactive substrates into
samples of the library, and detecting bioactive fluorescence
corresponding to the product of a desired activity as described
herein using a fluorescent analyzer, e.g., a flow cytometry device,
a CCD, a fluorometer, or a spectrophotometer.
[0358] Libraries can also be biased towards nucleic acids which
have specified characteristics, e.g., hybridization to a selected
nucleic acid probe. For example, application WO 99/10539 proposes
that polynucleotides encoding a desired activity (e.g., an
enzymatic activity, for example: a lipase, an esterase, a protease,
a glycosidase, a glycosyl transferase, a phosphatase, a kinase, an
oxygenase, a peroxidase, a hydrolase, a hydratase, a nitrilase, a
transaminase, an amidase or an acylase) can be identified from
among genomic DNA sequences in the following manner. Single
stranded DNA molecules from a population of genomic DNA are
hybridized to a ligand-conjugated probe. The genomic DNA can be
derived from either a cultivated or uncultivated microorganism, or
from an environmental sample. Alternatively, the genomic DNA can be
derived from a multicellular organism, or a tissue derived
therefrom. Second strand synthesis can be conducted directly from
the hybridization probe used in the capture, with or without prior
release from the capture medium or by a wide variety of other
strategies known in the art. Alternatively, the isolated
single-stranded genomic DNA population can be fragmented without
further cloning and used directly in, e.g., a recombination-based
approach, that employs a single-stranded template, as described
above.
[0359] "Non-Stochastic" methods of generating nucleic acids and
polypeptides are alleged in Short "Non-Stochastic Generation of
Genetic Vaccines and Enzymes" WO 00/46344. These methods, including
proposed non-stochastic polynucleotide reassembly and
site-saturation mutagenesis methods be applied to the present
invention as well. Random or semi-random mutagenesis using doped or
degenerate oligonucleotides is also described in, e.g., Arkin and
Youvan (1992) "Optimizing nucleotide mixtures to encode specific
subsets of amino acids for semi-random mutagenesis" Biotechnology
10:297-300; Reidhaar-Olson et al. (1991) "Random mutagenesis of
protein sequences using oligonucleotide cassettes" Methods Enzymol.
208:564-86; Lim and Sauer (1991) "The role of internal packing
interactions in determining the structure and stability of a
protein" J. Mol. Biol. 219:359-76; Breyer and Sauer (1989)
"Mutational analysis of the fine specificity of binding of
monoclonal antibody 51F to lambda repressor" J. Biol. Chem.
264:13355-60); and "Walk-Through Mutagenesis" (Crea, R; U.S. Pat.
Nos. 5,830,650 and 5,798,208, and EP Patent 0527809 B1.
[0360] It will readily be appreciated that any of the above
described techniques suitable for enriching a library prior to
diversification can also be used to screen the products, or
libraries of products, produced by the diversity generating
methods.
[0361] Kits for mutagenesis, library construction and other
diversity generation methods are also commercially available. For
example, kits are available from, e.g., Stratagene (e.g.,
QuickChange.TM. site-directed mutagenesis kit; and Chameleon.TM.
double-stranded, site-directed mutagenesis kit), Bio/Can
Scientific, Bio-Rad (e.g., using the Kunkel method described
above), Boehringer Mannheim Corp., Clonetech Laboratories, DNA
Technologies, Epicentre Technologies (e.g., 5 prime 3 prime kit);
Genpak Inc, Lemargo Inc, Life Technologies (Gibco BRL), New England
Biolabs, Pharmacia Biotech, Promega Corp., Quantum Biotechnologies,
Amersham International plc (e.g., using the Eckstein method above),
and Anglian Biotechnology Ltd (e.g., using the Carter/Winter method
above).
[0362] The above references provide many mutational formats,
including recombination, recursive recombination, recursive
mutation and combinations or recombination with other forms of
mutagenesis, as well as many modifications of these formats.
Regardless of the diversity generation format that is used, the
nucleic acids of the invention can be recombined (with each other,
or with related (or even unrelated) sequences) to produce a diverse
set of recombinant nucleic acids, including, e.g., sets of
homologous nucleic acids, as well as corresponding
polypeptides.
[0363] A recombinant nucleic acid produced by recombining one or
more polynucleotide sequences of the invention with one or more
additional nucleic acids using any of the above-described formats
alone or in combination also forms a part of the invention. The one
or more additional nucleic acids may include another polynucleotide
of the invention; optionally, alternatively, or in addition, the
one or more additional nucleic acid can include, e.g., a nucleic
acid encoding a naturally-occurring interferon-alpha or a
subsequence thereof, or any homologous interferon-alpha or
subsequence thereof (e.g., as found in GenBank or other available
literature), or, e.g., any other homologous or non-homologous
nucleic acid or fragments thereof (certain recombination formats
noted above, notably those performed synthetically or in silico, do
not require homology for recombination).
Therapeutic Uses
[0364] Various interferon-alpha polypeptides and interferon-alpha
conjugates have been approved or are in clinical development for
treatment of a variety of diseases or conditions such as Chronic
Hepatitis C, Chronic Hepatitis B, Hairy Cell Leukemia, Malignant
Melanoma, Follicular Lymphoma, Condylomata Acuminata, AIDS-related
Kaposi's Sarcoma, Non-Hodgkin's Lymphoma, Chronic Melogenous
Leukemia, Basal Cell Carcinoma, Multiple Myeloma, carcinoid tumors,
bladder cancer, Crohn's disease, Cutaneous T Cell Lymphoma, Renal
Cell Carcinoma, Multiple Sclerosis, and AIDS. Accordingly, the
present invention contemplates the use of a composition comprising
one or more polypeptide or conjugate of the invention (i.e., a
"composition of the invention") to treat a disease or condition
which is responsive to an interferon-alpha polypeptide and/or an
interferon-alpha conjugate, such as a condition described above, or
any other disease or condition which is responsive to a polypeptide
or a conjugate of the invention.
Treatment of Viral Infections and Conditions Associated with Viral
Infection
[0365] In one aspect, the invention provides a method for treating
a subject infected with a virus, comprising administering to the
subject a composition of the invention in an amount effective to
decrease the level of the virus in the subject and/or to ameliorate
a symptom or condition associated with the viral infection.
Exemplary viral infections contemplated for treatment methods of
the invention include, but are not limited to, infection by a virus
of the Flaviviridae family, such as, for example, Hepatitis C
Virus, Yellow Fever Virus, West Nile Virus, Japanese Encephalitis
Virus, Dengue Virus, and Bovine Viral Diarrhea Virus; infection by
a virus of the Hepadnaviridae family, such as, for example,
Hepatitis B Virus; infection by a virus of the Picornaviridae
family, such as, for example, Encephalomyocarditis Virus, Human
Rhinovirus, and Hepatitis A Virus; infection by a virus of the
Retroviridae family, such as, for example, Human Immunodeficiency
Virus, Simian Immunodeficiency Virus, Human T-Lymphotropic Virus,
and Rous Sarcoma Virus; infection by a virus of the Coronaviridae
family, such as, for example, SARS coronavirus; infection by a
virus of the Rhabdoviridae family, such as, for example, Rabies
Virus and Vesicular Stomatitis Virus; infection by a virus of the
Paramyxoviridae family, such as, for example, Respiratory Syncytial
Virus and Parainfluenza Virus; infection by a virus of the
Papillomaviridae family, such as, for example, Human
Papillomavirus; and infection by a virus of the Herpesviridae
family, such as, for example, Herpes Simplex Virus.
[0366] The following provides non-limiting examples for treatment
of exemplary viral infections and diseases and conditions
associated with such infections, using polypeptides and conjugates
of the invention, including suggested dosing schedules for
polypeptides and conjugates of the invention and approaches to
monitoring the efficacy of such treatments. The dosing schedules of
polypeptides or conjugates of the invention for the treatment of
other viral infections and diseases and conditions associated with
viral infections, and approaches to monitoring the efficacy of such
treatments, is ascertainable by one skilled in the art.
Hepatitis C Virus
[0367] In one aspect the invention provides a method of treating a
patient infected with Hepatitis C Virus (HCV), comprising
administering to the patient an effective amount of a composition
of the invention comprising one or more polypeptide or conjugate of
the invention. The invention also provides a composition for use in
treating a patient infected with HCV, comprising one or more
polypeptide or conjugate of the invention and a pharmaceutically
acceptable carrier or excipient. A patient diagnosed as infected
with HCV includes a patient exhibiting HCV RNA in the blood and/or
exhibiting anti-HCV antibody in the serum.
[0368] A composition comprising a polypeptide of the invention will
generally be administered at a dose and frequency similar to what
is employed in HCV therapeutic regimens using clinically-approved
interferon-alpha polypeptides, such as, e.g. ROFERON.RTM.-A
(Interferon alfa-2a, recombinant; Hoffmann-La Roche Inc.),
INTRON.RTM. A (Interferon alfa-2b, recombinant; Schering
Corporation), and INFERGEN.RTM. (interferon alfacon-1; InterMune,
Inc.). Exemplary recommended dosing schedules of ROFERON or INTRON
A for the treatment of chronic HCV is 3 million IU (approximately
15 micrograms (mcg)) three times a week by subcutaneous injection
for, e.g., 24 to 48 weeks. An exemplary recommended dosing schedule
of INFERGEN for the treatment of chronic HCV is 9 mcg three times a
week by subcutaneous injection for, e.g., 24 to 48 weeks. Depending
on a number of factors (including but not limited to the activity
and the pharmacokinetics of the polypeptide of the invention and
the size and health of the patient), the polypeptide may be
administered in lower amounts (such as, for example, about 2, 3, 4,
5, 6, 7, or 8 mcg) and/or less frequently (such as once per week or
twice per week) than described above.
[0369] Likewise, a composition comprising a conjugate of the
invention will generally be administered at a dose and frequency
similar to what is employed in HCV therapeutic regimens using
clinically-approved interferon-alpha conjugates, such as, e.g.,
PEGASYS.RTM. (Peginterferon alfa-2a; Hoffmann-La Roche, Inc.) or
PEG-INTRON.RTM. (peginterferon alfa-2b; Schering Corporation). An
exemplary recommended dosing schedule of PEGASYS for the treatment
of chronic HCV is 180 mcg once weekly by subcutaneous injection
for, e.g., 24 to 48 weeks. Depending on a number of factors
(including but not limited to the molecular weight, activity, and
pharmacokinetics of the conjugate of the invention and the size and
health of the patient), the conjugate may be administered in lower
amounts (such as, for example, about 25, 50, 75, 100, 125, or 150
mcg) and/or less frequently (such as once every 10 days, or once
every 2 weeks) than described above.
[0370] In some instances the polypeptide or conjugate of the
invention is administered in combination with one or more
additional therapeutic agent(s). For example, the polypeptide or
conjugate of the invention may be administered in combination with
a small-molecule antiviral drug such as Ribavirin, which is sold
under the names COPEGUS.RTM. (Hoffmann-La Roche, Inc) and
REBETOL.RTM. (Schering Corporation). Alternatively, or in addition
to a small-molecule antiviral drug, the polypeptide or conjugate of
the invention may be administered in combination with one or more
additional cytokine, such as, for example, IFN-gamma, which is sold
under the name Actimmune.RTM. (interferon gamma-1b; InterMune,
Inc.), IL-2, which is sold under the name PROLEUKIN.RTM. IL-2
(aldesleukin recombinant human interleukin-2 (rhIL-2); Chiron
Corp.), or IL-12 (interleukin-12).
[0371] The precise amount and frequency of administration of the
polypeptide or conjugate of the invention will depend on a number
of factors such as the specific activity and the pharmacokinetic
properties of the polypeptide or the conjugate, as well as the
nature of the condition being treated (such as, the genotype of the
Hepatitis C virus being treated), among other factors known to
those of skill in the art. Normally, the dose should be capable of
preventing or lessening the severity or spread of the indication
being treated. Such a dose may be termed an "effective" or
"therapeutically effective" amount. It will be apparent to those of
skill in the art that an effective amount of a polypeptide,
conjugate or composition of the invention depends, inter alia, upon
the condition being treated, the dose, the administration schedule,
whether the polypeptide or conjugate or composition is administered
alone or in combination with other therapeutic agents, the serum
half-life and other pharmacokinetic properties of the polypeptide,
conjugate or composition, as well as the size, age, and general
health of the patient. The dosage and frequency of administration
is ascertainable by one skilled in the art using known
techniques.
[0372] The effectiveness of treatment may be determined by
measuring viral load, for example by determining the titer or level
of virus in serum or plasma using methods known in the art, such
as, e.g., by monitoring viral RNA levels using quantitative
PCR-based tests, such as the COBAS AMPLICOR.RTM. HCV Test, v2.0 or
the COBAS AMPLICOR HCV MONITOR.RTM. Test, v2.0 (both from Roche
Diagnostics). In some instances, an effective amount of a
composition of the invention is one that is sufficient to achieve a
reduction in viral load by at least 2 log units, at least 3 log
units, at least 4 log units, at least 5 log units, at least 6 log
units or at least 7 log units over the course of treatment,
compared to the viral load prior to treatment (which is generally
in the range of 10.sup.5-10.sup.7 copies of HCV RNA/ml for chronic
HCV patients). In some instances an effective amount of a
composition of the invention is an amount that is sufficient to
reduce viral load to levels which are essentially undetectable,
such as, for example, less than about 500 copies/ml serum or less
than about 100 copies/ml serum. The invention includes a method of
reducing the level of HCV RNA in serum of a patient infected with
HCV, comprising administering to the patient a composition of the
invention in an amount effective to reduce the level of HCV RNA
compared to the HCV RNA level present prior to the start of
treatment.
[0373] The effectiveness of treatment may alternatively or in
addition be determined by measuring a parameter indicative of a
condition associated with HCV infection, such as, e.g., liver
damage. For example, the level of serum alanine aminotransferase
(ALT) may be measured using a standard assay. In general, an ALT
level of less than about 50 international units/ml (IU/ml) serum is
considered normal. A higher ALT level may be indicative of ongoing
liver damage. In some instances, an effective amount of a
composition of the invention is an amount effective to reduce ALT
level, in a patient with a higher than normal ALT level, to less
than about 50 IU/ml of serum. Thus, the invention includes a method
of reducing the serum ALT level of a patient infected with HCV
exhibiting an initial ALT level greater than 50 IU/ml, comprising
administering to the patient a composition of the invention in an
amount effective to reduce the ALT level to less than about 50
IU/ml.
Human Immunodeficiency Virus
[0374] In another aspect the invention provides a method of
treating a patient infected with Human Immunodeficiency Virus
(HIV), such as HIV-1 or HIV-2, or a disease or condition associated
with HIV infection, such as, for example, AIDS-related Kaposi's
sarcoma, comprising administering to the patient an effective
amount of a composition of the invention comprising one or more
polypeptide or conjugate of the invention, optionally in
association with other antiviral therapeutic agents as described
below. The invention also provides a composition for use in
treating a patient infected with HIV or a disease or condition
associated with HIV infection, comprising one or more polypeptide
or conjugate of the invention and a pharmaceutically acceptable
carrier or excipient. A patient diagnosed as infected with HIV
includes a patient exhibiting detectable levels of HIV RNA or
proviral DNA in the blood, and/or exhibiting detectable levels of
p24 antigen or anti-HIV antibody in serum.
[0375] A composition comprising a polypeptide of the invention will
generally be administered at a dose and frequency similar to what
is employed in HIV therapeutic regimens using interferon-alpha
polypeptides such as, e.g. ROFERON.RTM.-A (Interferon alfa-2a,
recombinant; Hoffmann-La Roche Inc.), INTRON.RTM. A (Interferon
alfa-2b, recombinant; Schering Corporation), and INFERGEN.RTM.
(interferon alfacon-1; InterMune, Inc.). As was noted above,
exemplary recommended dosing schedules of ROFERON or INTRON A for
the treatment of chronic HCV is 3 million IU (approximately 15
micrograms (mcg)) three times a week by subcutaneous injection for,
e.g., 24 to 48 weeks, and a exemplary recommended dosing schedule
of INFERGEN for the treatment of chronic HCV is 9 mcg three times a
week by subcutaneous injection for, e.g., 24 to 48 weeks. An
exemplary recommended dosing schedule of ROFERON for the treatment
of AIDS-related Kaposi's sarcoma is 36 million units daily for 10
to 12 weeks, then 36 million units 3 times a week. An exemplary
recommended dosing schedule of INTRON A for the treatment of
AIDS-related Kaposi's sarcoma is 30 million IU/m2 three times a
week administered subcutaneously. Such dosing schedules provide
useful ranges for dosage of a polypeptide of the invention for the
treatment of HIV or a disease or condition associated with HIV
infection. Depending on a number of factors (including but not
limited to the activity and the pharmacokinetics of the polypeptide
of the invention and the size, age and health of the patient), the
polypeptide of the invention may be administered in lower amounts
and/or less frequently than described above.
[0376] Likewise, a composition comprising a conjugate of the
invention will generally be administered at a dose and frequency
similar to what is employed in HIV therapeutic regimens using
interferon-alpha conjugates, such as, e.g., PEGASYS.RTM.
(Peginterferon alfa-2a; Hoffmann-La Roche, Inc.) or PEG-INTRON.RTM.
(peginterferon alfa-2b; Schering Corporation). An exemplary dosing
schedule of PEG-INTRON for the treatment of HIV is between about
1.0 mcg/kg/week and 3.0 mcg/kg/week by subcutaneous injection for,
e.g., 24 to 48 weeks. Such a dosing schedule provides a useful
range for dosage of a conjugate of the invention for the treatment
of HIV. Depending on a number of factors (including but not limited
to the molecular weight, activity, and pharmacokinetics of the
conjugate of the invention and the size, age and health of the
patient), the conjugate may be administered in lower amounts (such
as, for example, about 0.1, 0.25, 0.50, or 0.75 mcg/kg/week) and/or
less frequently (such as once every 10 days, or once every 2 weeks)
than described above.
[0377] In some instances the polypeptide or conjugate of the
invention is administered in combination with one or more
additional therapeutic agent(s). Current clinical treatments of
HIV-1 infection in man include multi-drug combination therapies
generally termed Highly Active Antiretroviral Therapy ("HAART").
The polypeptide or conjugate of the invention may thus be
administered in combination with HAART or other antiviral
therapeutic compounds. Typical components of HAART, which involve
various combinations of nucleoside reverse transcriptase inhibitors
("NRTI"), non-nucleoside reverse transcriptase inhibitors ("NNRTI")
and HIV protease inhibitors ("PI"), are described, for example, in
A. M. Vandamme et al. (1998) Antiviral Chemistry &
Chemotherapy, 9:187-203; "Drugs for HIV Infection" in The Medical
Letter Vol. 39 (Issue 1015) Dec. 5, 1997, pages 111-116; and
published United States Patent Application US 20020182179 A1; each
of which is incorporated by reference herein. If the HIV-infected
patient is also infected with HCV, the polypeptide or conjugate of
the invention may be administered in combination with an antiviral
drug such as Ribavirin, which is sold under the names COPEGUS.RTM.
(Hoffmann-La Roche, Inc) and REBETOL.RTM. (Schering Corporation),
along with HAART.
[0378] The precise amount and frequency of administration of the
polypeptide or conjugate of the invention, and administration of
additional therapeutic agents such as HAART and/or Ribavirin, will
depend on a number of factors such as the specific activity and the
pharmacokinetic properties of the polypeptide or the conjugate, as
well as the nature of the condition being treated (such as, the
presence of additional viral infections such as HCV), among other
factors known to those of skill in the art. Normally, the dose
should be capable of preventing or lessening the severity or spread
of the indication being treated. Such a dose may be termed an
"effective" or "therapeutically effective" amount. It will be
apparent to those of skill in the art that an effective amount of a
polypeptide, conjugate or composition of the invention depends,
inter alia, upon the condition being treated, the dose, the
administration schedule, whether the polypeptide or conjugate or
composition is administered alone or in combination with other
therapeutic agents, the serum half-life and other pharmacokinetic
properties of the polypeptide, conjugate or composition, as well as
the size, age, and general health of the patient. The dosage and
frequency of administration is ascertainable by one skilled in the
art using known techniques.
[0379] In addition to general uses described above, a polypeptide
or conjugate of the invention may be administered to the following
subsets of patients infected with HIV: as an adjuvant therapy, for
example to HAART as described above; as monotherapy or combination
therapy in early stage patients when the viral load is generally
high; as a combined anti-viral and immunodulatory agent for
patients undergoing structured treatment interruptions (STI) or
"drug holidays"; as salvage therapy in patients whose HAART options
are limited; as an antiviral method of treatment to keep viral load
in check without initiating HAART therapy in order to delay the
appearance of HAART resistant virus.
[0380] The effectiveness of treatment may be determined by
measuring viral load, for example by determining the titer or level
of virus in serum or plasma using methods known in the art, such
as, e.g., by monitoring HIV-1 viral RNA levels using quantitative
RT-PCR based tests, such as the AMPLICOR HIV-1 MONITOR.RTM. Test,
v1.5 (Roche Diagnostics). In some instances, an effective amount of
a composition of the invention is one that is sufficient to achieve
a reduction in viral load by at least 0.5 log units, at least 1 log
unit, at least 2 log units, at least 3 log units, at least 4 log
units, at least 5 log units, at least 6 log units or at least 7 log
units over the course of treatment, compared to the viral load
prior to treatment. In some instances an effective amount of a
composition of the invention is an amount that is sufficient to
reduce viral load to levels which are essentially undetectable,
such as, for example, less than about 50-100 copies HIV-1 RNA per
ml serum. The invention includes a method of reducing the level of
HIV RNA in serum of a patient infected with HIV, comprising
administering to the patient a composition of the invention in an
amount effective to reduce the level of HIV RNA compared to the HIV
RNA level present prior to the start of treatment.
[0381] The effectiveness of treatment may alternatively or in
addition be determined by a serum markers for HIV replication, such
as the presence of HIV p24 antigen in the blood. In some instances
an effective amount of a composition of the invention is an amount
that is sufficient to reduce the level of p24 antigen in the blood
to 50%, 25%, 10% or 5% of the level present prior to the start of
treatment. In some instances an effective amount of a composition
of the invention is an amount that is sufficient to reduce the
level of p24 antigen to a level which is essentially undetectable.
The invention includes a method of reducing the level of p24
antigen in serum of a patient infected with HIV, comprising
administering to the patient a composition of the invention in an
amount effective to reduce the level of p24 antigen compared to the
p24 antigen level present prior to the start of treatment.
Hepatitis B Virus
[0382] In another aspect, the invention provides a method of
treating a patient infected with Hepatitis B Virus (HBV),
comprising administering to the patient an effective amount of a
composition of the invention comprising one or more polypeptide or
conjugate of the invention. The invention also provides a
composition for use in treating a patient infected with HBV,
comprising one or more polypeptide or conjugate of the invention
and a pharmaceutically acceptable carrier or excipient.
[0383] A patient diagnosed as infected with HBV exhibits detectable
hepatitis B surface antigen (HBsAg) in the serum. Chronic HBV
infection is further categorized as either "replicative" or
"non-replicative". In replicative infection, the patient usually
has a relatively high serum concentration of viral DNA and
detectable HBeAg, which is an alternatively processed protein of
the HBV pre-core gene that is synthesized under conditions of high
viral replication. However, in rare strains of HBV with mutations
in the pre-core gene, replicative infection can occur in the
absence of detectable serum HBeAg. Patients with chronic hepatitis
B and replicative infection have a generally worse prognosis and a
greater chance of developing cirrhosis and/or hepatocellular
carcinoma than those without HBeAg. In non-replicative infection,
the rate of viral replication in the liver is low, serum HBV DNA
concentration is generally low and hepatitis Be antigen (HBeAg) is
not detected.
[0384] A composition comprising a polypeptide of the invention will
generally be administered at a dose and frequency similar to what
is employed in HBV therapeutic regimens using clinically-approved
interferon-alpha polypeptides, such as, e.g. INTRON.RTM. A
(Interferon alfa-2b, recombinant; Schering Corporation). An
exemplary recommended dosing schedule of INTRON A for the treatment
of chronic HBV in adults is 30 to 35 million IU per week by
subcutaneous or intramuscular injection, either as 5 million IU per
day (qd) or as 10 million IU three times per week (tiw) for 16
weeks. Depending on a number of factors (including, but not limited
to, the activity and the pharmacokinetics of the polypeptide of the
invention, and the size and health of the patient), the polypeptide
of the invention may be administered in lower amounts (such as, for
example, about 5, 10, 15, 20, or 25 million IU per week) and/or
less frequently (such as once per week or twice per week) than
described above.
[0385] Likewise, a composition comprising a conjugate of the
invention will generally be administered at a dose and frequency
similar to what is employed in HBV therapeutic regimens using
interferon-alpha conjugates currently undergoing clinical trials,
such as, e.g., PEGASYS.RTM. (Peginterferon alfa-2a; Hoffmann-La
Roche, Inc.). Exemplary dosing schedules of PEGASYS for the
treatment of chronic HBV is between 90 mcg-270 mcg injected once
per week for a total of 24 weeks. Depending on a number of factors
(including but not limited to the molecular weight, activity, and
pharmacokinetics of the conjugate of the invention and the size and
health of the patient), the conjugate may be administered in lower
amounts (such as, for example, about 25, 50, 75, 100, 125, 150, or
200 mcg) and/or less frequently (such as once every 10 days, or
once every 2 weeks) than described above.
[0386] In some instances the polypeptide or conjugate of the
invention is administered in combination with one or more
additional therapeutic agent(s). For example, the polypeptide or
conjugate of the invention may be administered in combination with
antiviral drugs such as lamivudine (also known as 3TC), which is
sold under the name Epivir-HBV.RTM. (GlaxoSmithKline), or adefovir
dipivoxil, which is sold under the name Hepsera.RTM. (Gilead
Sciences).
[0387] The precise amount and frequency of administration of the
polypeptide or conjugate of the invention will depend on a number
of factors such as the specific activity and the pharmacokinetic
properties of the polypeptide or the conjugate, as well as the
nature of the condition being treated (such as, e.g., in the case
of chronic HBV infection, whether the infection is replicative or
non-replicative), among other factors known to those of skill in
the art. Normally, the dose should be capable of preventing or
lessening the severity or spread of the indication being treated.
Such a dose may be termed an "effective" or "therapeutically
effective" amount. It will be apparent to those of skill in the art
that an effective amount of a polypeptide, conjugate or composition
of the invention depends, inter alia, upon the condition being
treated, the dose, the administration schedule, whether the
polypeptide or conjugate or composition is administered alone or in
combination with other therapeutic agents, the serum half-life and
other pharmacokinetic properties of the polypeptide, conjugate or
composition, as well as the size, age, and general health of the
patient. The dosage and frequency of administration is
ascertainable by one skilled in the art using known techniques.
[0388] The effectiveness of treatment may be determined for example
by measuring the viral load, e.g. the level of viral DNA in serum
or plasma, using methods known in the art. Methods for monitoring
HBV DNA levels include quantitative PCR-based tests, such as the
COBAS AMPLICOR HBV MONITOR.RTM. Test, v2.0 or the AMPLICOR HBV
MONITOR.RTM. Test, v2.0 (both from Roche Diagnostics). In some
instances an effective amount of a composition of the invention is
an amount that is sufficient to reduce viral DNA to, e.g., less
than about 500,000 copies/ml serum or less than about 100,000
copies/ml serum or less than about 10,000 copies/ml serum, or to
levels which are essentially undetectable (such as, for example,
less than about 1000 copies/ml serum, less than about 500 copies/ml
serum, or less than about 200 copies/ml serum). The invention
includes a method of reducing the level of HBV DNA in serum of a
patient infected with HBV, comprising administering to the patient
a composition of the invention in an amount effective to reduce the
level of HBV DNA compared to the HBV DNA level present prior to the
start of treatment.
[0389] The effectiveness of treatment may alternatively or in
addition be determined by measuring other serum markers for HBV
replication, such as HBeAg. In some instances an effective amount
of a composition of the invention is an amount that is sufficient
to reduce the level of HBeAg in serum to 50%, 25%, 10% or 5% of the
level present prior to the start of treatment. In some instances an
effective amount of a composition of the invention is an amount
that is sufficient to reduce the level of HBeAg to a level which is
essentially undetectable. The invention includes a method of
reducing the level of HBeAg in serum of a patient infected with
HBV, comprising administering to the patient a composition of the
invention in an amount effective to reduce the level of HBeAg
compared to the HBeAg level present prior to the start of
treatment.
[0390] As discussed above, another serum marker indicative of HBV
infection is HBsAg. Thus, the effectiveness of treatment may
alternatively or in addition be determined by measuring the level
of HBsAg in the serum. In some instances an effective amount of a
composition of the invention is an amount that is sufficient to
reduce the level of HBsAg in serum to 50%, 25%, 10% or 5% of the
level present prior to the start of treatment. In some instances an
effective amount of a composition of the invention is an amount
that is sufficient to reduce level of HBsAg to a level which is
essentially undetectable. The invention includes a method of
reducing the level of HBsAg in serum of a patient infected with
HBV, comprising administering to the patient a composition of the
invention in an amount effective to reduce the level of HBsAg
compared to the HBsAg level present prior to the start of
treatment.
[0391] The effectiveness of treatment may alternatively or in
addition be determined by measuring a parameter indicative of a
condition associated with HBV infection, such as, e.g., liver
damage. For example, the level of serum alanine aminotransferase
(ALT) may be measured using a standard assay. In general, an ALT
level of less than about 50 international units/ml (IU/ml) serum is
considered normal. A higher ALT level may be indicative of ongoing
liver damage. In some instances, an effective amount of a
composition of the invention is an amount effective to reduce ALT
level, in a patient with a higher than normal ALT level, to less
than about 50 IU/ml of serum. Thus, the invention includes a method
of reducing the serum ALT level of a patient infected with HBV
exhibiting an initial ALT level greater than 50 IU/ml, comprising
administering to the patient a composition of the invention in an
amount effective to reduce the ALT level to less than about 50
IU/ml.
Human T-Lymphotropic Virus
[0392] In another aspect the invention provides a method of
treating a patient infected with a Human T-Lymphotropic Virus, such
as Human T-Lymphotropic Virus type 1 (HTLV-1), or a disease or
condition associated with HTLV-1 infection, such as, for example,
adult T-cell leukemia/lymphoma (ATLL), HTLV-1-associated myelopathy
(HAM), Tropical Spastic Paraparesis (TSP), uveitis, or arthropathy.
The method comprises administering to the patient an effective
amount of a composition of the invention comprising one or more
polypeptide or conjugate of the invention. The invention also
provides a composition for use in treating a patient infected with
HTLV-1, or a disease or condition associated with HTLV-1 infection,
the composition comprising one or more polypeptide or conjugate of
the invention and a pharmaceutically acceptable carrier or
excipient. A patient diagnosed with HTLV-1 infection includes a
patient exhibiting HTLV-1 proviral DNA in the blood and/or antibody
to an HTLV-1 antigen in the serum.
[0393] A composition comprising a polypeptide of the invention will
generally be administered at a dose and frequency similar to what
is employed in HCV or oncology therapeutic regimens using
clinically-approved interferon-alpha polypeptides, such as, e.g.
ROFERON.RTM.-A (Interferon alfa-2a, recombinant; Hoffmann-La Roche
Inc.) and INTRON.RTM. A (Interferon alfa-2b, recombinant; Schering
Corporation). Exemplary recommended dosing schedules of ROFERON or
INTRON A for the treatment of chronic HCV is 3 million IU
(approximately 15 micrograms (mcg)) three times a week by
subcutaneous injection for, e.g., 24 to 48 weeks. An exemplary
recommended dosing schedule of ROFERON for the treatment of
hairy-cell leukemia is 3-5 million units daily by subcutaneous
injection for 16 to 24 weeks, then 3 million units 3 times a week
for maintenance. An exemplary recommended dosing schedule of INTRON
A for the treatment of hairy-cell leukemia is 2 million IU/m2
(square meter of body surface) administered subcutaneously 3 times
a week for 6 months. Such dosing schedules provide useful ranges
for dosage of a polypeptide of the invention for the treatment of
HTLV-1 infection, or a disease or condition associated with HTLV-1
infection such as adult T-cell leukemia/lymphoma (ATLL),
HTLV-1-associated myelopathy (HAM), or Tropical Spastic Paraparesis
(TSP). Depending on a number of factors (including but not limited
to the activity and the pharmacokinetics of the polypeptide of the
invention and the size, age and health of the patient), the
polypeptide may be administered in lower amounts and/or less
frequently than described above.
[0394] Likewise, a composition comprising a conjugate of the
invention will generally be administered at a dose and frequency
similar to what is employed in HCV therapeutic or oncology
therapeutic regimens using clinically-approved interferon-alpha
conjugates, such as, e.g., PEGASYS.RTM. (Peginterferon alfa-2a;
Hoffmann-La Roche, Inc.) or PEG-INTRON.RTM. (peginterferon alfa-2b;
Schering Corporation). An exemplary recommended dosing schedule of
PEGASYS for the treatment of chronic HCV is 180 mcg once weekly by
subcutaneous injection for, e.g., 24 to 48 weeks. An exemplary
recommended dosing schedule of PEG-INTRON for the treatment of
chronic myelogenous leukemia is 6 mcg/kg body weight once weekly by
subcutaneous injection for, e.g., 52 weeks. Such dosing schedules
provide useful ranges for dosage of a conjugate of the invention
for the treatment of HTLV-1 infection, or a disease or condition
associated with HTLV-1 infection such as adult T-cell
leukemia/lymphoma (ATLL), HTLV-1-associated myelopathy (HAM), or
Tropical Spastic Paraparesis (TSP). Depending on a number of
factors (including but not limited to the molecular weight,
activity, and pharmacokinetics of the conjugate of the invention
and the size, age and health of the patient), the conjugate may be
administered in lower amounts and/or less frequently than described
above.
[0395] In some instances the polypeptide or conjugate of the
invention is administered in combination with one or more
additional therapeutic agent(s). For example, the polypeptide or
conjugate of the invention may be administered in combination with
an antiretroviral drug such as zidovudine (AZT) and/or lamivudine
(3TC). It may also be administered in combination with peripheral
blood stem cell transplantation, conventional chemotherapy, or high
dose chemotherapy with autologous or allogeneic bone marrow
transplantation. Alternatively, the polypeptide or conjugate of the
invention may be combined with other immunotherapy, for example
with anti-interleukin-2 receptor monoclonal antibodies or injection
of cytotoxic T-cells directed against virus antigens.
[0396] The precise amount and frequency of administration of the
polypeptide or conjugate of the invention will depend on a number
of factors such as the specific activity and the pharmacokinetic
properties of the polypeptide or the conjugate, as well as the
nature of the condition being treated, among other factors known to
those of skill in the art. Normally, the dose should be capable of
preventing or lessening the severity or spread of the indication
being treated. Such a dose may be termed an "effective" or
"therapeutically effective" amount. It will be apparent to those of
skill in the art that an effective amount of a polypeptide,
conjugate or composition of the invention depends, inter alia, upon
the condition being treated, the dose, the administration schedule,
whether the polypeptide or conjugate or composition is administered
alone or in combination with other therapeutic agents, the serum
half-life and other pharmacokinetic properties of the polypeptide,
conjugate or composition, as well as the size, age, and general
health of the patient. The dosage and frequency of administration
is ascertainable by one skilled in the art using known
techniques.
[0397] The effectiveness of treatment may be determined by
measuring the HTLV-1 viral load, such as, for example, measuring
the level of HTLV-1 proviral DNA in the blood using methods known
in the art, for example by quantitative PCR as described by Saito
et al., (2004) J. Infect Dis. 189(1):29-40. In some instances, an
effective amount of a composition of the invention is one that is
sufficient to achieve a reduction in viral load by at least 0.5 log
unit, such as at least 1 log unit, at least 2 log units, at least 3
log units, at least 4 log units, at least 5 log units, at least 6
log units, or at least 7 log units over the course of treatment,
compared to the viral load prior to treatment. In some instances an
effective amount of a composition of the invention is an amount
that is sufficient to reduce viral load to levels which are
essentially undetectable. The invention includes a method of
reducing the level of HTLV-1 proviral DNA in blood of a patient
infected with HTLV-1, comprising administering to the patient a
composition of the invention in an amount effective to reduce the
level of HTLV-1 proviral DNA compared to that present prior to the
start of treatment.
[0398] The effectiveness of treatment may alternatively or in
addition be determined by measuring titer of an anti-HTLV-1
antibody in the serum, using methods known in the art, such as, for
example, by commercially-available tests such as INNO-LIA.TM. HTLV
I/II (Innogenetics; Gent Belgium) and Abbott HTLV-I/HTLV-II EIA
(Abbott Laboratories; Abbott Park, Ill.). In some instances an
effective amount of a composition of the invention is an amount
that is sufficient to reduce the titer of an anti-HTLV-1 antibody
in the serum to 50%, 25%, 10% or 5% of the titer present prior to
the start of treatment. In some instances an effective amount of a
composition of the invention is an amount that is sufficient to
reduce the titer of an anti-HTLV-1 antibody in the serum to a level
which is essentially undetectable. The invention includes a method
of reducing the titer of an anti-HTLV-1 antibody in the serum of a
patient infected with HTLV-1, comprising administering to the
patient a composition of the invention in an amount effective to
reduce the titer of the anti-HTLV-1 antibody in the serum compared
to that present prior to the start of treatment.
Human Papillomavirus
[0399] In another aspect the invention provides a method of
treating a patient infected with a Human Papillomavirus (HPV), or a
disease or condition associated with HPV infection, such as, for
example, warts of the hands and feet, or lesions of the mucous
membranes of the oral, anal and genital cavities. While some types
of HPV are relatively harmless, other types are spread through
sexual contact and give rise to genital or venereal warts (termed
condylomata acuminata) which may give rise to cervical cancer and
other genital cancers. The method comprises administering to the
patient infected with HPV an effective amount of a composition of
the invention comprising one or more polypeptide or conjugate of
the invention. The invention also provides a composition for use in
treating a patient infected with HPV, or a disease or condition
associated with HPV infection, the composition comprising one or
more polypeptide or conjugate of the invention and a
pharmaceutically acceptable carrier or excipient. A patient
diagnosed with HPV infection includes a patient exhibiting HPV
viral DNA in biopsied tissue (such as genital tissue), and
sometimes (but not always) exhibiting visible lesions, e.g. on
genital tissues.
[0400] A composition comprising a polypeptide of the invention will
generally be administered at a dose and frequency similar to what
is employed in HPV therapeutic regimens using clinically-approved
interferon-alpha polypeptides, such as, for example, INTRON.RTM. A
(Interferon alfa-2b, recombinant; Schering Corporation). A
recommended dose of INTRON A for the treatment of condylomata
acuminata is 1.0 million IU injected into each lesion, for up to 5
lesions, using a tuberculin or similar syringe and a 25- to
30-gauge needle, three times per week on alternate days, for 3
weeks. Patients with 6 to 10 condylomata may receive a second
(sequential) course of treatment at the above dosage schedule, to
treat up to five additional condylomata per course of treatment.
Patients with greater than 10 condylomata may receive additional
sequences depending on how large a number of condylomata are
present. The interferon may alternatively or in addition be applied
topically, e.g. in a cream or ointment form (as described for
example in Stentella et al. (1996) Clin. Exp. Obstet. Gynecol.
23(1):29-36). Such dosing schedules provide useful ranges for
dosage of a polypeptide of the invention for the treatment of HPV
infection, or a disease or condition associated with HPV infection
such as condylomata acuminata. Depending on a number of factors
(including but not limited to the activity and the pharmacokinetics
of the polypeptide of the invention and the size, age and health of
the patient), the polypeptide of the invention may be administered
in lower amounts and/or less frequently than described above.
Likewise, a composition comprising a conjugate of the invention
will generally be administered, e.g. intralesionally or topically,
at a dose effective to reduce the amount of HPV viral DNA in the
effected tissues or to reduce the size/or number of genital lesions
in the infected individual.
[0401] In some instances the polypeptide or conjugate of the
invention is administered in combination with one or more
additional therapeutic agent(s). For example, the polypeptide or
conjugate of the invention may be administered in combination with
an anti-HPV therapeutic such as Podofilox (Condylox) and/or
Podophyllin (Pododerm, Podocon-25).
[0402] The precise amount and frequency of administration of the
polypeptide or conjugate of the invention will depend on a number
of factors such as the specific activity and the pharmacokinetic
properties of the polypeptide or the conjugate, as well as the
nature of the condition being treated, among other factors known to
those of skill in the art. Normally, the dose should be capable of
preventing or lessening the severity or spread of the indication
being treated. Such a dose may be termed an "effective" or
"therapeutically effective" amount. It will be apparent to those of
skill in the art that an effective amount of a polypeptide,
conjugate or composition of the invention depends, inter alia, upon
the condition being treated, the dose, the administration schedule,
whether the polypeptide or conjugate or composition is administered
alone or in combination with other therapeutic agents, the serum
half-life and other pharmacokinetic properties of the polypeptide,
conjugate or composition, as well as the size, age, and general
health of the patient. The dosage and frequency of administration
is ascertainable by one skilled in the art using known
techniques.
[0403] The effectiveness of treatment may be determined by
measuring the HPV viral load, such as, for example, measuring the
level of HPV viral DNA in biopsied tissue. In some instances, an
effective amount of a composition of the invention is one that is
sufficient to achieve a reduction in viral load by at least 0.5 log
unit, such as at least 1 log unit, at least 2 log units, at least 3
log units, at least 4 log units, at least 5 log units, at least 6
log units, or at least 7 log units over the course of treatment,
compared to the viral load prior to treatment. In some instances an
effective amount of a composition of the invention is an amount
that is sufficient to reduce viral load to levels which are
essentially undetectable. The invention includes a method of
reducing the level of HPV viral DNA in tissue of a patient infected
with HPV, comprising administering to the patient a composition of
the invention in an amount effective to reduce the level of HPV
viral DNA compared to that present prior to the start of
treatment.
[0404] The effectiveness of treatment may alternatively or in
addition be determined by observing the size or number of genital
lesions (condylomata) in the infected individual. In some instances
an effective amount of a composition of the invention is an amount
that is sufficient to reduce the size and/or number of condylomata
in the infected individual. The invention includes a method of
reducing reduce the size and/or number of condylomata in a patient
infected with HPV, comprising administering to the patient a
composition of the invention in an amount effective to reduce the
size and/or number of condylomata in the patient compared to those
present prior to the start of treatment.
Formulations and Routes of Administration
[0405] Therapeutic formulations of the polypeptide or conjugate of
the invention are typically administered in a composition that
includes one or more pharmaceutically acceptable carriers or
excipients. Such pharmaceutical compositions may be prepared in a
manner known per se in the art to result in a polypeptide
pharmaceutical that is sufficiently storage-stable and is suitable
for administration to humans or animals.
Drug form
[0406] The polypeptide or conjugate of the invention can be used
"as is" and/or in a salt form thereof. Suitable salts include, but
are not limited to, salts with alkali metals or alkaline earth
metals, such as sodium, potassium, calcium and magnesium, as well
as e.g. zinc salts. These salts or complexes may by present as a
crystalline and/or amorphous structure.
Excipients
[0407] "Pharmaceutically acceptable" means a carrier or excipient
that at the dosages and concentrations employed does not cause any
untoward effects in the patients to whom it is administered. Such
pharmaceutically acceptable carriers and excipients are well known
in the art (see Remington's Pharmaceutical Sciences, 18th edition,
A. R. Gennaro, Ed., Mack Publishing Company (1990); Pharmaceutical
Formulation Development of Peptides and Proteins, S. Frokjaer and
L. Hovgaard, Eds., Taylor & Francis (2000); and Handbook of
Pharmaceutical Excipients, 3rd edition, A. Kibbe, Ed.,
Pharmaceutical Press (2000)).
Mix of Drugs
[0408] The composition of the invention may be administered alone
or in conjunction with other therapeutic agents. Ribavirin, for
example, is often co-administered with IFN-alpha and has been shown
to increase efficacy in antiviral treatments, such as HCV
treatment. A variety of small molecules are being developed against
both viral targets (viral proteases, viral polymerase, assembly of
viral replication complexes) and host targets (host proteases
required for viral processing, host kinases required for
phosphorylation of viral targets such as NS5A and inhibitors of
host factors required to efficiently utilize the viral IRES). Other
cytokines may be co-administered, such as for example IL-2, IL-12,
IL-23, IL-27, or IFN-gamma. These agents may be incorporated as
part of the same pharmaceutical composition or may be administered
separately from the polypeptide or conjugate of the invention,
either concurrently or in accordance with another treatment
schedule. In addition, the polypeptide, conjugate or composition of
the invention may be used as an adjuvant to other therapies.
Patients
[0409] A "patient" for the purposes of the present invention
includes both humans and other mammals. Thus the methods are
applicable to both human therapy and veterinary applications.
Types of Composition and Administration Route
[0410] The pharmaceutical composition comprising the polypeptide or
conjugate of the invention may be formulated in a variety of forms,
e.g. as a liquid, gel, lyophilized, or as a compressed solid. The
preferred form will depend upon the particular indication being
treated and will be apparent to one skilled in the art.
[0411] The administration of the formulations of the present
invention can be performed in a variety of ways, including, but not
limited to, orally, subcutaneously, intravenously, intracerebrally,
intranasally, transdermally, intraperitoneally, intramuscularly,
intrapulmonary, vaginally, rectally, intraocularly, or in any other
acceptable manner. The formulations can be administered
continuously by infusion, although bolus injection is acceptable,
using techniques well known in the art, such as pumps (e.g.,
subcutaneous osmotic pumps) or implantation. In some instances the
formulations may be directly applied as a solution, cream,
ointment, or spray.
Parenterals
[0412] An example of a pharmaceutical composition is a solution
designed for parenteral administration. Although in many cases
pharmaceutical solution formulations are provided in liquid form,
appropriate for immediate use, such parenteral formulations may
also be provided in frozen or in lyophilized form. In the former
case, the composition must be thawed prior to use. The latter form
is often used to enhance the stability of the active compound
contained in the composition under a wider variety of storage
conditions, as it is recognized by those skilled in the art that
lyophilized preparations are generally more stable than their
liquid counterparts. Such lyophilized preparations are
reconstituted prior to use by the addition of one or more suitable
pharmaceutically acceptable diluents such as sterile water for
injection or sterile physiological saline solution.
[0413] Parenterals may be prepared for storage as lyophilized
formulations or aqueous solutions by mixing, as appropriate, the
polypeptide having the desired degree of purity with one or more
pharmaceutically acceptable carriers, excipients or stabilizers
typically employed in the art (all of which are termed
"excipients"), for example buffering agents, stabilizing agents,
preservatives, isotonifiers, non-ionic detergents, antioxidants
and/or other miscellaneous additives.
[0414] Buffering agents help to maintain the pH in the range which
approximates physiological conditions. They are typically present
at a concentration ranging from about 2 mM to about 50 mM. Suitable
buffering agents for use with the present invention include both
organic and inorganic acids and salts thereof such as citrate
buffers (e.g., monosodium citrate-disodium citrate mixture, citric
acid-trisodium citrate mixture, citric acid-monosodium citrate
mixture, etc.), succinate buffers (e.g., succinic acid-monosodium
succinate mixture, succinic acid-sodium hydroxide mixture, succinic
acid-disodium succinate mixture, etc.), tartrate buffers (e.g.,
tartaric acid-sodium tartrate mixture, tartaric acid-potassium
tartrate mixture, tartaric acid-sodium hydroxide mixture, etc.),
fumarate buffers (e.g., fumaric acid-monosodium fumarate mixture,
fumaric acid-disodium fumarate mixture, monosodium
fumarate-disodium fumarate mixture, etc.), gluconate buffers (e.g.,
gluconic acid-sodium gluconate mixture, gluconic acid-sodium
hydroxide mixture, gluconic acid-potassium gluconate mixture,
etc.), oxalate buffer (e.g., oxalic acid-sodium oxalate mixture,
oxalic acid-sodium hydroxide mixture, oxalic acid-potassium oxalate
mixture, etc.), lactate buffers (e.g., lactic acid-sodium lactate
mixture, lactic acid-sodium hydroxide mixture, lactic
acid-potassium lactate mixture, etc.) and acetate buffers (e.g.,
acetic acid-sodium acetate mixture, acetic acid-sodium hydroxide
mixture, etc.). Additional possibilities are phosphate buffers,
histidine buffers and trimethylamine salts such as Tris.
[0415] Preservatives are added to retard microbial growth, and are
typically added in amounts of about 0.2%-1% (w/v). Suitable
preservatives for use with the present invention include phenol,
benzyl alcohol, meta-cresol, methyl paraben, propyl paraben,
octadecyldimethylbenzyl ammonium chloride, benzalkonium halides
(e.g. benzalkonium chloride, bromide or iodide), hexamethonium
chloride, alkyl parabens such as methyl or propyl paraben,
catechol, resorcinol, cyclohexanol and 3-pentanol.
[0416] Isotonicifiers are added to ensure isotonicity of liquid
compositions and include polyhydric sugar alcohols, preferably
trihydric or higher sugar alcohols, such as glycerin, erythritol,
arabitol, xylitol, sorbitol and mannitol. Polyhydric alcohols can
be present in an amount between 0.1% and 25% by weight, typically
1% to 5%, taking into account the relative amounts of the other
ingredients.
[0417] Stabilizers refer to a broad category of excipients which
can range in function from a bulking agent to an additive which
solubilizes the therapeutic agent or helps to prevent denaturation
or adherence to the container wall. Typical stabilizers can be
polyhydric sugar alcohols (enumerated above); amino acids such as
arginine, lysine, glycine, glutamine, asparagine, histidine,
alanine, ornithine, L-leucine, 2-phenylalanine, glutamic acid,
threonine, etc., organic sugars or sugar alcohols, such as lactose,
trehalose, stachyose, mannitol, sorbitol, xylitol, ribitol,
myoinisitol, galactitol, glycerol and the like, including cyclitols
such as inositol; polyethylene glycol; amino acid polymers;
sulfur-containing reducing agents, such as urea, glutathione,
thioctic acid, sodium thioglycolate, thioglycerol,
.alpha.-monothioglycerol and sodium thiosulfate; low molecular
weight polypeptides (i.e. <10 residues); proteins such as human
serum albumin, bovine serum albumin, gelatin or immunoglobulins;
hydrophilic polymers such as polyvinylpyrrolidone; monosaccharides
such as xylose, mannose, fructose and glucose; disaccharides such
as lactose, maltose and sucrose; trisaccharides such as raffinose,
and polysaccharides such as dextran. Stabilizers are typically
present in the range of from 0.1 to 10,000 parts by weight based on
the active protein weight.
[0418] Non-ionic surfactants or detergents (also known as "wetting
agents") may be present to help solubilize the therapeutic agent as
well as to protect the therapeutic polypeptide against
agitation-induced aggregation, which also permits the formulation
to be exposed to shear surface stress without causing denaturation
of the polypeptide. Suitable non-ionic surfactants include
polysorbates (20, 80, etc.), polyoxamers (184, 188 etc.),
Pluronic.RTM. polyols, polyoxyethylene sorbitan monoethers
(Tween.RTM.-20, Tween.RTM.-80, etc.).
[0419] Additional miscellaneous excipients include bulking agents
or fillers (e.g. starch), chelating agents (e.g. EDTA),
antioxidants (e.g., ascorbic acid, methionine, vitamin E) and
cosolvents.
[0420] The active ingredient may also be entrapped in microcapsules
prepared, for example, by coascervation techniques or by
interfacial polymerization, for example hydroxymethylcellulose,
gelatin or poly-(methylmethacylate) microcapsules, in colloidal
drug delivery systems (for example liposomes, albumin microspheres,
microemulsions, nano-particles and nanocapsules) or in
macroemulsions. Such techniques are disclosed in Remington's
Pharmaceutical Sciences, supra.
[0421] In one aspect of the invention the composition is a liquid
composition, such as an aqueous composition, and comprises a
sulfoalkyl ether cyclodextrin derivative of the formula ##STR1##
wherein n is 4, 5 or 6; R.sub.1, R.sub.2, R.sub.3, R.sub.4,
R.sub.5, R.sub.6, R.sub.7, R.sub.8, and R.sub.9 are each,
independently, --O-- or a --O--(C.sub.2-C.sub.6 alkyl)-SO.sub.3--
group, wherein at least one of R.sub.1, R.sub.2 or R.sub.3 is
independently a --O--(C.sub.2-C.sub.6 alkyl)-SO.sub.3-- group; and
S.sub.1, S.sub.2, S.sub.3, S.sub.4, S.sub.5, S.sub.6, S.sub.7,
S.sub.8, and S.sub.9 are each, independently, a pharmaceutically
acceptable cation, including H.sup.+.
[0422] It should be noted that when n=4, the sulfoalkyl ether
cyclodextrin may also be referred to as a .alpha.-sulfoalkyl ether
cyclodextrin. In a similar way, when n=5, the term
.beta.-sulfoalkyl ether cyclodextrin may be employed and when n=6,
the sulfoalkyl ether cyclodextrin may also be referred to as a
.gamma.-sulfoalkyl ether cyclodextrin.
[0423] In a further embodiment, n is 5 or 6. In a preferred
embodiment n=6.
[0424] In a still further embodiment R.sub.1, R.sub.2 or R.sub.3 is
independently selected from the group consisting of
--OCH.sub.2CH.sub.2CH.sub.2SO.sub.3--,
--OCH.sub.2CH.sub.2CH.sub.2CH.sub.2SO.sub.3-- and
--OCH.sub.2CH.sub.2CH.sub.2CH.sub.2CH.sub.2SO.sub.3--. Most
preferably, R.sub.1, R.sub.2 or R.sub.3 is independently
--OCH.sub.2CH.sub.2CH.sub.2CH.sub.2SO.sub.3--.
[0425] In a further embodiment S.sub.1, S.sub.2, S.sub.3, S.sub.4,
S.sub.5, S.sub.6, S.sub.7, S.sub.8, and S.sub.9 are each,
independently, a pharmaceutically acceptable cation selected from
H.sup.+, alkali metals (e.g. Li.sup.+, Na+, K.sup.+), alkaline
earth metals (e.g., Ca.sup.+2, Mg.sup.+2), ammonium ions and amine
cations such as the cations of (C.sub.1-C.sub.6) alkylamines,
piperidine, pyrazine, (C.sub.1-C.sub.6) alkanolamine and
(C.sub.4-C.sub.8)cycloalkanolamine. Most preferably, S.sub.1,
S.sub.2, S.sub.3, S.sub.4, S.sub.5, S.sub.6, S.sub.7, S.sub.8, and
S.sub.9 are each, independently, a pharmaceutically acceptable
cation selected from the group consisting of H.sup.+, Li.sup.+,
Na.sup.+, K.sup.+, in particular Na.sup.+.
[0426] The sulfoalkyl ether cyclodextrin may contain from 1 to 18
sulfoalkyl groups (when n=4), from 1-21 sulfoalkyl groups (when
n=5) or from 1-21 (when n=6). In a preferred embodiment of the
invention n=5 and the sulfoalkylether derivative comprises, on
average, 2-20 sulfoalkyl groups (in particular sulfobutyl groups),
such as 3-10 sulfoalkyl groups (in particular sulfobutyl groups),
more preferably 4-9 sulfoalkyl groups (in particular sulfobutyl
groups), even more preferably 5-9 sulfoalkyl groups (in particular
sulfobutyl groups), such as 6-8 sulfoalkyl groups (in particular
sulfobutyl groups), e.g. 7 sulfoalkyl groups (in particular
sulfobutyl groups).
[0427] In some instances the sulfoalkyl ether cyclodextrin
derivative is a salt, in particular a sodium salt, of
.beta.-cyclodextrin sulfobutyl ether (i.e. n=5), which on average
contains 7 sulfobutyl groups. This sulfoalkyl ether cyclodextrin
derivative is also termed SBE7-.beta.-CD and is available as
Captisol.RTM. (Cyclex, Overland Park, Kans.).
[0428] The term "C.sub.1-C.sub.6 alkyl" represents a branched or
straight alkyl group having from one to six carbon atoms. Typical
C.sub.1-C.sub.6 alkyl groups include, but are not limited to,
methyl ethyl, n-propyl, iso-propyl, n-butyl, iso-butyl, sec-butyl,
tert-butyl, n-pentyl, iso-pentyl, n-hexyl and iso-hexyl.
[0429] The term "C.sub.2-C.sub.6 alkyl" represents a branched or
straight alkyl group having from two to six carbon atoms. Typical
C.sub.2-C.sub.6 alkyl groups include, but are not limited to,
ethyl, n-propyl, iso-propyl, n-butyl, iso-butyl, sec-butyl,
tert-butyl, n-pentyl, iso-pentyl, n-hexyl and iso-hexyl.
[0430] Further details concerning compositions comprising the
polypeptides disclosed herein and sulfoalkyl ether cyclodextrin
derivatives can be found in WO 03/002152, particularly the section
entitled "The sulfoalkyl ether cyclodextrin derivative" on pp.
37-49, incorporated herein by reference.
[0431] Parenteral formulations to be used for in vivo
administration must be sterile. This is readily accomplished, for
example, by filtration through sterile filtration membranes.
Sustained Release Preparations
[0432] Suitable examples of sustained-release preparations include
semi-permeable matrices of solid hydrophobic polymers containing
the polypeptide or conjugate, the matrices having a suitable form
such as a film or microcapsules. Examples of sustained-release
matrices include polyesters, hydrogels (for example,
poly(2-hydroxyethyl-methacrylate) or poly(vinylalcohol)),
polylactides, copolymers of L-glutamic acid and ethyl-L-glutamate,
non-degradable ethylene-vinyl acetate, degradable lactic
acid-glycolic acid copolymers such as the ProLease.RTM. technology
or Lupron Depot.RTM. (injectable microspheres composed of lactic
acid-glycolic acid copolymer and leuprolide acetate), and
poly-D-(-)-3-hydroxybutyric acid. While polymers such as
ethylene-vinyl acetate and lactic acid-glycolic acid enable release
of molecules for long periods such as up to or over 100 days,
certain hydrogels release proteins for shorter time periods. When
encapsulated polypeptides remain in the body for a long time, they
may denature or aggregate as a result of exposure to moisture at
37.degree. C., resulting in a loss of biological activity and
possible changes in immunogenicity. Rational strategies can be
devised for stabilization depending on the mechanism involved. For
example, if the aggregation mechanism is discovered to be
intermolecular S--S bond formation through thio-disulfide
interchange, stabilization may be achieved by modifying sulfhydryl
residues, lyophilizing from acidic solutions, controlling moisture
content, using appropriate additives, and developing specific
polymer matrix compositions.
Oral Administration
[0433] For oral administration, the pharmaceutical composition may
be in solid or liquid form, e.g. in the form of a capsule, tablet,
suspension, emulsion or solution. The pharmaceutical composition is
preferably made in the form of a dosage unit containing a given
amount of the active ingredient. A suitable daily dose for a human
or other mammal may vary widely depending on the condition of the
patient and other factors, but can be determined by persons skilled
in the art using routine methods.
[0434] Solid dosage forms for oral administration may include
capsules, tablets, suppositories, powders and granules. In such
solid dosage forms, the active compound may be admixed with at
least one inert diluent such as sucrose, lactose, or starch. Such
dosage forms may also comprise, as is normal practice, additional
substances, e.g. lubricating agents such as magnesium stearate. In
the case of capsules, tablets and pills, the dosage forms may also
comprise buffering agents. Tablets and pills can additionally be
prepared with enteric coatings.
[0435] The polypeptides or conjugates may be admixed with adjuvants
such as lactose, sucrose, starch powder, cellulose esters of
alkanoic acids, stearic acid, talc, magnesium stearate, magnesium
oxide, sodium and calcium salts of phosphoric and sulphuric acids,
acacia, gelatin, sodium alginate, polyvinyl-pyrrolidine, and/or
polyvinyl alcohol, and tableted or encapsulated for conventional
administration. Alternatively, they may be dissolved in saline,
water, polyethylene glycol, propylene glycol, ethanol, oils (such
as corn oil, peanut oil, cottonseed oil or sesame oil), tragacanth
gum, and/or various buffers. Other adjuvants and modes of
administration are well known in the pharmaceutical art. The
carrier or diluent may include time delay material, such as
glyceryl monostearate or glyceryl distearate alone or with a wax,
or other materials well known in the art.
[0436] The pharmaceutical compositions may be subjected to
conventional pharmaceutical operations such as sterilization and/or
may contain conventional adjuvants such as preservatives,
stabilizers, wetting agents, emulsifiers, buffers, fillers, etc.,
e.g. as disclosed elsewhere herein.
[0437] Liquid dosage forms for oral administration may include
pharmaceutically acceptable emulsions, solutions, suspensions,
syrups and elixirs containing inert diluents commonly used in the
art, such as water. Such compositions may also comprise adjuvants
such as wetting agents, sweeteners, flavoring agents and perfuming
agents.
Pulmonary Delivery
[0438] Formulations suitable for use with a nebulizer, either jet
or ultrasonic, will typically comprise the polypeptide or conjugate
dissolved in water at a concentration of, e.g., about 0.01 to 25 mg
of conjugate per mL of solution, preferably about 0.1 to 10 mg/mL.
The formulation may also include a buffer and a simple sugar (e.g.,
for protein stabilization and regulation of osmotic pressure),
and/or human serum albumin ranging in concentration from 0.1 to 10
mg/ml. Examples of buffers that may be used are sodium acetate,
citrate and glycine. Preferably, the buffer will have a composition
and molarity suitable to adjust the solution to a pH in the range
of 3 to 9. Generally, buffer molarities of from 1 mM to 50 mM are
suitable for this purpose. Examples of sugars which can be utilized
are lactose, maltose, mannitol, sorbitol, trehalose, and xylose,
usually in amounts ranging from 1% to 10% by weight of the
formulation.
[0439] The nebulizer formulation may also contain a surfactant to
reduce or prevent surface induced aggregation of the protein caused
by atomization of the solution in forming the aerosol. Various
conventional surfactants can be employed, such as polyoxyethylene
fatty acid esters and alcohols, and polyoxyethylene sorbitan fatty
acid esters. Amounts will generally range between 0.001% and 4% by
weight of the formulation. An especially preferred surfactant for
purposes of this invention is polyoxyethylene sorbitan
monooleate.
[0440] Specific formulations and methods of generating suitable
dispersions of liquid particles of the invention are described in
WO 94/20069, U.S. Pat. No. 5,915,378, U.S. Pat. No. 5,960,792, U.S.
Pat. No. 5,957,124, U.S. Pat. No. 5,934,272, U.S. Pat. No.
5,915,378, U.S. Pat. No. 5,855,564, U.S. Pat. No. 5,826,570 and
U.S. Pat. No. 5,522,385 which are hereby incorporated by
reference.
[0441] Formulations for use with a metered dose inhaler device will
generally comprise a finely divided powder. This powder may be
produced by lyophilizing and then milling a liquid conjugate
formulation and may also contain a stabilizer such as human serum
albumin (HSA). Typically, more than 0.5% (w/w) HSA is added.
Additionally, one or more sugars or sugar alcohols may be added to
the preparation if necessary. Examples include lactose maltose,
mannitol, sorbitol, sorbitose, trehalose, xylitol, and xylose. The
amount added to the formulation can range from about 0.01 to 200%
(w/w), preferably from approximately 1 to 50%, of the conjugate
present. Such formulations are then lyophilized and milled to the
desired particle size.
[0442] The properly sized particles are then suspended in a
propellant with the aid of a surfactant. The propellant may be any
conventional material employed for this purpose, such as a
chlorofluorocarbon, a hydrochlorofluorocarbon, a hydrofluorocarbon,
or a hydrocarbon, including trichlorofluoromethane,
dichlorodifluoromethane, dichlorotetrafluoroethanol, and
1,1,1,2-tetrafluoroethane, or combinations thereof. Suitable
surfactants include sorbitan trioleate and soya lecithin. Oleic
acid may also be useful as a surfactant. This mixture is then
loaded into the delivery device. An example of a commercially
available metered dose inhaler suitable for use in the present
invention is the Ventolin metered dose inhaler, manufactured by
Glaxo Inc., Research Triangle Park, N.C., USA.
[0443] Formulations for powder inhalers will comprise a finely
divided dry powder containing conjugate and may also include a
bulking agent, such as lactose, sorbitol, sucrose, or mannitol in
amounts which facilitate dispersal of the powder from the device,
e.g., 50% to 90% by weight of the formulation. The particles of the
powder shall have aerodynamic properties in the lung corresponding
to particles with a density of about 1 g/cm.sup.2 having a median
diameter less than 10 micrometers, preferably between 0.5 and 5
micrometers, most preferably of between 1.5 and 3.5 micrometers. An
example of a powder inhaler suitable for use in accordance with the
teachings herein is the Spinhaler powder inhaler, manufactured by
Fisons Corp., Bedford, Mass., USA.
[0444] The powders for these devices may be generated and/or
delivered by methods disclosed in U.S. Pat. No. 5,997,848, U.S.
Pat. No. 5,993,783, U.S. Pat. No. 5,985,248, U.S. Pat. No.
5,976,574, U.S. Pat. No. 5,922,354, U.S. Pat. No. 5,785,049 and
U.S. Pat. No. 5,654,007.
[0445] Mechanical devices designed for pulmonary delivery of
therapeutic products, include but are not limited to nebulizers,
metered dose inhalers, and powder inhalers, all of which are
familiar to those of skill in the art. Specific examples of
commercially available devices suitable for the practice of this
invention are the Ultravent nebulizer, manufactured by
Mallinckrodt, Inc., St. Louis, Mo., USA; the Acorn II nebulizer,
manufactured by Marquest Medical Products, Englewood, Colo., USA;
the Ventolin metered dose inhaler, manufactured by Glaxo Inc.,
Research Triangle Park, N.C., USA; the Spinhaler powder inhaler,
manufactured by Fisons Corp., Bedford, Mass., USA the "standing
cloud" device of Nektar Therapeutics, Inc., San Carlos, Calif.,
USA; the AIR inhaler manufactured by Alkermes, Cambridge, Mass.,
USA; and the AERx pulmonary drug delivery system manufactured by
Aradigm Corporation, Hayward, Calif., USA.
Kits
[0446] The present invention also provides kits including the
polypeptides, conjugates, polynucleotides, expression vectors,
cells, methods, compositions, and systems, and apparatuses of the
invention. Kits of the invention optionally comprise at least one
of the following of the invention: (1) an apparatus, system, system
component, or apparatus component as described herein; (2) at least
one kit component comprising a polypeptide or conjugate or
polynucleotide of the invention; a plasmid expression vector
encoding a polypeptide of the invention; a cell expressing a
polypeptide of the invention; or a composition comprising at least
one of any such component; (3) instructions for practicing any
method described herein, including a therapeutic or prophylactic
method, instructions for using any component identified in (2) or
any composition of any such component; and/or instructions for
operating any apparatus, system or component described herein; (4)
a container for holding said at least one such component or
composition, and (5) packaging materials.
[0447] In a further aspect, the present invention provides for the
use of any apparatus, component, composition, or kit described
above and herein, for the practice of any method or assay described
herein, and/or for the use of any apparatus, component,
composition, or kit to practice any assay or method described
herein.
EXAMPLES
[0448] The following examples are offered to illustrate the present
invention, but not to limit the spirit or scope of the present
invention in any way.
Materials and Methods
I. Determination of Surface-Accessible Residues
[0449] Accessible Surface Area (ASA)
[0450] The computer program Access (B. Lee and F. M. Richards, J.
Mol. Biol. 55: 379-400 (1971)) version 2 (.COPYRGT.1983 Yale
University) was used to compute the accessible surface area (ASA)
of the individual atoms in the structure. This method typically
uses a probe-size of 1.4 .ANG. and defines the Accessible Surface
Area (ASA) as the area formed by the center of the probe. Prior to
this calculation all water molecules and all hydrogen atoms should
be removed from the coordinate set, as should other atoms not
directly related to the protein.
[0451] Fractional ASA of Side Chain
[0452] The fractional ASA of the side chain atoms is computed by
division of the sum of the ASA of the atoms in the side chain by a
value representing the ASA of the side chain atoms of that residue
type in an extended ALA-x-ALA tripeptide. See Hubbard, Campbell
& Thornton (1991) J. Mol. Biol. 220, 507-530. For this example
the CA atom is regarded as a part of the side chain of glycine
residues but not for the remaining residues. The following values
are used as standard 100% ASA for the side chain (Table 6):
TABLE-US-00006 Ala 69.23 .ANG..sup.2 Arg 200.35 .ANG..sup.2 Asn
106.25 .ANG..sup.2 Asp 102.06 .ANG..sup.2 Cys 96.69 .ANG..sup.2 Gln
140.58 .ANG..sup.2 Glu 134.61 .ANG..sup.2 Gly 32.28 .ANG..sup.2 His
147.00 .ANG..sup.2 Ile 137.91 .ANG..sup.2 Leu 140.76 .ANG..sup.2
Lys 162.50 .ANG..sup.2 Met 156.08 .ANG..sup.2 Phe 163.90
.ANG..sup.2 Pro 119.65 .ANG..sup.2 Ser 78.16 .ANG..sup.2 Thr 101.67
.ANG..sup.2 Trp 210.89 .ANG..sup.2 Tyr 176.61 .ANG..sup.2 Val
114.14 .ANG..sup.2
[0453] Residues not detected in the structure are defined as having
100% exposure as they are thought to reside in flexible regions. In
the case where an ensemble of NMR structures is analyzed, the
average ASA value of the ensemble is used.
[0454] Determination of Surface Exposed Residues when No
Three-Dimensional Structure is Available:
[0455] When no three-dimensional structure is available or if the
structure is not detailed enough to determine surface accessibility
(e.g. if only the position of the CA atoms is known) the surface
accessibility may be inferred from a sequence alignment created as
follows: [0456] A: If the structure is known but not detailed
enough to determine surface accessibility: [0457] The low detail
structure is included in a structure-based sequence alignment to
the known structures of the sequence family using the MODELER
program available from Molecular Simulations, Inc. [0458] B: If no
structure is known: [0459] The sequence is aligned to a predefined
sequence alignment, including the sequences of the known structures
of the sequence family, that may be prepared using the
"profile/structure alignment" option of the program ClustalW
(Thompson et al. (1994) Nucleic Acids Research 22:4673-4680).
[0460] From the sequence alignment obtained in A or B, residues in
the sequence to be analyzed at positions equivalent to residues
exposed in at least one of the other sequences having a known
structure are defined as being exposed. The degree of exposure is
taken to be the largest value for the equivalent residues in the
other sequences. In cases where the sequence to be analyzed is at
an insertion (i.e. there are no equivalent residues in the other
sequences) this residue is defined as being fully exposed, as it
most probably is located in a turn/loop region. In cases where a
low detailed structure exists, those residues not observed in the
structure are defined as being fully exposed, as they are thought
to be in flexible regions.
[0461] Determining Distances Between Atoms:
[0462] The distance between atoms is readily determined using
molecular graphics software, e.g. InsightII.RTM. 98.0 from
Molecular Simulations, Inc.
II. PROTEIN EXPRESSION AND PURIFICATION
[0463] A. Expression and Purification from CHO Cells
[0464] Some polypeptides of the invention were produced in Chinese
Hamster Ovary (CHO) K1 cells (ATCC: CCL-61) that were stably
transfected and selected with G418 to establish clonal cell
lines.
[0465] 1. CHO Expression Construct:
[0466] Nucleic acids encoding polypeptides of the invention were
cloned into a CHO expression vector, under control of the SV40
promoter and in-frame with a sequence which encodes an N-terminal
leader sequence, and, optionally, one or two a C-terminal tag
sequences. The leader sequence was either a generic leader
sequence, IFN alpha 6 leader or a modified IFN alpha 6 leader
sequence, and C-terminal tags included an E-tag (Amersham
Biosciences) &/or a His-tag. Plasmid production was in XL1-Blue
cells.
[0467] 2. Selection of stable subclones expressing IFN-alpha
polypeptides:
[0468] Materials:
[0469] Culture medium: DMEM-F12 with G418, FBS and Penicillin,
Streptomycin and Glutamine (PSG; Gibco/Invitrogen);
[0470] 1.times.PBS (Gibco/Invitrogen);
[0471] Trypsin/EDTA
[0472] Anti E-Tag Antibody-HRP conjugate (Amersham BioSciences)
[0473] ECL Plus Western Blotting detection Reagents (Amersham
BioSciences)
[0474] Procedure: Stable transfectants were generated under
selection with G418 in DMEM/F-12 medium with FBS and penicillin.
Cells were split into T175 flasks with 50 ml of selection medium
and incubated in a 37.degree. C. CO.sub.2 incubator for .about.24
hr. or until cells reached 80% confluence. Cells were harvested by
washing with PBS followed by addition of 2.5 ml Trypsin/EDTA and
incubation at 37.degree. C. for 3-5 min. Cells were collected and
recovered by centrifugation at 1000 g for 30 min in a Beckman Model
bench top centrifuge. Cells were washed once in PBS and resuspended
in 3 ml PBS with 1% FBS. The cell density was determined and
adjusted to 1.times.10.sup.6 cell/ml with PBS/FBS. For each
IFN-alpha, polypeptide cells were sorted in a DakoCytomation MoFlo
sorter into 2-5 96 well plates containing 200 ml of selection
medium. The plates were incubated in a 37.degree. C. incubator for
10-14 days to allow the sorted cells to grow. Two subclones were
selected for each IFN-alpha polypeptide for high level expression
first by dot blot analysis and subsequently confirmed by Western
blot analysis using an anti-E tag antibody-HRP conjugate and
chemiluminescent detection.
[0475] 3. Protein Expression:
[0476] Materials:
[0477] DMEM-F12 medium (Gibco/Invitrogen)
[0478] Ultra CHO medium (BioWhittaker)
[0479] CHO III A medium (Gibco/Invitrogen)
[0480] Ex-Cyte Growth Enhancing Media supplement (Serologicals
Proteins)
[0481] ITSA (Insulin, Transferrin, Selenium supplement for adherent
culture; Gibco/Invitrogen)
[0482] Penicillin/Streptomycin (P/S)
[0483] FBS, PBS, Trypsin
[0484] Procedure:
[0485] Day 1: Cells from one T-175 flask were transferred to one
roller bottle (1700 cm.sup.2) in 300 ml DMEM-F12 with 10% FBS and
1.times.P/S and grown in a 37.degree. C. CO.sub.2 incubator.
[0486] Day 3: Medium was changed to 300 ml fresh
DMEM-F12-FBS-P/S.
[0487] Day 5: The medium was changed to 300 ml Ultra CHO with
1/1000 Ex-Cyte and P/S.
[0488] Day 7: The media was replaced with 300 ml CHO III A+P/S
production medium.
[0489] Supernatants were harvested on Day 8, 9 and 10. The
supernatants were centrifuged at 2000 g for 20 min in a Beckman
Coulter Allegra 6R bench top centrifuge and filtered using a
0.2.mu. PES bottle top sterile filter and stored at 4.degree. C.
for purification.
[0490] 4. Protein Purification:
[0491] Some polypeptides of the invention were expressed as fusion
proteins containing a 13 amino acid E-tag sequence at the
C-terminus. Such polypeptides were purified using an E-tag affinity
column, as follows.
[0492] Materials: Recombinant Phage Antibody System Purification
Module (Amersham BioSciences, Cat. No. 17-1362-01). Purification
kit contains a 16 mm diameter.times.25 mm height (5 ml bed volume)
anti E-Tag column and associated buffers.
[0493] Procedure: Supernatants collected from CHO-HK1 cells in
roller bottles were clarified using a combination of centrifugation
at 2800.times.g for 20 min and filtration using a 0.2.mu. PES
bottle top filter module. Supernatants were loaded onto the E-tag
column equilibrated in RPAS binding buffer at 150 cm/h (5 ml/min).
The column was washed with 5 CV (column volume) of binding buffer
and the protein was eluted at 75 cm/h (2.5 ml/min) with RPAS
elution buffer. Elution fractions were neutralized with 0.05
volumes of 1M Tris-Cl pH 8.0, dialyzed into PBS, concentrated to
0.1-1.5 mg/ml and stored frozen in aliquots at -80.degree. C.
Samples for assays were formulated at 50 .mu.g/ml in PBS with 0.5%
BSA and stored frozen in aliquots at -80.degree. C. Samples were
routinely analyzed by SDS-PAGE followed by Coomassie staining using
materials, reagents and protocols obtained from Invitrogen. Protein
concentrations were routinely determined by the BCA assay using an
IFN-alpha standard, the concentration of which had been verified by
amino acid analysis.
B. Expression and Purification from E. coli
[0494] Some polypeptides of the invention were produced in E. coli
as inclusion bodies, which were purified and refolded as
follows.
[0495] 1. E. coli Expression Construct
[0496] In some instances, nucleic acids encoding polypeptides of
the invention were modified for improved expression in E. coli.
Such modifications comprised replacing rare Arg-Arg codon pairs
AGGAGG at nucleotide positions 34-39, and AGGAGA at nucleotide
positions 64-69 (position numbering relative to SEQ ID NO:18), each
with Arg-Arg codon pairs such as CGTCGC which are preferred in E.
coli, and adding a methionine codon (ATG) to the 5' end of the
coding sequence. The coding sequence was placed into the pET-42
expression vector (Novagen) under control of a T7 promoter with
kanamycin selection marker, or into the pQE80-Kan expression vector
(Qiagen) under the control of a T5 promoter with kanamycin
selection marker.
[0497] 2. Protein Expression
[0498] pET-42 vectors containing interferon coding sequences were
transformed into an E. coli strain such as BL21(DE3) using standard
methods, and plated on to agar plates containing 50 .mu.g/ml
kanamycin and incubated at 37.degree. C. After 18-24 h, three
separate colonies were picked and transferred into tubes containing
5 ml of 2.times.YT with 50 .mu.g/ml kanamycin and incubated
overnight at 37.degree. C. The overnight culture was used to
inoculate 2 sets of flasks containing 100 ml of 2.times.YT with 50
.mu.g/ml kanamycin. The growth of the culture at 37.degree. C. was
monitored at OD.sub.600. The culture was induced at an OD.sub.600
of 0.5-0.8 with 1 mM IPTG for 3 h at 37.degree. C. IPTG induced
cultures were analyzed for expression by SDS-PAGE by lysing
pelleted cells in SDS sample buffer. The corresponding uninduced
sets of cultures were used to prepare frozen stocks by addition of
25% glycerol and freezing cells in 1 ml aliquots at -80.degree. C.
pQE80-Kan vectors containing interferon coding sequences were
transformed into E. coli strains W3 110 or W3 110-fhuA. Expression
was verified as described above.
[0499] Larger scale shake flask expression was performed by
inoculating 4.times.1L 2.times.YT media+kanamycin with 25 ml of an
overnight culture. Cultures were monitored at OD600 and induced
with 1 mM IPTG at 0.5-0.8 OD units. After 3 h of induction cells
are harvested by centrifugation at 5000 g and stored frozen at
-80.degree. C. Cells were disrupted using 2-3 passes through a
French press or a APV 1000 homogenizer at 10,000 psi and processed
as described under "Isolation of IB" and subsequent sections.
[0500] Fed-batch fermentation was conducted at 10 L scale in a
B.Braun bioreactor in Terrific Broth (TB) medium supplemented with
trace element solution and 40 mg/L kanamycin. Fermentation was
initiated by inoculating the bioreactor with a 400 ml overnight
culture in TB medium. During the initial growth phase the dissolved
oxygen (DO) was maintained at 50% by varying the agitation rate.
When the OD600 of the culture reached 5.0, the glycerol/amino acid
feed was initiated at 0.5 ml/min and the agitation was set to 1000
rpm. The feed rate was adjusted to maintain the DO at 40% for the
rest of the fermentation process. When the OD600 reached 25 the
culture was induced by addition of IPTG to a final concentration of
1 mM. Three hours post induction the cells were harvested by
centrifugation at 10000.times.g in a Beckman centrifuge and the
cell paste was stored at -60.degree. C. The OD600 at harvest was
typically around 35-40.
[0501] 3. Isolation, Solubilization, Sulfonation and Refolding of
Inclusion Bodies (IB)
[0502] For isolating IB the thawed cell paste was resuspended in
1.times.PBS at 10 ml per gram of cell paste and mixed until a
uniform slurry was obtained. The cells were disrupted by two passes
through a microfluidizer at 17,000-19,000 psi. The cell lysate was
adjusted to 1% Triton-X100, mixed for 10 min and the IB were
recovered by centrifugation at 10,000 g for 60 min at 2-8.degree.
C. The IB pellet was washed once by resuspending in PBS with 1%
Triton-X100 and recovered by centrifugation as above and stored
frozen at -80.degree. C.
[0503] For solubilizing IB, the IB pellet was resuspended in Urea
buffer (50 mM Tris-Cl, 200 mM NaCl, 8 M urea, 2 mM DTT, 1 mM EDTA,
pH 8.0) at 10 ml buffer per gram of cell paste. The suspension was
mixed for 30 min and centrifuged at 10,000 g for 30 min at
2-8.degree. C. The pellet was washed once by resuspending in the
Urea buffer without DTT, centrifuged as above and washed twice with
water. The washed pellet was solubilized in Guanidine buffer (50 mM
Tris-Cl, 200 mM NaCl, 8 M guanidine-HCl, 1 mM EDTA, pH 8.0) at 10
ml buffer per gram pellet, mixed for 30-60 min and centrifuged at
10,000 g for 30 min at 2-8.degree. C. The supernatant containing
the solubilized IFN was adjusted to 10 mg/ml sodium sulfite and 5
mg/ml sodium tetrathionate to initiate the sulfonation process
which was performed at 2-8.degree. C. for 16 hours.
Post-sulfonation the IFN solution was diluted 2-fold with water and
the IFN pellet was recovered by centrifugation at 10,000 g at
2-8.degree. C. The pellet was washed twice with water and
resuspended in Guanidine buffer as above. The protein concentration
of the sulfonated IFN was determined by absorbance at 280 nm.
[0504] The refolding process was initiated by diluting the
sulfonated IFN in Guanidine buffer at 2-8.degree. C. to a final
concentration of 100 mg/ml in Refolding buffer (50 mM Tris-Cl, 20
mM NaCl, 2 mM reduced glutathione, 1 mM oxidized glutathione).
Refolding was performed at 2-8.degree. C. with slow mixing for 6-8
h, followed by addition of CuSO.sub.4 to a final concentration of 2
mM followed by an additional refolding period of 16-20 h. The
progress of the refolding reaction was monitored by SDS-PAGE and
reverse phase HPLC.
[0505] 4. Purification
[0506] The refolded IFN was purified using three chromatography
steps. The refolding solution was adjusted to 80% ammonium sulfate
(weight/volume), filtered through a 0.2 .mu.M filter and loaded at
200 cm/h onto a 20 ml Butyl Sepharose Fast Flow Hydrophobic
Interaction Chromatography (HIC) column (Amersham Biosciences)
equilibrated in Equil buffer (50 mM Tris-Cl, 0.8 M ammonium
sulfate, pH 8.0). The HIC column was washed with 8 column volumes
(CV) of Equil buffer, followed by 8 CV of Wash buffer (50 mM
Tris-Cl, 0.5 M ammonium sulfate, pH 8.0). IFN was eluted with 15%
ammonium sulfate in 50 mM Tris-Cl, pH 8.0.
[0507] The HIC pool was adjusted to 50 mM sodium acetate using 0.5
M sodium acetate, pH 4.5 stock and diluted two fold. This adjusted
pool was loaded at 90 cm/h on to a 5 ml HiTrap CM Sepharose Fast
Flow column (Amersham Biosciences), equilibrated in 50 mM sodium
acetate, 100 mM NaCl, pH 5.0. Post loading the column was washed
with 5 CV under equilibration buffer conditions and IFN was eluted
using a 20 CV gradient from 100-650 mM NaCl. Peak fractions
containing IFN were pooled based on absorbance at 280 nm.
[0508] The CM Sepharose pool was adjusted to 20 mM 1,3
diaminopropane using a 2 M 1,3 diaminopropane stock to set pH at
.about.10. The sample was loaded at 200 cm/h on to a 5 ml HiTrap Q
Sepharose Fast Flow column (Amersham Biosciences), equilibrated in
50 mM 1,3 diaminopropane, 100 mM NaCl, pH 10.0. Post loading the
column was washed with 5 CV of buffer under equilibration
conditions and IFN was eluted using a 20 CV gradient from 100-500
mM NaCl. The fractions containing IFN are pooled based on
absorbance at 280 nm and immediately dialyzed against 250-500
volumes of 50 mM sodium acetate, 150 mM NaCl, pH 5.0 or 50 mM
sodium borate, pH 9.0. Post-dialysis the IFN samples were sterile
filtered using a 0.2 .mu.M filter in a biosafety cabinet, the
concentration was measured by absorbance at 280 nm and the samples
are stored at 2-8.degree. C. for periods up to a week or in
aliquots at -80.degree. C. for extended storage. For activity
assays the sample was generally formulated in 0.5% BSA, PBS pH 7.4
at 5, 20 and 50 ug/ml, and stored frozen in aliquots at -80.degree.
C.
III. Activity Assays
[0509] A. EMCV-HuH7 Antiviral Assay
[0510] Provided below is an exemplary assay for antiviral activity
of interferon-alpha polypeptides and conjugates of the invention.
The assay is a cell-based dose-response assay used to assess the
anti-viral potency of a drug, and is sometimes referred to as
"protection from cytopathic effect" (or PCPE) assay. Briefly, cells
are incubated with drug and exposed to virus. In the absence of
drug, cells exposed to virus die. With increasing concentrations of
drug, an increasing proportion of cells survive. The number of
surviving cells can be measured directly (e.g., by visual counts)
or indirectly by estimating metabolic rate. For example, metabolic
dyes such as MTT or WST-1 may be used as an indirect measure of
cell survival. Live cells metabolize such dyes to form metabolic
products which can be quantified by spectrophotometry (optical
density).
[0511] Materials:
[0512] Cells:
[0513] HuH7 Cells: Human hepatoma cell line (obtained from Dr.
Michael Lai, USC-Surgery Department, Los Angeles, Calif.). The cell
line may also be obtained from the Cell Bank of the Japanese
Collection of Research Bioresources (JCRB)/ Health Science Research
Resources Bank (HSRRB), Osaka, Japan. The HuH7 cell line was
originally established in the laboratory of Dr. J. Sato (Okayama
University School of Medicine) from a 57-year old Japanese male
with well-differentiated hepatocellular carcinoma (Nakabayashi, H.,
et al. (1982) Cancer Res. 42(9):3858-63). The cell line is negative
for Hepatitis B surface antigen.
[0514] VERO Cells: African green monkey kidney cell line (ATCC #
CCL-81)
[0515] L-929 Cells: Murine fibroblast cell line (ATCC # CCL-1)
[0516] Virus:
[0517] Encephalomyocarditis virus (EMCV): tissue culture adapted
strain (ATCC # VR-129B). High titer viral stocks were produced
in-house by passage in VERO cells
[0518] Complete Media:
[0519] Dulbecco's Modified Eagle Medium (DMEM, Gibco Cat. No.
11965-092)
[0520] 10% Fetal Bovine Serum (FBS, Hyclone Cat. No.
SH30071.03)
[0521] 1.times.Penicillin-streptomycin (PS, Gibco Cat. No.
15140-122)
[0522] Reduced Serum Media:
[0523] Dulbecco's Modified Eagle Medium (DMEM, Gibco Cat. No.
11965-092)
[0524] 2% Fetal Bovine Serum (FBS, Hyclone Cat. No. SH30071.03)
[0525] 1.times.Penicillin-streptomycin (PS, Gibco Cat. No.
15140-122)
[0526] Trypsin/EDTA (Gibco Cat. No. 25300-054)
[0527] WST-1 (Roche; Cat. No. 1644 807)
[0528] HuH7 cells were maintained in Complete Media at 37.degree.
C. in a humidified 5% CO.sub.2 incubator. The cells were harvested
with trypsin and split twice weekly when confluent to a final
density of 1-2.times.10.sup.6 cells per 25 ml in a T175 flask. One
day prior to the assay, the cells were trypsinized and seeded into
new T175 flasks at a density of 4.5.times.10.sup.6 cells per 25 ml
to ensure that the cells were in log phase prior to the assay.
[0529] Procedure:
[0530] A high titer EMCV virus stock was amplified in VERO cells.
The lethal concentration at which 95% of the cells were killed
(LC.sub.95) was determined by an EMCV viral killing curve on HuH7
cell monolayers. Briefly, HuH7 cells were plated on day one in
96-well microtiter plates at 6.times.10.sup.4 cells per well. Virus
was serial diluted 1:3 in DMEM+2% FBS with 10 dilution points and
added to the cells on day two. Twenty-four hours post-infection,
cell survival was determined by a tetrazolium salt metabolism
assay, WST-1 (Roche). The LC.sub.95 determined for the HuH7-EMCV
assay corresponded to an MOI of 0.034 (PFU/cell). Titer of the
virus stock was determined by a standard plaque assay on L929
cells.
[0531] On day one of the assay, log phase HuH7 cells were harvested
with trypsin, resuspended in Reduced Serum Media and concentrated
by centrifugation. The cell pellets, corresponding to 5 T175 flasks
of cells, were resuspended in 10 ml of Reduced Serum Media,
filtered through a 40 micron Nylon cell strainer and counted with a
hemocytometer. Cell viability was determined by trypan blue
exclusion. The cells were resuspended in Reduced Serum Media to a
final density of 6.times.10.sup.5 cells/ml. One hundred microliters
of the diluted cells were added to each well of a 96-well assay
plates (6.times.10.sup.4 cells/well) and the plates were incubated
at 37.degree. C. in a humidified 5% CO.sub.2 incubator for 4
hours.
[0532] The potencies of "reference" interferon alphas and
interferon-alpha polypeptides of the invention (also called "test
samples") were determined by dose-response analysis. There was
generally one test sample and one reference IFN-alpha per plate,
each with three replicate curves of IFN-alpha treated/EMCV
challenged cells and two replicate curves of IFN-alpha treatment
alone. The later was assayed to control for potential
antiproliferative effects of IFN-alpha on HuH7 cells. The
dose-response curves for the reference IFN-alphas generally
consisted of 8 three-fold dilutions ranging from 100 ng/ml to 0.05
ng/ml. For the IFN-alpha test samples, the three-fold dilutions
generally ranged from 5 ng/ml to 0.002 ng/ml. Eight wells each of
cells treated with virus but no IFN-alpha and cells alone were also
run as controls.
[0533] The IFN-alpha dilutions were prepared using Reduced Serum
Media. One hundred microliters of the diluted IFN-alpha
preparations were transferred to the assay plates. The assay plates
were incubated at 37.degree. C. in a humidified 5% CO.sub.2
incubator for 16 hours.
[0534] On day two, the cells were challenged with EMC virus. Medium
was aspirated from each well of the assay plate. The EMCV stock was
diluted 1:5400 in DMEM+2% FBS. One hundred microliters of the
diluted virus, corresponding to 0.034 viral particles per cell, was
added to each well. The cells were incubated with virus at
37.degree. C. in a humidified 5% CO.sub.2 incubator for 24
hours.
[0535] On day three, the number of viable cells in each well was
quantified by WST-1 assay. Medium was aspirated from each well of
the assay plate. The WST-1 reagent was diluted 1:20 in Reduced
Serum Media, 100 .mu.l of the diluted WST-1 reagent was added to
each well and the cells were incubated at 37.degree. C. in a
humidified 5% CO.sub.2 incubator for 60 minutes. The number of
viable cells in each well was quantified by measuring OD at 450 nm
on a plate reader.
[0536] Analysis:
[0537] The antiviral potency of the IFN-alpha reference and test
samples were calculated with the equation:
[0538] Antiviral potency=(Viable cells.sub.C+I+V-Viable
cells.sub.C+V)/(Viable cells.sub.C+I-Viable cells.sub.C+V)*
100%
[0539] where C+V=HuH7 cells+EMCV, C+I=HuH7 cells+IFN-.alpha., and
C+I+V=HuH7 cells+IFN-.alpha.+EMCV
[0540] Dose-response curves were analyzed by non-linear regression
using GraphPad Prism 4 (GraphPad Software Inc.) The following
equation was used for the curve fits: Y = Bottom + ( Top - Bottom )
1 + 10 ( Log .times. .times. EC50 - X ) HillSlope ##EQU1##
[0541] Bottom is the Y value at the bottom plateau; Top is the Y
value at the top plateau, and LogEC50 is the X value when the
response is halfway between Bottom and Top. The Levenberg-Marquardt
method was used as optimization algorithm.
[0542] B. T.sub.H1 Differentiation Assay
[0543] Provided below is an exemplary assay for T.sub.H1
differentiation activity of interferon-alpha polypeptides and
conjugates of the invention.
[0544] Assay Procedure:
[0545] Human buffy coats (25-30 ml) containing leukocytes and
erythrocytes prepared from 500 ml blood were collected from
Stanford Blood Bank the day of assay initiation and kept at room
temperature. Each buffy coat was carefully transferred to a T75
flask and diluted to 100 ml with PBS. For each buffy coat, 13 ml of
Histopaque/Ficoll (Sigma H8889) was pipetted into four 50 ml
centrifuge tubes, and 25 ml of diluted blood sample was carefully
overlaid on top of the Histopaque/Ficoll without disrupting the
interface. The tubes were then centrifuged (20.degree. C., 2500
rpm) for 20 minutes. Using 3 ml plastic transfer pipettes, the top
plasma was removed to the mononuclear cell layer, followed by
transfer of the PBMCs to two 50 ml conical tubes (cells from 2
Histopaque/Ficoll/buffy coat tubes to one tube). The PBMCs were
then diluted to 50 ml/tube with PBS and centrifuged (20.degree. C.,
1000 rpm) for 10 minutes to remove platelets. After removal of PBS,
the PBMCs and remaining RBCs were mixed to prevent aggregation. 5
ml of RBC lysis buffer (ammonium chloride buffer) was added and two
tubes of cells were combined to one tube. Each tube, now containing
the total PBMC and RBC isolate from one donor, was incubated at
room temperature for 10 min. Potential clots of blood cells were
removed by filtering the cells with a cell strainer (70 um, Falcon
Cat. No. 2350). PBS was added to a total volume of 50 ml followed
by centrifugation (20.degree. C., 1000 rpm) for 10 min. The cell
number was finally counted using a hemocytometer.
[0546] Next, a fraction of each PBMC preparation was prestained and
analyzed by FACS to select PBMC preparations with a percentage of
naive Th0 cells above 15%. Two ml of PBMCs were stained with 20
.mu.l FITC-conjugated anti-human CD45RA (Pharmigen, Cat. No.
555488), 20 .mu.l Cy-chrome conjugated anti-human CD4 (Pharmigen,
Cat. No. 555348), 10 .mu.l PE-conjugated anti-human CD8 (Pharmigen,
Cat. No. 555367), 10 .mu.l PE-conjugated anti-human CD14
(Pharmigen, Cat. No. 555398), and 10 .mu.l PE-conjugated anti-human
CD20 (Pharmigen, Cat. No. 555623), and incubated on ice for 45
minutes. The cells were washed with PBS, resuspended in 1 ml PBS,
and filtered with a 40 .mu.m cell strainer (Falcon, Cat. No. 2340).
The percentage of naive T.sub.H0 cells (positive to CD4 and CD45RA
and negative to CD8, CD14, CD20) were quantified by FACS, and PBMC
preparations with more than 15% naive T.sub.H0 cells were selected
for the assay.
[0547] The selected PBMC preparations were stained with 800 .mu.l
FITC-conjugated anti-human CD45RA, 800 .mu.l Cy-chrome conjugated
anti-human CD4, 500 .mu.l PE-conjugated anti-human CD8, 200 .mu.l
PE-conjugated anti-human CD14, and 200 .mu.l PE-conjugated
anti-human CD20, and incubated on ice for 60 minutes. The cells
were washed with PBS, PI was added, and the cells were diluted with
20 ml/ml PBS followed by filtering with a 40 .mu.m cell strainer.
The cells were FACS sorted, and 1.times.10.sup.4 naive T.sub.H0
cells (positive to CD4 and CD45RA and negative to CD8, CD14, CD20)
were transferred by MOFLO into each well of 96 well round bottom
plates, containing 160 .mu.l DMEM plus Penicillin-streptomycin plus
2 mM Glutamine and 10% Fetal Bovine Serum (Hyclone Cat. No.
SH30071.03).
[0548] Twenty .mu.l Dynabeads CD3/CD28 T cell expander (Dynal, Cat.
No. 111.32) were added to each well. The stimulatory effect of the
Dynabeads was calibrated prior to the experiment to avoid
lot-to-lot variance. Next, 20 .mu.l/well of protein samples were
added to the assay plates. Generally, concentration ranges for IL-4
and IL-12 standards (obtained from R&D Systems) were from 0.04
pg/ml to 10 ng/ml, and concentration ranges for IFN-alpha test
samples and IFN- alpha reference sample were from 0.76 pg/ml to 200
ng/ml.
[0549] The cells were incubated at 37.degree. C. in a humidified 5%
CO.sub.2 incubator for 7 days. Supernatants from each well were
harvested to determine the degree of T.sub.H1 expansion through
quantification of the IFN-.gamma. content, using a standard
ELISA.
[0550] Analysis:
[0551] Response .dbd.IFN-.gamma. concentrations in pg/ml.
[0552] The following equation was used for curve fitting: Y =
Bottom + ( Top - Bottom ) 1 + 10 ( Log .times. .times. EC50 - X )
HillSlope ##EQU2##
[0553] The variable Bottom is the Y value at the bottom plateau;
Top is the Y value at the top plateau, and LogEC50 is the X value
when the response is halfway between Bottom and Top. The
Levenberg-Marquardt method was used as optimization algorithm.
[0554] C. Daudi Antiproliferation Assay
[0555] Provided below is an exemplary assay for antiproliferative
activity of interferon-alpha polypeptides and conjugates of the
invention.
[0556] Cell Line Maintenance:
[0557] Daudi Burkitt's lymphoma cells grown in suspension were
maintained in T175 tissue culture flasks, containing 50 ml culture
medium (RPMI+10% Fetal Bovine Serum (FBS, Hyclone Cat. No.
SH30071.03)+1.times.Penicillin-streptomycin (PS, Gibco Cat. No.
15140-122)+2 mM Glutamine), at 37.degree. C. in a humidified 5%
CO.sub.2 incubator. The cells were split 1:10 when confluent.
[0558] Assay procedure:
[0559] Daudi cells were spun down and washed with 1.times.PBS. The
cell number was adjusted to 10.sup.5 cells/ml. 80 .mu.l culture
medium was added to each well in 96 well round bottom assay plates
followed by transfer of 100 .mu.l cells (10.sup.4 cells/well) to
each well.
[0560] Eleven dilutions of the IFN-alpha reference material and
IFN-alpha test samples, ranging from 200 ng/ml to 0.2 pg/ml (4-fold
dilutions), were prepared in dilution plates using culture medium.
Twenty .mu.l of the diluted IFN-alpha preparations were then
transferred to the assay plates.
[0561] The cells were incubated at 37.degree. C. in a humidified 5%
CO.sub.2 incubator. After 48 hours, 1 .mu.Ci of methyl-.sup.3H
thymidine (Amersham Pharmacia, Cat. No. TRK758) was added to each
well followed by incubation for 24 hours at 37.degree. C. in a
humidified 5% CO.sub.2 incubator. The cells were harvested on the
following day and incorporation of thymidine was determined.
[0562] Analysis:
[0563] The EC.sub.50 of the IFN-alpha reference and samples were
calculated using the equation: Y = Bottom + ( Top - Bottom ) 1 + 10
( Log .times. .times. EC50 - X ) HillSlope ##EQU3##
[0564] where Bottom is the Y value at the bottom plateau; Top is
the Y value at the top plateau, and LogEC50 is the X value when the
response is halfway between Bottom and Top. The Levenberg-Marquardt
method was used as optimization algorithm.
Example 1
Determination of Surface-Accessible Residues of
Interferon-Alphas
Surface exposure of human interferon-.alpha.2a residues:
[0565] Based on the 24 NMR structures of human interferon-alpha 2a
reported by Klaus et al., J. Mol. Biol., 274: 661-675 (1997), the
fractional ASA of side chains was calculated. The sequence
numbering used below is based on the mature sequence of the human
interferon-alpha 2a protein (identified herein as SEQ ID
NO:32+R23K). It is noted that this structure contains two
disulphide bridges involving Cys1-Cys98 and Cys29-Cys138,
respectively. By computing the ASA and the fractional ASA and
taking the average of the 24 structures, focusing on the ASA of the
side chains, it was determined that the following residues have
more than 25% fractional ASA: D2, L3, P4, Q5, T6, H7, S8, L9, G10,
R12, R13, M16, A19, Q20, R22, 23,124, S25, L26, F27, S28, L30, K31,
R33, H34, D35, G37, Q40, E41, E42, G44, N45, Q46, Q48, K49, A50,
E51, E58, Q61, Q62, N65, S68, T69, K70, D71, S73, A74, D77, E78,
T79, L80, D82, K83, T86, Y89, Q90, N93, D94, E96, A97, V99, 1100,
Q101, G102, V103, G104, T106, E107, T108, P109, L110, M111, K112,
E113, D114, L117, R120, K121, Q124, R125, T127, L128, K131, E132,
K133, K134, Y135, S136, P137, C138, A145, M148, R149, S152, L153,
N156, Q158, E159, S160, L161, R162, S163, K164 and E165, with
position numbering relative to that of the interferon-alpha 2a
sequence identified herein as SEQ ID NO:32+R23K.
[0566] The following residues were determined to have on average
more than 50% fractional ASA of their side chain: D2, L3, P4, Q5,
T6, H7, S8, L9, R12, R13, M16, A19, S25, F27, S28, K31, R33, H34,
D35, G37, E41, G44, N45, Q46, Q48, K49, N65, K70, A74, D77, E78,
T79, D82, K83, T86, Y89, Q90, N93, D94, 1100, Q101, G102, G104,
T106, E107, T108, P109, L110, E113, D114, L117, R120, K121, Q124,
R125, L128, K131, E132, K134, P137, R149, E159, L161, R162, S163,
K164 and E165, with position numbering relative to that of the
interferon-alpha 2a sequence identified herein as SEQ ID
NO:32+R23K.
Surface exposure of residues corresponding to SEQ ID NO:1:
[0567] Owing to an insertion of an amino acid after position 44 of
the human interferon-alpha 2 subtypes--such as, for example,
interferon-alpha 2b (SEQ ID NO:32) and interferon-alpha 2a (SEQ ID
NO:32+R23K)--in many interferon alpha sequences, including all of
the known human interferon alpha sequences (apart from the
IFN-alpha 2 subtypes) and certain polypeptides of the invention,
the position numbering of the surface-exposed residues will be
shifted by one residue past position number 44 in, for example, the
sequences shown in the alignment FIG. 2, relative to the numbering
of the sequence denoted hIFNalpha 2b (SEQ ID NO:32).
[0568] Based on the above analysis, the following positions,
numbered relative to SEQ ID NO:1, are considered to contain amino
acid residues having more than 25% fractional ASA: positions 2, 3,
4, 5, 6, 7, 8, 9, 10, 12, 13, 16, 19, 20, 22, 23, 24, 25, 26, 27,
28, 30, 31, 33, 34, 35, 37, 40, 41, 42, 44, 46, 47, 49, 50, 51, 52,
59, 62, 63, 66, 69, 70, 71, 72, 74, 75, 78, 79, 80, 81, 83, 84, 87,
90, 91, 94, 95, 97, 98, 100, 101, 102, 103, 104, 105, 107, 108,
109, 110, 111, 112, 113, 114, 115, 118, 121, 122, 125, 126, 128,
129, 132, 133, 134, 135, 136, 137, 138, 139, 146, 149, 150, 153,
154, 157, 159, 160, 161, 162, 163, 164, 165, and 166.
[0569] Likewise, the following positions, again numbered relative
to SEQ ID NO:1, are considered contain amino acid residues having
on average more than 50% fractional ASA of their side chain: 2, 3,
4, 5, 6, 7, 8, 9, 12, 13, 16, 19, 25, 27, 28, 31, 33, 34, 35, 37,
41, 44, 46, 47, 49, 50, 66, 71, 75, 78, 79, 80, 83, 84, 87, 90, 91,
94, 95, 101, 102, 103, 105, 107, 108, 109, 110, 111, 114, 115, 118,
121, 122, 125, 126, 129, 132, 133, 135, 138, 150, 160, 162, 163,
164, 165, and 166.
Example 2
Antiviral Activities of Interferon-Alpha Polypeptides
[0570] Patients with chronic HCV infection have initial viral loads
in the range of 10.sup.4-10.sup.7 copies of HCV RNA/ml. Upon
treatment with IFN-alpha, the viral load characteristically
undergoes two distinct log-linear phases of decline reflecting two
distinct mechanisms (FIG. 1B). The initial drop in viral load
occurs in about the first two days and is believed to be due to the
reduction in rate of virus production by infected liver cells in
the face of the IFN-alpha therapy.
[0571] The major technical challenge with HCV is that the virus
cannot be grown in vitro and has only recently been cultured in
tractable animal models. There are, however, viruses that replicate
in vitro which are considered to be useful surrogates for HCV viral
replication. In vitro surrogate assays believed to be predictive of
in vivo HCV antiviral activity include the assay described above,
which measures the ability of test molecules to protect cells from
the cytopathic effect of viral infection, using EMC RNA virus
(EMCV) in the human liver-derived cell line HuH7.
[0572] Antiviral activities of some IFN-alpha polypeptides of the
invention were assayed in the EMCV/HuH7 antiviral assay described
in the Materials and Methods section above. Some such polypeptides
exhibited antiviral activities about equal to or greater than that
of a reference molecule, e.g., huIFN-alpha 2b (SEQ ID NO:32) or
huIFN-alpha 2a (SEQ ID NO:32+R23K), as evidenced by the EC.sub.50
(the concentration of sample which yields half-maximal protective
response in the assay) of the polypeptide of the invention being
about equal to or less than the EC.sub.50 of the reference
molecule. Some such polypeptides of the invention exhibited at
least about a 1.5-fold higher, at least about a two-fold higher, at
least about a four-fold higher, at least about a five-fold higher,
or at least about a ten-fold higher antiviral activity than the
reference molecule (as evidenced by the EC.sub.50 of the
polypeptide being about 0.66.times. or lower, about 0.5.times. or
lower, about 0.25.times. or lower, about 0.2.times. or lower, or
about 0.1.times. or lower than the EC.sub.50 of the reference
molecule, respectively).
[0573] Table 7 below shows relative antiviral activities of
exemplary IFN-alpha polypeptides of the invention, in comparison to
IFN-alpha Con1 and human IFN-alpha 2b assayed under the same
conditions, expressed as antiviral activity relative to huIFN-alpha
2b (EC.sub.50 huIFN-alpha 2b/EC.sub.50 sample). TABLE-US-00007
TABLE 7 Antiviral activity Sample relative name Sequence to
hulFN.alpha.-2.beta. B9x14 SEQ ID NO:3 .gtoreq. 10 B9x25 SEQ ID
NO:12 .gtoreq. 10 B9x16 SEQ ID NO:3 .gtoreq. 10 + H47Q B9x28 SEQ ID
NO:12 .gtoreq. 10 + E133K, A140S B9x23 SEQ ID NO.12 .gtoreq. 10 +
H47Q B9x18 SEQ ID NO:3 .gtoreq. 10 + H47Q, V51T, F55S, L56V, Y58H
B9x22 SEQ ID NO.12 .gtoreq. 10 + V51T, F55S, L56V, Y58H B9x11 SEQ
ID NO:3 .gtoreq. 10 + E133K, A140S B9x17 SEQ ID NO:3 .gtoreq. 10 +
V51T, F55S, L56V, Y58H B9x27 SEQ ID NO.12 .gtoreq. 10 + H47Q,
E133K, A140S B9x12 SEQ ID NO:3 .gtoreq. 10 + H47Q, E133K, A140S
B9x21 SEQ ID NO:12 .gtoreq. 10 + H47Q, V51T, F55S, L56V, Y58H B9x26
SEQ ID NO:12 .gtoreq. 5 + V51T, F55S, L56V, Y58H, E133K, A140S
B9x24 SEQ ID NO:12 .gtoreq. 5 + H47Q, V51T, F55S, L56V, Y58H,
E133K, A140S B9x15 SEQ ID NO:3 .gtoreq. 5 + H47Q, V51T, F55S, L56V,
Y58H, E133K, A140S 25Ep05 SEQ ID NO:12 .gtoreq. 2.5 + H47Q, V51T,
F55S, L56V, Y58H, N72D, N95D, F154L, K160E, R161S, R164S
IFN.alpha.-Con1 SEQ ID NO:43 .about.2 hulFN.alpha.-2.beta. SEQ ID
NO:32 1
Example 3
T.sub.H1 Differentiation Activities of Interferon-Alpha
Polypeptides
[0574] Patients with chronic HCV infection have initial viral loads
in the range of 10.sup.4-10.sup.7 copies of HCV RNA/ml. Upon
treatment with IFN-alpha, the viral load characteristically
undergoes two distinct log-linear phases of decline reflecting two
distinct mechanisms (FIG. 1). The initial drop in viral load occurs
in about the first two days and is believed to be due to the
reduction in rate of virus production by infected liver cells in
response to IFN-alpha therapy. This reaches a new steady state
after about two days at which time a second, less rapid, log linear
phase of viral clearance is observed. This second phase is believed
to be due to killing of infected liver cells by antigen specific T
cells. IFN-alpha therapy is believed to play a key role in this
biological response through the stimulation of antigen specific T
cells to differentiate into T.sub.H1 cells.
[0575] Without being limited to a particular theory, it is proposed
that an interferon-alpha with an improved ability to stimulate
differentiation of T.sub.H0 cells to T.sub.H1 cells may exhibit a
more robust second phase of viral clearance, and may therefore have
improved efficacy in viral clearance. Based on this working
hypothesis, an assay was developed to measure T.sub.H1
differentiation activity of interferon-alphas on naive T.sub.H0
cells isolated from blood donors.
[0576] T.sub.H1 differentiation activities of some IFN-alpha
polypeptides of the invention were assayed as described in the
Materials and Methods section above. Some such polypeptides of the
invention exhibit T.sub.H1 differentiation activities about equal
to that of a reference molecule, e.g., huIFN-alpha 2b (SEQ ID
NO:32) or huIFN-alpha 2a (SEQ ID NO:32+R23K), as evidenced by the
EC.sub.50 (the concentration of sample which yields half-maximal
production of interferon-gamma in the assay) of the polypeptide
being about equal to the EC.sub.50 of the reference molecule. Some
polypeptides of the invention exhibited T.sub.H1 differentiation
activities greater than that of the reference molecule, as
evidenced by the EC.sub.50 of the polypeptide being lower than the
EC.sub.50 of the reference molecule.
Example 4
Antiproliferative Activities of Interferon-Alpha Polypeptides
[0577] IFN-alpha inhibits proliferation of many cell types,
although the antiproliferative effects often occur at higher doses
than are required for the antiviral response. Daudi cells are a
human derived EVB-transformed B cell line that is IFN-alpha
sensitive. This IFN-alpha responsive cell line serves as a useful
probe of the antiproliferative effects of the IFN-alpha
polypeptides of the invention. Furthermore, antiproliferative
activity of IFN-alpha on megakaryocytes and neutrophils at high
dose is believed to contribute to thrombocytopenia and neutropenia,
respectively. The Daudi antiproliferation assay may serve as a
useful surrogate assay for antiproliferative effects on these other
lymphoid cell types.
[0578] Antiproliferative activities of some IFN-alpha polypeptides
of the invention were assayed as described in the Materials and
Methods section above. Some polypeptides of the invention exhibit
antiproliferative activities about equal to that of a reference
molecule, such as huIFN-alpha 2b (SEQ ID NO:32) or huIFN-alpha 2a
(SEQ ID NO:32+R23K), as evidenced by the EC.sub.50 (the
concentration which yields half-maximal thymidine incorporation in
the assay) of the polypeptide being about equal to the EC.sub.50 of
the reference molecule. Some polypeptides of the invention exhibit
antiproliferative activities about equal to or greater than that of
the reference molecule, as evidenced by the EC.sub.50 of the
polypeptide being about equal to or less than (e.g., about
0.75-fold, about 0.5-fold, or about 0.25-fold) the EC.sub.50 of the
reference molecule. Some polypeptides of the invention exhibit
antiproliferative activities which are about equal to or less than
that of the reference molecule, as evidenced by the EC.sub.50 of
the polypeptide being about equal to or greater than the EC.sub.50
of the reference molecule. Some such polypeptides of the invention
exhibit about a 0.75-fold or lower, about a 0.66-fold or lower,
about a 0.5-fold or lower, about a 0.25-fold or lower, about a
0.2-fold or lower, or about a 0.1-fold or lower antiproliferative
activity than that of the reference molecule (as evidenced by the
EC.sub.50 of the polypeptide being at least about 1.3-fold greater,
at least about 1.5-fold greater, at least about 2-fold greater, at
least about 4-fold greater, at least about 5-fold greater, or at
least about 10-fold greater than the EC.sub.50 of the reference
molecule, respectively); such polypeptides of the invention
nevertheless exhibit a measurable antiproliferative activity.
[0579] Table 8 below shows relative antiproliferative activities of
several exemplary polypeptides of the invention, in comparison to
human IFN-alpha 2b and IFN-alpha Con1 assayed under the same
conditions, expressed as antiproliferative activity relative to
huIFN-alpha 2b (EC.sub.50 huIFN-alpha 2b/EC.sub.50 sample).
TABLE-US-00008 TABLE 8 Antiproliferative Sample activity relative
name Sequence to hulFN.alpha.-2.beta. B9x14 SEQ ID NO:3 .ltoreq.
0.5 B9x25 SEQ ID NO:12 .ltoreq. 0.5 B9x16 SEQ ID NO:3 .ltoreq. 0.25
+ H47Q B9x23 SEQ ID NO:12 .ltoreq. 0.5 + H47Q B9x18 SEQ ID NO:3
.ltoreq. 0.25 + H47Q, V51T, F55S, L56V, Y58H B9x22 SEQ ID NO:12
.ltoreq. 0.5 + V51T, F55S, L56V, Y58H B9x17 SEQ ID NO:3 .ltoreq.
0.5 + V51T, F55S, L56V, Y58H B9x27 SEQ ID NO:12 .ltoreq. 0.25 +
H47Q, E133K, A140S B9x21 SEQ ID NO:12 .ltoreq. 0.5 + H47Q, V51T,
F55S, L56V, Y58H B9x26 SEQ ID NO:12 .ltoreq. 0.25 + V51T, F55S,
L56V, Y58H, E133K, A140S B9x24 SEQ ID NO:12 .ltoreq. 0.25 + H47Q,
V51T, F55S, L56V, Y58H, E133K, A140S B9x15 SEQ ID NO:3 .ltoreq.
0.25 + H47Q, V51T, F55S, L56V, Y58H, E133K, A140S IFN.alpha.-Con1
SEQ ID NO:43 .about.1.5 hulFN.alpha.-2.beta. SEQ ID NO:32 1
Example 5
PEGylation of Interferon-Alpha Polypeptides
Cys-PEGylation
[0580] A polypeptide of the invention which contains a free
cysteine, (such as, for example, B9.times.14CHO6 (SEQ ID NO:49),
which contains a cysteine at position 164), may be
cysteine-PEGylated as follows. The polypeptide is first partially
reduced with an equimolar concentration of TCEP
(Triscarboxyethylphosphine) at 4.degree. C. for 30 min in 50 mM
MES, 100 mM NaCl, pH 6.0. The reduced polypeptide is then reacted
with a 4 fold molar excess of mPEG-MAL reagent (with a PEG moiety
such as a 20 kDa or 30 kDa linear mPEG, or a 40 kDa branched mPEG2)
for 1 h at 4.degree. C. under the same conditions. The PEGylated
reaction mixture is loaded on to a SP-Sepharose HP column
equilibrated with 50 mM MES, pH 6.0, 100 mM NaCl. After a 10 CV
(column volume) wash step a gradient from 0-600 mM NaCl is applied
to fractionate the PEGylated and unPEGylated fractions. Fractions
are collected and aliquots are analyzed by SDS-PAGE. Fractions
containing monoPEGylated species are pooled and formulated for
assays for interferon-alpha activity as described above.
Lys-PEGylation
[0581] A polypeptide of the invention comprising the sequence SEQ
ID NO:47 was buffer-exchanged into 50 mM sodium borate pH 9 by
dialysis or gel filtration, and was concentrated to between 1 and 5
mg/ml. The solution was cooled to 2-8.degree. C. A 3- to 4-times
molar excess of dry powdered NHS-mPEG2 40 kDa (Nektar; Huntsville,
Ala.) over protein was added to the protein solution and stirred
using a stir bar. The stir speed was kept as high as possible
without frothing. The reaction was complete after about 1 h, after
which the reaction was diluted 8-10 fold with 50 mm sodium acetate,
100 mm NaCl pH 5 (optionally including 0.05% TWEEN80). The sample
was filtered and loaded onto a HiTrap SPFF column equilibrated with
50 mm sodium acetate, 100 mm NaCl pH 5. The column was washed
extensively with 10-15 column volumes of starting buffer. A 100 mm
to 1 M NaCl gradient in the same buffer was used to separate
PEGylated proteins from non-PEGylated protein. Fractions containing
PEGylated proteins were pooled and formulated for interferon-alpha
activity assays, and in some instances were further characterized
by amino acid analysis and/or MALDI-TOF mass spectrometry.
Preliminary experiments indicate that a polypeptide of the
invention comprising the sequence SEQ ID NO:47, when
lysine-PEGylated as described above, exhibits over 10-fold higher
antiviral activity than a lysine-PEGylated huIFN-alpha 2a
conjugate.
N-Terminal PEGylation
[0582] A polypeptide of the invention comprising the sequence SEQ
ID NO:47 was initially buffer exchanged into 50 mM sodium borate pH
9 by dialysis or gel filtration, and concentrated to between 1 and
5 mg/ml, after which the polypeptide was buffer exchanged into 100
mM sodium phosphate pH 4 by dialysis. A precipitate that on
occasion formed during dialysis redissolved readily. The protein
solution was cooled to 4.degree. C. A 4 to 10 times molar excess of
dry powdered mPEG2-butylALD 40 kDa (Nektar; Huntsville, Ala.) over
protein was added to the protein solution and stirred using a stir
bar. The stir speed was kept as high as possible without frothing.
Subsequently a 1/10 vol of 200 mM NaCNBH.sub.3 in 100 mM potassium
phosphate pH 4 was added. The reaction was complete after several
hours but could be left overnight. The solution was diluted 5-10
fold with 50 mM sodium acetate, 100 mM NaCl pH 4 (optionally
including 0.05% TWEEN80). The sample was filtered and loaded onto a
HiTrap SPFF column equilibrated with 50 mM sodium acetate, 100 mM
NaCl pH 4. The column was washed extensively with 10-15 column
volumes of starting buffer. A 100 mm to 1 M NaCl gradient in the
same buffer was used to separate PEGylated proteins from
non-PEGylated protein. Fractions containing PEGylated protein were
pooled and formulated for interferon-alpha activity assays, and in
some instances were further characterized by amino acid analysis
and/or MALDI-TOF mass spectrometry. Preliminary experiments
indicate that a polypeptide of the invention comprising the
sequence SEQ ID NO:47, when N-terminally PEGylated as described
above, exhibits over 10-fold higher antiviral activity than a
lysine-PEGylated huIFN-alpha 2a conjugate.
Example 6
In Vivo Assays
Measurement of Half-Life of a Polypeptide or Conjugate of the
Invention
[0583] Measurement of biological or serum half-life may be carried
out in a number of ways described in the literature. For example,
biological half-life may be determined using an ELISA method to
detect serum levels of interferon-alpha after e.g. subcutaneous or
intramuscular administration. Use of an ELISA method to determine
the pharmacokinetics of interferon-alpha administered
subcutaneously is e.g. described by Rostaing et al. (1998), J. Am.
Soc. Nephrol. 9(12): 2344-48. Merimsky et al. (1991), Cancer
Chemother. Pharmacol. 27(5); 406-8, describe the determination of
the serum level of an interferon-alpha administered
intramuscularly.
Determining In Vitro Immunogenicity
[0584] Reduced immunogenicity of a polypeptide or conjugate of the
invention can be determined by use of an ELISA method measuring the
immunoreactivity of the molecule relative to a reference molecule
or preparation, typically a known interferon-alpha protein. The
ELISA method is based on antibodies from patients treated with the
reference protein. The immunogenicity is considered to be reduced
when the polypeptide or conjugate of the invention has a
statistically significant lower response in the assay than the
reference molecule or preparation.
[0585] While the foregoing invention has been described in some
detail for purposes of clarity and understanding, it will be clear
to one skilled in the art from a reading of this disclosure that
various changes in form and detail can be made without departing
from the true scope of the invention. It is understood that the
examples and embodiments described herein are for illustrative
purposes only and that various modifications or changes in light
thereof will be suggested to persons skilled in the art and are to
be included within the spirit and purview of this application and
scope of the appended claims. For example, all the techniques and
apparatus described above may be used in various combinations. All
publications, patents, patent applications, and/or other documents
cited in this application are incorporated herein by reference in
their entirety for all purposes to the same extent as if each
individual publication, patent, patent application, and/or other
document were individually indicated to be incorporated herein by
reference in its entirety for all purposes.
Sequence CWU 1
1
104 1 166 PRT Artificial Sequence IFNalpha B9x11 1 Cys Asp Leu Pro
Gln Thr His Ser Leu Gly His Arg Arg Thr Met Met 1 5 10 15 Leu Leu
Ala Gln Met Arg Arg Ile Ser Leu Phe Ser Cys Leu Lys Asp 20 25 30
Arg His Asp Phe Arg Phe Pro Gln Glu Glu Phe Asp Gly Asn His Phe 35
40 45 Gln Lys Val Gln Ala Ile Phe Leu Phe Tyr Glu Met Met Gln Gln
Thr 50 55 60 Phe Asn Leu Phe Ser Thr Lys Asn Ser Ser Ala Ala Trp
Asp Glu Thr 65 70 75 80 Leu Leu Glu Lys Phe Tyr Ile Glu Leu Phe Gln
Gln Met Asn Asp Leu 85 90 95 Glu Ala Cys Val Met Gln Glu Val Gly
Val Glu Glu Thr Pro Leu Met 100 105 110 Asn Val Asp Ser Ile Leu Ala
Val Arg Lys Tyr Phe Gln Arg Ile Thr 115 120 125 Leu Tyr Leu Thr Lys
Lys Lys Tyr Ser Pro Cys Ser Trp Glu Val Val 130 135 140 Arg Ala Glu
Ile Met Arg Ser Phe Ser Phe Ser Thr Asn Leu Gln Lys 145 150 155 160
Arg Leu Arg Arg Lys Glu 165 2 166 PRT Artificial Sequence IFNalpha
B9x12 2 Cys Asp Leu Pro Gln Thr His Ser Leu Gly His Arg Arg Thr Met
Met 1 5 10 15 Leu Leu Ala Gln Met Arg Arg Ile Ser Leu Phe Ser Cys
Leu Lys Asp 20 25 30 Arg His Asp Phe Arg Phe Pro Gln Glu Glu Phe
Asp Gly Asn Gln Phe 35 40 45 Gln Lys Val Gln Ala Ile Phe Leu Phe
Tyr Glu Met Met Gln Gln Thr 50 55 60 Phe Asn Leu Phe Ser Thr Lys
Asn Ser Ser Ala Ala Trp Asp Glu Thr 65 70 75 80 Leu Leu Glu Lys Phe
Tyr Ile Glu Leu Phe Gln Gln Met Asn Asp Leu 85 90 95 Glu Ala Cys
Val Met Gln Glu Val Gly Val Glu Glu Thr Pro Leu Met 100 105 110 Asn
Val Asp Ser Ile Leu Ala Val Arg Lys Tyr Phe Gln Arg Ile Thr 115 120
125 Leu Tyr Leu Thr Lys Lys Lys Tyr Ser Pro Cys Ser Trp Glu Val Val
130 135 140 Arg Ala Glu Ile Met Arg Ser Phe Ser Phe Ser Thr Asn Leu
Gln Lys 145 150 155 160 Arg Leu Arg Arg Lys Glu 165 3 166 PRT
Artificial Sequence B9x14 IFNalpha 3 Cys Asp Leu Pro Gln Thr His
Ser Leu Gly His Arg Arg Thr Met Met 1 5 10 15 Leu Leu Ala Gln Met
Arg Arg Ile Ser Leu Phe Ser Cys Leu Lys Asp 20 25 30 Arg His Asp
Phe Arg Phe Pro Gln Glu Glu Phe Asp Gly Asn His Phe 35 40 45 Gln
Lys Val Gln Ala Ile Phe Leu Phe Tyr Glu Met Met Gln Gln Thr 50 55
60 Phe Asn Leu Phe Ser Thr Lys Asn Ser Ser Ala Ala Trp Asp Glu Thr
65 70 75 80 Leu Leu Glu Lys Phe Tyr Ile Glu Leu Phe Gln Gln Met Asn
Asp Leu 85 90 95 Glu Ala Cys Val Met Gln Glu Val Gly Val Glu Glu
Thr Pro Leu Met 100 105 110 Asn Val Asp Ser Ile Leu Ala Val Arg Lys
Tyr Phe Gln Arg Ile Thr 115 120 125 Leu Tyr Leu Thr Glu Lys Lys Tyr
Ser Pro Cys Ala Trp Glu Val Val 130 135 140 Arg Ala Glu Ile Met Arg
Ser Phe Ser Phe Ser Thr Asn Leu Gln Lys 145 150 155 160 Arg Leu Arg
Arg Lys Glu 165 4 166 PRT Artificial Sequence IFNalpha B9x15 4 Cys
Asp Leu Pro Gln Thr His Ser Leu Gly His Arg Arg Thr Met Met 1 5 10
15 Leu Leu Ala Gln Met Arg Arg Ile Ser Leu Phe Ser Cys Leu Lys Asp
20 25 30 Arg His Asp Phe Arg Phe Pro Gln Glu Glu Phe Asp Gly Asn
Gln Phe 35 40 45 Gln Lys Thr Gln Ala Ile Ser Val Phe His Glu Met
Met Gln Gln Thr 50 55 60 Phe Asn Leu Phe Ser Thr Lys Asn Ser Ser
Ala Ala Trp Asp Glu Thr 65 70 75 80 Leu Leu Glu Lys Phe Tyr Ile Glu
Leu Phe Gln Gln Met Asn Asp Leu 85 90 95 Glu Ala Cys Val Met Gln
Glu Val Gly Val Glu Glu Thr Pro Leu Met 100 105 110 Asn Val Asp Ser
Ile Leu Ala Val Arg Lys Tyr Phe Gln Arg Ile Thr 115 120 125 Leu Tyr
Leu Thr Lys Lys Lys Tyr Ser Pro Cys Ser Trp Glu Val Val 130 135 140
Arg Ala Glu Ile Met Arg Ser Phe Ser Phe Ser Thr Asn Leu Gln Lys 145
150 155 160 Arg Leu Arg Arg Lys Glu 165 5 166 PRT Artificial
Sequence IFNalpha B9x16 5 Cys Asp Leu Pro Gln Thr His Ser Leu Gly
His Arg Arg Thr Met Met 1 5 10 15 Leu Leu Ala Gln Met Arg Arg Ile
Ser Leu Phe Ser Cys Leu Lys Asp 20 25 30 Arg His Asp Phe Arg Phe
Pro Gln Glu Glu Phe Asp Gly Asn Gln Phe 35 40 45 Gln Lys Val Gln
Ala Ile Phe Leu Phe Tyr Glu Met Met Gln Gln Thr 50 55 60 Phe Asn
Leu Phe Ser Thr Lys Asn Ser Ser Ala Ala Trp Asp Glu Thr 65 70 75 80
Leu Leu Glu Lys Phe Tyr Ile Glu Leu Phe Gln Gln Met Asn Asp Leu 85
90 95 Glu Ala Cys Val Met Gln Glu Val Gly Val Glu Glu Thr Pro Leu
Met 100 105 110 Asn Val Asp Ser Ile Leu Ala Val Arg Lys Tyr Phe Gln
Arg Ile Thr 115 120 125 Leu Tyr Leu Thr Glu Lys Lys Tyr Ser Pro Cys
Ala Trp Glu Val Val 130 135 140 Arg Ala Glu Ile Met Arg Ser Phe Ser
Phe Ser Thr Asn Leu Gln Lys 145 150 155 160 Arg Leu Arg Arg Lys Glu
165 6 166 PRT Artificial Sequence IFNalpha B9x17 6 Cys Asp Leu Pro
Gln Thr His Ser Leu Gly His Arg Arg Thr Met Met 1 5 10 15 Leu Leu
Ala Gln Met Arg Arg Ile Ser Leu Phe Ser Cys Leu Lys Asp 20 25 30
Arg His Asp Phe Arg Phe Pro Gln Glu Glu Phe Asp Gly Asn His Phe 35
40 45 Gln Lys Thr Gln Ala Ile Ser Val Phe His Glu Met Met Gln Gln
Thr 50 55 60 Phe Asn Leu Phe Ser Thr Lys Asn Ser Ser Ala Ala Trp
Asp Glu Thr 65 70 75 80 Leu Leu Glu Lys Phe Tyr Ile Glu Leu Phe Gln
Gln Met Asn Asp Leu 85 90 95 Glu Ala Cys Val Met Gln Glu Val Gly
Val Glu Glu Thr Pro Leu Met 100 105 110 Asn Val Asp Ser Ile Leu Ala
Val Arg Lys Tyr Phe Gln Arg Ile Thr 115 120 125 Leu Tyr Leu Thr Glu
Lys Lys Tyr Ser Pro Cys Ala Trp Glu Val Val 130 135 140 Arg Ala Glu
Ile Met Arg Ser Phe Ser Phe Ser Thr Asn Leu Gln Lys 145 150 155 160
Arg Leu Arg Arg Lys Glu 165 7 166 PRT Artificial Sequence IFNalpha
B9x18 7 Cys Asp Leu Pro Gln Thr His Ser Leu Gly His Arg Arg Thr Met
Met 1 5 10 15 Leu Leu Ala Gln Met Arg Arg Ile Ser Leu Phe Ser Cys
Leu Lys Asp 20 25 30 Arg His Asp Phe Arg Phe Pro Gln Glu Glu Phe
Asp Gly Asn Gln Phe 35 40 45 Gln Lys Thr Gln Ala Ile Ser Val Phe
His Glu Met Met Gln Gln Thr 50 55 60 Phe Asn Leu Phe Ser Thr Lys
Asn Ser Ser Ala Ala Trp Asp Glu Thr 65 70 75 80 Leu Leu Glu Lys Phe
Tyr Ile Glu Leu Phe Gln Gln Met Asn Asp Leu 85 90 95 Glu Ala Cys
Val Met Gln Glu Val Gly Val Glu Glu Thr Pro Leu Met 100 105 110 Asn
Val Asp Ser Ile Leu Ala Val Arg Lys Tyr Phe Gln Arg Ile Thr 115 120
125 Leu Tyr Leu Thr Glu Lys Lys Tyr Ser Pro Cys Ala Trp Glu Val Val
130 135 140 Arg Ala Glu Ile Met Arg Ser Phe Ser Phe Ser Thr Asn Leu
Gln Lys 145 150 155 160 Arg Leu Arg Arg Lys Glu 165 8 166 PRT
Artificial Sequence IFNalpha B9x21 8 Cys Asp Leu Pro Gln Thr His
Ser Leu Ser Asn Arg Arg Thr Leu Met 1 5 10 15 Leu Met Ala Gln Met
Arg Arg Ile Ser Pro Phe Ser Cys Leu Lys Asp 20 25 30 Arg His Asp
Phe Gly Phe Pro Glu Glu Glu Phe Asp Gly His Gln Phe 35 40 45 Gln
Lys Thr Gln Ala Ile Ser Val Leu His Glu Leu Ile Gln Gln Thr 50 55
60 Phe Asn Leu Phe Ser Thr Lys Asn Ser Ser Ala Ala Trp Asp Glu Thr
65 70 75 80 Leu Leu Glu Lys Phe Tyr Ile Glu Leu Phe Gln Gln Met Asn
Asn Leu 85 90 95 Glu Ala Cys Val Ile Gln Glu Val Gly Val Glu Glu
Ile Ala Leu Met 100 105 110 Asn Val Asp Ser Ile Leu Ala Val Arg Lys
Tyr Phe Arg Arg Ile Thr 115 120 125 Leu Tyr Leu Thr Glu Lys Lys Tyr
Ser Pro Cys Ala Trp Glu Val Val 130 135 140 Arg Ala Glu Ile Met Arg
Ser Phe Ser Phe Ser Thr Asn Leu Gln Lys 145 150 155 160 Arg Leu Arg
Arg Lys Glu 165 9 166 PRT Artificial Sequence IFNalpha B9x22 9 Cys
Asp Leu Pro Gln Thr His Ser Leu Ser Asn Arg Arg Thr Leu Met 1 5 10
15 Leu Met Ala Gln Met Arg Arg Ile Ser Pro Phe Ser Cys Leu Lys Asp
20 25 30 Arg His Asp Phe Gly Phe Pro Glu Glu Glu Phe Asp Gly His
His Phe 35 40 45 Gln Lys Thr Gln Ala Ile Ser Val Leu His Glu Leu
Ile Gln Gln Thr 50 55 60 Phe Asn Leu Phe Ser Thr Lys Asn Ser Ser
Ala Ala Trp Asp Glu Thr 65 70 75 80 Leu Leu Glu Lys Phe Tyr Ile Glu
Leu Phe Gln Gln Met Asn Asn Leu 85 90 95 Glu Ala Cys Val Ile Gln
Glu Val Gly Val Glu Glu Ile Ala Leu Met 100 105 110 Asn Val Asp Ser
Ile Leu Ala Val Arg Lys Tyr Phe Arg Arg Ile Thr 115 120 125 Leu Tyr
Leu Thr Glu Lys Lys Tyr Ser Pro Cys Ala Trp Glu Val Val 130 135 140
Arg Ala Glu Ile Met Arg Ser Phe Ser Phe Ser Thr Asn Leu Gln Lys 145
150 155 160 Arg Leu Arg Arg Lys Glu 165 10 166 PRT Artificial
Sequence IFNalpha B9x23 10 Cys Asp Leu Pro Gln Thr His Ser Leu Ser
Asn Arg Arg Thr Leu Met 1 5 10 15 Leu Met Ala Gln Met Arg Arg Ile
Ser Pro Phe Ser Cys Leu Lys Asp 20 25 30 Arg His Asp Phe Gly Phe
Pro Glu Glu Glu Phe Asp Gly His Gln Phe 35 40 45 Gln Lys Val Gln
Ala Ile Phe Leu Leu Tyr Glu Leu Ile Gln Gln Thr 50 55 60 Phe Asn
Leu Phe Ser Thr Lys Asn Ser Ser Ala Ala Trp Asp Glu Thr 65 70 75 80
Leu Leu Glu Lys Phe Tyr Ile Glu Leu Phe Gln Gln Met Asn Asn Leu 85
90 95 Glu Ala Cys Val Ile Gln Glu Val Gly Val Glu Glu Ile Ala Leu
Met 100 105 110 Asn Val Asp Ser Ile Leu Ala Val Arg Lys Tyr Phe Arg
Arg Ile Thr 115 120 125 Leu Tyr Leu Thr Glu Lys Lys Tyr Ser Pro Cys
Ala Trp Glu Val Val 130 135 140 Arg Ala Glu Ile Met Arg Ser Phe Ser
Phe Ser Thr Asn Leu Gln Lys 145 150 155 160 Arg Leu Arg Arg Lys Glu
165 11 166 PRT Artificial Sequence IFNalpha B9x24 11 Cys Asp Leu
Pro Gln Thr His Ser Leu Ser Asn Arg Arg Thr Leu Met 1 5 10 15 Leu
Met Ala Gln Met Arg Arg Ile Ser Pro Phe Ser Cys Leu Lys Asp 20 25
30 Arg His Asp Phe Gly Phe Pro Glu Glu Glu Phe Asp Gly His Gln Phe
35 40 45 Gln Lys Thr Gln Ala Ile Ser Val Leu His Glu Leu Ile Gln
Gln Thr 50 55 60 Phe Asn Leu Phe Ser Thr Lys Asn Ser Ser Ala Ala
Trp Asp Glu Thr 65 70 75 80 Leu Leu Glu Lys Phe Tyr Ile Glu Leu Phe
Gln Gln Met Asn Asn Leu 85 90 95 Glu Ala Cys Val Ile Gln Glu Val
Gly Val Glu Glu Ile Ala Leu Met 100 105 110 Asn Val Asp Ser Ile Leu
Ala Val Arg Lys Tyr Phe Arg Arg Ile Thr 115 120 125 Leu Tyr Leu Thr
Lys Lys Lys Tyr Ser Pro Cys Ser Trp Glu Val Val 130 135 140 Arg Ala
Glu Ile Met Arg Ser Phe Ser Phe Ser Thr Asn Leu Gln Lys 145 150 155
160 Arg Leu Arg Arg Lys Glu 165 12 166 PRT Artificial Sequence
IFNalpha B9x25 12 Cys Asp Leu Pro Gln Thr His Ser Leu Ser Asn Arg
Arg Thr Leu Met 1 5 10 15 Leu Met Ala Gln Met Arg Arg Ile Ser Pro
Phe Ser Cys Leu Lys Asp 20 25 30 Arg His Asp Phe Gly Phe Pro Glu
Glu Glu Phe Asp Gly His His Phe 35 40 45 Gln Lys Val Gln Ala Ile
Phe Leu Leu Tyr Glu Leu Ile Gln Gln Thr 50 55 60 Phe Asn Leu Phe
Ser Thr Lys Asn Ser Ser Ala Ala Trp Asp Glu Thr 65 70 75 80 Leu Leu
Glu Lys Phe Tyr Ile Glu Leu Phe Gln Gln Met Asn Asn Leu 85 90 95
Glu Ala Cys Val Ile Gln Glu Val Gly Val Glu Glu Ile Ala Leu Met 100
105 110 Asn Val Asp Ser Ile Leu Ala Val Arg Lys Tyr Phe Arg Arg Ile
Thr 115 120 125 Leu Tyr Leu Thr Glu Lys Lys Tyr Ser Pro Cys Ala Trp
Glu Val Val 130 135 140 Arg Ala Glu Ile Met Arg Ser Phe Ser Phe Ser
Thr Asn Leu Gln Lys 145 150 155 160 Arg Leu Arg Arg Lys Glu 165 13
166 PRT Artificial Sequence IFNalpha B9x26 13 Cys Asp Leu Pro Gln
Thr His Ser Leu Ser Asn Arg Arg Thr Leu Met 1 5 10 15 Leu Met Ala
Gln Met Arg Arg Ile Ser Pro Phe Ser Cys Leu Lys Asp 20 25 30 Arg
His Asp Phe Gly Phe Pro Glu Glu Glu Phe Asp Gly His His Phe 35 40
45 Gln Lys Thr Gln Ala Ile Ser Val Leu His Glu Leu Ile Gln Gln Thr
50 55 60 Phe Asn Leu Phe Ser Thr Lys Asn Ser Ser Ala Ala Trp Asp
Glu Thr 65 70 75 80 Leu Leu Glu Lys Phe Tyr Ile Glu Leu Phe Gln Gln
Met Asn Asn Leu 85 90 95 Glu Ala Cys Val Ile Gln Glu Val Gly Val
Glu Glu Ile Ala Leu Met 100 105 110 Asn Val Asp Ser Ile Leu Ala Val
Arg Lys Tyr Phe Arg Arg Ile Thr 115 120 125 Leu Tyr Leu Thr Lys Lys
Lys Tyr Ser Pro Cys Ser Trp Glu Val Val 130 135 140 Arg Ala Glu Ile
Met Arg Ser Phe Ser Phe Ser Thr Asn Leu Gln Lys 145 150 155 160 Arg
Leu Arg Arg Lys Glu 165 14 166 PRT Artificial Sequence IFNalpha
B9x27 14 Cys Asp Leu Pro Gln Thr His Ser Leu Ser Asn Arg Arg Thr
Leu Met 1 5 10 15 Leu Met Ala Gln Met Arg Arg Ile Ser Pro Phe Ser
Cys Leu Lys Asp 20 25 30 Arg His Asp Phe Gly Phe Pro Glu Glu Glu
Phe Asp Gly His Gln Phe 35 40 45 Gln Lys Val Gln Ala Ile Phe Leu
Leu Tyr Glu Leu Ile Gln Gln Thr 50 55 60 Phe Asn Leu Phe Ser Thr
Lys Asn Ser Ser Ala Ala Trp Asp Glu Thr 65 70 75 80 Leu Leu Glu Lys
Phe Tyr Ile Glu Leu Phe Gln Gln Met Asn Asn Leu 85 90 95 Glu Ala
Cys Val Ile Gln Glu Val Gly Val Glu Glu Ile Ala Leu Met 100 105 110
Asn Val Asp Ser Ile Leu Ala Val Arg Lys Tyr Phe Arg Arg Ile Thr 115
120 125 Leu Tyr Leu Thr Lys Lys Lys Tyr Ser Pro Cys Ser Trp Glu Val
Val 130 135 140 Arg Ala Glu Ile Met Arg Ser Phe Ser Phe Ser Thr Asn
Leu Gln Lys 145 150 155 160 Arg Leu Arg Arg Lys Glu 165 15 166 PRT
Artificial Sequence IFNalpha B9x28 15 Cys Asp Leu Pro Gln Thr His
Ser Leu Ser Asn Arg Arg Thr Leu Met 1 5 10 15 Leu Met Ala Gln Met
Arg Arg Ile Ser Pro Phe Ser Cys Leu Lys Asp 20 25
30 Arg His Asp Phe Gly Phe Pro Glu Glu Glu Phe Asp Gly His His Phe
35 40 45 Gln Lys Val Gln Ala Ile Phe Leu Leu Tyr Glu Leu Ile Gln
Gln Thr 50 55 60 Phe Asn Leu Phe Ser Thr Lys Asn Ser Ser Ala Ala
Trp Asp Glu Thr 65 70 75 80 Leu Leu Glu Lys Phe Tyr Ile Glu Leu Phe
Gln Gln Met Asn Asn Leu 85 90 95 Glu Ala Cys Val Ile Gln Glu Val
Gly Val Glu Glu Ile Ala Leu Met 100 105 110 Asn Val Asp Ser Ile Leu
Ala Val Arg Lys Tyr Phe Arg Arg Ile Thr 115 120 125 Leu Tyr Leu Thr
Lys Lys Lys Tyr Ser Pro Cys Ser Trp Glu Val Val 130 135 140 Arg Ala
Glu Ile Met Arg Ser Phe Ser Phe Ser Thr Asn Leu Gln Lys 145 150 155
160 Arg Leu Arg Arg Lys Glu 165 16 498 DNA Artificial Sequence
IFNalpha B9x11 coding sequence 16 tgtgatctgc ctcagaccca cagcctgggt
cacaggagga ccatgatgct cctggcacaa 60 atgaggagaa tctctctttt
ctcctgtctg aaggacagac atgacttcag atttccccag 120 gaggagtttg
atggcaacca cttccagaag gttcaagcta tcttcctttt ctatgagatg 180
atgcagcaga ccttcaacct cttcagcaca aagaactcat ctgctgcttg ggatgagacc
240 ctcctagaaa aattctacat tgaacttttc cagcaaatga atgacctgga
agcctgcgtg 300 atgcaggagg ttggagtgga agagactccc ctgatgaatg
tggactccat cctggctgtg 360 aggaaatact ttcaaagaat cactctttat
ctgacaaaga agaagtatag cccttgttcc 420 tgggaggttg tcagagcaga
aatcatgaga tctttctctt tttcaacaaa cttgcaaaaa 480 agattaagga ggaaggaa
498 17 498 DNA Artificial Sequence IFNalpha B9x12 coding sequence
17 tgtgatctgc ctcagaccca cagcctgggt cacaggagga ccatgatgct
cctggcacaa 60 atgaggagaa tctctctttt ctcctgtctg aaggacagac
atgacttcag atttccccag 120 gaggagtttg atggcaacca gttccagaag
gttcaagcta tcttcctttt ctatgagatg 180 atgcagcaga ccttcaacct
cttcagcaca aagaactcat ctgctgcttg ggatgagacc 240 ctcctagaaa
aattctacat tgaacttttc cagcaaatga atgacctgga agcctgcgtg 300
atgcaggagg ttggagtgga agagactccc ctgatgaatg tggactccat cctggctgtg
360 aggaaatact ttcaaagaat cactctttat ctgacaaaga agaagtatag
cccttgttcc 420 tgggaggttg tcagagcaga aatcatgaga tctttctctt
tttcaacaaa cttgcaaaaa 480 agattaagga ggaaggaa 498 18 498 DNA
Artificial Sequence IFNalpha B9x14 coding sequence 18 tgtgatctgc
ctcagaccca cagcctgggt cacaggagga ccatgatgct cctggcacaa 60
atgaggagaa tctctctttt ctcctgtctg aaggacagac atgacttcag atttccccag
120 gaggagtttg atggcaacca cttccagaag gttcaagcta tcttcctttt
ctatgagatg 180 atgcagcaga ccttcaacct cttcagcaca aagaactcat
ctgctgcttg ggatgagacc 240 ctcctagaaa aattctacat tgaacttttc
cagcaaatga atgacctgga agcctgcgtg 300 atgcaggagg ttggagtgga
agagactccc ctgatgaatg tggactccat cctggctgtg 360 aggaaatact
ttcaaagaat cactctttat ctgacagaga agaagtatag cccttgtgcc 420
tgggaggttg tcagagcaga aatcatgaga tctttctctt tttcaacaaa cttgcaaaaa
480 agattaagga ggaaggaa 498 19 498 DNA Artificial Sequence IFNalpha
B9x15 coding sequence 19 tgtgatctgc ctcagaccca cagcctgggt
cacaggagga ccatgatgct cctggcacaa 60 atgaggagaa tctctctttt
ctcctgtctg aaggacagac atgacttcag atttccccag 120 gaggagtttg
atggcaacca gttccagaag actcaagcta tctctgtctt ccatgagatg 180
atgcagcaga ccttcaacct cttcagcaca aagaactcat ctgctgcttg ggatgagacc
240 ctcctagaaa aattctacat tgaacttttc cagcaaatga atgacctgga
agcctgcgtg 300 atgcaggagg ttggagtgga agagactccc ctgatgaatg
tggactccat cctggctgtg 360 aggaaatact ttcaaagaat cactctttat
ctgacaaaga agaagtatag cccttgttcc 420 tgggaggttg tcagagcaga
aatcatgaga tctttctctt tttcaacaaa cttgcaaaaa 480 agattaagga ggaaggaa
498 20 498 DNA Artificial Sequence IFNalpha B9x16 coding sequence
20 tgtgatctgc ctcagaccca cagcctgggt cacaggagga ccatgatgct
cctggcacaa 60 atgaggagaa tctctctttt ctcctgtctg aaggacagac
atgacttcag atttccccag 120 gaggagtttg atggcaacca gttccagaag
gttcaagcta tcttcctttt ctatgagatg 180 atgcagcaga ccttcaacct
cttcagcaca aagaactcat ctgctgcttg ggatgagacc 240 ctcctagaaa
aattctacat tgaacttttc cagcaaatga atgacctgga agcctgcgtg 300
atgcaggagg ttggagtgga agagactccc ctgatgaatg tggactccat cctggctgtg
360 aggaaatact ttcaaagaat cactctttat ctgacagaga agaagtatag
cccttgtgcc 420 tgggaggttg tcagagcaga aatcatgaga tctttctctt
tttcaacaaa cttgcaaaaa 480 agattaagga ggaaggaa 498 21 498 DNA
Artificial Sequence IFNalpha B9x17 coding sequence 21 tgtgatctgc
ctcagaccca cagcctgggt cacaggagga ccatgatgct cctggcacaa 60
atgaggagaa tctctctttt ctcctgtctg aaggacagac atgacttcag atttccccag
120 gaggagtttg atggcaacca cttccagaag actcaagcta tctctgtctt
ccatgagatg 180 atgcagcaga ccttcaacct cttcagcaca aagaactcat
ctgctgcttg ggatgagacc 240 ctcctagaaa aattctacat tgaacttttc
cagcaaatga atgacctgga agcctgcgtg 300 atgcaggagg ttggagtgga
agagactccc ctgatgaatg tggactccat cctggctgtg 360 aggaaatact
ttcaaagaat cactctttat ctgacagaga agaagtatag cccttgtgcc 420
tgggaggttg tcagagcaga aatcatgaga tctttctctt tttcaacaaa cttgcaaaaa
480 agattaagga ggaaggaa 498 22 498 DNA Artificial Sequence IFNalpha
B9x18 coding sequence 22 tgtgatctgc ctcagaccca cagcctgggt
cacaggagga ccatgatgct cctggcacaa 60 atgaggagaa tctctctttt
ctcctgtctg aaggacagac atgacttcag atttccccag 120 gaggagtttg
atggcaacca gttccagaag actcaagcta tctctgtctt ccatgagatg 180
atgcagcaga ccttcaacct cttcagcaca aagaactcat ctgctgcttg ggatgagacc
240 ctcctagaaa aattctacat tgaacttttc cagcaaatga atgacctgga
agcctgcgtg 300 atgcaggagg ttggagtgga agagactccc ctgatgaatg
tggactccat cctggctgtg 360 aggaaatact ttcaaagaat cactctttat
ctgacagaga agaagtatag cccttgtgcc 420 tgggaggttg tcagagcaga
aatcatgaga tctttctctt tttcaacaaa cttgcaaaaa 480 agattaagga ggaaggaa
498 23 498 DNA Artificial Sequence IFNalpha B9x21 coding sequence
23 tgtgatctgc ctcagaccca cagcctgagt aacaggagga ctctgatgct
catggcacaa 60 atgaggagaa tctctccttt ctcctgcctg aaggacagac
atgatttcgg attccccgag 120 gaggagtttg atggccacca gttccagaag
actcaagcca tctctgtcct ccatgagctg 180 atccagcaga ccttcaatct
cttcagcaca aagaactcat ctgctgcttg ggatgagacc 240 ctcctagaaa
aattctacat tgaacttttc cagcaaatga ataacctgga agcatgtgtg 300
atacaggagg ttggggtgga agagattgcc ctgatgaatg tggactccat cctggctgtg
360 aggaaatact tccgaagaat cactctctat ctgacagaga agaaatacag
cccttgtgcc 420 tgggaggttg tcagagcaga aatcatgaga tctttctctt
tttcaacaaa cttgcaaaaa 480 agattaagga ggaaggaa 498 24 498 DNA
Artificial Sequence IFNalpha B9x22 coding sequence 24 tgtgatctgc
ctcagaccca cagcctgagt aacaggagga ctctgatgct catggcacaa 60
atgaggagaa tctctccttt ctcctgcctg aaggacagac atgatttcgg attccccgag
120 gaggagtttg atggccacca cttccagaag actcaagcca tctctgtcct
ccatgagctg 180 atccagcaga ccttcaatct cttcagcaca aagaactcat
ctgctgcttg ggatgagacc 240 ctcctagaaa aattctacat tgaacttttc
cagcaaatga ataacctgga agcatgtgtg 300 atacaggagg ttggggtgga
agagattgcc ctgatgaatg tggactccat cctggctgtg 360 aggaaatact
tccgaagaat cactctctat ctgacagaga agaaatacag cccttgtgcc 420
tgggaggttg tcagagcaga aatcatgaga tctttctctt tttcaacaaa cttgcaaaaa
480 agattaagga ggaaggaa 498 25 498 DNA Artificial Sequence IFNalpha
B9x23 coding sequence 25 tgtgatctgc ctcagaccca cagcctgagt
aacaggagga ctctgatgct catggcacaa 60 atgaggagaa tctctccttt
ctcctgcctg aaggacagac atgatttcgg attccccgag 120 gaggagtttg
atggccacca gttccagaag gttcaagcca tcttccttct ctatgagctg 180
atccagcaga ccttcaatct cttcagcaca aagaactcat ctgctgcttg ggatgagacc
240 ctcctagaaa aattctacat tgaacttttc cagcaaatga ataacctgga
agcatgtgtg 300 atacaggagg ttggggtgga agagattgcc ctgatgaatg
tggactccat cctggctgtg 360 aggaaatact tccgaagaat cactctctat
ctgacagaga agaaatacag cccttgtgcc 420 tgggaggttg tcagagcaga
aatcatgaga tctttctctt tttcaacaaa cttgcaaaaa 480 agattaagga ggaaggaa
498 26 498 DNA Artificial Sequence IFNalpha B9x24 coding sequence
26 tgtgatctgc ctcagaccca cagcctgagt aacaggagga ctctgatgct
catggcacaa 60 atgaggagaa tctctccttt ctcctgcctg aaggacagac
atgatttcgg attccccgag 120 gaggagtttg atggccacca gttccagaag
actcaagcca tctctgtcct ccatgagctg 180 atccagcaga ccttcaatct
cttcagcaca aagaactcat ctgctgcttg ggatgagacc 240 ctcctagaaa
aattctacat tgaacttttc cagcaaatga ataacctgga agcatgtgtg 300
atacaggagg ttggggtgga agagattgcc ctgatgaatg tggactccat cctggctgtg
360 aggaaatact tccgaagaat cactctctat ctgacaaaga agaaatacag
cccttgttcc 420 tgggaggttg tcagagcaga aatcatgaga tctttctctt
tttcaacaaa cttgcaaaaa 480 agattaagga ggaaggaa 498 27 498 DNA
Artificial Sequence IFNalpha B9x25 coding sequence 27 tgtgatctgc
ctcagaccca cagcctgagt aacaggagga ctctgatgct catggcacaa 60
atgaggagaa tctctccttt ctcctgcctg aaggacagac atgatttcgg attccccgag
120 gaggagtttg atggccacca cttccagaag gttcaagcca tcttccttct
ctatgagctg 180 atccagcaga ccttcaatct cttcagcaca aagaactcat
ctgctgcttg ggatgagacc 240 ctcctagaaa aattctacat tgaacttttc
cagcaaatga ataacctgga agcatgtgtg 300 atacaggagg ttggggtgga
agagattgcc ctgatgaatg tggactccat cctggctgtg 360 aggaaatact
tccgaagaat cactctctat ctgacagaga agaaatacag cccttgtgcc 420
tgggaggttg tcagagcaga aatcatgaga tctttctctt tttcaacaaa cttgcaaaaa
480 agattaagga ggaaggaa 498 28 498 DNA Artificial Sequence IFNalpha
B9x26 coding sequence 28 tgtgatctgc ctcagaccca cagcctgagt
aacaggagga ctctgatgct catggcacaa 60 atgaggagaa tctctccttt
ctcctgcctg aaggacagac atgatttcgg attccccgag 120 gaggagtttg
atggccacca cttccagaag actcaagcca tctctgtcct ccatgagctg 180
atccagcaga ccttcaatct cttcagcaca aagaactcat ctgctgcttg ggatgagacc
240 ctcctagaaa aattctacat tgaacttttc cagcaaatga ataacctgga
agcatgtgtg 300 atacaggagg ttggggtgga agagattgcc ctgatgaatg
tggactccat cctggctgtg 360 aggaaatact tccgaagaat cactctctat
ctgacaaaga agaaatacag cccttgttcc 420 tgggaggttg tcagagcaga
aatcatgaga tctttctctt tttcaacaaa cttgcaaaaa 480 agattaagga ggaaggaa
498 29 498 DNA Artificial Sequence IFNalpha B9x27 coding sequence
29 tgtgatctgc ctcagaccca cagcctgagt aacaggagga ctctgatgct
catggcacaa 60 atgaggagaa tctctccttt ctcctgcctg aaggacagac
atgatttcgg attccccgag 120 gaggagtttg atggccacca gttccagaag
gttcaagcca tcttccttct ctatgagctg 180 atccagcaga ccttcaatct
cttcagcaca aagaactcat ctgctgcttg ggatgagacc 240 ctcctagaaa
aattctacat tgaacttttc cagcaaatga ataacctgga agcatgtgtg 300
atacaggagg ttggggtgga agagattgcc ctgatgaatg tggactccat cctggctgtg
360 aggaaatact tccgaagaat cactctctat ctgacaaaga agaaatacag
cccttgttcc 420 tgggaggttg tcagagcaga aatcatgaga tctttctctt
tttcaacaaa cttgcaaaaa 480 agattaagga ggaaggaa 498 30 498 DNA
Artificial Sequence IFNalpha B9x28 coding sequence 30 tgtgatctgc
ctcagaccca cagcctgagt aacaggagga ctctgatgct catggcacaa 60
atgaggagaa tctctccttt ctcctgcctg aaggacagac atgatttcgg attccccgag
120 gaggagtttg atggccacca cttccagaag gttcaagcca tcttccttct
ctatgagctg 180 atccagcaga ccttcaatct cttcagcaca aagaactcat
ctgctgcttg ggatgagacc 240 ctcctagaaa aattctacat tgaacttttc
cagcaaatga ataacctgga agcatgtgtg 300 atacaggagg ttggggtgga
agagattgcc ctgatgaatg tggactccat cctggctgtg 360 aggaaatact
tccgaagaat cactctctat ctgacaaaga agaaatacag cccttgttcc 420
tgggaggttg tcagagcaga aatcatgaga tctttctctt tttcaacaaa cttgcaaaaa
480 agattaagga ggaaggaa 498 31 166 PRT Homo sapiens mature huIFN
alpha-1a 31 Cys Asp Leu Pro Glu Thr His Ser Leu Asp Asn Arg Arg Thr
Leu Met 1 5 10 15 Leu Leu Ala Gln Met Ser Arg Ile Ser Pro Ser Ser
Cys Leu Met Asp 20 25 30 Arg His Asp Phe Gly Phe Pro Gln Glu Glu
Phe Asp Gly Asn Gln Phe 35 40 45 Gln Lys Ala Pro Ala Ile Ser Val
Leu His Glu Leu Ile Gln Gln Ile 50 55 60 Phe Asn Leu Phe Thr Thr
Lys Asp Ser Ser Ala Ala Trp Asp Glu Asp 65 70 75 80 Leu Leu Asp Lys
Phe Cys Thr Glu Leu Tyr Gln Gln Leu Asn Asp Leu 85 90 95 Glu Ala
Cys Val Met Gln Glu Glu Arg Val Gly Glu Thr Pro Leu Met 100 105 110
Asn Ala Asp Ser Ile Leu Ala Val Lys Lys Tyr Phe Arg Arg Ile Thr 115
120 125 Leu Tyr Leu Thr Glu Lys Lys Tyr Ser Pro Cys Ala Trp Glu Val
Val 130 135 140 Arg Ala Glu Ile Met Arg Ser Leu Ser Leu Ser Thr Asn
Leu Gln Glu 145 150 155 160 Arg Leu Arg Arg Lys Glu 165 32 165 PRT
Homo sapiens mature huIFN alpha-2b 32 Cys Asp Leu Pro Gln Thr His
Ser Leu Gly Ser Arg Arg Thr Leu Met 1 5 10 15 Leu Leu Ala Gln Met
Arg Arg Ile Ser Leu Phe Ser Cys Leu Lys Asp 20 25 30 Arg His Asp
Phe Gly Phe Pro Gln Glu Glu Phe Gly Asn Gln Phe Gln 35 40 45 Lys
Ala Glu Thr Ile Pro Val Leu His Glu Met Ile Gln Gln Ile Phe 50 55
60 Asn Leu Phe Ser Thr Lys Asp Ser Ser Ala Ala Trp Asp Glu Thr Leu
65 70 75 80 Leu Asp Lys Phe Tyr Thr Glu Leu Tyr Gln Gln Leu Asn Asp
Leu Glu 85 90 95 Ala Cys Val Ile Gln Gly Val Gly Val Thr Glu Thr
Pro Leu Met Lys 100 105 110 Glu Asp Ser Ile Leu Ala Val Arg Lys Tyr
Phe Gln Arg Ile Thr Leu 115 120 125 Tyr Leu Lys Glu Lys Lys Tyr Ser
Pro Cys Ala Trp Glu Val Val Arg 130 135 140 Ala Glu Ile Met Arg Ser
Phe Ser Leu Ser Thr Asn Leu Gln Glu Ser 145 150 155 160 Leu Arg Ser
Lys Glu 165 33 166 PRT Homo sapiens mature huIFN alpha-4b 33 Cys
Asp Leu Pro Gln Thr His Ser Leu Gly Asn Arg Arg Ala Leu Ile 1 5 10
15 Leu Leu Ala Gln Met Gly Arg Ile Ser His Phe Ser Cys Leu Lys Asp
20 25 30 Arg His Asp Phe Gly Phe Pro Glu Glu Glu Phe Asp Gly His
Gln Phe 35 40 45 Gln Lys Thr Gln Ala Ile Ser Val Leu His Glu Met
Ile Gln Gln Thr 50 55 60 Phe Asn Leu Phe Ser Thr Glu Asp Ser Ser
Ala Ala Trp Glu Gln Ser 65 70 75 80 Leu Leu Glu Lys Phe Ser Thr Glu
Leu Tyr Gln Gln Leu Asn Asp Leu 85 90 95 Glu Ala Cys Val Ile Gln
Glu Val Gly Val Glu Glu Thr Pro Leu Met 100 105 110 Asn Val Asp Ser
Ile Leu Ala Val Arg Lys Tyr Phe Gln Arg Ile Thr 115 120 125 Leu Tyr
Leu Thr Glu Lys Lys Tyr Ser Pro Cys Ala Trp Glu Val Val 130 135 140
Arg Ala Glu Ile Met Arg Ser Leu Ser Phe Ser Thr Asn Leu Gln Lys 145
150 155 160 Arg Leu Arg Arg Lys Asp 165 34 166 PRT Homo sapiens
mature huIFN alpha-5 34 Cys Asp Leu Pro Gln Thr His Ser Leu Ser Asn
Arg Arg Thr Leu Met 1 5 10 15 Ile Met Ala Gln Met Gly Arg Ile Ser
Pro Phe Ser Cys Leu Lys Asp 20 25 30 Arg His Asp Phe Gly Phe Pro
Gln Glu Glu Phe Asp Gly Asn Gln Phe 35 40 45 Gln Lys Ala Gln Ala
Ile Ser Val Leu His Glu Met Ile Gln Gln Thr 50 55 60 Phe Asn Leu
Phe Ser Thr Lys Asp Ser Ser Ala Thr Trp Asp Glu Thr 65 70 75 80 Leu
Leu Asp Lys Phe Tyr Thr Glu Leu Tyr Gln Gln Leu Asn Asp Leu 85 90
95 Glu Ala Cys Met Met Gln Glu Val Gly Val Glu Asp Thr Pro Leu Met
100 105 110 Asn Val Asp Ser Ile Leu Thr Val Arg Lys Tyr Phe Gln Arg
Ile Thr 115 120 125 Leu Tyr Leu Thr Glu Lys Lys Tyr Ser Pro Cys Ala
Trp Glu Val Val 130 135 140 Arg Ala Glu Ile Met Arg Ser Phe Ser Leu
Ser Ala Asn Leu Gln Glu 145 150 155 160 Arg Leu Arg Arg Lys Glu 165
35 166 PRT Homo sapiens mature huIFN alpha-6 35 Cys Asp Leu Pro Gln
Thr His Ser Leu Gly His Arg Arg Thr Met Met 1 5 10 15 Leu Leu Ala
Gln Met Arg Arg Ile Ser Leu Phe Ser Cys Leu Lys Asp 20 25 30 Arg
His Asp Phe Arg Phe Pro Gln Glu Glu Phe Asp Gly Asn Gln Phe 35 40
45 Gln Lys Ala Glu Ala Ile Ser Val Leu His Glu Val Ile Gln Gln Thr
50 55 60 Phe Asn Leu Phe Ser Thr Lys Asp Ser Ser Val Ala Trp Asp
Glu Arg 65 70 75 80 Leu Leu Asp Lys Leu Tyr Thr Glu Leu Tyr Gln Gln
Leu Asn Asp Leu 85 90 95 Glu Ala Cys Val Met Gln Glu Val Trp Val
Gly Gly Thr Pro Leu Met 100 105 110 Asn Glu Asp Ser Ile Leu Ala Val
Arg Lys Tyr Phe Gln Arg Ile Thr 115 120 125 Leu Tyr Leu Thr Glu Lys
Lys Tyr Ser Pro Cys Ala Trp Glu Val Val 130 135 140 Arg Ala Glu Ile
Met Arg Ser Phe Ser Ser Ser Arg Asn Leu Gln Glu 145 150 155
160 Arg Leu Arg Arg Lys Glu 165 36 166 PRT Homo sapiens mature
huIFN alpha-7a 36 Cys Asp Leu Pro Gln Thr His Ser Leu Arg Asn Arg
Arg Ala Leu Ile 1 5 10 15 Leu Leu Ala Gln Met Gly Arg Ile Ser Pro
Phe Ser Cys Leu Lys Asp 20 25 30 Arg His Glu Phe Arg Phe Pro Glu
Glu Glu Phe Asp Gly His Gln Phe 35 40 45 Gln Lys Thr Gln Ala Ile
Ser Val Leu His Glu Met Ile Gln Gln Thr 50 55 60 Phe Asn Leu Phe
Ser Thr Glu Asp Ser Ser Ala Ala Trp Glu Gln Ser 65 70 75 80 Leu Leu
Glu Lys Phe Ser Thr Glu Leu Tyr Gln Gln Leu Asn Asp Leu 85 90 95
Glu Ala Cys Val Ile Gln Glu Val Gly Val Glu Glu Thr Pro Leu Met 100
105 110 Asn Glu Asp Phe Ile Leu Ala Val Arg Lys Tyr Phe Gln Arg Ile
Thr 115 120 125 Leu Tyr Leu Met Glu Lys Lys Tyr Ser Pro Cys Ala Trp
Glu Val Val 130 135 140 Arg Ala Glu Ile Met Arg Ser Phe Ser Phe Ser
Thr Asn Leu Lys Lys 145 150 155 160 Gly Leu Arg Arg Lys Asp 165 37
166 PRT Homo sapiens mature huIFN alpha-8b 37 Cys Asp Leu Pro Gln
Thr His Ser Leu Gly Asn Arg Arg Ala Leu Ile 1 5 10 15 Leu Leu Ala
Gln Met Arg Arg Ile Ser Pro Phe Ser Cys Leu Lys Asp 20 25 30 Arg
His Asp Phe Glu Phe Pro Gln Glu Glu Phe Asp Asp Lys Gln Phe 35 40
45 Gln Lys Ala Gln Ala Ile Ser Val Leu His Glu Met Ile Gln Gln Thr
50 55 60 Phe Asn Leu Phe Ser Thr Lys Asp Ser Ser Ala Ala Leu Asp
Glu Thr 65 70 75 80 Leu Leu Asp Glu Phe Tyr Ile Glu Leu Asp Gln Gln
Leu Asn Asp Leu 85 90 95 Glu Ser Cys Val Met Gln Glu Val Gly Val
Ile Glu Ser Pro Leu Met 100 105 110 Tyr Glu Asp Ser Ile Leu Ala Val
Arg Lys Tyr Phe Gln Arg Ile Thr 115 120 125 Leu Tyr Leu Thr Glu Lys
Lys Tyr Ser Ser Cys Ala Trp Glu Val Val 130 135 140 Arg Ala Glu Ile
Met Arg Ser Phe Ser Leu Ser Ile Asn Leu Gln Lys 145 150 155 160 Arg
Leu Lys Ser Lys Glu 165 38 166 PRT Homo sapiens mature huIFN
alpha-10a 38 Cys Asp Leu Pro Gln Thr His Ser Leu Gly Asn Arg Arg
Ala Leu Ile 1 5 10 15 Leu Leu Gly Gln Met Gly Arg Ile Ser Pro Phe
Ser Cys Leu Lys Asp 20 25 30 Arg His Asp Phe Arg Ile Pro Gln Glu
Glu Phe Asp Gly Asn Gln Phe 35 40 45 Gln Lys Ala Gln Ala Ile Ser
Val Leu His Glu Met Ile Gln Gln Thr 50 55 60 Phe Asn Leu Phe Ser
Thr Glu Asp Ser Ser Ala Ala Trp Glu Gln Ser 65 70 75 80 Leu Leu Glu
Lys Phe Ser Thr Glu Leu Tyr Gln Gln Leu Asn Asp Leu 85 90 95 Glu
Ala Cys Val Ile Gln Glu Val Gly Val Glu Glu Thr Pro Leu Met 100 105
110 Asn Glu Asp Ser Ile Leu Ala Val Arg Lys Tyr Phe Gln Arg Ile Thr
115 120 125 Leu Tyr Leu Ile Glu Arg Lys Tyr Ser Pro Cys Ala Trp Glu
Val Val 130 135 140 Arg Ala Glu Ile Met Arg Ser Leu Ser Phe Ser Thr
Asn Leu Gln Lys 145 150 155 160 Arg Leu Arg Arg Lys Asp 165 39 166
PRT Homo sapiens mature huIFN alpha-14a 39 Cys Asn Leu Ser Gln Thr
His Ser Leu Asn Asn Arg Arg Thr Leu Met 1 5 10 15 Leu Met Ala Gln
Met Arg Arg Ile Ser Pro Phe Ser Cys Leu Lys Asp 20 25 30 Arg His
Asp Phe Glu Phe Pro Gln Glu Glu Phe Asp Gly Asn Gln Phe 35 40 45
Gln Lys Ala Gln Ala Ile Ser Val Leu His Glu Met Met Gln Gln Thr 50
55 60 Phe Asn Leu Phe Ser Thr Lys Asn Ser Ser Ala Ala Trp Asp Glu
Thr 65 70 75 80 Leu Leu Glu Lys Phe Tyr Ile Glu Leu Phe Gln Gln Met
Asn Asp Leu 85 90 95 Glu Ala Cys Val Ile Gln Glu Val Gly Val Glu
Glu Thr Pro Leu Met 100 105 110 Asn Glu Asp Ser Ile Leu Ala Val Arg
Lys Tyr Phe Gln Arg Ile Thr 115 120 125 Leu Tyr Leu Met Glu Lys Lys
Tyr Ser Pro Cys Ala Trp Glu Val Val 130 135 140 Arg Ala Glu Ile Met
Arg Ser Phe Ser Phe Ser Thr Asn Leu Gln Lys 145 150 155 160 Arg Leu
Arg Arg Lys Asp 165 40 166 PRT Homo sapiens mature huIFN alpha-16
40 Cys Asp Leu Pro Gln Thr His Ser Leu Gly Asn Arg Arg Ala Leu Ile
1 5 10 15 Leu Leu Ala Gln Met Gly Arg Ile Ser His Phe Ser Cys Leu
Lys Asp 20 25 30 Arg Tyr Asp Phe Gly Phe Pro Gln Glu Val Phe Asp
Gly Asn Gln Phe 35 40 45 Gln Lys Ala Gln Ala Ile Ser Ala Phe His
Glu Met Ile Gln Gln Thr 50 55 60 Phe Asn Leu Phe Ser Thr Lys Asp
Ser Ser Ala Ala Trp Asp Glu Thr 65 70 75 80 Leu Leu Asp Lys Phe Tyr
Ile Glu Leu Phe Gln Gln Leu Asn Asp Leu 85 90 95 Glu Ala Cys Val
Thr Gln Glu Val Gly Val Glu Glu Ile Ala Leu Met 100 105 110 Asn Glu
Asp Ser Ile Leu Ala Val Arg Lys Tyr Phe Gln Arg Ile Thr 115 120 125
Leu Tyr Leu Met Gly Lys Lys Tyr Ser Pro Cys Ala Trp Glu Val Val 130
135 140 Arg Ala Glu Ile Met Arg Ser Phe Ser Phe Ser Thr Asn Leu Gln
Lys 145 150 155 160 Gly Leu Arg Arg Lys Asp 165 41 166 PRT Homo
sapiens mature huIFN alpha-17b 41 Cys Asp Leu Pro Gln Thr His Ser
Leu Gly Asn Arg Arg Ala Leu Ile 1 5 10 15 Leu Leu Ala Gln Met Gly
Arg Ile Ser Pro Phe Ser Cys Leu Lys Asp 20 25 30 Arg His Asp Phe
Gly Leu Pro Gln Glu Glu Phe Asp Gly Asn Gln Phe 35 40 45 Gln Lys
Thr Gln Ala Ile Ser Val Leu His Glu Met Ile Gln Gln Thr 50 55 60
Phe Asn Leu Phe Ser Thr Glu Asp Ser Ser Ala Ala Trp Glu Gln Ser 65
70 75 80 Leu Leu Glu Lys Phe Ser Thr Glu Leu Tyr Gln Gln Leu Asn
Asn Leu 85 90 95 Glu Ala Cys Val Ile Gln Glu Val Gly Met Glu Glu
Thr Pro Leu Met 100 105 110 Asn Glu Asp Ser Ile Leu Ala Val Arg Lys
Tyr Phe Gln Arg Ile Thr 115 120 125 Leu Tyr Leu Thr Glu Lys Lys Tyr
Ser Pro Cys Ala Trp Glu Val Val 130 135 140 Arg Ala Glu Ile Met Arg
Ser Leu Ser Phe Ser Thr Asn Leu Gln Lys 145 150 155 160 Ile Leu Arg
Arg Lys Asp 165 42 166 PRT Homo sapiens mature huIFN alpha-21 42
Cys Asp Leu Pro Gln Thr His Ser Leu Gly Asn Arg Arg Ala Leu Ile 1 5
10 15 Leu Leu Ala Gln Met Gly Arg Ile Ser Pro Phe Ser Cys Leu Lys
Asp 20 25 30 Arg His Asp Phe Gly Phe Pro Gln Glu Glu Phe Asp Gly
Asn Gln Phe 35 40 45 Gln Lys Ala Gln Ala Ile Ser Val Leu His Glu
Met Ile Gln Gln Thr 50 55 60 Phe Asn Leu Phe Ser Thr Lys Asp Ser
Ser Ala Thr Trp Glu Gln Ser 65 70 75 80 Leu Leu Glu Lys Phe Ser Thr
Glu Leu Asn Gln Gln Leu Asn Asp Leu 85 90 95 Glu Ala Cys Val Ile
Gln Glu Val Gly Val Glu Glu Thr Pro Leu Met 100 105 110 Asn Val Asp
Ser Ile Leu Ala Val Lys Lys Tyr Phe Gln Arg Ile Thr 115 120 125 Leu
Tyr Leu Thr Glu Lys Lys Tyr Ser Pro Cys Ala Trp Glu Val Val 130 135
140 Arg Ala Glu Ile Met Arg Ser Phe Ser Leu Ser Lys Ile Phe Gln Glu
145 150 155 160 Arg Leu Arg Arg Lys Glu 165 43 167 PRT Artificial
Sequence IFNalpha-Con1 43 Met Cys Asp Leu Pro Gln Thr His Ser Leu
Gly Asn Arg Arg Ala Leu 1 5 10 15 Ile Leu Leu Ala Gln Met Arg Arg
Ile Ser Pro Phe Ser Cys Leu Lys 20 25 30 Asp Arg His Asp Phe Gly
Phe Pro Gln Glu Glu Phe Asp Gly Asn Gln 35 40 45 Phe Gln Lys Ala
Gln Ala Ile Ser Val Leu His Glu Met Ile Gln Gln 50 55 60 Thr Phe
Asn Leu Phe Ser Thr Lys Asp Ser Ser Ala Ala Trp Asp Glu 65 70 75 80
Ser Leu Leu Glu Lys Phe Tyr Thr Glu Leu Tyr Gln Gln Leu Asn Asp 85
90 95 Leu Glu Ala Cys Val Ile Gln Glu Val Gly Val Glu Glu Thr Pro
Leu 100 105 110 Met Asn Val Asp Ser Ile Leu Ala Val Lys Lys Tyr Phe
Gln Arg Ile 115 120 125 Thr Leu Tyr Leu Thr Glu Lys Lys Tyr Ser Pro
Cys Ala Trp Glu Val 130 135 140 Val Arg Ala Glu Ile Met Arg Ser Phe
Ser Leu Ser Thr Asn Leu Gln 145 150 155 160 Glu Arg Leu Arg Arg Lys
Glu 165 44 166 PRT Artificial Sequence IFNalpha B9x14C2a 44 Cys Asp
Leu Pro Gln Thr His Ser Leu Gly His Arg Arg Thr Met Met 1 5 10 15
Leu Leu Ala Gln Met Arg Arg Ile Ser Leu Phe Ser Cys Leu Lys Asp 20
25 30 Arg His Asp Phe Arg Phe Pro Gln Glu Glu Phe Asp Gly Asn His
Phe 35 40 45 Gln Lys Val Gln Ala Ile Phe Leu Phe Tyr Glu Met Met
Gln Gln Thr 50 55 60 Phe Asn Leu Phe Ser Thr Lys Asn Ser Ser Ala
Ala Trp Asp Glu Thr 65 70 75 80 Leu Leu Glu Lys Phe Tyr Ile Glu Leu
Phe Gln Gln Met Asn Asp Leu 85 90 95 Glu Ala Cys Val Met Gln Glu
Val Gly Val Glu Glu Thr Pro Leu Met 100 105 110 Asn Val Asp Ser Ile
Leu Ala Val Arg Lys Tyr Phe Gln Arg Ile Thr 115 120 125 Leu Tyr Leu
Thr Glu Lys Lys Tyr Ser Pro Cys Ala Trp Glu Val Val 130 135 140 Arg
Ala Glu Ile Met Arg Ser Phe Ser Leu Ser Thr Asn Leu Gln Glu 145 150
155 160 Ser Leu Arg Ser Lys Glu 165 45 167 PRT Artificial Sequence
IFNalpha B9x14CHO1 45 Cys Asp Leu Pro Gln Thr His Ser Leu Gly His
Arg Arg Thr Met Met 1 5 10 15 Leu Leu Ala Gln Met Arg Arg Ile Ser
Leu Phe Ser Cys Leu Lys Asp 20 25 30 Arg His Asp Phe Arg Phe Pro
Gln Glu Glu Phe Asp Gly Asn His Phe 35 40 45 Gln Lys Val Gln Ala
Ile Phe Leu Phe Tyr Glu Met Met Gln Gln Thr 50 55 60 Phe Asn Leu
Phe Ser Thr Lys Asn Ser Ser Ala Ala Trp Asp Glu Thr 65 70 75 80 Leu
Leu Glu Lys Phe Tyr Ile Glu Leu Phe Gln Gln Met Asn Asp Leu 85 90
95 Glu Ala Cys Val Met Gln Glu Val Gly Val Glu Glu Thr Pro Leu Met
100 105 110 Asn Val Asp Ser Ile Leu Ala Val Arg Lys Tyr Phe Gln Arg
Ile Thr 115 120 125 Leu Tyr Leu Thr Glu Lys Lys Tyr Ser Pro Cys Ala
Trp Glu Val Val 130 135 140 Arg Ala Glu Ile Met Arg Ser Phe Ser Phe
Ser Thr Asn Leu Gln Lys 145 150 155 160 Arg Leu Arg Arg Lys Glu Cys
165 46 166 PRT Artificial Sequence IFNalpha B9x14CHO3 46 Cys Asp
Leu Pro Gln Thr His Ser Leu Gly His Arg Arg Thr Met Met 1 5 10 15
Leu Leu Ala Gln Met Arg Arg Ile Ser Leu Phe Ser Cys Leu Lys Asp 20
25 30 Arg His Asp Phe Arg Phe Pro Gln Glu Glu Phe Asp Gly Asn His
Phe 35 40 45 Gln Lys Val Gln Ala Ile Phe Leu Phe Tyr Glu Met Met
Gln Gln Thr 50 55 60 Phe Asn Leu Phe Ser Thr Lys Asp Ser Ser Ala
Ala Trp Asp Glu Thr 65 70 75 80 Leu Leu Glu Lys Phe Tyr Ile Glu Leu
Phe Gln Gln Met Asn Asp Leu 85 90 95 Glu Ala Cys Val Met Gln Glu
Val Gly Val Glu Glu Thr Pro Leu Met 100 105 110 Asn Val Asp Ser Ile
Leu Ala Val Arg Lys Tyr Phe Gln Arg Ile Thr 115 120 125 Leu Tyr Leu
Thr Glu Lys Lys Tyr Ser Pro Cys Ala Trp Glu Val Val 130 135 140 Arg
Ala Glu Ile Met Arg Ser Phe Ser Phe Ser Thr Asn Leu Gln Lys 145 150
155 160 Arg Leu Arg Arg Lys Glu 165 47 166 PRT Artificial Sequence
IFNalpha B9x14CHO4 47 Cys Asp Leu Pro Gln Thr His Ser Leu Gly His
Arg Arg Thr Met Met 1 5 10 15 Leu Leu Ala Gln Met Arg Arg Ile Ser
Leu Phe Ser Cys Leu Lys Asp 20 25 30 Arg His Asp Phe Arg Phe Pro
Gln Glu Glu Phe Asp Gly Asn His Phe 35 40 45 Gln Lys Val Gln Ala
Ile Phe Leu Phe Tyr Glu Met Met Gln Gln Thr 50 55 60 Phe Asn Leu
Phe Ser Thr Lys Asp Ser Ser Ala Ala Trp Asp Glu Thr 65 70 75 80 Leu
Leu Glu Lys Phe Tyr Ile Glu Leu Phe Gln Gln Met Asn Asp Leu 85 90
95 Glu Ala Cys Val Met Gln Glu Val Gly Val Glu Glu Thr Pro Leu Met
100 105 110 Asn Val Asp Ser Ile Leu Ala Val Arg Lys Tyr Phe Gln Arg
Ile Thr 115 120 125 Leu Tyr Leu Thr Glu Lys Lys Tyr Ser Pro Cys Ala
Trp Glu Val Val 130 135 140 Arg Ala Glu Ile Met Arg Ser Phe Ser Phe
Ser Thr Asn Leu Gln Glu 145 150 155 160 Ser Leu Arg Ser Lys Glu 165
48 166 PRT Artificial Sequence IFNalpha B9x14CHO5 48 Cys Asp Leu
Pro Gln Thr His Ser Leu Gly His Arg Arg Thr Met Met 1 5 10 15 Leu
Leu Ala Gln Met Arg Arg Ile Ser Leu Phe Ser Cys Leu Lys Asp 20 25
30 Arg His Asp Phe Arg Phe Pro Gln Glu Glu Phe Asp Gly Asn His Phe
35 40 45 Gln Lys Val Gln Ala Ile Phe Leu Phe Tyr Glu Met Met Gln
Gln Thr 50 55 60 Phe Asn Leu Phe Ser Thr Lys Asp Ser Ser Ala Ala
Trp Asp Glu Thr 65 70 75 80 Leu Leu Glu Lys Phe Tyr Ile Glu Leu Phe
Gln Gln Met Asn Asp Leu 85 90 95 Glu Ala Cys Val Met Gln Glu Val
Gly Val Glu Glu Thr Pro Leu Met 100 105 110 Asn Val Asp Ser Ile Leu
Ala Val Arg Lys Tyr Phe Gln Arg Ile Thr 115 120 125 Leu Tyr Leu Thr
Glu Lys Lys Tyr Ser Pro Cys Ala Trp Glu Val Val 130 135 140 Arg Ala
Glu Ile Met Arg Ser Phe Ser Phe Ser Thr Asn Leu Gln Glu 145 150 155
160 Cys Leu Arg Ser Lys Glu 165 49 166 PRT Artificial Sequence
IFNalpha B9x14CHO6 49 Cys Asp Leu Pro Gln Thr His Ser Leu Gly His
Arg Arg Thr Met Met 1 5 10 15 Leu Leu Ala Gln Met Arg Arg Ile Ser
Leu Phe Ser Cys Leu Lys Asp 20 25 30 Arg His Asp Phe Arg Phe Pro
Gln Glu Glu Phe Asp Gly Asn His Phe 35 40 45 Gln Lys Val Gln Ala
Ile Phe Leu Phe Tyr Glu Met Met Gln Gln Thr 50 55 60 Phe Asn Leu
Phe Ser Thr Lys Asp Ser Ser Ala Ala Trp Asp Glu Thr 65 70 75 80 Leu
Leu Glu Lys Phe Tyr Ile Glu Leu Phe Gln Gln Met Asn Asp Leu 85 90
95 Glu Ala Cys Val Met Gln Glu Val Gly Val Glu Glu Thr Pro Leu Met
100 105 110 Asn Val Asp Ser Ile Leu Ala Val Arg Lys Tyr Phe Gln Arg
Ile Thr 115 120 125 Leu Tyr Leu Thr Glu Lys Lys Tyr Ser Pro Cys Ala
Trp Glu Val Val 130 135 140 Arg Ala Glu Ile Met Arg Ser Phe Ser Phe
Ser Thr Asn Leu Gln Glu 145 150 155 160 Ser Leu Arg Cys Lys Glu 165
50 166 PRT Artificial Sequence IFNalpha 14Ep01 50 Cys Asp Leu Pro
Gln Thr His Ser Leu Gly His Arg Arg Thr Met Met 1 5 10 15 Leu Leu
Ala Gln Met Arg
Arg Ile Ser Leu Phe Ser Cys Leu Lys Asp 20 25 30 Arg His Asp Phe
Arg Phe Pro Gln Glu Glu Phe Asp Gly Asn Gln Phe 35 40 45 Gln Lys
Ala Gln Ala Ile Ser Val Leu His Glu Met Met Gln Gln Thr 50 55 60
Phe Asn Leu Phe Ser Thr Lys Asp Ser Ser Ala Ala Trp Asp Glu Thr 65
70 75 80 Leu Leu Glu Lys Phe Tyr Ile Glu Leu Phe Gln Gln Met Asn
Asp Leu 85 90 95 Glu Ala Cys Val Met Gln Glu Val Gly Val Glu Glu
Thr Pro Leu Met 100 105 110 Asn Val Asp Ser Ile Leu Ala Val Arg Lys
Tyr Phe Gln Arg Ile Thr 115 120 125 Leu Tyr Leu Thr Glu Lys Lys Tyr
Ser Pro Cys Ala Trp Glu Val Val 130 135 140 Arg Ala Glu Ile Met Arg
Ser Phe Ser Leu Ser Thr Asn Leu Gln Glu 145 150 155 160 Ser Leu Arg
Ser Lys Glu 165 51 166 PRT Artificial Sequence IFNalpha 14Ep02 51
Cys Asp Leu Pro Gln Thr His Ser Leu Gly His Arg Arg Thr Met Met 1 5
10 15 Leu Leu Ala Gln Met Arg Arg Ile Ser Leu Phe Ser Cys Leu Lys
Asp 20 25 30 Arg His Asp Phe Arg Phe Pro Gln Glu Glu Phe Asp Gly
Asn His Phe 35 40 45 Gln Lys Val Gln Ala Ile Phe Leu Phe Tyr Glu
Met Ile Gln Gln Thr 50 55 60 Phe Asn Leu Phe Ser Thr Lys Asp Ser
Ser Ala Ala Trp Asp Glu Thr 65 70 75 80 Leu Leu Asp Lys Phe Tyr Ile
Glu Leu Phe Gln Gln Leu Asn Asp Leu 85 90 95 Glu Ala Cys Val Thr
Gln Glu Val Gly Val Glu Glu Ile Ala Leu Met 100 105 110 Asn Glu Asp
Ser Ile Leu Ala Val Arg Lys Tyr Phe Gln Arg Ile Thr 115 120 125 Leu
Tyr Leu Thr Glu Lys Lys Tyr Ser Pro Cys Ala Trp Glu Val Val 130 135
140 Arg Ala Glu Ile Met Arg Ser Phe Ser Leu Ser Thr Asn Leu Gln Glu
145 150 155 160 Ser Leu Arg Ser Lys Glu 165 52 166 PRT Artificial
Sequence IFNalpha 14Ep03 52 Cys Asp Leu Pro Gln Thr His Ser Leu Gly
His Arg Arg Thr Met Met 1 5 10 15 Leu Leu Ala Gln Met Arg Arg Ile
Ser Leu Phe Ser Cys Leu Lys Asp 20 25 30 Arg His Asp Phe Arg Phe
Pro Gln Glu Glu Phe Asp Gly Asn His Phe 35 40 45 Gln Lys Val Gln
Ala Ile Phe Leu Phe Tyr Glu Met Met Gln Gln Thr 50 55 60 Phe Asn
Leu Phe Ser Thr Lys Asp Ser Ser Ala Ala Trp Asp Glu Thr 65 70 75 80
Leu Leu Glu Lys Phe Tyr Ile Glu Leu Phe Gln Gln Met Asn Asp Leu 85
90 95 Glu Ala Cys Val Ile Gln Glu Val Gly Val Glu Glu Thr Pro Leu
Met 100 105 110 Asn Val Asp Ser Ile Leu Ala Val Arg Lys Tyr Phe Gln
Arg Ile Thr 115 120 125 Leu Tyr Leu Thr Glu Lys Lys Tyr Ser Pro Cys
Ala Trp Glu Val Val 130 135 140 Arg Ala Glu Ile Met Arg Ser Phe Ser
Leu Ser Thr Asn Leu Gln Glu 145 150 155 160 Ser Leu Arg Ser Lys Glu
165 53 166 PRT Artificial Sequence IFNalpha 14Ep04 53 Cys Asp Leu
Pro Gln Thr His Ser Leu Gly His Arg Arg Thr Met Met 1 5 10 15 Leu
Leu Ala Gln Met Arg Arg Ile Ser Leu Phe Ser Cys Leu Lys Asp 20 25
30 Arg His Asp Phe Arg Phe Pro Gln Glu Glu Phe Asp Gly Asn His Phe
35 40 45 Gln Lys Val Gln Ala Ile Phe Leu Phe Tyr Glu Met Met Gln
Gln Thr 50 55 60 Phe Asn Leu Phe Ser Thr Lys Asp Ser Ser Ala Ala
Trp Asp Glu Thr 65 70 75 80 Leu Leu Glu Lys Phe Tyr Ile Glu Leu Phe
Gln Gln Met Asn Asp Leu 85 90 95 Glu Ala Cys Val Met Gln Glu Val
Gly Val Glu Glu Thr Pro Leu Met 100 105 110 Asn Val Asp Ser Ile Leu
Ala Val Arg Lys Tyr Phe Gln Arg Ile Thr 115 120 125 Leu Tyr Leu Thr
Glu Lys Lys Tyr Ser Pro Cys Ala Trp Glu Val Val 130 135 140 Arg Ala
Glu Ile Met Arg Ser Phe Ser Leu Ser Thr Asn Leu Gln Glu 145 150 155
160 Ser Leu Arg Ser Lys Glu 165 54 166 PRT Artificial Sequence
IFNalpha 14Ep05 54 Cys Asp Leu Pro Gln Thr His Ser Leu Gly His Arg
Arg Thr Met Met 1 5 10 15 Leu Leu Ala Gln Met Arg Arg Ile Ser Leu
Phe Ser Cys Leu Lys Asp 20 25 30 Arg His Asp Phe Arg Phe Pro Gln
Glu Glu Phe Asp Gly Asn Gln Phe 35 40 45 Gln Lys Ala Gln Ala Ile
Ser Val Leu His Glu Met Met Gln Gln Thr 50 55 60 Phe Asn Leu Phe
Ser Thr Lys Asp Ser Ser Ala Ala Trp Asp Glu Thr 65 70 75 80 Leu Leu
Glu Lys Phe Tyr Ile Glu Leu Phe Gln Gln Met Asn Asp Leu 85 90 95
Glu Ala Cys Val Ile Gln Glu Val Gly Val Glu Glu Thr Pro Leu Met 100
105 110 Asn Val Asp Ser Ile Leu Ala Val Arg Lys Tyr Phe Gln Arg Ile
Thr 115 120 125 Leu Tyr Leu Thr Glu Lys Lys Tyr Ser Pro Cys Ala Trp
Glu Val Val 130 135 140 Arg Ala Glu Ile Met Arg Ser Phe Ser Leu Ser
Thr Asn Leu Gln Glu 145 150 155 160 Ser Leu Arg Ser Lys Glu 165 55
166 PRT Artificial Sequence IFNalpha 14EF 55 Cys Asp Leu Pro Gln
Thr His Ser Leu Gly His Arg Arg Thr Met Met 1 5 10 15 Leu Leu Ala
Gln Met Arg Arg Ile Ser Leu Phe Ser Cys Leu Lys Asp 20 25 30 Arg
His Asp Phe Arg Phe Pro Gln Glu Glu Phe Asp Gly Asn Gln Phe 35 40
45 Gln Lys Ala Gln Ala Ile Ser Val Leu His Glu Met Ile Gln Gln Thr
50 55 60 Phe Asn Leu Phe Ser Thr Lys Asp Ser Ser Ala Ala Trp Asp
Glu Thr 65 70 75 80 Leu Leu Asp Lys Phe Tyr Ile Glu Leu Phe Gln Gln
Leu Asn Asp Leu 85 90 95 Glu Ala Cys Val Thr Gln Glu Val Gly Val
Glu Glu Ile Ala Leu Met 100 105 110 Asn Glu Asp Ser Ile Leu Ala Val
Arg Lys Tyr Phe Gln Arg Ile Thr 115 120 125 Leu Tyr Leu Thr Glu Lys
Lys Tyr Ser Pro Cys Ala Trp Glu Val Val 130 135 140 Arg Ala Glu Ile
Met Arg Ser Phe Ser Leu Ser Thr Asn Leu Gln Glu 145 150 155 160 Ser
Leu Arg Ser Lys Glu 165 56 166 PRT Artificial Sequence IFNalpha
B9x14EP04C31 56 Cys Asp Leu Pro Gln Thr His Ser Leu Gly His Arg Arg
Thr Met Met 1 5 10 15 Leu Leu Ala Gln Met Arg Arg Ile Ser Leu Phe
Ser Cys Leu Cys Asp 20 25 30 Arg His Asp Phe Arg Phe Pro Gln Glu
Glu Phe Asp Gly Asn His Phe 35 40 45 Gln Lys Val Gln Ala Ile Phe
Leu Phe Tyr Glu Met Met Gln Gln Thr 50 55 60 Phe Asn Leu Phe Ser
Thr Lys Asp Ser Ser Ala Ala Trp Asp Glu Thr 65 70 75 80 Leu Leu Glu
Lys Phe Tyr Ile Glu Leu Phe Gln Gln Met Asn Asp Leu 85 90 95 Glu
Ala Cys Val Met Gln Glu Val Gly Val Glu Glu Thr Pro Leu Met 100 105
110 Asn Val Asp Ser Ile Leu Ala Val Arg Lys Tyr Phe Gln Arg Ile Thr
115 120 125 Leu Tyr Leu Thr Glu Lys Lys Tyr Ser Pro Cys Ala Trp Glu
Val Val 130 135 140 Arg Ala Glu Ile Met Arg Ser Phe Ser Leu Ser Thr
Asn Leu Gln Glu 145 150 155 160 Ser Leu Arg Ser Lys Glu 165 57 166
PRT Artificial Sequence IFNalpha B9x14CH04C31 57 Cys Asp Leu Pro
Gln Thr His Ser Leu Gly His Arg Arg Thr Met Met 1 5 10 15 Leu Leu
Ala Gln Met Arg Arg Ile Ser Leu Phe Ser Cys Leu Cys Asp 20 25 30
Arg His Asp Phe Arg Phe Pro Gln Glu Glu Phe Asp Gly Asn His Phe 35
40 45 Gln Lys Val Gln Ala Ile Phe Leu Phe Tyr Glu Met Met Gln Gln
Thr 50 55 60 Phe Asn Leu Phe Ser Thr Lys Asp Ser Ser Ala Ala Trp
Asp Glu Thr 65 70 75 80 Leu Leu Glu Lys Phe Tyr Ile Glu Leu Phe Gln
Gln Met Asn Asp Leu 85 90 95 Glu Ala Cys Val Met Gln Glu Val Gly
Val Glu Glu Thr Pro Leu Met 100 105 110 Asn Val Asp Ser Ile Leu Ala
Val Arg Lys Tyr Phe Gln Arg Ile Thr 115 120 125 Leu Tyr Leu Thr Glu
Lys Lys Tyr Ser Pro Cys Ala Trp Glu Val Val 130 135 140 Arg Ala Glu
Ile Met Arg Ser Phe Ser Phe Ser Thr Asn Leu Gln Glu 145 150 155 160
Ser Leu Arg Ser Lys Glu 165 58 166 PRT Artificial Sequence IFNalpha
B9x14CH04C46 58 Cys Asp Leu Pro Gln Thr His Ser Leu Gly His Arg Arg
Thr Met Met 1 5 10 15 Leu Leu Ala Gln Met Arg Arg Ile Ser Leu Phe
Ser Cys Leu Lys Asp 20 25 30 Arg His Asp Phe Arg Phe Pro Gln Glu
Glu Phe Asp Gly Cys His Phe 35 40 45 Gln Lys Val Gln Ala Ile Phe
Leu Phe Tyr Glu Met Met Gln Gln Thr 50 55 60 Phe Asn Leu Phe Ser
Thr Lys Asp Ser Ser Ala Ala Trp Asp Glu Thr 65 70 75 80 Leu Leu Glu
Lys Phe Tyr Ile Glu Leu Phe Gln Gln Met Asn Asp Leu 85 90 95 Glu
Ala Cys Val Met Gln Glu Val Gly Val Glu Glu Thr Pro Leu Met 100 105
110 Asn Val Asp Ser Ile Leu Ala Val Arg Lys Tyr Phe Gln Arg Ile Thr
115 120 125 Leu Tyr Leu Thr Glu Lys Lys Tyr Ser Pro Cys Ala Trp Glu
Val Val 130 135 140 Arg Ala Glu Ile Met Arg Ser Phe Ser Phe Ser Thr
Asn Leu Gln Glu 145 150 155 160 Ser Leu Arg Ser Lys Glu 165 59 166
PRT Artificial Sequence IFNalpha B9x14CH04C71 59 Cys Asp Leu Pro
Gln Thr His Ser Leu Gly His Arg Arg Thr Met Met 1 5 10 15 Leu Leu
Ala Gln Met Arg Arg Ile Ser Leu Phe Ser Cys Leu Lys Asp 20 25 30
Arg His Asp Phe Arg Phe Pro Gln Glu Glu Phe Asp Gly Asn His Phe 35
40 45 Gln Lys Val Gln Ala Ile Phe Leu Phe Tyr Glu Met Met Gln Gln
Thr 50 55 60 Phe Asn Leu Phe Ser Thr Cys Asp Ser Ser Ala Ala Trp
Asp Glu Thr 65 70 75 80 Leu Leu Glu Lys Phe Tyr Ile Glu Leu Phe Gln
Gln Met Asn Asp Leu 85 90 95 Glu Ala Cys Val Met Gln Glu Val Gly
Val Glu Glu Thr Pro Leu Met 100 105 110 Asn Val Asp Ser Ile Leu Ala
Val Arg Lys Tyr Phe Gln Arg Ile Thr 115 120 125 Leu Tyr Leu Thr Glu
Lys Lys Tyr Ser Pro Cys Ala Trp Glu Val Val 130 135 140 Arg Ala Glu
Ile Met Arg Ser Phe Ser Phe Ser Thr Asn Leu Gln Glu 145 150 155 160
Ser Leu Arg Ser Lys Glu 165 60 166 PRT Artificial Sequence IFNalpha
B9x14CH04C75 60 Cys Asp Leu Pro Gln Thr His Ser Leu Gly His Arg Arg
Thr Met Met 1 5 10 15 Leu Leu Ala Gln Met Arg Arg Ile Ser Leu Phe
Ser Cys Leu Lys Asp 20 25 30 Arg His Asp Phe Arg Phe Pro Gln Glu
Glu Phe Asp Gly Asn His Phe 35 40 45 Gln Lys Val Gln Ala Ile Phe
Leu Phe Tyr Glu Met Met Gln Gln Thr 50 55 60 Phe Asn Leu Phe Ser
Thr Lys Asp Ser Ser Cys Ala Trp Asp Glu Thr 65 70 75 80 Leu Leu Glu
Lys Phe Tyr Ile Glu Leu Phe Gln Gln Met Asn Asp Leu 85 90 95 Glu
Ala Cys Val Met Gln Glu Val Gly Val Glu Glu Thr Pro Leu Met 100 105
110 Asn Val Asp Ser Ile Leu Ala Val Arg Lys Tyr Phe Gln Arg Ile Thr
115 120 125 Leu Tyr Leu Thr Glu Lys Lys Tyr Ser Pro Cys Ala Trp Glu
Val Val 130 135 140 Arg Ala Glu Ile Met Arg Ser Phe Ser Phe Ser Thr
Asn Leu Gln Glu 145 150 155 160 Ser Leu Arg Ser Lys Glu 165 61 166
PRT Artificial Sequence IFNalpha B9x14CH04C79 61 Cys Asp Leu Pro
Gln Thr His Ser Leu Gly His Arg Arg Thr Met Met 1 5 10 15 Leu Leu
Ala Gln Met Arg Arg Ile Ser Leu Phe Ser Cys Leu Lys Asp 20 25 30
Arg His Asp Phe Arg Phe Pro Gln Glu Glu Phe Asp Gly Asn His Phe 35
40 45 Gln Lys Val Gln Ala Ile Phe Leu Phe Tyr Glu Met Met Gln Gln
Thr 50 55 60 Phe Asn Leu Phe Ser Thr Lys Asp Ser Ser Ala Ala Trp
Asp Cys Thr 65 70 75 80 Leu Leu Glu Lys Phe Tyr Ile Glu Leu Phe Gln
Gln Met Asn Asp Leu 85 90 95 Glu Ala Cys Val Met Gln Glu Val Gly
Val Glu Glu Thr Pro Leu Met 100 105 110 Asn Val Asp Ser Ile Leu Ala
Val Arg Lys Tyr Phe Gln Arg Ile Thr 115 120 125 Leu Tyr Leu Thr Glu
Lys Lys Tyr Ser Pro Cys Ala Trp Glu Val Val 130 135 140 Arg Ala Glu
Ile Met Arg Ser Phe Ser Phe Ser Thr Asn Leu Gln Glu 145 150 155 160
Ser Leu Arg Ser Lys Glu 165 62 166 PRT Artificial Sequence IFNalpha
B9x14CH04C107 62 Cys Asp Leu Pro Gln Thr His Ser Leu Gly His Arg
Arg Thr Met Met 1 5 10 15 Leu Leu Ala Gln Met Arg Arg Ile Ser Leu
Phe Ser Cys Leu Lys Asp 20 25 30 Arg His Asp Phe Arg Phe Pro Gln
Glu Glu Phe Asp Gly Asn His Phe 35 40 45 Gln Lys Val Gln Ala Ile
Phe Leu Phe Tyr Glu Met Met Gln Gln Thr 50 55 60 Phe Asn Leu Phe
Ser Thr Lys Asp Ser Ser Ala Ala Trp Asp Glu Thr 65 70 75 80 Leu Leu
Glu Lys Phe Tyr Ile Glu Leu Phe Gln Gln Met Asn Asp Leu 85 90 95
Glu Ala Cys Val Met Gln Glu Val Gly Val Cys Glu Thr Pro Leu Met 100
105 110 Asn Val Asp Ser Ile Leu Ala Val Arg Lys Tyr Phe Gln Arg Ile
Thr 115 120 125 Leu Tyr Leu Thr Glu Lys Lys Tyr Ser Pro Cys Ala Trp
Glu Val Val 130 135 140 Arg Ala Glu Ile Met Arg Ser Phe Ser Phe Ser
Thr Asn Leu Gln Glu 145 150 155 160 Ser Leu Arg Ser Lys Glu 165 63
166 PRT Artificial Sequence IFNalpha B9x14CH04C122 63 Cys Asp Leu
Pro Gln Thr His Ser Leu Gly His Arg Arg Thr Met Met 1 5 10 15 Leu
Leu Ala Gln Met Arg Arg Ile Ser Leu Phe Ser Cys Leu Lys Asp 20 25
30 Arg His Asp Phe Arg Phe Pro Gln Glu Glu Phe Asp Gly Asn His Phe
35 40 45 Gln Lys Val Gln Ala Ile Phe Leu Phe Tyr Glu Met Met Gln
Gln Thr 50 55 60 Phe Asn Leu Phe Ser Thr Lys Asp Ser Ser Ala Ala
Trp Asp Glu Thr 65 70 75 80 Leu Leu Glu Lys Phe Tyr Ile Glu Leu Phe
Gln Gln Met Asn Asp Leu 85 90 95 Glu Ala Cys Val Met Gln Glu Val
Gly Val Glu Glu Thr Pro Leu Met 100 105 110 Asn Val Asp Ser Ile Leu
Ala Val Arg Cys Tyr Phe Gln Arg Ile Thr 115 120 125 Leu Tyr Leu Thr
Glu Lys Lys Tyr Ser Pro Cys Ala Trp Glu Val Val 130 135 140 Arg Ala
Glu Ile Met Arg Ser Phe Ser Phe Ser Thr Asn Leu Gln Glu 145 150 155
160 Ser Leu Arg Ser Lys Glu 165 64 166 PRT Artificial Sequence
IFNalpha B9x14CH04C134 64 Cys Asp Leu Pro Gln Thr His Ser Leu Gly
His Arg Arg Thr Met Met 1 5 10 15 Leu Leu Ala Gln Met Arg Arg Ile
Ser Leu Phe Ser Cys Leu Lys Asp 20 25 30 Arg His Asp Phe Arg Phe
Pro Gln Glu Glu Phe Asp Gly Asn His Phe 35 40 45 Gln
Lys Val Gln Ala Ile Phe Leu Phe Tyr Glu Met Met Gln Gln Thr 50 55
60 Phe Asn Leu Phe Ser Thr Lys Asp Ser Ser Ala Ala Trp Asp Glu Thr
65 70 75 80 Leu Leu Glu Lys Phe Tyr Ile Glu Leu Phe Gln Gln Met Asn
Asp Leu 85 90 95 Glu Ala Cys Val Met Gln Glu Val Gly Val Glu Glu
Thr Pro Leu Met 100 105 110 Asn Val Asp Ser Ile Leu Ala Val Arg Lys
Tyr Phe Gln Arg Ile Thr 115 120 125 Leu Tyr Leu Thr Glu Cys Lys Tyr
Ser Pro Cys Ala Trp Glu Val Val 130 135 140 Arg Ala Glu Ile Met Arg
Ser Phe Ser Phe Ser Thr Asn Leu Gln Glu 145 150 155 160 Ser Leu Arg
Ser Lys Glu 165 65 160 PRT Artificial Sequence IFNalpha
B9x14Ep04(161-166 65 Cys Asp Leu Pro Gln Thr His Ser Leu Gly His
Arg Arg Thr Met Met 1 5 10 15 Leu Leu Ala Gln Met Arg Arg Ile Ser
Leu Phe Ser Cys Leu Lys Asp 20 25 30 Arg His Asp Phe Arg Phe Pro
Gln Glu Glu Phe Asp Gly Asn His Phe 35 40 45 Gln Lys Val Gln Ala
Ile Phe Leu Phe Tyr Glu Met Met Gln Gln Thr 50 55 60 Phe Asn Leu
Phe Ser Thr Lys Asp Ser Ser Ala Ala Trp Asp Glu Thr 65 70 75 80 Leu
Leu Glu Lys Phe Tyr Ile Glu Leu Phe Gln Gln Met Asn Asp Leu 85 90
95 Glu Ala Cys Val Met Gln Glu Val Gly Val Glu Glu Thr Pro Leu Met
100 105 110 Asn Val Asp Ser Ile Leu Ala Val Arg Lys Tyr Phe Gln Arg
Ile Thr 115 120 125 Leu Tyr Leu Thr Glu Lys Lys Tyr Ser Pro Cys Ala
Trp Glu Val Val 130 135 140 Arg Ala Glu Ile Met Arg Ser Phe Ser Leu
Ser Thr Asn Leu Gln Glu 145 150 155 160 66 164 PRT Artificial
Sequence IFNalpha B9x14Ep04(165-166 66 Cys Asp Leu Pro Gln Thr His
Ser Leu Gly His Arg Arg Thr Met Met 1 5 10 15 Leu Leu Ala Gln Met
Arg Arg Ile Ser Leu Phe Ser Cys Leu Lys Asp 20 25 30 Arg His Asp
Phe Arg Phe Pro Gln Glu Glu Phe Asp Gly Asn His Phe 35 40 45 Gln
Lys Val Gln Ala Ile Phe Leu Phe Tyr Glu Met Met Gln Gln Thr 50 55
60 Phe Asn Leu Phe Ser Thr Lys Asp Ser Ser Ala Ala Trp Asp Glu Thr
65 70 75 80 Leu Leu Glu Lys Phe Tyr Ile Glu Leu Phe Gln Gln Met Asn
Asp Leu 85 90 95 Glu Ala Cys Val Met Gln Glu Val Gly Val Glu Glu
Thr Pro Leu Met 100 105 110 Asn Val Asp Ser Ile Leu Ala Val Arg Lys
Tyr Phe Gln Arg Ile Thr 115 120 125 Leu Tyr Leu Thr Glu Lys Lys Tyr
Ser Pro Cys Ala Trp Glu Val Val 130 135 140 Arg Ala Glu Ile Met Arg
Ser Phe Ser Leu Ser Thr Asn Leu Gln Glu 145 150 155 160 Ser Leu Arg
Ser 67 159 PRT Artificial Sequence IFNalpha
B9x14Ep04(1-4D44*(161-166 67 Gln Thr His Ser Leu Gly His Arg Arg
Thr Met Met Leu Leu Ala Gln 1 5 10 15 Met Arg Arg Ile Ser Leu Phe
Ser Cys Leu Lys Asp Arg His Asp Phe 20 25 30 Arg Phe Pro Gln Glu
Glu Phe Gly Asn His Phe Gln Lys Val Gln Ala 35 40 45 Ile Phe Leu
Phe Tyr Glu Met Met Gln Gln Thr Phe Asn Leu Phe Ser 50 55 60 Thr
Lys Asp Ser Ser Ala Ala Trp Asp Glu Thr Leu Leu Glu Lys Phe 65 70
75 80 Tyr Ile Glu Leu Phe Gln Gln Met Asn Asp Leu Glu Ala Cys Val
Met 85 90 95 Gln Glu Val Gly Val Glu Glu Thr Pro Leu Met Asn Val
Asp Ser Ile 100 105 110 Leu Ala Val Arg Lys Tyr Phe Gln Arg Ile Thr
Leu Tyr Leu Thr Glu 115 120 125 Lys Lys Tyr Ser Pro Cys Ala Trp Glu
Val Val Arg Ala Glu Ile Met 130 135 140 Arg Ser Phe Ser Leu Ser Thr
Asn Leu Gln Glu Ser Leu Arg Ser 145 150 155 68 166 PRT Artificial
Sequence IFNalpha B9x14CHO4NP1 68 Cys Asp Leu Pro Gln Thr His Ser
Leu Gly His Arg Arg Thr Met Met 1 5 10 15 Leu Leu Ala Gln Met Arg
Arg Ile Ser Leu Phe Ser Cys Leu Lys Asp 20 25 30 Arg Gln Asp Phe
Arg Phe Pro Gln Glu Glu Phe Asp Gly Asn His Phe 35 40 45 Gln Lys
Val Gln Ala Ile Phe Leu Phe Tyr Glu Met Met Gln Gln Thr 50 55 60
Phe Asn Leu Phe Ser Thr Lys Asp Ser Ser Ala Ala Trp Asp Glu Thr 65
70 75 80 Leu Leu Glu Lys Phe Tyr Ile Glu Leu Phe Gln Gln Met Asn
Asp Leu 85 90 95 Glu Ala Cys Val Met Gln Glu Val Gly Val Glu Glu
Thr Pro Leu Met 100 105 110 Asn Val Asp Ser Ile Leu Ala Val Arg Lys
Tyr Phe Gln Arg Ile Thr 115 120 125 Leu Tyr Leu Thr Glu Lys Lys Tyr
Ser Pro Cys Ala Trp Glu Val Val 130 135 140 Arg Ala Glu Ile Met Arg
Ser Phe Ser Phe Ser Thr Asn Leu Gln Glu 145 150 155 160 Ser Leu Arg
Ser Lys Glu 165 69 166 PRT Artificial Sequence IFNalpha
B9x14CHO4NP2 69 Cys Asp Leu Pro Gln Thr His Ser Leu Gly His Arg Arg
Thr Met Met 1 5 10 15 Leu Leu Ala Gln Met Arg Arg Ile Ser Leu Phe
Ser Cys Leu Lys Asp 20 25 30 Arg Gln Asp Phe Arg Phe Pro Gln Glu
Glu Phe Asp Gly Asn Gln Phe 35 40 45 Gln Lys Val Gln Ala Ile Phe
Leu Phe Tyr Glu Met Met Gln Gln Thr 50 55 60 Phe Asn Leu Phe Ser
Thr Lys Asp Ser Ser Ala Ala Trp Asp Glu Thr 65 70 75 80 Leu Leu Glu
Lys Phe Tyr Ile Glu Leu Phe Gln Gln Met Asn Asp Leu 85 90 95 Glu
Ala Cys Val Met Gln Glu Val Gly Val Glu Glu Thr Pro Leu Met 100 105
110 Asn Val Asp Ser Ile Leu Ala Val Arg Lys Tyr Phe Gln Arg Ile Thr
115 120 125 Leu Tyr Leu Thr Glu Lys Lys Tyr Ser Pro Cys Ala Trp Glu
Val Val 130 135 140 Arg Ala Glu Ile Met Arg Ser Phe Ser Phe Ser Thr
Asn Leu Gln Glu 145 150 155 160 Ser Leu Arg Ser Lys Glu 165 70 166
PRT Artificial Sequence IFNalpha B9x14CHO8 70 Cys Asp Leu Pro Gln
Thr His Ser Leu Gly His Arg Arg Thr Met Met 1 5 10 15 Leu Leu Ala
Gln Met Arg Arg Ile Ser Leu Phe Ser Cys Leu Arg Asp 20 25 30 Arg
His Asp Phe Arg Phe Pro Gln Glu Glu Phe Asp Gly Asn His Phe 35 40
45 Gln Lys Val Gln Ala Ile Phe Leu Phe Tyr Glu Met Met Gln Gln Thr
50 55 60 Phe Asn Leu Phe Ser Thr Lys Asp Ser Ser Ala Ala Trp Asp
Glu Thr 65 70 75 80 Leu Leu Glu Lys Phe Tyr Ile Glu Leu Phe Gln Gln
Met Asn Asp Leu 85 90 95 Glu Ala Cys Val Met Gln Glu Val Gly Val
Glu Glu Thr Pro Leu Met 100 105 110 Asn Val Asp Ser Ile Leu Ala Val
Arg Lys Tyr Phe Gln Arg Ile Thr 115 120 125 Leu Tyr Leu Thr Glu Lys
Lys Tyr Ser Pro Cys Ala Trp Glu Val Val 130 135 140 Arg Ala Glu Ile
Met Arg Ser Phe Ser Phe Ser Thr Asn Leu Gln Glu 145 150 155 160 Ser
Leu Arg Ser Lys Glu 165 71 166 PRT Artificial Sequence IFNalpha
B9x14CHO9 71 Cys Asp Leu Pro Gln Thr His Ser Leu Gly His Arg Arg
Thr Met Met 1 5 10 15 Leu Leu Ala Gln Met Arg Arg Ile Ser Leu Phe
Ser Cys Leu Lys Asp 20 25 30 Arg His Asp Phe Arg Phe Pro Gln Glu
Glu Phe Asp Gly Asn His Phe 35 40 45 Gln Arg Val Gln Ala Ile Phe
Leu Phe Tyr Glu Met Met Gln Gln Thr 50 55 60 Phe Asn Leu Phe Ser
Thr Lys Asp Ser Ser Ala Ala Trp Asp Glu Thr 65 70 75 80 Leu Leu Glu
Lys Phe Tyr Ile Glu Leu Phe Gln Gln Met Asn Asp Leu 85 90 95 Glu
Ala Cys Val Met Gln Glu Val Gly Val Glu Glu Thr Pro Leu Met 100 105
110 Asn Val Asp Ser Ile Leu Ala Val Arg Lys Tyr Phe Gln Arg Ile Thr
115 120 125 Leu Tyr Leu Thr Glu Lys Lys Tyr Ser Pro Cys Ala Trp Glu
Val Val 130 135 140 Arg Ala Glu Ile Met Arg Ser Phe Ser Phe Ser Thr
Asn Leu Gln Glu 145 150 155 160 Ser Leu Arg Ser Lys Glu 165 72 166
PRT Artificial Sequence IFNalpha B9x14CHO10 72 Cys Asp Leu Pro Gln
Thr His Ser Leu Gly His Arg Arg Thr Met Met 1 5 10 15 Leu Leu Ala
Gln Met Arg Arg Ile Ser Leu Phe Ser Cys Leu Lys Asp 20 25 30 Arg
His Asp Phe Arg Phe Pro Gln Glu Glu Phe Asp Gly Asn His Phe 35 40
45 Gln Lys Val Gln Ala Ile Phe Leu Phe Tyr Glu Met Met Gln Gln Thr
50 55 60 Phe Asn Leu Phe Ser Thr Arg Asp Ser Ser Ala Ala Trp Asp
Glu Thr 65 70 75 80 Leu Leu Glu Lys Phe Tyr Ile Glu Leu Phe Gln Gln
Met Asn Asp Leu 85 90 95 Glu Ala Cys Val Met Gln Glu Val Gly Val
Glu Glu Thr Pro Leu Met 100 105 110 Asn Val Asp Ser Ile Leu Ala Val
Arg Lys Tyr Phe Gln Arg Ile Thr 115 120 125 Leu Tyr Leu Thr Glu Lys
Lys Tyr Ser Pro Cys Ala Trp Glu Val Val 130 135 140 Arg Ala Glu Ile
Met Arg Ser Phe Ser Phe Ser Thr Asn Leu Gln Glu 145 150 155 160 Ser
Leu Arg Ser Lys Glu 165 73 166 PRT Artificial Sequence IFNalpha
B9x14CHO11 73 Cys Asp Leu Pro Gln Thr His Ser Leu Gly His Arg Arg
Thr Met Met 1 5 10 15 Leu Leu Ala Gln Met Arg Arg Ile Ser Leu Phe
Ser Cys Leu Lys Asp 20 25 30 Arg His Asp Phe Arg Phe Pro Gln Glu
Glu Phe Asp Gly Asn His Phe 35 40 45 Gln Lys Val Gln Ala Ile Phe
Leu Phe Tyr Glu Met Met Gln Gln Thr 50 55 60 Phe Asn Leu Phe Ser
Thr Lys Asp Ser Ser Ala Ala Trp Asp Glu Thr 65 70 75 80 Leu Leu Glu
Arg Phe Tyr Ile Glu Leu Phe Gln Gln Met Asn Asp Leu 85 90 95 Glu
Ala Cys Val Met Gln Glu Val Gly Val Glu Glu Thr Pro Leu Met 100 105
110 Asn Val Asp Ser Ile Leu Ala Val Arg Lys Tyr Phe Gln Arg Ile Thr
115 120 125 Leu Tyr Leu Thr Glu Lys Lys Tyr Ser Pro Cys Ala Trp Glu
Val Val 130 135 140 Arg Ala Glu Ile Met Arg Ser Phe Ser Phe Ser Thr
Asn Leu Gln Glu 145 150 155 160 Ser Leu Arg Ser Lys Glu 165 74 166
PRT Artificial Sequence IFNalpha B9x14CHO12 74 Cys Asp Leu Pro Gln
Thr His Ser Leu Gly His Arg Arg Thr Met Met 1 5 10 15 Leu Leu Ala
Gln Met Arg Arg Ile Ser Leu Phe Ser Cys Leu Lys Asp 20 25 30 Arg
His Asp Phe Arg Phe Pro Gln Glu Glu Phe Asp Gly Asn His Phe 35 40
45 Gln Lys Val Gln Ala Ile Phe Leu Phe Tyr Glu Met Met Gln Gln Thr
50 55 60 Phe Asn Leu Phe Ser Thr Lys Asp Ser Ser Ala Ala Trp Asp
Glu Thr 65 70 75 80 Leu Leu Glu Lys Phe Tyr Ile Glu Leu Phe Gln Gln
Met Asn Asp Leu 85 90 95 Glu Ala Cys Val Met Gln Glu Val Gly Val
Glu Glu Thr Pro Leu Met 100 105 110 Asn Val Asp Ser Ile Leu Ala Val
Arg Arg Tyr Phe Gln Arg Ile Thr 115 120 125 Leu Tyr Leu Thr Glu Lys
Lys Tyr Ser Pro Cys Ala Trp Glu Val Val 130 135 140 Arg Ala Glu Ile
Met Arg Ser Phe Ser Phe Ser Thr Asn Leu Gln Glu 145 150 155 160 Ser
Leu Arg Ser Lys Glu 165 75 166 PRT Artificial Sequence IFNalpha
B9x14CHO13 75 Cys Asp Leu Pro Gln Thr His Ser Leu Gly His Arg Arg
Thr Met Met 1 5 10 15 Leu Leu Ala Gln Met Arg Arg Ile Ser Leu Phe
Ser Cys Leu Lys Asp 20 25 30 Arg His Asp Phe Arg Phe Pro Gln Glu
Glu Phe Asp Gly Asn His Phe 35 40 45 Gln Lys Val Gln Ala Ile Phe
Leu Phe Tyr Glu Met Met Gln Gln Thr 50 55 60 Phe Asn Leu Phe Ser
Thr Lys Asp Ser Ser Ala Ala Trp Asp Glu Thr 65 70 75 80 Leu Leu Glu
Lys Phe Tyr Ile Glu Leu Phe Gln Gln Met Asn Asp Leu 85 90 95 Glu
Ala Cys Val Met Gln Glu Val Gly Val Glu Glu Thr Pro Leu Met 100 105
110 Asn Val Asp Ser Ile Leu Ala Val Arg Lys Tyr Phe Gln Arg Ile Thr
115 120 125 Leu Tyr Leu Thr Glu Arg Lys Tyr Ser Pro Cys Ala Trp Glu
Val Val 130 135 140 Arg Ala Glu Ile Met Arg Ser Phe Ser Phe Ser Thr
Asn Leu Gln Glu 145 150 155 160 Ser Leu Arg Ser Lys Glu 165 76 166
PRT Artificial Sequence IFNalpha B9x14CHO14 76 Cys Asp Leu Pro Gln
Thr His Ser Leu Gly His Arg Arg Thr Met Met 1 5 10 15 Leu Leu Ala
Gln Met Arg Arg Ile Ser Leu Phe Ser Cys Leu Lys Asp 20 25 30 Arg
His Asp Phe Arg Phe Pro Gln Glu Glu Phe Asp Gly Asn His Phe 35 40
45 Gln Lys Val Gln Ala Ile Phe Leu Phe Tyr Glu Met Met Gln Gln Thr
50 55 60 Phe Asn Leu Phe Ser Thr Lys Asp Ser Ser Ala Ala Trp Asp
Glu Thr 65 70 75 80 Leu Leu Glu Lys Phe Tyr Ile Glu Leu Phe Gln Gln
Met Asn Asp Leu 85 90 95 Glu Ala Cys Val Met Gln Glu Val Gly Val
Glu Glu Thr Pro Leu Met 100 105 110 Asn Val Asp Ser Ile Leu Ala Val
Arg Lys Tyr Phe Gln Arg Ile Thr 115 120 125 Leu Tyr Leu Thr Glu Lys
Arg Tyr Ser Pro Cys Ala Trp Glu Val Val 130 135 140 Arg Ala Glu Ile
Met Arg Ser Phe Ser Phe Ser Thr Asn Leu Gln Glu 145 150 155 160 Ser
Leu Arg Ser Lys Glu 165 77 166 PRT Artificial Sequence IFNalpha
B9x14CHO15 77 Cys Asp Leu Pro Gln Thr His Ser Leu Gly His Arg Arg
Thr Met Met 1 5 10 15 Leu Leu Ala Gln Met Arg Arg Ile Ser Leu Phe
Ser Cys Leu Lys Asp 20 25 30 Arg His Asp Phe Arg Phe Pro Gln Glu
Glu Phe Asp Gly Asn His Phe 35 40 45 Gln Lys Val Gln Ala Ile Phe
Leu Phe Tyr Glu Met Met Gln Gln Thr 50 55 60 Phe Asn Leu Phe Ser
Thr Lys Asp Ser Ser Ala Ala Trp Asp Glu Thr 65 70 75 80 Leu Leu Glu
Lys Phe Tyr Ile Glu Leu Phe Gln Gln Met Asn Asp Leu 85 90 95 Glu
Ala Cys Val Met Gln Glu Val Gly Val Glu Glu Thr Pro Leu Met 100 105
110 Asn Val Asp Ser Ile Leu Ala Val Arg Lys Tyr Phe Gln Arg Ile Thr
115 120 125 Leu Tyr Leu Thr Glu Lys Lys Tyr Ser Pro Cys Ala Trp Glu
Val Val 130 135 140 Arg Ala Glu Ile Met Arg Ser Phe Ser Phe Ser Thr
Asn Leu Gln Glu 145 150 155 160 Ser Leu Arg Ser Arg Glu 165 78 166
PRT Artificial Sequence IFNalpha B9x14CHO16 78 Cys Asp Leu Pro Gln
Thr His Ser Leu Gly His Arg Arg Thr Met Met 1 5 10 15 Leu Leu Ala
Gln Met Arg Arg Ile Ser Leu Phe Ser Cys Leu Lys Asp 20 25 30 Arg
His Asp Phe Arg Phe Pro Gln Glu Glu Phe Asp Gly Asn His Phe 35 40
45 Gln Lys Val Gln Ala Ile Phe Leu Phe Tyr Glu Met Met Gln Gln Thr
50 55 60 Phe Asn Leu Phe Ser Thr Lys Asp Ser Ser Ala Ala Trp Asp
Glu Thr 65 70 75 80 Leu Leu Glu Lys Phe Tyr Ile Glu Leu Phe Gln Gln
Met Asn Asp Leu
85 90 95 Glu Ala Cys Val Met Gln Glu Val Gly Val Glu Glu Thr Pro
Leu Met 100 105 110 Asn Val Asp Ser Ile Leu Ala Val Arg Arg Tyr Phe
Gln Arg Ile Thr 115 120 125 Leu Tyr Leu Thr Glu Lys Arg Tyr Ser Pro
Cys Ala Trp Glu Val Val 130 135 140 Arg Ala Glu Ile Met Arg Ser Phe
Ser Phe Ser Thr Asn Leu Gln Glu 145 150 155 160 Ser Leu Arg Ser Lys
Glu 165 79 166 PRT Artificial Sequence IFNalpha B9x14CHO17 79 Cys
Asp Leu Pro Gln Thr His Ser Leu Gly His Arg Arg Thr Met Met 1 5 10
15 Leu Leu Ala Gln Met Arg Arg Ile Ser Leu Phe Ser Cys Leu Arg Asp
20 25 30 Arg His Asp Phe Arg Phe Pro Gln Glu Glu Phe Asp Gly Asn
His Phe 35 40 45 Gln Lys Val Gln Ala Ile Phe Leu Phe Tyr Glu Met
Met Gln Gln Thr 50 55 60 Phe Asn Leu Phe Ser Thr Lys Asp Ser Ser
Ala Ala Trp Asp Glu Thr 65 70 75 80 Leu Leu Glu Lys Phe Tyr Ile Glu
Leu Phe Gln Gln Met Asn Asp Leu 85 90 95 Glu Ala Cys Val Met Gln
Glu Val Gly Val Glu Glu Thr Pro Leu Met 100 105 110 Asn Val Asp Ser
Ile Leu Ala Val Arg Lys Tyr Phe Gln Arg Ile Thr 115 120 125 Leu Tyr
Leu Thr Glu Lys Arg Tyr Ser Pro Cys Ala Trp Glu Val Val 130 135 140
Arg Ala Glu Ile Met Arg Ser Phe Ser Phe Ser Thr Asn Leu Gln Glu 145
150 155 160 Ser Leu Arg Ser Lys Glu 165 80 166 PRT Artificial
Sequence IFNalpha B9x14CHO18 80 Cys Asp Leu Pro Gln Thr His Ser Leu
Gly His Arg Arg Thr Met Met 1 5 10 15 Leu Leu Ala Gln Met Arg Arg
Ile Ser Leu Phe Ser Cys Leu Arg Asp 20 25 30 Arg His Asp Phe Arg
Phe Pro Gln Glu Glu Phe Asp Gly Asn His Phe 35 40 45 Gln Lys Val
Gln Ala Ile Phe Leu Phe Tyr Glu Met Met Gln Gln Thr 50 55 60 Phe
Asn Leu Phe Ser Thr Lys Asp Ser Ser Ala Ala Trp Asp Glu Thr 65 70
75 80 Leu Leu Glu Lys Phe Tyr Ile Glu Leu Phe Gln Gln Met Asn Asp
Leu 85 90 95 Glu Ala Cys Val Met Gln Glu Val Gly Val Glu Glu Thr
Pro Leu Met 100 105 110 Asn Val Asp Ser Ile Leu Ala Val Arg Arg Tyr
Phe Gln Arg Ile Thr 115 120 125 Leu Tyr Leu Thr Glu Lys Lys Tyr Ser
Pro Cys Ala Trp Glu Val Val 130 135 140 Arg Ala Glu Ile Met Arg Ser
Phe Ser Phe Ser Thr Asn Leu Gln Glu 145 150 155 160 Ser Leu Arg Ser
Lys Glu 165 81 166 PRT Artificial Sequence IFNalpha B9x14CHO18NP2
81 Cys Asp Leu Pro Gln Thr His Ser Leu Gly His Arg Arg Thr Met Met
1 5 10 15 Leu Leu Ala Gln Met Arg Arg Ile Ser Leu Phe Ser Cys Leu
Arg Asp 20 25 30 Arg Gln Asp Phe Arg Phe Pro Gln Glu Glu Phe Asp
Gly Asn Gln Phe 35 40 45 Gln Lys Val Gln Ala Ile Phe Leu Phe Tyr
Glu Met Met Gln Gln Thr 50 55 60 Phe Asn Leu Phe Ser Thr Lys Asp
Ser Ser Ala Ala Trp Asp Glu Thr 65 70 75 80 Leu Leu Glu Lys Phe Tyr
Ile Glu Leu Phe Gln Gln Met Asn Asp Leu 85 90 95 Glu Ala Cys Val
Met Gln Glu Val Gly Val Glu Glu Thr Pro Leu Met 100 105 110 Asn Val
Asp Ser Ile Leu Ala Val Arg Arg Tyr Phe Gln Arg Ile Thr 115 120 125
Leu Tyr Leu Thr Glu Lys Lys Tyr Ser Pro Cys Ala Trp Glu Val Val 130
135 140 Arg Ala Glu Ile Met Arg Ser Phe Ser Phe Ser Thr Asn Leu Gln
Glu 145 150 155 160 Ser Leu Arg Ser Lys Glu 165 82 164 PRT
Artificial Sequence IFNalpha B9x14CHO18NP2(165-166 82 Cys Asp Leu
Pro Gln Thr His Ser Leu Gly His Arg Arg Thr Met Met 1 5 10 15 Leu
Leu Ala Gln Met Arg Arg Ile Ser Leu Phe Ser Cys Leu Arg Asp 20 25
30 Arg Gln Asp Phe Arg Phe Pro Gln Glu Glu Phe Asp Gly Asn Gln Phe
35 40 45 Gln Lys Val Gln Ala Ile Phe Leu Phe Tyr Glu Met Met Gln
Gln Thr 50 55 60 Phe Asn Leu Phe Ser Thr Lys Asp Ser Ser Ala Ala
Trp Asp Glu Thr 65 70 75 80 Leu Leu Glu Lys Phe Tyr Ile Glu Leu Phe
Gln Gln Met Asn Asp Leu 85 90 95 Glu Ala Cys Val Met Gln Glu Val
Gly Val Glu Glu Thr Pro Leu Met 100 105 110 Asn Val Asp Ser Ile Leu
Ala Val Arg Arg Tyr Phe Gln Arg Ile Thr 115 120 125 Leu Tyr Leu Thr
Glu Lys Lys Tyr Ser Pro Cys Ala Trp Glu Val Val 130 135 140 Arg Ala
Glu Ile Met Arg Ser Phe Ser Phe Ser Thr Asn Leu Gln Glu 145 150 155
160 Ser Leu Arg Ser 83 166 PRT Artificial Sequence IFNalpha
B9x25CHO1 83 Cys Asp Leu Pro Gln Thr His Ser Leu Ser Asn Arg Arg
Thr Leu Met 1 5 10 15 Leu Met Ala Gln Met Arg Arg Ile Ser Pro Phe
Ser Cys Leu Lys Asp 20 25 30 Arg His Asp Phe Gly Phe Pro Glu Glu
Glu Phe Asp Gly His His Phe 35 40 45 Gln Lys Val Gln Ala Ile Phe
Leu Leu Tyr Glu Leu Ile Gln Gln Thr 50 55 60 Phe Asn Leu Phe Ser
Thr Lys Asp Ser Ser Ala Ala Trp Asp Glu Thr 65 70 75 80 Leu Leu Glu
Lys Phe Tyr Ile Glu Leu Phe Gln Gln Met Asn Asn Leu 85 90 95 Glu
Ala Cys Val Ile Gln Glu Val Gly Val Glu Glu Ile Ala Leu Met 100 105
110 Asn Val Asp Ser Ile Leu Ala Val Arg Lys Tyr Phe Arg Arg Ile Thr
115 120 125 Leu Tyr Leu Thr Glu Lys Lys Tyr Ser Pro Cys Ala Trp Glu
Val Val 130 135 140 Arg Ala Glu Ile Met Arg Ser Phe Ser Phe Ser Thr
Asn Leu Gln Lys 145 150 155 160 Arg Leu Arg Arg Lys Glu 165 84 166
PRT Artificial Sequence IFNalpha B9x25CHO2 84 Cys Asp Leu Pro Gln
Thr His Ser Leu Ser Asn Arg Arg Thr Leu Met 1 5 10 15 Leu Met Ala
Gln Met Arg Arg Ile Ser Pro Phe Ser Cys Leu Lys Asp 20 25 30 Arg
His Asp Phe Gly Phe Pro Glu Glu Glu Phe Asp Gly His His Phe 35 40
45 Gln Lys Val Gln Ala Ile Phe Leu Leu Tyr Glu Leu Ile Gln Gln Thr
50 55 60 Phe Asn Leu Phe Ser Thr Lys Asp Ser Ser Ala Ala Trp Asp
Glu Thr 65 70 75 80 Leu Leu Glu Lys Phe Tyr Ile Glu Leu Phe Gln Gln
Met Asn Asn Leu 85 90 95 Glu Ala Cys Val Ile Gln Glu Val Gly Val
Glu Glu Ile Ala Leu Met 100 105 110 Asn Val Asp Ser Ile Leu Ala Val
Arg Lys Tyr Phe Arg Arg Ile Thr 115 120 125 Leu Tyr Leu Thr Glu Lys
Lys Tyr Ser Pro Cys Ala Trp Glu Val Val 130 135 140 Arg Ala Glu Ile
Met Arg Ser Phe Ser Leu Ser Thr Asn Leu Gln Glu 145 150 155 160 Ser
Leu Arg Ser Lys Glu 165 85 166 PRT Artificial Sequence IFNalpha
B9x25CHO3 85 Cys Asp Leu Pro Gln Thr His Ser Leu Ser Asn Arg Arg
Thr Leu Met 1 5 10 15 Leu Met Ala Gln Met Arg Arg Ile Ser Pro Phe
Ser Cys Leu Lys Asp 20 25 30 Arg His Asp Phe Gly Phe Pro Glu Glu
Glu Phe Asp Gly His His Phe 35 40 45 Gln Lys Val Gln Ala Ile Phe
Leu Leu Tyr Glu Leu Ile Gln Gln Thr 50 55 60 Phe Asn Leu Phe Ser
Thr Lys Asp Ser Ser Ala Ala Trp Asp Glu Thr 65 70 75 80 Leu Leu Glu
Lys Phe Tyr Ile Glu Leu Phe Gln Gln Met Asn Asn Leu 85 90 95 Glu
Ala Cys Val Ile Gln Glu Val Gly Val Glu Glu Ile Ala Leu Met 100 105
110 Asn Val Asp Ser Ile Leu Ala Val Arg Lys Tyr Phe Arg Arg Ile Thr
115 120 125 Leu Tyr Leu Thr Glu Lys Lys Tyr Ser Pro Cys Ala Trp Glu
Val Val 130 135 140 Arg Ala Glu Ile Met Arg Ser Phe Ser Leu Ser Thr
Asn Leu Gln Glu 145 150 155 160 Cys Leu Arg Ser Lys Glu 165 86 166
PRT Artificial Sequence IFNalpha B9x25CHO4 86 Cys Asp Leu Pro Gln
Thr His Ser Leu Ser Asn Arg Arg Thr Leu Met 1 5 10 15 Leu Met Ala
Gln Met Arg Arg Ile Ser Pro Phe Ser Cys Leu Lys Asp 20 25 30 Arg
His Asp Phe Gly Phe Pro Glu Glu Glu Phe Asp Gly His His Phe 35 40
45 Gln Lys Val Gln Ala Ile Phe Leu Leu Tyr Glu Leu Ile Gln Gln Thr
50 55 60 Phe Asn Leu Phe Ser Thr Lys Asp Ser Ser Ala Ala Trp Asp
Glu Thr 65 70 75 80 Leu Leu Glu Lys Phe Tyr Ile Glu Leu Phe Gln Gln
Met Asn Asn Leu 85 90 95 Glu Ala Cys Val Ile Gln Glu Val Gly Val
Glu Glu Ile Ala Leu Met 100 105 110 Asn Val Asp Ser Ile Leu Ala Val
Arg Lys Tyr Phe Arg Arg Ile Thr 115 120 125 Leu Tyr Leu Thr Glu Lys
Lys Tyr Ser Pro Cys Ala Trp Glu Val Val 130 135 140 Arg Ala Glu Ile
Met Arg Ser Phe Ser Leu Ser Thr Asn Leu Gln Glu 145 150 155 160 Ser
Leu Arg Cys Lys Glu 165 87 166 PRT Artificial Sequence IFNalpha
B9x25Ep01 87 Cys Asp Leu Pro Gln Thr His Ser Leu Ser Asn Arg Arg
Thr Leu Met 1 5 10 15 Leu Met Ala Gln Met Arg Arg Ile Ser Pro Phe
Ser Cys Leu Lys Asp 20 25 30 Arg His Asp Phe Gly Phe Pro Glu Glu
Glu Phe Asp Gly His Gln Phe 35 40 45 Gln Lys Thr Gln Ala Ile Ser
Val Leu His Glu Leu Ile Gln Gln Thr 50 55 60 Phe Asn Leu Phe Ser
Thr Lys Asp Ser Ser Ala Ala Trp Asp Glu Thr 65 70 75 80 Leu Leu Glu
Lys Phe Tyr Ile Glu Leu Phe Gln Gln Met Asn Asn Leu 85 90 95 Glu
Ala Cys Val Ile Gln Glu Val Gly Val Glu Glu Ile Ala Leu Met 100 105
110 Asn Val Asp Ser Ile Leu Ala Val Arg Lys Tyr Phe Arg Arg Ile Thr
115 120 125 Leu Tyr Leu Thr Glu Lys Lys Tyr Ser Pro Cys Ala Trp Glu
Val Val 130 135 140 Arg Ala Glu Ile Met Arg Ser Phe Ser Phe Ser Thr
Asn Leu Gln Lys 145 150 155 160 Arg Leu Arg Arg Lys Glu 165 88 166
PRT Artificial Sequence IFNalpha B9x25Ep02 88 Cys Asp Leu Pro Gln
Thr His Ser Leu Ser Asn Arg Arg Thr Leu Met 1 5 10 15 Ile Met Ala
Gln Met Gly Arg Ile Ser Pro Phe Ser Cys Leu Lys Asp 20 25 30 Arg
His Asp Phe Gly Phe Pro Glu Glu Glu Phe Asp Gly His Gln Phe 35 40
45 Gln Lys Thr Gln Ala Ile Ser Val Leu His Glu Leu Ile Gln Gln Thr
50 55 60 Phe Asn Leu Phe Ser Thr Lys Asp Ser Ser Ala Ala Trp Asp
Glu Thr 65 70 75 80 Leu Leu Glu Lys Phe Tyr Ile Glu Leu Phe Gln Gln
Met Asn Asn Leu 85 90 95 Glu Ala Cys Val Ile Gln Glu Val Gly Val
Glu Glu Ile Ala Leu Met 100 105 110 Asn Val Asp Ser Ile Leu Ala Val
Arg Lys Tyr Phe Arg Arg Ile Thr 115 120 125 Leu Tyr Leu Thr Glu Lys
Lys Tyr Ser Pro Cys Ala Trp Glu Val Val 130 135 140 Arg Ala Glu Ile
Met Arg Ser Phe Ser Leu Ser Thr Asn Leu Gln Glu 145 150 155 160 Ser
Leu Arg Ser Lys Glu 165 89 166 PRT Artificial Sequence IFNalpha
B9x25Ep03 89 Cys Asn Leu Ser Gln Thr His Ser Leu Asn Asn Arg Arg
Thr Leu Met 1 5 10 15 Leu Met Ala Gln Met Arg Arg Ile Ser Pro Phe
Ser Cys Leu Lys Asp 20 25 30 Arg His Asp Phe Gly Phe Pro Glu Glu
Glu Phe Asp Gly His Gln Phe 35 40 45 Gln Lys Thr Gln Ala Ile Ser
Val Leu His Glu Leu Ile Gln Gln Thr 50 55 60 Phe Asn Leu Phe Ser
Thr Lys Asp Ser Ser Ala Ala Trp Asp Glu Thr 65 70 75 80 Leu Leu Glu
Lys Phe Tyr Ile Glu Leu Phe Gln Gln Met Asn Asn Leu 85 90 95 Glu
Ala Cys Val Ile Gln Glu Val Gly Val Glu Glu Ile Ala Leu Met 100 105
110 Asn Val Asp Ser Ile Leu Ala Val Arg Lys Tyr Phe Arg Arg Ile Thr
115 120 125 Leu Tyr Leu Thr Glu Lys Lys Tyr Ser Pro Cys Ala Trp Glu
Val Val 130 135 140 Arg Ala Glu Ile Met Arg Ser Phe Ser Leu Ser Thr
Asn Leu Gln Glu 145 150 155 160 Ser Leu Arg Ser Lys Glu 165 90 166
PRT Artificial Sequence IFNalpha B9x25Ep04 90 Cys Asp Leu Pro Gln
Thr His Ser Leu Ser Asn Arg Arg Thr Leu Met 1 5 10 15 Leu Met Ala
Gln Met Arg Arg Ile Ser Pro Phe Ser Cys Leu Lys Asp 20 25 30 Arg
His Asp Phe Gly Phe Pro Glu Glu Glu Phe Asp Gly His Gln Phe 35 40
45 Gln Lys Thr Gln Ala Ile Ser Val Leu His Glu Met Ile Gln Gln Thr
50 55 60 Phe Asn Leu Phe Ser Thr Lys Asp Ser Ser Ala Ala Trp Asp
Glu Thr 65 70 75 80 Leu Leu Glu Lys Phe Tyr Ile Glu Leu Phe Gln Gln
Met Asn Asn Leu 85 90 95 Glu Ala Cys Val Ile Gln Glu Val Gly Val
Glu Glu Ile Ala Leu Met 100 105 110 Asn Val Asp Ser Ile Leu Ala Val
Arg Lys Tyr Phe Arg Arg Ile Thr 115 120 125 Leu Tyr Leu Thr Glu Lys
Lys Tyr Ser Pro Cys Ala Trp Glu Val Val 130 135 140 Arg Ala Glu Ile
Met Arg Ser Phe Ser Leu Ser Thr Asn Leu Gln Glu 145 150 155 160 Ser
Leu Arg Ser Lys Glu 165 91 166 PRT Artificial Sequence IFNalpha
B9x25Ep05 91 Cys Asp Leu Pro Gln Thr His Ser Leu Ser Asn Arg Arg
Thr Leu Met 1 5 10 15 Leu Met Ala Gln Met Arg Arg Ile Ser Pro Phe
Ser Cys Leu Lys Asp 20 25 30 Arg His Asp Phe Gly Phe Pro Glu Glu
Glu Phe Asp Gly His Gln Phe 35 40 45 Gln Lys Thr Gln Ala Ile Ser
Val Leu His Glu Leu Ile Gln Gln Thr 50 55 60 Phe Asn Leu Phe Ser
Thr Lys Asp Ser Ser Ala Ala Trp Asp Glu Thr 65 70 75 80 Leu Leu Glu
Lys Phe Tyr Ile Glu Leu Phe Gln Gln Met Asn Asp Leu 85 90 95 Glu
Ala Cys Val Ile Gln Glu Val Gly Val Glu Glu Ile Ala Leu Met 100 105
110 Asn Val Asp Ser Ile Leu Ala Val Arg Lys Tyr Phe Arg Arg Ile Thr
115 120 125 Leu Tyr Leu Thr Glu Lys Lys Tyr Ser Pro Cys Ala Trp Glu
Val Val 130 135 140 Arg Ala Glu Ile Met Arg Ser Phe Ser Leu Ser Thr
Asn Leu Gln Glu 145 150 155 160 Ser Leu Arg Ser Lys Glu 165 92 166
PRT Artificial Sequence IFNalpha B9x25Ep06 92 Cys Asp Leu Pro Gln
Thr His Ser Leu Ser Asn Arg Arg Thr Leu Met 1 5 10 15 Leu Met Ala
Gln Met Arg Arg Ile Ser Pro Phe Ser Cys Leu Lys Asp 20 25 30 Arg
His Asp Phe Gly Phe Pro Glu Glu Glu Phe Asp Gly His Gln Phe 35 40
45 Gln Lys Thr Gln Ala Ile Ser Val Leu His Glu Leu Ile Gln Gln Thr
50 55 60 Phe Asn Leu Phe Ser Thr Lys Asp Ser Ser Ala Ala Trp Asp
Glu Thr 65 70 75 80 Leu Leu Asp Lys Phe Tyr Ile Glu Leu Phe Gln Gln
Leu Asn Asp Leu 85 90 95 Glu Ala Cys Val Thr Gln Glu Val Gly Val
Glu Glu Ile Ala Leu Met 100 105 110 Asn Glu Asp Ser Ile Leu Ala Val
Arg Lys Tyr Phe Arg Arg
Ile Thr 115 120 125 Leu Tyr Leu Thr Glu Lys Lys Tyr Ser Pro Cys Ala
Trp Glu Val Val 130 135 140 Arg Ala Glu Ile Met Arg Ser Phe Ser Leu
Ser Thr Asn Leu Gln Glu 145 150 155 160 Ser Leu Arg Ser Lys Glu 165
93 166 PRT Artificial Sequence IFNalpha B9x25Ep07 93 Cys Asp Leu
Pro Gln Thr His Ser Leu Ser Asn Arg Arg Thr Leu Met 1 5 10 15 Leu
Met Ala Gln Met Arg Arg Ile Ser Pro Phe Ser Cys Leu Lys Asp 20 25
30 Arg His Asp Phe Gly Phe Pro Glu Glu Glu Phe Asp Gly His Gln Phe
35 40 45 Gln Lys Thr Gln Ala Ile Ser Val Leu His Glu Leu Ile Gln
Gln Thr 50 55 60 Phe Asn Leu Phe Ser Thr Lys Asp Ser Ser Ala Ala
Trp Asp Glu Thr 65 70 75 80 Leu Leu Glu Lys Phe Tyr Ile Glu Leu Phe
Gln Gln Met Asn Asn Leu 85 90 95 Glu Ala Cys Val Ile Gln Glu Val
Gly Val Glu Glu Ile Ala Leu Met 100 105 110 Asn Val Asp Ser Ile Leu
Ala Val Arg Lys Tyr Phe Gln Arg Ile Thr 115 120 125 Leu Tyr Leu Thr
Glu Lys Lys Tyr Ser Pro Cys Ala Trp Glu Val Val 130 135 140 Arg Ala
Glu Ile Met Arg Ser Phe Ser Leu Ser Thr Asn Leu Gln Glu 145 150 155
160 Ser Leu Arg Ser Lys Glu 165 94 166 PRT Artificial Sequence
IFNalpha B9x25Ep08 94 Cys Asp Leu Pro Gln Thr His Ser Leu Ser Asn
Arg Arg Thr Leu Met 1 5 10 15 Leu Met Ala Gln Met Arg Arg Ile Ser
Pro Phe Ser Cys Leu Lys Asp 20 25 30 Arg His Asp Phe Gly Phe Pro
Glu Glu Glu Phe Asp Gly His Gln Phe 35 40 45 Gln Lys Thr Gln Ala
Ile Ser Val Leu His Glu Leu Ile Gln Gln Thr 50 55 60 Phe Asn Leu
Phe Ser Thr Lys Asp Ser Ser Ala Ala Trp Asp Glu Thr 65 70 75 80 Leu
Leu Glu Lys Phe Tyr Ile Glu Leu Phe Gln Gln Met Asn Asn Leu 85 90
95 Glu Ala Cys Val Ile Gln Glu Val Gly Val Glu Glu Ile Ala Leu Met
100 105 110 Asn Val Asp Ser Ile Leu Ala Val Arg Lys Tyr Phe Arg Arg
Ile Thr 115 120 125 Leu Tyr Leu Thr Glu Lys Lys Tyr Ser Pro Cys Ala
Trp Glu Val Val 130 135 140 Arg Ala Glu Ile Met Arg Ser Phe Ser Leu
Ser Thr Asn Leu Gln Glu 145 150 155 160 Ser Leu Arg Ser Lys Glu 165
95 166 PRT Artificial Sequence IFNalpha B9x25Ep10 95 Cys Asp Leu
Pro Gln Thr His Ser Leu Ser Asn Arg Arg Thr Leu Met 1 5 10 15 Ile
Met Ala Gln Met Gly Arg Ile Ser Pro Phe Ser Cys Leu Lys Asp 20 25
30 Arg His Asp Phe Gly Phe Pro Glu Glu Glu Phe Asp Gly His His Phe
35 40 45 Gln Lys Val Gln Ala Ile Phe Leu Leu Tyr Glu Leu Ile Gln
Gln Thr 50 55 60 Phe Asn Leu Phe Ser Thr Lys Asp Ser Ser Ala Ala
Trp Asp Glu Thr 65 70 75 80 Leu Leu Glu Lys Phe Tyr Ile Glu Leu Phe
Gln Gln Met Asn Asn Leu 85 90 95 Glu Ala Cys Val Ile Gln Glu Val
Gly Val Glu Glu Ile Ala Leu Met 100 105 110 Asn Val Asp Ser Ile Leu
Ala Val Arg Lys Tyr Phe Arg Arg Ile Thr 115 120 125 Leu Tyr Leu Thr
Glu Lys Lys Tyr Ser Pro Cys Ala Trp Glu Val Val 130 135 140 Arg Ala
Glu Ile Met Arg Ser Phe Ser Leu Ser Thr Asn Leu Gln Glu 145 150 155
160 Ser Leu Arg Ser Lys Glu 165 96 166 PRT Artificial Sequence
IFNalpha B9x25Ep11 96 Cys Asn Leu Ser Gln Thr His Ser Leu Asn Asn
Arg Arg Thr Leu Met 1 5 10 15 Leu Met Ala Gln Met Arg Arg Ile Ser
Pro Phe Ser Cys Leu Lys Asp 20 25 30 Arg His Asp Phe Gly Phe Pro
Glu Glu Glu Phe Asp Gly His His Phe 35 40 45 Gln Lys Val Gln Ala
Ile Phe Leu Leu Tyr Glu Leu Ile Gln Gln Thr 50 55 60 Phe Asn Leu
Phe Ser Thr Lys Asp Ser Ser Ala Ala Trp Asp Glu Thr 65 70 75 80 Leu
Leu Glu Lys Phe Tyr Ile Glu Leu Phe Gln Gln Met Asn Asn Leu 85 90
95 Glu Ala Cys Val Ile Gln Glu Val Gly Val Glu Glu Ile Ala Leu Met
100 105 110 Asn Val Asp Ser Ile Leu Ala Val Arg Lys Tyr Phe Arg Arg
Ile Thr 115 120 125 Leu Tyr Leu Thr Glu Lys Lys Tyr Ser Pro Cys Ala
Trp Glu Val Val 130 135 140 Arg Ala Glu Ile Met Arg Ser Phe Ser Leu
Ser Thr Asn Leu Gln Glu 145 150 155 160 Ser Leu Arg Ser Lys Glu 165
97 166 PRT Artificial Sequence IFNalpha B9x25Ep12 97 Cys Asp Leu
Pro Gln Thr His Ser Leu Ser Asn Arg Arg Thr Leu Met 1 5 10 15 Leu
Met Ala Gln Met Arg Arg Ile Ser Pro Phe Ser Cys Leu Lys Asp 20 25
30 Arg His Asp Phe Gly Phe Pro Glu Glu Glu Phe Asp Gly His His Phe
35 40 45 Gln Lys Val Gln Ala Ile Phe Leu Leu Tyr Glu Leu Ile Gln
Gln Thr 50 55 60 Phe Asn Leu Phe Ser Thr Lys Asp Ser Ser Ala Ala
Trp Asp Glu Thr 65 70 75 80 Leu Leu Glu Lys Phe Tyr Ile Glu Leu Phe
Gln Gln Met Asn Asn Leu 85 90 95 Glu Ala Cys Val Ile Gln Glu Val
Gly Val Glu Glu Ile Ala Leu Met 100 105 110 Asn Val Asp Ser Ile Leu
Ala Val Arg Lys Tyr Phe Gln Arg Ile Thr 115 120 125 Leu Tyr Leu Thr
Glu Lys Lys Tyr Ser Pro Cys Ala Trp Glu Val Val 130 135 140 Arg Ala
Glu Ile Met Arg Ser Phe Ser Leu Ser Thr Asn Leu Gln Glu 145 150 155
160 Ser Leu Arg Ser Lys Glu 165 98 166 PRT Artificial Sequence
IFNalpha B9x25Ep13 98 Cys Asp Leu Pro Gln Thr His Ser Leu Ser Asn
Arg Arg Thr Leu Met 1 5 10 15 Ile Met Ala Gln Met Gly Arg Ile Ser
Pro Phe Ser Cys Leu Lys Asp 20 25 30 Arg His Asp Phe Gly Phe Pro
Glu Glu Glu Phe Asp Gly His His Phe 35 40 45 Gln Lys Val Gln Ala
Ile Phe Leu Leu Tyr Glu Leu Ile Gln Gln Thr 50 55 60 Phe Asn Leu
Phe Ser Thr Lys Asp Ser Ser Ala Ala Trp Asp Glu Thr 65 70 75 80 Leu
Leu Glu Lys Phe Tyr Ile Glu Leu Phe Gln Gln Met Asn Asn Leu 85 90
95 Glu Ala Cys Val Ile Gln Glu Val Gly Val Glu Glu Ile Ala Leu Met
100 105 110 Asn Val Asp Ser Ile Leu Ala Val Arg Lys Tyr Phe Gln Arg
Ile Thr 115 120 125 Leu Tyr Leu Thr Glu Lys Lys Tyr Ser Pro Cys Ala
Trp Glu Val Val 130 135 140 Arg Ala Glu Ile Met Arg Ser Phe Ser Leu
Ser Thr Asn Leu Gln Glu 145 150 155 160 Ser Leu Arg Ser Lys Glu 165
99 166 PRT Artificial Sequence IFNalpha B9x25Ep14 99 Cys Asn Leu
Ser Gln Thr His Ser Leu Asn Asn Arg Arg Thr Leu Met 1 5 10 15 Leu
Met Ala Gln Met Arg Arg Ile Ser Pro Phe Ser Cys Leu Lys Asp 20 25
30 Arg His Asp Phe Gly Phe Pro Glu Glu Glu Phe Asp Gly His His Phe
35 40 45 Gln Lys Val Gln Ala Ile Phe Leu Leu Tyr Glu Leu Ile Gln
Gln Thr 50 55 60 Phe Asn Leu Phe Ser Thr Lys Asp Ser Ser Ala Ala
Trp Asp Glu Thr 65 70 75 80 Leu Leu Glu Lys Phe Tyr Ile Glu Leu Phe
Gln Gln Met Asn Asn Leu 85 90 95 Glu Ala Cys Val Ile Gln Glu Val
Gly Val Glu Glu Ile Ala Leu Met 100 105 110 Asn Val Asp Ser Ile Leu
Ala Val Arg Lys Tyr Phe Gln Arg Ile Thr 115 120 125 Leu Tyr Leu Thr
Glu Lys Lys Tyr Ser Pro Cys Ala Trp Glu Val Val 130 135 140 Arg Ala
Glu Ile Met Arg Ser Phe Ser Leu Ser Thr Asn Leu Gln Glu 145 150 155
160 Ser Leu Arg Ser Lys Glu 165 100 166 PRT Artificial Sequence
IFNalpha B9x25Ep15 100 Cys Asp Leu Pro Gln Thr His Ser Leu Ser Asn
Arg Arg Thr Leu Met 1 5 10 15 Leu Met Ala Gln Met Arg Arg Ile Ser
Pro Phe Ser Cys Leu Lys Asp 20 25 30 Arg His Asp Phe Gly Phe Pro
Glu Glu Glu Phe Asp Gly His Gln Phe 35 40 45 Gln Lys Thr Gln Ala
Ile Ser Val Leu His Glu Leu Ile Gln Gln Thr 50 55 60 Phe Asn Leu
Phe Ser Thr Lys Asp Ser Ser Ala Ala Trp Asp Glu Thr 65 70 75 80 Leu
Leu Glu Lys Phe Tyr Ile Glu Leu Phe Gln Gln Met Asn Asn Leu 85 90
95 Glu Ala Cys Val Ile Gln Glu Val Gly Val Glu Glu Ile Ala Leu Met
100 105 110 Asn Val Asp Ser Ile Leu Ala Val Arg Lys Tyr Phe Arg Arg
Ile Thr 115 120 125 Leu Tyr Leu Thr Glu Lys Lys Tyr Ser Pro Cys Ala
Trp Glu Val Val 130 135 140 Arg Ala Glu Ile Met Arg Ser Phe Ser Leu
Ser Thr Asn Leu Gln Glu 145 150 155 160 Ser Leu Arg Ser Lys Glu 165
101 166 PRT Artificial Sequence IFNalpha B9x25Ep16 101 Cys Asp Leu
Pro Gln Thr His Ser Leu Ser Asn Arg Arg Thr Leu Met 1 5 10 15 Ile
Met Ala Gln Met Gly Arg Ile Ser Pro Phe Ser Cys Leu Lys Asp 20 25
30 Arg His Asp Phe Gly Phe Pro Glu Glu Glu Phe Asp Gly His Gln Phe
35 40 45 Gln Lys Thr Gln Ala Ile Ser Val Leu His Glu Leu Ile Gln
Gln Thr 50 55 60 Phe Asn Leu Phe Ser Thr Lys Asp Ser Ser Ala Ala
Trp Asp Glu Thr 65 70 75 80 Leu Leu Glu Lys Phe Tyr Ile Glu Leu Phe
Gln Gln Met Asn Asn Leu 85 90 95 Glu Ala Cys Val Ile Gln Glu Val
Gly Val Glu Glu Ile Ala Leu Met 100 105 110 Asn Val Asp Ser Ile Leu
Ala Val Arg Lys Tyr Phe Gln Arg Ile Thr 115 120 125 Leu Tyr Leu Thr
Glu Lys Lys Tyr Ser Pro Cys Ala Trp Glu Val Val 130 135 140 Arg Ala
Glu Ile Met Arg Ser Phe Ser Leu Ser Thr Asn Leu Gln Glu 145 150 155
160 Ser Leu Arg Ser Lys Glu 165 102 166 PRT Artificial Sequence
IFNalpha B9x25Ep17 102 Cys Asn Leu Ser Gln Thr His Ser Leu Asn Asn
Arg Arg Thr Leu Met 1 5 10 15 Leu Met Ala Gln Met Arg Arg Ile Ser
Pro Phe Ser Cys Leu Lys Asp 20 25 30 Arg His Asp Phe Gly Phe Pro
Glu Glu Glu Phe Asp Gly His Gln Phe 35 40 45 Gln Lys Thr Gln Ala
Ile Ser Val Leu His Glu Leu Ile Gln Gln Thr 50 55 60 Phe Asn Leu
Phe Ser Thr Lys Asp Ser Ser Ala Ala Trp Asp Glu Thr 65 70 75 80 Leu
Leu Glu Lys Phe Tyr Ile Glu Leu Phe Gln Gln Met Asn Asn Leu 85 90
95 Glu Ala Cys Val Ile Gln Glu Val Gly Val Glu Glu Ile Ala Leu Met
100 105 110 Asn Val Asp Ser Ile Leu Ala Val Arg Lys Tyr Phe Gln Arg
Ile Thr 115 120 125 Leu Tyr Leu Thr Glu Lys Lys Tyr Ser Pro Cys Ala
Trp Glu Val Val 130 135 140 Arg Ala Glu Ile Met Arg Ser Phe Ser Leu
Ser Thr Asn Leu Gln Glu 145 150 155 160 Ser Leu Arg Ser Lys Glu 165
103 166 PRT Artificial Sequence IFNalpha B9x25EF1 103 Cys Asp Leu
Pro Gln Thr His Ser Leu Ser Asn Arg Arg Thr Leu Met 1 5 10 15 Ile
Met Ala Gln Met Gly Arg Ile Ser Pro Phe Ser Cys Leu Lys Asp 20 25
30 Arg His Asp Phe Gly Phe Pro Glu Glu Glu Phe Asp Gly His Gln Phe
35 40 45 Gln Lys Thr Gln Ala Ile Ser Val Leu His Glu Met Ile Gln
Gln Thr 50 55 60 Phe Asn Leu Phe Ser Thr Lys Asp Ser Ser Ala Ala
Trp Asp Glu Thr 65 70 75 80 Leu Leu Asp Lys Phe Tyr Ile Glu Leu Phe
Gln Gln Leu Asn Asp Leu 85 90 95 Glu Ala Cys Val Thr Gln Glu Val
Gly Val Glu Glu Ile Ala Leu Met 100 105 110 Asn Glu Asp Ser Ile Leu
Ala Val Arg Lys Tyr Phe Gln Arg Ile Thr 115 120 125 Leu Tyr Leu Thr
Glu Lys Lys Tyr Ser Pro Cys Ala Trp Glu Val Val 130 135 140 Arg Ala
Glu Ile Met Arg Ser Phe Ser Leu Ser Thr Asn Leu Gln Glu 145 150 155
160 Ser Leu Arg Ser Lys Glu 165 104 166 PRT Artificial Sequence
IFNalpha B9x25EF2 104 Cys Asn Leu Ser Gln Thr His Ser Leu Asn Asn
Arg Arg Thr Leu Met 1 5 10 15 Leu Met Ala Gln Met Arg Arg Ile Ser
Pro Phe Ser Cys Leu Lys Asp 20 25 30 Arg His Asp Phe Gly Phe Pro
Glu Glu Glu Phe Asp Gly His Gln Phe 35 40 45 Gln Lys Thr Gln Ala
Ile Ser Val Leu His Glu Met Ile Gln Gln Thr 50 55 60 Phe Asn Leu
Phe Ser Thr Lys Asp Ser Ser Ala Ala Trp Asp Glu Thr 65 70 75 80 Leu
Leu Asp Lys Phe Tyr Ile Glu Leu Phe Gln Gln Leu Asn Asp Leu 85 90
95 Glu Ala Cys Val Thr Gln Glu Val Gly Val Glu Glu Ile Ala Leu Met
100 105 110 Asn Glu Asp Ser Ile Leu Ala Val Arg Lys Tyr Phe Gln Arg
Ile Thr 115 120 125 Leu Tyr Leu Thr Glu Lys Lys Tyr Ser Pro Cys Ala
Trp Glu Val Val 130 135 140 Arg Ala Glu Ile Met Arg Ser Phe Ser Leu
Ser Thr Asn Leu Gln Glu 145 150 155 160 Ser Leu Arg Ser Lys Glu
165
* * * * *