Note: Descriptions are shown in the official language in which they were submitted.
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IFN-ALPHA HOMOLOGUES
COPYRIGHT NOTIFICATION
Pursuant to 37 C.F.R. 1.71(e), a portion of this patent document 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 the patent
disclosure, as it
appears in the Patent and Trademark Office patent file or records, but
otherwise reserves
all copyright rights whatsoever.
CROSS REFERENCE TO RELATED APPLICATIONS
This application is a continuation-in-part application of and claims the
benefit of and priority to U.S. Patent Application Serial No. 09/145,483,
filed October 7,
1999, the disclosure of which is incorporated herein by reference in its
entirety for all
purposes.
FIELD OF THE INVENTION
The present invention relates to the generation of new interferon-alpha
homologues.
BACKGROUND OF THE INVENTION
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 that share 85-98% sequence identity at the amino acid level
(Henco, K. et
al. (1985) J. Mol. Biol. 185:227-260).
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).
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Interferon-alphas are also useful against various types of viral infections
(Finter, N.B. et al. (1991) Drugs 42(5):749). Interferon-alphas have shown
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, GL et al. (1989) N.
England J.
Med. 321:1501). 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).
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 26:1125-121S; 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,
neurological
disorders including hallucination, fever, hepatic enzyme elevation, and
leukopenia
(Pontzer, C.H. et al. (1991) Cancer Res. 51:5304; Oldham, 1985, supra).
The existence of abundant naturally occurring sequence diversity within the
interferon-alphas (and hence a large sequence space of recombinants) along
with the
intricacy of interferon-alpha/receptor interactions and variety of therapeutic
and
prophylactic activities creates an opportunity for the construction of
superior interferon
homologues.
SUMMARY OF THE INVENTION
The invention provides novel interferon-alpha (IFN-alpha or IFN-a)
homologue polypeptides, nucleic acids encoding the polypeptides and
complementary
nucleotide sequences thereof, fragments of said polypeptides and nucleic
acids, antibodies
to the polypeptides, and uses therefor, data sets containing character strings
of interferon-
alpha homologue sequences, and automated systems for using the character
strings.
In one aspect, the invention includes an isolated or recombinant interferon-
alpha nucleic acid homologue. Included are a polynucleotide sequences selected
from
SEQ ll~ NO: l to SEQ )D N0:35, or to SEQ >D N0:72 to SEQ )D N0:78, and
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complementary polynucleotide sequences thereof. Polynucleotide sequences
encoding a
polypeptide selected from SEQ ID N0:36 to SEQ ID N0:81 or from SEQ B7 N0:79 to
SEQ 1D N0:85, and complementary polynucleotide sequences thereof are also a
feature of
the invention. Similarly, a polynucleotide sequence which hybridizes under
highly
stringent conditions over substantially the entire length of any of the
preceding
polynucleotide sequences is a feature of the present invention. In addition, a
polynucleotide sequence comprising a nucleotide fragment of any of the
preceding
polynucleotide sequences which nucleotide fragment encodes a polypeptide
having an
antiproliferative activity in a human Daudi cell line- based cell
proliferation assay is a
feature of the invention. Similarly, a polynucleotide sequence comprising a
nucleotide
fragment of any of the polynucleotide sequences of the invention described
above and
below which encodes a polypeptide having antiviral activity in a murine cell
line/EMCV -
based assay is a feature of the invention.
The invention also includes an isolated or recombinant nucleic acid,
comprising a polynucleotide sequence encoding a polypeptide, wherein the
polypeptide
comprises the amino acid sequence: CDLPQTHSLG-X11-X12-RA-Xls-X16-LL-X19-QM-
X22-R-X24-S-X26-FS CLKDR-X34-DFG-X3 s-P-X4o-EEFD-X4s-X46-X4~-FQ-Xso-Xs 1-QAI-
Xss-Xs6-Xsr~-X6o-X61-QQT~-X6~-FSTK-X~2-S S-X~s-X~6-W-X~ s-X~9-Xso-LL-Xs3-K-
Xss-Xs6-T-Xa s-L-X~o-QQLN-X9s-LEAC V-X 101-Q-X 1 o3-V-X 1 os-X 1 o6-X 1 o7-X 1
os-TPLMN-
X114-D-X116-ILAV-X121-KY-X124-QRITLYL-Xlsz-E-X134-KYSPC-Xl4o-
WEVVRAE>ZVVIRSFSFSTNLQKRLRRKE, or a conservatively substituted variation
thereof, where X11 is N or D; X12 is R, S, or K; Xls is L or M; X16 is I, M,
or V; X19 is A or
G; X22 is G or R; X24 is I or T; X26 is P or H; X34 is H, Y or Q; X3s is F or
L; X4o is Q or R;
X4s is G or S; X46 is N or H; X4~ is Q or R; Xso is K or R; Xsl is A or T; Xss
is S or F; Xs6
is V or A; Xs~ is L or F; X6o is M or I; X61 is I or M; X6~ is L or F; X~2 is
D or N; X~s is A
or V; X~6 is A or T; X~s is E or D; X~9 is Q or E; Xgo is S, R, T, or N; Xs3
is E or D; Xss is
F or L; Xs6 is S or Y; Xss is E or G; X9o is Y, H, N; X9s is D, E, or N; Xlol
is I, M, or V;
Xlo3isEorG;XlosisGorW;Xlo6isVorM;Xlo~isE,G,orK;XlosisEorG;X114isV,
E, or G; X116 is S or P; X121 is K or R; X124 is F or L; Xl3z is T, I, or M;
Xls4 is K or R; and
Xl4o is A or S. Each of the single letters of this amino acid sequence
represents a
particular amino acid residue according to standard practice known to those of
ordinary
skill in the art.
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A polypeptide having any of the preceding sequences, such as those
embodied in SEQ ID N0:36 to SEQ ID N0:54, is also a feature of the invention.
In other embodiments, the encoded polypeptide comprises an amino acid
sequence selected from the group consisting of SEQ ID N0:36 to SEQ >D N0:54;
and the
nucleic acid comprises a polynucleotide sequence selected from the group
consisting of
SEQ ID NO:1 to SEQ ID N0:19.
The invention also provides polypeptide fragments of any of SEQ NOS:36-
70 and SEQ ID NOS:72-79. In one aspect of the invention, such a polypeptide
fragment
exhibits an antiproliferative activity in a human Daudi cell line- based cell
proliferation
assay or an antiviral activity in a murine cell line/EMCV - based assay, or
both said
activities. The human Daudi cell line- based cell proliferation assay and
antiviral activity
in a murine cell line/EMCV - based assay are described in greater detail
below. In yet
another aspect, the invention provides a polynucleotide sequence comprising a
nucleotide
fragment of any nucleic acid of the invention described above and below,
wherein said
nucleotide fragment encodes a polypeptide fragment that exhibits an
antiproliferative
activity in a human Daudi cell line- based cell proliferation assay or an
antiviral activity in
a murine cell line/EMCV - based assay, or both activities, as is described in
greater detail
below.
The invention also includes an isolated or recombinant nucleic acid
comprising a polynucleotide sequence encoding a polypeptide, wherein the
polypeptide
comprises an amino acid sequence comprising at least 20 contiguous amino acids
of any
one of SEQ ID NOS:36-70. In other embodiments, the polypeptide of the
invention
comprises an amino acid sequence comprising one or more of amino acid residues
(Tyr or
Gln)34, G1y37, Phe38, Lys7l, A1a76, Tyr90, Ilel32, Argl34, Phe152, Lys160, and
GIu166, wherein the numbering of the amino acid residues corresponds to the
numbering
of residues in the amino acid sequence of SEQ ID N0:36. In various
embodiments, the
encoded polypeptide of the invention comprises at least 30, at least 50, at
least 70, at least
75, at least 100, at least 110, at least 120, at least 130, at least 140, at
least 150, at least
155, at least 160, or at least 165 contiguous amino acid residues of any one
of SEQ ID
NOS:36-70. In other embodiments, the encoded polypeptide is at least 150, at
least 155,
at least 160, at least 163, or at least 165 amino acids in length. In another
embodiment, the
encoded polypeptide is about 166 amino acids in length. In yet other
embodiments, the
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encoded polypeptide comprises an amino acid sequence selected from SEQ ID
N0:36,
SEQ ID N0:37, SEQ ID N0:39, SEQ ID N0:40, SEQ ID N0:41, SEQ D7 N0:42, SEQ
D7 N0:45, and SEQ ID N0:46.
In other embodiments, the invention provides a nucleic acid that comprises
a polynucleotide sequence selected from SEQ )D NO:1, SEQ )D N0:2, SEQ ID N0:3,
SEQ ID N0:4, SEQ ID N0:5, SEQ ID N0:6, SEQ >D N0:7, SEQ B7 NO:10, and SEQ ID
NO:11.
In other embodiments, the polypeptide encoded by any nucleic acid or the
invention described herein or a fragment thereof may have antiproliferative
activity in a
human Daudi cell line - based assay, or antiviral activity in a human WISH
cell/EMCV-
based assay. In other embodiments, the encoded polypeptide has
antiproliferative activity
of at least about 8.3x106 units/milligram in the human Daudi cell line - based
assay (1 unit
is the amount of protein in milligram (mg) required to induce 50%
antiproliferative
activity), or antiviral activity of at about least 2.1x10' units/milligram
(mg) in the human
WISH cell/EMCV-based assay (1 unit is the amount of protein in mg required to
induce
50% antiviral activity). In other embodiments, the encoded polypeptide can
bind to a type
I interferon receptor, preferably a human type I interferon receptor, more
preferably a
human (e.g., type I) interferon-alpha receptor.
The invention also includes a cell comprising any nucleic acid of the
invention described herein, or which expresses any polypeptide of the
invention noted
herein. In one embodiment, the cell expresses a polypeptide encoded by the
nucleic acid
of the invention as described herein.
The invention also includes a vector comprising any nucleic acid of the
invention described above and below. The vector can comprise a plasmid, a
cosmid, a
phage, or a virus; the vector can be, e.g., an expression vector, a cloning
vector, a
packaging vector, an integration vector, or the like. The invention also
includes a cell
transduced by a vector of the invention. The invention also includes
compositions
comprising any nucleic acid of the invention described above and below, and an
excipient,
preferably a pharmaceutically acceptable excipient. Cells and transgenic
animals which
include any polypeptide or nucleic acid of the invention described above and
below, e.g.,
produced by transduction of vector, are a feature of the invention.
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The invention also includes compositions produced by digesting one or
more of the nucleic acids of the invention described above or below with a
restriction
endonuclease, an RNAse, or a DNAse; and, compositions produced by incubating
one or
more nucleic acids described above or below in the presence of
deoxyribonucelotide
triphosphates and a nucleic acid polymerase, e.g., a thermostable polymerase.
The invention also includes compositions comprising two or more nucleic
acids described above or below. The composition may comprise a library of
nucleic acids,
where the library contains at least about 5, 10, 20 or 50 nucleic acids.
In another aspect, the invention includes an isolated or recombinant
polypeptide encoded by any nucleic acid described above or below. In one
embodiment,
the polypeptide may comprise a sequence selected from SEQ ID N0:36 to SEQ >D
N0:70, or SEQ 117 N0:79 to SEQ >D N0:85.
The invention also includes a polypeptide comprising at least 50 contiguous
amino acids of a protein encoded by a polynucleotide sequence, the
polynucleotide
sequence selected from the group consisting of: (a) SEQ ID NO:1 to SEQ ID
N0:35 or
SEQ >D N0:72 to SEQ 1D N0:78; (b) a polynucleotide sequence that encodes a
polypeptide selected from SEQ 1D N0:36 to SEQ ID N0:70 or SEQ ID N0:79 to SEQ
ID
N0:85; and (c) a complementary sequence of a polynucleotide sequence which
hybridizes
under highly stringent conditions over substantially the entire length of
polynucleotide
sequence (a) or (b). In various embodiments, the polypeptide comprises at
least about 70,
100, 120, 130, 140, 150, 155, 160, 165, or 166 contiguous amino acids of the
encoded
protein.
The invention also includes an isolated or recombinant polypeptide
comprising an amino acid sequence comprising at least 50 contiguous amino acid
residues
of any one of SEQ )D NOS:36-70, and one or more of amino acids A1a19, (Tyr or
Gln)34,
G1y37, Phe38, Lys7l, A1a76, Tyr90, I1e132, Arg134, Phe152, Lys160, and Glul66,
where
the numbering of the amino acids corresponds to that of SEQ 1D N0:36. In
various
embodiments, the polypeptide comprises at least about 50, 70, 75, 100, 110,
120, 130, 140
150, 155, 160, 163, 165, or 166 contiguous amino acids of any one of SEQ ID
NOS:36-70.
In more preferred embodiments, the polypeptide comprises at least about 50,
70, 75, 100,
110, 120, 130, 140, 150, 155, 160, 163, 165, or 166 contiguous amino acid
residues of any
one of SEQ )D N0:36, SEQ >D N0:37, SEQ >D N0:39, SEQ >D N0:40, SEQ >D N0:41,
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SEQ ID N0:42, SEQ >D N0:45, or SEQ ID N0:46. In other embodiments, the
polypeptide of the invention is at least about 50, 70, 75, 100, 110, 120, 130,
140, 150, 155,
160, 163, 165, or 166 amino acid residues in length, or is preferably 166
amino acids in
length. Longer polypeptides, e.g., which comprise purification tags or the
like, are also
contemplated. Such polypeptides may display antiproliferative activities in
human Daudi
cell-line based assay and/or antiviral activities in a human WISH cell/EMCV-
based assay.
The invention also includes a polypeptide which specifically binds
polyclonal antisera raised against at least one antigen, said at least one
antigen comprising
a polypeptide sequence selected from an amino acid sequence set forth in SEQ
ID N0:36
to SEQ >D N0:70 or SEQ 1D N0:79 to SEQ >D N0:85 or a fragment thereof. In
particular, the invention provides polypeptides which bind a polyclonal
antisera raised
against at least one antigen, wherein said at least one antigen comprises at
least one amino
acid sequence set forth in SEQ 117 N0:36 to SEQ ID N0:70 or SEQ ID N0:79 to
SEQ >D
N0:85, or a fragment of any of these amino sequences, wherein the polyclonal
antisera is
subtracted with one or more known interferon-alpha polypeptides or proteins,
including,
e.g., a polypeptide or protein encoded by a nucleic acid having or
corresponding to one
or more of the following GenBank~ accession numbers: J00210 (alpha-D), J00207
(Alpha-a), X02958 (Alpha-6), X02956 (Alpha-5), V00533 (alpha-H), V00542 (alpha-
14),
V00545 (IFN-1B), X03125 (alpha-8), X02957 (alpha-16), V00540 (alpha-21),
X02955
(alpha-4b), V00532 (alpha-C), X02960 (alpha-7), X02961 (alpha-10 pseudogene),
80067
(Gx-1), I01614, I01787, I07821, M12350 (alpha-F), M38289, V00549 (alpha-2a),
and
I08313 (alpha-Con1), and other similar or homologous interferon-alpha nucleic
acid
sequences presented in GenBank.
Any polypeptide described above or below optionally has antiproliferative
activity in a human Daudi cell line - based assay and/or in an antiviral
activity in a human
WISH cell/BMCV-based assay. Any polypeptide described above or below can have
antiproliferative activity of at least about 8.3x106units/mg in the human
Daudi cell line -
based assay or antiviral activity of at least about 2.1x 10' units/mg in the
human WISH
cell/EMCV-based assay. In other embodiments, any polypeptide described above
or
below can bind to a type I interferon receptor, preferably a human type I
interferon
receptor, more preferably a human interferon-alpha receptor.
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In other embodiments, any polypeptide described above or below may
further include a secretion/localization sequence, e.g., a signal sequence, an
organelle
targeting sequence, a membrane localization sequence, and the like. Any
polypeptide
described herein may further include a sequence that facilitates purification,
e.g., an
epitope tag (such as, a FLAG epitope), a polyhistidine tag, a GST fusion, and
the like.
The polypeptide optionally includes a methionine at the N-terminus. Any
polypeptide of
the invention described herein optionally includes one or more modified amino
acids,
such as a glycosylated amino acid, a PEG-ylated amino acid, a farnesylated
amino acid, an
acetylated amino acid, a biotinylated amino acid, a carboxylated amino acid, a
phosphorylated amino acid, an acylated amino acid, or the like.
The invention also includes compositions comprising any polypeptide
described herein in an excipient, preferably a pharmaceutically acceptable
excipient.
The invention also includes an antibody or antisera produced by
administering one or more of the polypeptides of the invention described
herein to a
mammal, wherein the antibody or antisera does not specifically bind to a known
alpha-
interferon polypeptide or protein, including, e.g., any polypeptide or protein
encoded by a
nucleic acid having or corresponding to one or more of the following GenBank
accession
numbers: J00210 (alpha-D), J00207 (Alpha-A), X02958 (Alpha-6), X02956 (Alpha-
5),
V00533 (alpha-H), V00542 (alpha-14), V00545 (IFN-1B), X03125 (alpha-8), X02957
(alpha-16), V00540 (alpha-21), X02955 (alpha-4b), V00532 (alpha-C), X02960
(alpha-7),
X02961 (alpha-10 pseudogene), 80067 (Gx-1), I01614, I01787, I07821, M12350
(alpha-
F), M38289, V00549 (alpha-2a), and I08313 (alpha-Conl), and other similar or
homologous interferon-alpha sequences presented in GenBank.
The invention also includes antibodies which specifically bind a
polypeptide comprising a sequence selected from SEQ >D N0:36 to SEQ >D N0:70
or
SEQ >Z7 N0:79 to SEQ >D N0:85. The antibodies are, e.g., polyclonal,
monoclonal,
chimeric, humanized, single chain, Fab fragments, fragments produced by an Fab
expression library, or the like.
Methods for producing the polypeptides of the invention are also included.
One such method comprises introducing into a population of cells any nucleic
acid
described herein, operatively linked to a regulatory sequence effective to
produce the
encoded polypeptide, culturing the cells in a culture medium to produce the
polypeptide,
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and optionally isolating the polypeptide from the cells or from the culture
medium. The
nucleic acid may be part of a vector, such as a recombinant expression vector.
The invention also includes a method of inhibiting growth of tumor cells,
by contacting the tumor cells with a polypeptide of the invention described
herein,
thereby inhibiting growth of the tumor cells. In one embodiment, the invention
includes a
method of inhibiting growth of population of tumor cells comprising contacting
the
population of tumor cells with an effective amount of a polypeptide of the
invention
sufficient to inhibit growth of tumor cells in said population of tumor cells,
thereby
inhibiting growth of tumor cells in said population of cells. In various
embodiments, the
tumor cells can be human carcinoma cells, human leukemia cells, human T-
lymphoma
cells, human melanoma cells, other human cancer cells as described herein, and
the like.
The tumor cells can be in vivo, ex vivo, or in vitro (e.g., cultured cells).
The invention also includes a method of inhibiting the replication of a virus
within one or more cells infected by the virus, by contacting one or more of
the infected
cells with an effective amount of a polypeptide of the invention as described
above and
below, wherein said amount is sufficient to inhibit viral replication in said
one or more
infected cells, thereby inhibiting replication of the virus in the one or more
cells. In
various embodiments, the virus can be an RNA virus, e.g., a human
immunodeficiency
virus or a hepatitis C virus, or a DNA virus, e.g., a hepatitis B virus. The
infected cells
can be in vivo, ex vivo, or in vitro (e.g., cultured cells).
The invention also includes a method of treating an autoimmune disorder
in a subject in need of such treatment, by administering to the subject an
effective amount
of a polypeptide of the invention as described herein sufficient to treat the
autoimmune
disorder. In various embodiments, the autoimmune disorder may be multiple
sclerosis,
rheumatoid arthritis, lupus erythematosus, type I diabetes, and the like. The
invention
also includes, in a method of treating a disorder treatable by administration
of interferon-
alpha to a subject, an improvement comprising administering to the subject an
effective
amount of a polypeptide of the invention as described herein sufficient to
treat said
disorder. The disorder treatable by administration of interferon-alpha
disorder may be
multiple sclerosis, rheumatoid arthritis, lupus erythematosus, type I
diabetes, AIDS or
A>DS-related complexes, or the like.
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In general, nucleic acids and proteins derived by mutation of the sequences
herein are a feature of the invention. Similarly, those produced by diversity
generation or
recursive sequence recombination (RSR) methods (e.g., DNA shuffling) are a
feature of
the invention. Mutation and recombination methods using the nucleic acids
described
herein are a feature of the invention. For example, one method of the
invention includes
recursively recombining one or more nucleic acid sequences of the invention as
described
above and below with one or more additional nucleic acids (including, but not
limited to,
those noted herein), each sequence of the one or more additional nucleic acids
encoding an
interferon-alpha homologue or an amino acid subsequence thereof. The
recombining steps
are optionally performed in vivo, ex vivo, in silico or in vitro. Said
recursive
recombination produces at least one library of recombinant interferon-alpha
homologue
nucleic acids. Also included in the invention are a recombinant interferon-
alpha
homologue nucleic acid produced by this method, a cell containing the
recombinant
interferon-alpha homologue nucleic acid, a nucleic acid library produced by
this recursive
recombination method, a composition comprising two or more of said recombinant
interferon-alpha nucleic acids, and a population of cells comprising such
recombinant
interferon-alpha nucleic acids or containing the library. In one embodiment,
the library
comprise at least ten such recombinant nucleic acids.
The invention also provides a method of producing a modified or
recombinant interferon-alpha homologue nucleic acid that comprises mutating a
nucleic
acid of the invention as described herein.
Also provided are nucleic acids that encode an interferon-alpha homologue
having an increased growth inhibition activity, cytostatic activity, or
cytotoxic activity
against a population of cells (e.g., cancer cells) relative to the growth
inhibition activity
cytostatic activity, or cytotoxic activity, respectively, of human interferon-
alpha 2a or
other known interferon-alpha against the population of cells.
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.
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BRIEF DESCRIPTION OF THE FIGURES
Figures lA-lE show an alignment of exemplary mature interferon
homologue polypeptide sequences (SEQ >D NOS: 36-70 and 79-85) according to the
invention.
Figure 2 shows antiproliferative activities in a human Daudi cell line -
based assay and antiviral activities in a human WISH celllEMCV-based assay of,
respectively, exemplary interferon homologues of the present invention
relative to the
respective antiproliferative and antiviral activities of two control
compounds, human
interferon alpha-2a ("IFN-a-2a" or "2a") and consensus human interferon ("IFN-
Conl" or
"Conl").
Figures 3A, 3B, and 3C illustrate activity profiles of IFN-alpha homologue
3DA11 (SEQ ID N0:40) and control interferons, human interferon alpha-2a (
"2a") and
consensus human interferon alpha ( "Conl"), against a panel of tumor cell
lines. Fig. 3A
shows the cell total growth inhibitory activity of IFN-alpha homologue 3DA11
and each
control IFN on each respective cell line as reflected in the GI50 value, which
is the
concentration (pg/ml) of interferon alpha homologue or control IFN alpha at
which growth
of a particular cell line is inhibited by 50%, as measured by a 50% reduction
in the net
protein/polypeptide increase in the interferon alpha homologue or control IFN
alpha at the
end of the incubation period.
Fig. 3B shows the cytostatic activity of IFN-alpha homologue 3DA11 and
each control 1FN on each cell line of the panel of cell lines. Cytostatic
activity refers to an
activity capable of suppressing growth and multiplication of cells. Cytostatic
activity is
assessed as a reflection of the concentration of IFN-alpha homologue 3DA11 or
control
IFN (p.g/ml) at which the growth and/or multiplication of cells of a
particular cell line is
completely inhibited or suppressed, such that the amount of cellular protein
at the end of
the incubation period equals the amount of cellular protein at the beginning
of the
incubation period ("total growth inhibition" or "TGI").
Fig. 3C illustrates the cytotoxic activity of IFN-alpha homologue 3DA11
and each control IFN on each respective cell line. The cytotoxicity of an
agent (e.g., an
IFN homologue or IFN compound) is the degree to which the agent possess a
specific
destructive action on certain cells or the possession of such action. The term
typically
refers to an agent capable of causing cell death and is used particularly in
referring to the
lysis of cells by immune phenomena and to agents of compounds that selectively
kill
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dividing cells. In Fig. 3C, cytotoxic activity is illustrated as LC50, the
concentration of
IFN-alpha homologue 3DAl 1 (~g/ml) at which a 50% reduction in the net protein
increase
in control cells (control IFN alpha) at the end of the incubation as compared
to that at the
beginning of the incubation period is observed, indicating a net loss of cells
following
addition of the particular interferon. Cytotoxic activity may be assessed as
the
concentration of IFN-alpha homologue 3DA11 at which, relative to the control
cells, 50%
of the total number of cells (i.e., total population) of a particular cell
line are destroyed or
killed.
Figs. 4A, 4B, 4C, and 4D show the cytostatic activity of selected
interferon-alpha homologues of the present invention relative to the
cytostatic activities of
two control interferon alphas, human interferon-alpha 2a ("2a") and consensus
human
interferon-alpha ("Conl"), against a leukemia cell line (RPMI-8226) (Fig. 4A),
a lung
cancer cell line (NCI-H23) (Fig. 4B), a renal cancer cell line (ACHN) (Fig.
4C), and an
ovarian cancer cell line (OVCAR-3) (Fig. 4D), respectively. Cytostatic
activity is
reflected by a TGI value for a particular interferon alpha (i.e., the
concentration of
interferon alpha at which cell growth of a cell line is totally inhibited,
wherein the amount
of cellular protein at the end of the incubation period equals the amount of
cellular protein
at the beginning of the incubation period).
Fig. 5 presents a comparison of the number of mice (out of a total number
of six mice) that survived following administration of doses of 2 ~.g, 10 fig,
and 50 ~g of
two exemplary IFN-alpha homologues of the present invention (designated "IFN-
CH2.2"
and "IFN-CH2.3"), doses of 2 ~,g, 10 fig, and 50 ~g of murine IFN-alpha-4, and
doses of 2
fig, 10 fig, and SO ~g of human IFN-alpha-2a, respectively. The results shown
in Fig. 5
demonstrate that in a murine model system, the improved in vitro antiviral
activity of these
two exemplary IFN-alpha homologues is maintained and sustained in vivo.
Phosphate-
buffered saline (PBS) is used as a control.
DETAILED DESCRIPTION OF THE INVENTION
DEFINITIONS
Unless otherwise defined herein or below 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 present
invention
belongs.
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A "polynucleotide sequence" is a nucleic acid (which is a polymer of
nucleotides (A,C,T,U,G, etc. or naturally occurring 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.
Similarly, an "amino acid sequence" is a polymer of amino acids (a protein,
polypeptide, etc.) or a character string representing an amino acid polymer,
depending on
context. Either the given nucleic acid or the complementary nucleic acid can
be
determined from any specified polynucleotide sequence.
A nucleic acid, protein, peptide, polypeptide, 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.,
polymerises 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 nucleic acid, polypeptide, 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 preferred embodiments, the
preparation consists
of more than 70%, typically more than 80%, or preferably more than 90% of the
isolated
species.
In one aspect, 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.
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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
polymerise 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.
A nucleic acid or polypeptide is "recombinant" when it is artificial or
engineered, or derived from an artificial or engineered protein or nucleic
acid. The term
"recombinant" when used with reference e.g., to a cell, nucleotide, vector, or
polypeptide
typically indicates that the cell, nucleotide, 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 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 (e.g., genes) that are not found in the native (non-recombinant)
form of the cell
or express native nucleic acid sequences (e.g., genes) that would be
abnormally expressed
under-expressed, or not expressed at all. The term "recombinant nucleic acid"
(e.g., DNA
or RNA) molecule means, for example, a nucleotide sequence that is not
naturally
occurring or is made by the combatant (for example, 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. 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
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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. A "recombinant polypeptide" or "recombinant protein" usually
refers to
polypeptide or protein, respectively, that results from a cloned or
recombinant gene or
nucleic acid.
A "subsequence" or "fragment" is any portion of an entire sequence, up to
and including the complete sequence.
Numbering of a given amino acid or nucleotide polymer "corresponds to
numbering" of a selected amino acid polymer or nucleic acid when the position
of any
given polymer component (amino acid residue, incorporated nucleotide, etc.) is
designated
by reference to the same residue position in the selected amino acid or
nucleotide, rather
than by the actual position of the component in the given polymer.
A vector is a composition for facilitating cell transduction by a selected
nucleic acid, or expression of the nucleic acid in the cell. Vectors include,
e.g., plasmids,
cosmids, viruses, YACs, bacteria, poly-lysine, etc. An "expression vector" is
a nucleic
acid construct, 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.
"Substantially an entire length of a polynucleotide or amino acid sequence"
refers to at least about 50%, at least about 60%, generally at least about
70%, generally at
least about 80%, or typically at least about 90%, 95,%, 96%, 97%, 98%, or 99%
or more
of a length of an amino acid sequence or nucleic acid sequence.
"A human alpha-interferon receptor" is a receptor which is naturally
activated in human cells by an alpha interferon.
"Naturally occurring" as applied to an object refers to the fact that the
object can be found in nature. For example, a polypeptide or polynucleotide
sequence that
is present in an organism, including viruses, that can be isolated from a
source in nature
and which has not been intentionally modified by man in the laboratory is
naturally
occurring. In one aspect, a "naturally occurring" nucleic acid (e.g., DNA or
RNA)
CA 02385045 2002-03-14
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molecule is a nucleic acid molecule that exists in the same state as it exists
in nature; that
is, the nucleic acid molecule is not isolated, recombinant, or cloned.
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 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 kD) and one "heavy" chain (about 50-70 kD). 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. Antibodies exist as intact
immunoglobulins or
as a number of well characterized fragments produced by digestion with various
peptidases. Thus, for example, pepsin digests an antibody below the disulfide
linkages in
the hinge region to produce F(ab)'2, a dimer of Fab which itself is a light
chain joined to
VH-CHI by a disulfide bond. The F(ab)'2 may be reduced under mild conditions
to break
the disulfide linkage in the hinge region thereby converting the (Fab')2 dimer
into an Fab'
monomer. The Fab' monomer is essentially an Fab with part of the hinge region
(see
Fundamental Immunology, W.E. Paul, ed., Raven Press, N.Y. (1993), for a more
detailed
description of other antibody fragments). While various antibody fragments are
defined in
terms of the digestion of an intact antibody, one of skill will appreciate
that such Fab'
fragments may be synthesized de novo either chemically or by utilizing
recombinant DNA
methodology. Thus, the term antibody, as used herein also includes antibody
fragments
either produced by the modification of whole antibodies or synthesized de novo
using
recombinant DNA methodologies. Antibodies include 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.
An "antigen-binding fragment" of an antibody is a peptide or polypeptide
fragment of the antibody which binds an antigen. An antigen-binding site is
formed by
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those amino acids of the antibody which contribute to, are involved in, or
affect the
binding of the antigen. See Scott, T.A. and Mercer, E.L, CONCISE ENCYCLOPEDIA:
BIOCHEMISTRY AND MOLECULAR BIOLOGY (de Gruyter, 3d ed. 1997) [hereinafter
"Scott,
CONCISE ENCYCLOPEDIA"] 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.
An "immunogen" refers to a substance that is capable of provoking an
immune response. Examples of immunogens include, e.g., antigens, autoantigens
that
play a role in induction of autoimmune diseases, and tumor-associated antigens
expressed
on cancer cells.
An "antigen" is 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.
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.
The term "homology" generally refers to the degree of similarity between
two or more structures. The term "homologous sequences" refers to regions in
macromolecules that have a similar order of monomers. When used in relation to
nucleic
acid sequences, the term "homology" refers to the degree of similarity between
two or
more nucleic acid sequences (e.g., genes) or fragments thereof. Typically, the
degree of
similarity between two or more nucleic acid sequences refers to the degree of
similarity of
the composition, order, or arrangement of two or more nucleotide bases (or
other
genotypic feature) of the two or more nucleic acid sequences. The term
"homologous
nucleic acids" generally refers to nucleic acids comprising nucleotide
sequences having a
degree of similarity in nucleotide base composition, arrangement, or order.
The two or
more nucleic acids may be of the same or different species or group. The term
"percent
homology" when used in relation to nucleic acid sequences, refers generally to
a percent
degree of similarity between the nucleotide sequences of two or more nucleic
acids.
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When used in relation to polypeptide (or protein) sequences, the term
"homology" refers to the degree of similarity between two or more polypeptide
(or
protein) sequences (e.g., genes) or fragments thereof. Typically, the degree
of similarity
between two or more polypeptide (or protein) sequences refers to the degree of
similarity
of the composition, order, or arrangement of two or more amino acid of the two
or more
polypeptides (or proteins). The two or more polypeptides (or proteins) may be
of the same
or different species or group. The term "percent homology" when used in
relation to
polypeptide (or protein) sequences, refers generally to a percent degree of
similarity
between the amino acid sequences of two or more polypeptide (or protein)
sequences. The
term "homologous polypeptides" or "homologous proteins" generally refers to
polypeptides or proteins, respectively, that have amino acid sequences and
functions that
are similar. Such homologous polypeptides or proteins may be related by having
amino
acid sequences and functions that are similar, but are derived or evolved from
different or
the same species using the techniques described herein.
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.,
monkey), mouse,
pig, cow, goat, 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.
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
pharmaceutically acceptable Garner.
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.
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
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activity" is an activity of an agent, such as a nucleic acid, vector, gene,
polypeptide,
protein, substance, 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.
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,
diminishes 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.
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.
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, New York, 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.
(supplemented through 1999) (hereinafter "Ausubel"), are used for recombinant
nucleic
acid methods, nucleic acid synthesis, cell culture methods, and transgene
incorporation,
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e.g., electroporation, injection, and lipofection. 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.
A variety of additional terms are defined or otherwise characterized herein.
POLYNUCLEOT>DES OF THE INVENTION
Interferon-alpha Homolo ug a Sequences
The invention provides isolated or recombinant interferon-alpha homologue
polypeptides, and isolated or recombinant polynucleotides encoding the
polypeptides.
As described in more detail below, in accordance with the present
invention, polynucleotide sequences which encode novel interferon-alpha
homologue
polypeptides, nucleotide sequences (e.g., subsequences) that encode fragments
of
interferon-alpha homologue polypeptides, and nucleotide sequences that encode
related
fusion polypeptides or proteins, or functional equivalents thereof, are
collectively referred
to herein as "interferon-alpha homologues," "interferon homologue nucleic
acids," "IFN-
alpha homologues," "IFN homologues," "IFN nucleic acids," "interferon
homologues,"
"interferon nucleic acids, " "recombinant interferon-alpha," "recombinant
interferon-alpha
nucleic acids," "nucleic acids of the invention," "polynucleotides of the
invention," or
"nucleotides of the invention." Polynucleotide, nucleotide are nucleic acid
fragments of
each of the preceding terms are also intended to be included and encompassed
in
polynucleotides, nucleotides, and nucleic acids of the invention. The term
"nucleic acid"
is used interchangeable with the term "nucleotide."
Polynucleotides encoding the polypeptides of the invention were
discovered in libraries of shuffled interferon-alpha related sequences. The
library
members were screened for antiproliferative activity against human tumor cell
lines and,
in some cases, assayed for antiviral activity against virus-infected human
cells. A subset
of the sequences provided herein were discovered in shuffled libraries
screened for
antiviral activity against virus-infected mouse cells. Coding sequences for
interferon
homologues were identified as described in the examples.
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Briefly, libraries of shuffled mature interferon-alpha coding sequences were
introduced into E. coli. Colonies were screened in a high-throughput
antiproliferative
activity assay against a human Daudi tumor cell line as described in Example
1, and
colonies expressing active polypeptides were selected, re-screened, and
expression levels
determined. DNA from selected colonies was isolated and re-shuffled to create
secondary
libraries. The secondary libraries were introduced into E. coli and screened
for
antiproliferative activity in the human Daudi cell line-based cell
proliferation assay. DNA
from colonies selected from the primary and secondary library screens were
transduced
into Chinese hamster ovary (CHO) cells, and stable cell lines were generated.
CHO-
expressed proteins were purified, quantitated, and assayed for
antiproliferative activity
using the human Daudi cell line, and optionally, for antiviral activity using
encephalomyocarditis virus (EMCV)-infected human WISH cells, as described in
Example 1. Exemplary shuffled nucleic acids which encode interferon-alpha
homologue
polypeptides having antiproliferative activity in the human Daudi cell line-
based assay are
identified herein as SEQ ID NO:1 to SEQ >D N0:35, which encode mature
interferon-
alpha homologue polypeptides identified herein as SEQ >17 N0:36 to SEQ ID
N0:70,
respectively. Libraries of shuffled mature interferon-alpha coding sequences
were also
screened in a high-throughput antiviral activity screen against EMCV-infected
mouse
cells. Exemplary shuffled nucleic acids which encode polypeptides having
antiviral
activity in the murine cell /EMCV-based assay are identified herein as SEQ ID
N0:72 to
SEQ ID N0:78, which encode mature interferon homologue polypeptides identified
herein as SEQ >D N0:79 to SEQ ID N0:85.
In another aspect, the invention provides an isolated or recombinant nucleic
acid that comprises a polynucleotide sequence selected from the group of: (a)
SEQ >D
NO:1 to SEQ >D N0:35, or a complementary polynucleotide sequence thereof; (b)
a
polynucleotide sequence encoding a polypeptide selected from SEQ >D N0:36 to
SEQ ID
N0:71, or a complementary polynucleotide sequence thereof; (c) a
polynucleotide
sequence which hybridizes under at least stringent or at least highly
stringent hybridization
conditions (or ultra-high stringent or ultra-ultra- high stringent
hybridization conditions)
over substantially the entire length of polynucleotide sequence (a) or (b), or
with a 50,
120, 130, 140, 145, 150, 155, 160, or 165 nucleotide base subsequence or
fragment of a
polynucleotide sequence of (a) or (b); and (d) a polynucleotide sequence
comprising a
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fragment of (a), (b), or (c), which fragment encodes all or a part of a
polypeptide having
an antiproliferative activity in a human Daudi cell line-based assay or an
antiviral activity
in an assay known in the art for measuring antiviral activity.
In another aspect, the invention provides an isolated or recombinant nucleic
acid that comprises a polynucleotide sequence selected from the group of: (a)
SEQ ID
N0:72 to SEQ m N0:78, or a complementary polynucleotide sequence thereof; (b)
a
polynucleotide sequence encoding a polypeptide selected from SEQ ll~ N0:79 to
SEQ ID
N0:85, or a complementary polynucleotide sequence thereof; (c) a
polynucleotide
sequence which hybridizes under at least stringent or at least highly
stringent hybridization
conditions (or ultra-high stringent or ultra-ultra- high stringent
hybridization conditions)
over substantially the entire length of polynucleotide sequence (a) or (b), or
with a 50,
120, 130, 140, 145, 150, 155, 160, or 165 nucleotide base subsequence or
fragment of a
polynucleotide sequence of (a) or (b); and (d) a polynucleotide sequence
comprising a
fragment of (a), (b), or (c), which fragment encodes all or a part of a
polypeptide having
an antiproliferative activity in a human Daudi cell line-based assay or an
antiviral activity
in a murine cell line/EMCV-based assay.
The present invention also includes a mature interferon-alpha homologue
polypeptide comprising the amino acid identified herein as SEQ ID N0:71 and a
polynucleotide sequence encoding said polypeptide or a fragment of said
polypeptide
having an antiproliferative activity in the human Daudi cell line-based assay
and/or an
antiviral activity in the murine cell /EMCV-based assay.
The invention also includes an isolated or recombinant nucleic acid
comprising a polynucleotide sequence encoding a polypeptide, wherein the
polypeptide
comprises the amino acid sequence: CDLPQTHSLG-X,~-X1z-RA-Xls-X~6-LL-X19-QM-
XZZ-R-X24-S-Xz6-FSCLKDR-X34-DFG-X3$-P-X4o-EEFD-X4s-X46-X47-FQ-Xso-Xsl-QAI-
Xs5-XSG-X57-~-XGO-X61-QQT~'X67-FSTK-X72-S S-X75-X76-w-X7g-X79-Xg0-LL-Xg3-K-
Xss-Xa6-T-Xs s-L-X~o-QQLN-X9s-LEAC V-X l o ~ -Q-X i os-V-X 1 os-X 1 o6-X ~ o7-
X 1 os-TPLMN-
X114-D-Xl 16-B-AV-X~ Z ~-KY-X1 z4-QRITLYL-Xl sz-E-Xl s4-KYSPC-Xl4o-
WEVVRAEIMRSFSFSTNLQKRLRRKE, or a conservatively substituted variation
thereof, where X~ 1 is N or D; XlZ is R, S, or K; Xls is L or M; X16 is I, M,
or V; X19 is A or
G; XZZ is G or R; X24 is I or T; X26 is P or H; X34 is H, Y or Q; X3s is F or
L; X4o is Q or R;
X4sisGorS;X46isNorH;X47isQorR;XsoisKorR;XslisAorT;XssisSorF;Xs6
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is V or A; X5~ is L or F; X6o is M or I; X61 is I or M; X~~ is L or F; X~2 is
D or N; X~5 is A
or V; X~6 is A or T; X~8 is E or D; X~9 is Q or E; X$o is S, R, T, or N; X83
is E or D; X85 is
Fort;Xg6isSorY;X$$isEorG;X~oisY,H,N;X95isD,E,orN;Xlo1 is I, M, or V;
XlosisEorG;X,osisGorW;Xlo6isVorM;Xlo~isE,G,orK;XloBisEorG;X11a1SV,
E, or G; X116 is S or P; X121 is K or R; Xl2a is F or L; Xl3a is T, I, or M;
Xl3a is K or R; and
Xlao is A or S. Each of the single letters of this amino acid sequence
represents a
particular amino acid residue according to standard practice known to those of
ordinary
skill in the art. Such polypeptides having an antiproliferative activity in
the human Daudi
cell line-based assay (e.g., at least about 8.3x106 units/mg) and/or an
antiviral activities in
a human WISH cell/EMCV-based assay (at least about 2.1x10 units/mg).
As described in greater detail below, the polynucleotides of the invention
are useful in for a variety of applications, including, but not limited to, as
therapeutic and
prophylactic agents in methods of in vivo and ex vivo treatment of a variety
of diseases,
disorders, and conditions in a variety of subjects; for use in in vitro
methods, such as
diagnostic methods, to detect, diagnose, and treat a variety of diseases,
disorders, and
conditions in a variety of subjects; for use in, e.g., gene therapy; as
therapeutics and
prophylactics, e.g., for use in methods of therapeutic and prophylactic
treatment of a
disease, disorder or condition; as immunogens; for use in diagnostic and
screening assays;
and as diagnostic probes for the presence of complementary or partially
complementary
nucleic acids (including for detection of IFN-alpha coding nucleic acids).
Making Polynucleotides of the Invention
Polynucleotides and oligonucleotides of the invention can be prepared by
standard solid-phase methods, according to known synthetic methods. Typically,
fragments of up to about 20, 30, 40, 50, 60, 70, 80, 90, and/or 100 nucleotide
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. In another aspect, nucleotide fragments of greater than 100
nucleotide bases
(e.g., 150, 180, 200, 210, 240, 270, 300, 330, 360, 390, 400, 420, 450, 465,
474, 470, 475,
489, 490, 495, 496 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. example, the polynucleotides and
oligonucleotides of the invention, including fragments thereof (and those as
described
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WO 01/25438 PCT/US00/27781
herein), can be prepared by chemical synthesis using, e.g., the classical
phosphoramidite
method described by Beaucage et al. (1981) Tetrahedron Letters 22:1859-69, or
the
method described by Matthes et al. (1984) EMBO J. 3:801-OS., 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 in appropriate vectors.
In addition, essentially any nucleic acid can be custom ordered from any of
a variety of commercial sources, such as The Midland Certified Reagent Company
(mcrc@oligos.com), The Great American Gene Company (http://www.genco.com),
ExpressGen Inc. (www.expressgen.com), Operon Technologies Inc. (Alameda, CA)
and
many others. Similarly, peptides and antibodies can be custom ordered from any
of a
variety of sources, such as PeptidoGenic (pkim@ccnet.com), HTI Bio-products,
inc.
(http://www.htibio.com), BMA Biomedicals Ltd. (U.K.), Bio.Synthesis, Inc., and
many
others.
Certain polynucleotides of the invention may also obtained by screening
cDNA libraries (e.g., libraries generated by recombining homologous nucleic
acids as in
typical diversity generation methods, such as, e.g., shuffling methods) using
oligonucleotide probes which can hybridize to or PCR-amplify polynucleotides
which
encode the interferon homologue 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, for example, Sambrook et al.,
Molecular
Cloning - A Laboratory Manual (2nd Ed.), Vols. 1-3, Cold Spring Harbor
Laboratory,
Cold Spring Harbor, New York, 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. (supplemented
through
1999) (hereinafter "Ausubel").
As described in more detail herein, the polynucleotides of the invention
include sequences which encode novel mature interferon-alpha homologues and
sequences
complementary to the coding sequences, and novel fragments of such coding
sequences
and complements thereof. The polynucleotides can be in the form of RNA or in
the form
of DNA, and include mRNA, cRNA, synthetic RNA and DNA, and cDNA. The
polynucleotides can be double-stranded or single-stranded, and if single-
stranded, can be
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WO 01/25438 PCT/US00/27781
the coding strand or the non-coding (anti-sense, complementary) strand. The
polynucleotides optionally include the coding sequence of an interferon-alpha
homologue
(i) in isolation, (ii) in combination with additional coding sequence, 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, 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 or host environment in
which the
interferon-alpha homologue coding sequence is a heterologous nucleic acid
sequence or
gene. Sequences 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.
The term DNA or RNA encoding the respective interferon-alpha
homologue polypeptide includes any oligodeoxynucleotide or
oligodeoxyribonucleotide
sequence which, upon expression in an appropriate host cell, results in
production of an
interferon-alpha homologue polypeptide of the invention. The DNA or RNA can be
produced in an appropriate host cell, or in a cell-free (in vitro) system, or
can be produced
synthetically (e.g., by an amplification technique such as PCR) or chemically.
Using Polynucleotides of the Invention
The polynucleotides of the invention have a variety of uses in, for example:
recombinant production (i.e., expression) of the interferon-alpha homologue
polypeptides
of the invention; as therapeutics and prophylactics, e.g., for use in methods
of therapeutic
and prophylactic treatment of a disease, disorder or condition; for use in,
gene therapy
methods and related applications;; as immunogens; for use in diagnostic and
screening
assays; as diagnostic probes for the presence of complementary or partially
complementary nucleic acids (including for detection of natural IFN- alpha
coding
nucleic acids); as substrates for further reactions, e.g., shuffling reactions
or mutation
reactions to produce new and/or improved IFN-alpha homologues, and the like.
EXPRESSION OF POLYPEPT>DES
In accordance with the present invention, polynucleotide sequences which
encode novel and/or mature interferon-alpha homologues, fragments of
interferon-alpha
proteins, related fusion proteins, or functional equivalents thereof, are
collectively referred
to herein as "interferon-alpha homologue polypeptides," "interferon-alpha
homologue
CA 02385045 2002-03-14
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proteins," or "interferon-alpha homologues," "interferon homologues," "IFN-
alpha
homologues," "IFN homologues", "IFN polypeptides," "IFN proteins"
"polypeptides of the
invention," or "proteins of the invention." Polypeptide or amino acid
fragments of each of
the preceding terms are also intended to be included and encompassed in the
polypeptides
or proteins of the invention. Such polynucleotide sequences of the invention
are used in
recombinant DNA (or RNA) molecules that direct the expression of the
interferon-alpha
homologue polypeptides in appropriate host cells. Due to the inherent
degeneracy of the
genetic code, other nucleic acid sequences which encode substantially the same
or a
functionally equivalent amino acid sequence are also used to clone and express
the
interferon homologues.
Modified Coding Sequences
As will be understood by those of skill in the art, it can be advantageous to
modify a coding sequence (including, e.g., a nucleotide sequence encoding an
interferon-
alpha homologue of the invention or a fragment thereof) 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 called 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
called "codon
optimization" or "controlling for species codon bias."
Optimized coding sequence containing codons preferred by a particular
prokaryotic or eukaryotic host (see also 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. cerevisiae and 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) Nuc. Acids Res. 24:216-218).
The polynucleotide sequences of the present invention can be engineered in
order to alter an interferon homologue coding sequence for a variety of
reasons, including
but not limited to, alterations which modify the cloning, processing and/or
expression of
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WO 01/25438 PCT/US00/27781
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 change codon preference, to introduce splice sites,
etc.
Vectors, Promoters and Expression S std
The present invention also includes recombinant constructs comprising one
or more of the nucleic acid sequences as broadly described herein ( e.g.,
those encoding an
interferon-alpha homologue of the invention or a fragment thereof). The
constructs
comprise a vector, such as, a plasmid, a cosmid, a phage, a virus (including a
retrovirus), 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 a preferred aspect of this embodiment, the construct
further
comprises regulatory sequences, including, for example, a promoter, operably
linked to the
sequence. Large numbers of suitable vectors and promoters are known to those
of skill in
the art, and are commercially available.
General texts which describe molecular biological techniques useful herein,
including the use of vectors, promoters and many other relevant topics,
include Juo, P-S.,
CONCISE DICTIONARY OF BIOMEDICAL AND MOLECULAR BIOLOGY (CRC Press 1996);
Singleton et al., DICTIONARY OF MICROBIOLOGY AND MOLECULAR BIOLOGY (2d ed.
1994);
THE CAMBRmGE DICTIONARY OF SCIENCE AND TECHNOLOGY (Walker ed., 1988); Hale &
Marham, THE HARPER COLLINS DICTIONARY OF BIOLOGY (1991); Scott and Mercer,
CONCISE ENCYCLOPEDIA OF BIOCHEMISTRY AND MOLECULAR BIOLOGY (3d ed. 1997);
Berger and Kimmel, Guide to Molecular Cloning Techniques, Methods in
Enzymology,
volume 152 Academic Press, Inc., San Diego, CA (hereinafter "Berger");
Sambrook et al.,
Molecular Cloning - A Laboratory Manual (2nd Ed.), Vol. 1-3, Cold Spring
Harbor
Laboratory, Cold Spring Harbor, New York, 1989 ("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. (supplemented
through
1999) ("Ausubel")). Examples of techniques sufficient to direct persons of
skill through
in vitro amplification methods, including the polymerise chain reaction (PCR),
the ligase
chain reaction (LCR), Q~i-replicase amplification and other RNA polymerise
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, as well as Mullis et al.
(1987) U.S.
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WO 01/25438 PCT/US00/27781
Patent No. 4,683,202; U.S. Pat. No. 4,683,195, issued July 28, 1997; PCR
Protocols: A
Guide to Methods and Applications (Innis et al., eds.) Academic Press Inc. San
Diego, CA
(1990) (Innis); Arnheim & Levinson (October 1, 1990) C&EN 36-47; The Journal
Of
NIH Research (1991) 3, 81-94; (Kwoh et al. (1989) Proc. Nat'l Acad. Sci. USA
86, 1173;
Guatelli et al. (1990) Proc. Nat'l Acad. Sci. USA 87, 1874; Lomell et al.
(1989) J. Clin.
Chem. 35, 1826; Landegren et al. (1988) Science 241, 1077-1080; Van Brunt
(1990)
Biotechnology 8, 291-294; Wu and Wallace (1989) Gene 4, 560; Barnnger et al.
(1990)
Gene 89, 117, and Sooknanan and Malek (1995) Biotechnology 13:563-564.
PCR generally refers to a procedure wherein minute amounts of a specific
piece of nucleic acid, RNA, and/or DNA, are amplified by methods well known in
the art
(see, e.g., U.S. Pat. No. 4,683,195 and other references above). Generally,
sequence
information from the ends of the region of interest or beyond is used, for
design of
oligonucleotide primers. Such primers will be identical or similar in sequence
to the
opposite strands of the template to be amplified. The 5' terminal nucleotides
of the
opposite strands may coincide with the ends of the amplified material. PCR may
be used
to amplify specific RNA or specific DNA sequences, recombinant DNA or RNA
sequences, DNA and RNA sequences from total genomic DNA, and cDNA transcribed
from total cellular RNA, bacteriophage or plasmid sequences, etc. PCR is one
example,
but not the only example, of a nucleic acid polymerise reaction method for
amplifying a
nucleic acid test sample comprising the use of a another (e.g., known) nucleic
acid as a
primer. 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 40kb 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
polymerise. See Ausubel, Sambrook and Bergen all supra.
The present invention also relates to host cells which are transduced with
vectors of the invention, and the production of polypeptides of the invention
(including
fragments thereof) by recombinant techniques. Host cells are genetically
engineered (i.e.,
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
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CA 02385045 2002-03-14
WO 01/25438 PCT/US00/27781
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 the interferon homologue gene. 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, 3d ed., Wiley-Liss, New York and the references
cited
therein.
The interferon homologue polypeptides and proteins 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 can
be found in
Payne et al. (1992) Plant Cell and Tissue Culture in Liquid Systems, John
Wiley & Sons,
Inc. New York, NY; Gamborg and Phillips (eds.) (1995) Plant Cell, Tissue and
Organ
Culture; Fundamental Methods, Springer Lab Manual, Springer-Verlag (Berlin
Heidelberg New York) and Atlas and Parks (eds.) The Handbook of
Microbiological
Media (1993) CRC Press, Boca Raton, FL.
The polynucleotides of the present invention 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, adenovirus, adeno-associated virus, retroviruses and
many others.
Any vector that transducers genetic material into a cell, and, if replication
is desired,
which is replicable and viable in the relevant host can be used.
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 PL 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. In
addition, the
expression vectors optionally comprise one or more selectable marker genes to
provide a
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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.
The vector containing the appropriate DNA sequence as described herein,
as well as an appropriate promoter or control sequence, may be employed to
transform an
appropriate host to permit the host to express the protein. 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 interferon homologues; for example, antigenic fragments of an
interferon
homologue may be produced in a bacterial or other expression system. The
invention is
not limited by the host cells employed.
In bacterial systems, a number of expression vectors may be selected
depending upon the use intended for the interferon homologue. For example,
when large
quantities of interferon homologue 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
interferon homologue 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 WI); and the like.
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 interferon homologue proteins of the
invention.
For reviews, see Ausubel et al. (supra) and Grant et al. (1987; Methods in
Enzymology
153:516-544).
In mammalian host cells, a number 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
CA 02385045 2002-03-14
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consisting of the late promoter and tripartite leader sequence. Insertion in a
nonessential
E1 or E3 region of the viral genome will result in a viable virus capable of
expressing
interferon homologue in infected host cells (Logan and Shenk (1984) Proc.
Natl. Acad.
Sci. 81:3655-3659). In addition, transcription enhancers, such as the rous
sarcoma virus
(RSV) enhancer, may be used to increase expression in mammalian host cells.
Additional Expression Elements
Specific initiation signals can aid in efficient translation of an interferon
homologue coding sequence. These signals can include, e.g., the ATG initiation
codon
and adjacent sequences. In cases where interferon homologue 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 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 may be enhanced by the inclusion of enhancers appropriate to the
cell system
in use (Scharf, D. et al. (1994) Results Probl. Cell Differ. 20:125-62;
Bittner et al. (1987)
Methods in Enzymol. 153:516-544).
Secretion/Localization Se uences
Polynucleotides 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 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. Polypeptides
expressed by
such polynucleotides of the invention may include the amino acid sequence
corresponding
to the secretion and/or localization sequence(s).
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Expression Hosts
In a further embodiment, the present invention relates to host cells
containing the above-described constructs. 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, or
other common techniques (Davis, L., Dibner, M., and Battey, I. (1986) Basic
Methods in
Molecular Biology). The cell may include a nucleic acid of the invention, said
nucleic
acid encoding a polypeptide, wherein said cells expresses a polypeptide (e.g.,
an
interferon-alpha homologue polypeptide having an antiviral or anti-
proliferative activity as
measured by the assays described herein). The invention also includes a vector
comprising any nucleic acid of the invention described herein and includes a
cell
transduced by such a vector. Furthermore, Cells and transgenic animals which
include any
polypeptide or nucleic acid above or throughout this specification, e.g.,
produced by
transduction of a vector of the invention, are an additional feature of the
invention.
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
CHO, HeLa,
BHK, MDCK, 293, WI38, 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.
For long-term, high-yield production of recombinant proteins, stable
expression can be used. 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
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stably transformed cells can be proliferated using tissue culture techniques
appropriate to
the cell type.
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 protein or fragment
thereof
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 mature
interferon homologues of the invention can be designed with signal sequences
which
direct secretion of the mature polypeptides through a prokaryotic or
eukaryotic cell
membrane.
Additional Polypeptide Sequences
The polynucleotides of the present invention may also comprise a coding
sequence fused in-frame to a marker sequence which, e.g., facilitates
purification of the
encoded polypeptide of the invention. Such purification facilitating domains
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
(Immunex Corp., Seattle, WA), and the like. The inclusion of a protease-
cleavable
polypeptide linker sequence between the purification domain and the interferon
homologue sequence is useful to facilitate purification. One expression vector
contemplated for 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 means for separating the interferon
homologue
polypeptide from the fusion protein. pGEX vectors (Promega; Madison, WI) may
also be
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
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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.
Polypeptide Production and Recovery
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.
Microbial cells
employed in expression of 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.
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, 4th 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, Payne et al. (1992) Plant Cell and Tissue Culture in Liquid
Systems, John
Wiley & Sons, Inc., New York, NY; Gamborg and Phillips (eds.) (1995) Plant
Cell,
Tissue and Organ Culture; Fundamental Methods Springer Lab Manual, Springer-
Verlag
(Berlin Heidelberg New York) 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, FL. Additional information for cell culture is
found in
available commercial literature such as the Life Science Research Cell Culture
Catalogue
(1998) from Sigma- Aldrich, Inc (St. Louis, MO) ("Sigma-LSRCCC") and, e.g.,
the Plant
Culture Catalogue and supplement (1997) also from Sigma-Aldrich, Inc. (St.
Louis, MO)
("Sigma-PCCS").
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
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ammonium sulfate or ethanol precipitation, acid extraction, anion or canon
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. 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.; and Bollag et al. (1996)
Protein
Methods, 2"d Edition, Wiley-Liss, NY; Walker (1996) The Protein Protocols
Handbook,
Humana Press, NJ, Harns and Angal (1990) Protein Purification Applications: A
Practical Approach, IRL Press at Oxford, Oxford, England; Harris and Angal,
Protein
Purif canon Methods: A Practical Approach, IRL Press at Oxford, Oxford,
England;
Scopes (1993) Protein Purification: Principles and Practice 3rd 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 SXstems
Cell-free transcription/translation systems can also be employed to produce
polypeptides using DNAs or RNAs 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.
Modified Amino Acids
Polypeptides of the invention may contain one or more modified amino
acids. The presence of modified amino acids may be advantageous in, for
example, (a)
increasing polypeptide serum half-life, (b) reducing polypeptide antigenicity,
(c)
increasing polypeptide storage stability. Amino acids) 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.
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Non-limiting examples of a modified amino acid include a glycosylated
amino acid, a sulfated amino acid, a prenylated (e.g., farnesylated,
geranylgeranylated)
amino acid, an acetylated amino acid, an acylated amino acid, a PEG-ylated
amino acid, a
biotinylated amino acid, a carboxylated amino acid, a phosphorylated amino
acid, and the
like. References adequate to guide one of skill in the modification of amino
acids are
replete throughout the literature. Example protocols are found in Walker
(1998) Protein
Protocols on CD-ROM Human Press, Towata, NJ.
The polynucleotides and polypeptides of the invention have a variety of
uses, including, but not limited to, for example: in recombinant production
(i.e.,
expression) of the recombinant interferon-alpha homologues of the invention;
as
therapeutic and prophylactic agents in methods of in vivo and ex vivo
treatment of a
variety of diseases, disorders, and conditions in a variety of subjects; for
use in in vitro
methods, such as diagnostic and screening methods, to detect, diagnose, and
treat a variety
of diseases, disorders, and conditions (e.g., cancers, viral-based disorders,
angiogenic-
based disorders) in a variety of subjects (e.g., mammals); as immunogens; in
gene therapy
methods and DNA- or RNA-based delivery methods to deliver or administer in
vivo, ex
vivo, or in vitro biologically active polypeptides of the invention to a
tissue, population or
cells, organ, graft, bodily system of a subject (e.g., organ system, lymphatic
system, blood
system, etc.); as DNA vaccines, multi-component vaccines for use in
prophylactic or
therapeutic treatment of a variety of diseases, disorders, or other conditions
(e.g., cancers,
viral-based disorders, angiogenic-based disorders) in a variety of subjects
(e.g.,
mammals); as adjuvants to enhance or augment an immune response in a subject;
as a
component of a multiple-step boosting vaccination method (e.g., a format
comprising a
prime vaccination by delivery of a DNA or RNA nucleotide (e.g., a nucleotide
encoding a
polypeptide of the invention or encoding another polypeptide) followed by a
second boost
of a polypeptide (e.g., a polypeptide of the invention or other polypeptide);
as diagnostic
probes for the presence of complementary or partially complementary nucleic
acids
(including for detection of natural interferon-alpha coding nucleic acids); as
substrates for
further reactions, e.g., shuffling reactions, mutation reactions, or other
diversity generation
reactions to produce new and/or improved interferon-alpha homologues and new
interferon-alpha nucleic acids encoding such homologues, e.g., to evolve novel
therapeutic
or prophylactic properties, and the like; for polymerise chain reactions (PCR)
or cloning
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WO 01/25438 PCT/US00/27781
methods, e.g., including digestion or ligation reactions, to identify new
and/or improved
naturally-occurring or non-naturally occurring IFN-alpha nucleic acids and
polypeptides
encoded therefrom. Polynucleotides which encode an interferon homologue of the
invention, or complements of the polynucleotides, are optionally administered
to a cell to
accomplish a therapeutically or prophylactically useful process or to express
a
therapeutically useful product in vivo, ex vivo, or in vitro. These
applications, including in
vivo or ex vivo applications, including, e.g., 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
or
prophylactically useful polypeptides, such as the interferon homologues of the
present
invention. Such methods include, for example, infecting with a retrovirus
comprising the
polynucleotides and/or polypeptides of the invention. Optionally, the
retrovirus further
comprises additional exogenous, e.g., therapeutic or prophylactic gene
construct,
sequences. In one aspect, the invention provides gene therapy methods of
prophylactically
or therapeutically treating a disease, disorder or condition in a subject in
need of such
treatment by administering in vivo, ex vivo, or in vitro one or more nucleic
acids of the
invention described herein to one or more cells of a subject, including an
organism or
mammal, including, e.g., a human, primate, mouse, pig, cow, goat, rabbit, rat,
guinea pig,
hamster, horse, sheep; or a non-mammalian vertebrate such as a bird (e.g., a
chicken or
duck) or a fish, or invertebrate, as described in more detail below.
In another aspect, the invention provides methods of prophylactically or
therapeutically treating a disease, disorder or condition in a subject in need
of such
treatment by administering in vivo, ex vivo, or in vitro one or more
polypeptides of the
invention described herein to one or more cells of a subject (including those
defined
herein), as described in more detail below.
Polypeptide Expression
Polynucleotides encoding interferon homologue polypeptides of the
invention are particularly useful for in vivo or ex vivo therapeutic or
prophylactic
applications, using techniques well known to those skilled in the art. For
example,
cultured cells are engineered ex vivo with a polynucleotide (DNA or RNA), with
the
engineered cells then being returned to the patient. Cells may also be
engineered in vivo
or ex vivo for expression of a polypeptide in vivo or ex vivo, respectively.
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A number of viral vectors suitable for organismal in vivo or ex vivo
transduction and expression are known. Such vectors include retroviral vectors
(see
Miller(1992) Curr. Top. Microbiol. Immunol. 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.
Gene therapy provides methods for combating chronic infectious diseases
(e.g., HIV infection, viral hepatitis, Herpes Simplex Virus (HSV), hepatitis B
(HepB),
dengue virus, etc.), as well as non-infectious diseases including cancer and
allergic
diseases and some forms of congenital defects such as enzyme deficiencies.
Several
approaches for introducing nucleic acids into cells in vivo, ex vivo and in
vitro have been
used. These 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. Nat'1 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.
Nat'l Acad.
Sci. (USA) 84:7851-7855).; adenoviral vector mediated gene delivery, e.g., to
treat cancer
(see, e.g., Chen et al. (1994) Proc. Nat'L 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._199 (Pt. 3):297-
306;
Addison et al. (1995) Proc. Nat'l 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
other
diseases. 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);
38
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Kolberg (1992) J. NIH Res. 4:43, and Cornetta et al. (1991) Hum. Gene Ther.
2:215).
Nucleic acid transport coupled to ligand-specific, canon-based transport
systems (Wu and
Wu (1988) J. Biol. Chem. 263:14621-14624) have 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 interferon homologues
herein into
the appropriate vectors.
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 Robbins (1996) Gene Therapy Protocols, Humana Press, NJ, and Joyner
(1993)
Gene Targeting: A Practical Approach, IRL Press, Oxford, England.
Antisense Technolo~y
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 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.
Pharmaceutical Compositions
The polynucleotides and polypeptides of the invention (including vectors,
cells, antibodies, etc., comprising polynucleotides or polypeptides of the
invention) may
be employed for therapeutic and prophylactic uses in combination with a
suitable
pharmaceutical Garner. Such compositions comprise a therapeutically or
prophylactically
effective amount of the polynucleotide or polypeptide of the invention, and a
pharmaceutically acceptable Garner or excipient. A pharmaceutically acceptable
Garner
encompasses any of the standard pharmaceutical carriers, buffers and
excipients. Such a
carrier or excipient includes, but is not limited to, saline, buffered saline
(e.g., phosphate-
buffered saline solution), dextrose, water, glycerol, ethanol, emulsions (such
as an
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oil/water or water/oil emulsion), various types of wetting agents and/or
adjuvants, and
combinations thereof. Suitable pharmaceutical carriers and agents are
described in
REMINGTON~S PHARMACEUTICAL SCIENCES (Mack Publishing Co., Easton, 19'h ed.
1995).
The formulation should suit the mode of administration of the active agent
(e.g.,
nucleotide, polypeptide, vector, cell, etc.). Methods of administering nucleic
acids,
polypeptides, vectors, cells, antibodies, and proteins are well known in the
art, and further
discussed below.
Use as Probes
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, 30, or 50 bases, which hybridize under at least highly stringent
(or ultra-high
stringent or ultra-ultra- high stringent conditions) conditions to an
interferon homologue
polynucleotide sequence described above. The polynucleotides may be used as
probes,
primers, sense and antisense agents, and the like, according to methods as
noted supra.
SEQUENCE VARIATIONS
Silent Variations
It will be appreciated by those skilled in the art that due to the degeneracy
of the genetic code, a multitude of nucleic acids sequences encoding
interferon homologue
polypeptides of the invention may be produced, some which may bear minimal
sequence
homology to the nucleic acid sequences explicitly disclosed herein.
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Table 1
Codon Table
Amino acids Codon
Alanine Ala A GCA GCC GCG GCU
Cysteine Cys C UGC UGU
Aspartic Asp D GAC GAU
acid
Glutamic Glu E GAA GAG
acid
PhenylalaninePhe F UUC UUU
Glycine Gly G GGA GGC GGG GGU
Histidine His H CAC CAU
IsoleucineIle I AUA AUC AUU
Lysine Lys K AAA AAG
Leucine Leu L UUA UUG CUA CUC CUG CUU
MethionineMet M AUG
AsparagineAsn 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
TryptophanTrp W UGG
Tyrosine T Y UAC UAU
r
For instance, inspection of the codon table (Table 1) shows that codons
AGA, AGG, CGA, CGC, CGG, and CGU all encode the amino acid arginine. Thus, at
every position in the nucleic acids of the invention 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. It is understood that U in an RNA
sequence
corresponds to T in a DNA sequence.
Using, as an example, the nucleic acid sequence corresponding to
nucleotides 1-15 of SEQ ID NO:1, TGT GAT CTG CCT CAG, a silent variation of
this
sequence includes TGC GAC TTA CCA CAA, both sequences which encode the amino
acid sequence CDLPQ, corresponding to amino acids 1-5 of SEQ >D N0:36.
Such "silent variations" are one species of "conservatively modified
variations," discussed below. One of skill will recognize that each codon in a
nucleic acid
(except AUG, which is ordinarily the only codon for methionine) can be
modified by
standard techniques to encode a functionally identical polypeptide.
Accordingly, each
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silent variation of a nucleic acid which encodes a polypeptide is implicit in
any described
sequence. The invention provides each and every possible variation of nucleic
acid
sequence encoding a polypeptide of the invention that could be made by
selecting
combinations based on possible codon choices. These combinations are made in
accordance with the standard triplet genetic code (e.g., as set forth in Table
1) as applied to
the nucleic acid sequence encoding an interferon homologue 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.
Conservative Variations
"Conservatively modified variations" or, simply, "conservative variations"
of a particular nucleic acid sequence refers to those nucleic acids which
encode identical
or essentially identical amino acid sequences, or, where the nucleic acid does
not encode
an amino acid sequence, to essentially identical sequences. One of skill will
recognize
that individual substitutions, deletions or additions which alter, add or
delete a single
amino acid or a small percentage of amino acids (typically less than 5%, more
typically
less than 4%, 3%, 2% or 1%) in an encoded sequence are "conservatively
modified
variations" where the alterations result in the deletion of an amino acid,
addition of an
amino acid, or substitution of an amino acid with a chemically similar amino
acid.
Conservative substitution tables providing functionally similar amino acids
are well known in the art. Table 2 sets forth six groups which contain amino
acids that are
"conservative substitutions" for one another.
Table 2
Conservative Substitution Groups
1 Alanine (A) Serine (S) Threonine (T)
2 Aspartic acid Glutamic acid (E)
(D)
3 Asparagine Glutamine (Q)
(N)
4 Arginine (R) Lysine (K)
5 Isoleucine Leucine (L) Methionine (M) Valine
(I) (V)
6 Phenylalanine Tyrosine (Y) Tryptophan (W)
(F)
Thus, "conservatively substituted variations" or "conservative
substitutions" of a listed polypeptide sequence of the present invention
include
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substitutions of a small percentage, typically less than 5%, more typically
less than 4%,
3%, 2% or 1%, of the amino acids of the polypeptide sequence, with a
conservatively
selected amino acid of the same conservative substitution group.
For example, a conservatively substituted variation of the polypeptide
identified herein as SEQ ID N0:36 will contain "conservative substitutions",
according to
the six groups defined above, in up to about 8 or 9 residues (i.e., about 5%
of the amino
acids) in the 166-amino acid polypeptide.
In a further example, if four conservative substitutions were localized in
the region corresponding to amino acid residues 141-166 of SEQ ID N0:36,
examples of
conservatively substituted variations of this region,
WEVVR AEIMR SFSFS TNLQK RLRRKE include:
WEVVR SEIMR SFSYS TNLQR RLRRKD and
WELVR AEIVR SFSFS TNLNK RLRKKE and the like, in accordance with the
conservative substitutions listed in Table 2 (in the above example,
conservative
substitutions are underlined). Listing of a protein sequence herein, in
conjunction with the
above substitution table, provides an express listing of all conservatively
substituted
proteins.
Finally, the addition of sequences which do not alter the encoded activity of
a nucleic acid molecule, such as the addition of a non-functional sequence, is
a
conservative variation of the basic nucleic acid.
One of ordinary skill will appreciate that many conservative variations of
the nucleic acid constructs which are disclosed yield a functionally identical
construct.
For example, as discussed above, owing to the degeneracy of the genetic code,
"silent
substitutions" (i.e., substitutions in a nucleic acid sequence which do not
result in an
alteration in an encoded polypeptide) are an implied feature of every nucleic
acid sequence
which encodes an amino acid. Similarly, "conservative amino acid
substitutions," in one
or a few amino acids in an amino acid sequence are substituted with different
amino acids
with highly similar properties, are also readily identified as being highly
similar to a
disclosed construct. Such conservative variations of each disclosed sequence
are a feature
of the present invention.
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Nucleic Acid Hybridization
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, New York), 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.
"Stringent hybridization wash 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 the hybridization of nucleic acids is found in Tijssen (1993), supra,
and in Hames
and Higgins 1 and Hames and Higgins 2, supra.
For purposes of the present invention, generally, "highly stringent"
hybridization and wash conditions are selected to be about 5° C or less
lower lower than
the thermal melting point (Tm) 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 Tm is the temperature (under defined ionic strength and pH) at which 50%
of the test
sequence hybridizes to a perfectly matched probe. Very stringent conditions
are selected
to be equal to the Tm for a particular probe.
The Tm is the temperature of the nucleic acid duplexes indicates the
temperature at which the duplex is 50% denatured under the given conditions
and its
represents a direct measure of the stability of the nucleic acid hybrid. Thus,
the Tm
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.
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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.
The Tm of a DNA-DNA duplex can be estimated using the following
equation:
Tn, (°C) = 81.5°C + 16.6 (logloM) + 0.41 (%G + C) - 0.72
(%f) - 500/n,
where M is the molarity of the monovalent canons (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.
The Tm of an RNA-DNA duplex can be estimated as follows:
T~, (°C) = 79.8°C + 18.5 (log~pM) + 0.58 (%G + C) - 11.8(%G
+ C)2 - 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 are typically accurate only for hybrid duplexes longer
than about 100-200 nucleotides. Id.
The Tm of nucleic acid sequences shorter than 50 nucleotides can be
calculated as follows:
T,t, (°C) = 4(G + C) + 2(A + T),
where A (adenine), C, T (thymine), and G are the numbers of the
corresponding nucleotides.
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 with 1 mg of heparin at
42°C, with
CA 02385045 2002-03-14
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the hybridization being carried out overnight. An example of stringent wash
conditions is
a 0.2x SSC wash at 65°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 2x SSC at
40°C for 15
minutes.
In general, a signal to noise ratio of 2.Sx-Sx (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.
As noted, "highly stringent" conditions are selected to be about 5° C
or less
lower than the thermal melting point (Tm) 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, supra.
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.
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WO 01/25438 PCT/US00/27781
Thus, one measure of stringent hybridization is the ability to hybridize to
one of the listed nucleic acids (e.g., nucleic acid sequences SEQ ID NO:1 to
SEQ ID
N0:35, and SEQ ID N0:72 to SEQ ID N0:78, and complementary polynucleotide
sequences thereof) under highly stringent conditions (or very stringent
conditions, or ultra-
s 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.
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 >D NO:1 to SEQ ID N0:35, SEQ ID
N0:72 to SEQ ID N0:78, 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 )D NO:1 to SEQ ID N0:35, SEQ B7 N0:72
to
SEQ ID N0:78, and complementary polynucleotide sequences thereof), with a
signal to
noise ratio that is at least 2.5x, and optionally Sx 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 a known alpha interferon, e.g., an alpha
interferon nucleic
acid that is present in a public database such as GenBankTM at the time of
filing of the
subject application. Examples of such unmatched target nucleic acids include,
e.g., those
with the following GenBank accession numbers: J00210 (alpha-D), J00207 (Alpha-
A),
X02958 (Alpha-6), X02956 (Alpha-5), V00533 (alpha-H), V00542 (alpha-14),
V00545
(IFN-1B), X03125 (alpha-8), X02957 (alpha-16), V00540 (alpha-21), X02955
(alpha-4b),
V00532 (alpha-C), X02960 (alpha-7), X02961 (alpha-10 pseudogene), 80067 (Gx-
1),
I01614, I01787, I07821, M12350 (alpha-F), M38289, V00549 (alpha-2a), and
I08313
(alpha-Conl). Additional such sequences can be identified in GenBank by one of
ordinary
skill in the art. Nomenclature of the human interferon genes and proteins is
discussed in
Diaz et al., (1996) J. Interferon and Cytokine Res. 16:179-180 and Allen et
al. (1996) J.
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WO 01/25438 PCT/US00/27781
Interferon and Cytokine Res. 16:181-184, respectively, each of which is
incorporated
herein by reference in its entirety for all purposes.
A test nucleic acid is said to specifically hybridize to a probe nucleic acid
when it hybridizes at least'h as well to the probe as to the perfectly matched
complementary target, i.e., with a signal to noise ratio at least'h 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 2.5x-lOx, typically 5x-lOx as high as that observed for hybridization to
any of the
unmatched target nucleic acids represented by GenBank accession numbers J00210
(alpha-D), J00207 (Alpha-A), X02958 (Alpha-6), X02956 (Alpha-5), V00533 (alpha-
H),
V00542 (alpha-14), V00545 (IFN-1B), X03125 (alpha-8), X02957 (alpha-16),
V00540
(alpha-21), X02955 (alpha-4b), V00532 (alpha-C), X02960 (alpha-7), X02961
(alpha-10
pseudogene), 80067 (Gx-1), I01614, I01787, I07821, M12350 (alpha-F), M38289,
V00549 (alpha-2a), and I08313 (alpha-Conl), or other similar interferon-alpha
sequences
presented in GenBank.
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 lOx as high as that observed for hybridization to any of the
unmatched target
nucleic acids represented by GenBank accession numbers J00210 (alpha-D),
J00207
(Alpha-A), X02958 (Alpha-6), X02956 (Alpha-5), V00533 (alpha-H), V00542 (alpha-
14),
V00545 (IFN-1B), X03125 (alpha-8), X02957 (alpha-16), V00540 (alpha-21),
X02955
(alpha-4b), V00532 (alpha-C), X02960 (alpha-7), X02961 (alpha-10 pseudogene),
80067
(Gx-1), I01614, I01787, I07821, M12350 (alpha-F), M38289, V00549 (alpha-2a),
and
I08313 (alpha-Conl), or other similar IFN-alpha sequences presented in GenBank
A target
nucleic acid which hybridizes to a probe under such conditions, with a signal
to noise ratio
of at least'/z that of the perfectly matched complementary target nucleic acid
is said to
bind to the probe under ultra-high stringency conditions.
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
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WO 01/25438 PCT/US00/27781
complementary target nucleic acid is at least 10x, 20X, 50X, 100X, or 500X or
more as
high as that observed for hybridization to any of the unmatched target nucleic
acids
represented by GenBank accession numbers J00210 (alpha-D), J00207 (Alpha-A),
X02958 (Alpha-6), X02956 (Alpha-5), V00533 (alpha-H), V00542 (alpha-14),
V00545
(IFN-1B), X03125 (alpha-8), X02957 (alpha-16), V00540 (alpha-21), X02955
(alpha-4b),
V00532 (alpha-C), X02960 (alpha-7), X02961 (alpha-10 pseudogene), 80067 (Gx-
1),
I01614, I01787, I07821, M12350 (alpha-F), M38289,V00549 (alpha-2a), and I08313
(alpha-Conl),or other similar interferon-alpha sequences presented in GenBank,
can be
identified. A target nucleic acid which hybridizes to a probe under such
conditions, with a
signal to noise ratio of at least'/a that of the perfectly matched
complementary target
nucleic acid is said to bind to the probe under ultra-ultra-high stringency
conditions.
Target nucleic acids which hybridize to the nucleic acids represented by
SEQ ID NO:1 to SEQ >D N0:35 and SEQ >D N0:72 to SEQ LD N0:78 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.
Nucleic acids which do not hybridize to each other under stringent
conditions are still substantially identical if the polypeptides which they
encode are
substantially identical. This occurs, e.g., when a copy of a nucleic acid is
created using the
maximum codon degeneracy permitted by the genetic code, or when antisera or
antiserum
generated against one or more of SEQ ID N0:36 to SEQ ID N0:70 and SEQ ID N0:79
to
SEQ ID N0:85 which has been subtracted using the polypeptides encoded by known
interferon-alpha sequences, including, e.g., the those encoded by the
following interferon-
alpha nucleic acid sequences in GenBank: Accession numbers J00210 (alpha-D),
J00207
(Alpha-A), X02958 (Alpha-6), X02956 (Alpha-5), V00533 (alpha-H), V00542 (alpha-
14),
V00545 (IFN-1B), X03125 (alpha-8), X02957 (alpha-16), V00540 (alpha-21),
X02955
(alpha-4b), V00532 (alpha-C), X02960 (alpha-7), X02961 (alpha-10 pseudogene),
80067
(Gx-1), I01614, I01787, I07821, M12350 (alpha-F), M38289, V00549 (alpha-2a),
and
I08313 (alpha-Conl), or other similar interferon-alpha sequences presented in
GenBank.
Further details on immunological identification of polypeptides of the
invention are found
below. Additionally, for distinguishing between duplexes with sequences of
less than
about 100 nucleotides, a TMAC1 hybridization procedure known to those of
ordinary skill
49
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WO 01/25438 PCT/US00/27781
in the art can be used. See, e.g., Sorg, U. et al. 1 Nucleic Acids Res. (Sept.
11, 1991)
19(17), incorporated herein by reference in its entirety for all purposes.
In one aspect, the invention provides a nucleic acid which comprises a
unique subsequence in a nucleic acid selected from SEQ ID NO: l to SEQ )D
N0:35 or
SEQ 1D N0:72 to SEQ 1D N0:78. The unique subsequence is unique as compared to
a
nucleic acid corresponding to any known interferon-alpha nucleic acid sequence
including, e.g., the known sequences represented by GenBank accession numbers
J00210
(alpha-D), J00207 (Alpha-A), X02958 (Alpha-6), X02956 (Alpha-5), V00533 (alpha-
H),
V00542 (alpha-14), V00545 (IFN-1B), X03125 (alpha-8), X02957 (alpha-16),
V00540
(alpha-21), X02955 (alpha-4b), V00532 (alpha-C), X02960 (alpha-7), X02961
(alpha-10
pseudogene), A12109, 80067 (Gx-1), I01614, I01787, I07821, M12350 (alpha-F),
M38289, V00549 (alpha-2a), and I08313 (alpha-Conl), or other similar
interferon-alpha
sequences presented in GenBank. Such unique subsequences can be determined by
aligning any of SEQ ID NO:1 to SEQ ID N0:35 or SEQ ID N0:72 to SEQ ID N0:78
against the complete set of nucleic acids corresponding to GenBank accession
numbers of
known interferon-alpha nucleic acid sequences, such as, e.g., J00210 (alpha-
D), J00207
(Alpha-A), X02958 (Alpha-6), X02956 (Alpha-5), V00533 (alpha-H), V00542 (alpha-
14),
V00545 (IFN-1B), X03125 (alpha-8), X02957 (alpha-16), V00540 (alpha-21),
X02955
(alpha-4b), V00532 (alpha-C), X02960 (alpha-7), X02961 (alpha-10 pseudogene),
A12109 (alpha-4B), 80067 (Gx-1), I01614, I01787, I07821, M12350 (alpha-F),
M38289,V00549 (alpha-2a), and I08313 (alpha-Conl), or other similar interferon-
alpha
sequences presented in GenBank. Alignment can be performed using the BLAST
algorithm set to default parameters. Any unique subsequence is useful, e.g.,
as a probe to
identify the nucleic acids of the invention.
Similarly, the invention includes a polypeptide which comprises a unique
amino acid subsequence in a polypeptide selected from: SEQ ID N0:36 to SEQ 1D
N0:70
or SEQ >D N0:79 to SEQ ID N0:85. Here, the unique subsequence is unique as
compared to an amino acid subsequence of a known interferon-alpha polypeptide
including, e.g., an amino acid subsequence of a polypeptide encoded by a known
interferon-alpha nucleic acid corresponding to any of GenBank accession
numbers:
J00210 (alpha-D), J00207 (Alpha-A), X02958 (Alpha-6), X02956 (Alpha-5), V00533
(alpha-H), V00542 (alpha-14), V00545 (IFN-1B), X03125 (alpha-8), X02957 (alpha-
16),
CA 02385045 2002-03-14
WO 01/25438 PCT/US00/27781
V00540 (alpha-21), X02955 (alpha-4b), V00532 (alpha-C), X02960 (alpha-7),
X02961
(alpha-10 pseudogene), 80067 (Gx-1), I01614, I01787, I07821, M12350 (alpha-F),
M38289, V00549 (alpha-2a), and I08313 (alpha-Conl), or other similar
interferon-alpha
nucleic acid or polypeptide sequences presented in GenBank. Here again, the
polypeptide
is aligned against the complete set of known interferon-alpha polypeptide
sequences, such
as those polypeptides encoded by nucleic acids corresponding to GenBank
accession
numbers J00210 (alpha-D), J00207 (Alpha-A), X02958 (Alpha-6), X02956 (Alpha-
5),
V00533 (alpha-H), V00542 (alpha-14), V00545 (IFN-1B), X03125 (alpha-8), X02957
(alpha-16), V00540 (alpha-21), X02955 (alpha-4b), V00532 (alpha-C), X02960
(alpha-7),
X02961 (alpha-10 pseudogene), 80067 (Gx-1), I01614, I01787, I07821, M12350
(alpha-
F), - M38289,V00549 (alpha-2a), and I08313 (alpha-Conl), (referred to as "the
control
polypeptides") (note that where the sequence corresponds to a non-translated
sequence
such as a pseudo gene, the corresponding polypeptide is generated simply by in
silico
translation of the nucleic acid sequence into an amino acid sequence, where
the reading
frame is selected to correspond to the reading frame of homologous alpha
interferon
nucleic acids) or other similar interferon-alpha nucleic acid or polypeptide
sequences
presented in GenBank.
In addition, the present invention provides a target nucleic acid which
hybridizes under at least stringent or highly stringent conditions (or
conditions of greater
stringency) to a unique coding oligonucleotide which encodes a unique
subsequence in a
polypeptide selected from: SEQ >D N0:36 to SEQ )D N0:70 or SEQ ID N0:79 to SEQ
>D N0:85, wherein the unique subsequence is unique as compared to a an amino
acid
subsequence of a known interferon-alpha polypeptide sequence shown in GenBank
or to a
polypeptide corresponding to any of the control polypeptides. Unique sequences
are
determined as noted above.
In one example, 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-lOx higher signal to noise ratio than
for
hybridization of the perfectly complementary oligonucleotide to a control
nucleic acid
corresponding to any of the control polypeptides. Conditions can be selected
such that
higher ratios of signal to noise are observed in the particular assay which is
used, e.g.,
about 15x, 20x, 30x, 50x or more. In this example, the target nucleic acid
hybridizes to
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the unique coding oligonucleotide with at least a 2x higher signal to noise
ratio as
compared to hybridization of the control nucleic acid to the coding
oligonucleotide.
Again, higher signal to noise ratios can be selected, e.g., about 2.5x, about
Sx, about 10x,
about 20x, about 30x, about SOx or more. The particular signal will depend on
the label
used in the relevant assay, e.g., a fluorescent label, a colorimetric label, a
radio active
label, or the like.
In another aspect, the invention provides a polypeptide that comprises
unique subsequence in a polypeptide selected from SEQ >D N0:36 to SEQ ID N0:70
and
SEQ ID N0:79 to SEQ >D N0:85, wherein the unique subsequence is unique as
compared
to a polypeptide sequence corresponding to a known interferon-alpha
polypeptide, such as,
e.g., an interferon-alpha polypeptide sequence present in GenBank.
SUBSTRATES AND FORMATS FOR SEQUENCE RECOMBINATION
The polynucleotides of the invention are useful as substrates for a variety of
recombination and recursive recombination (e.g., DNA shuffling) reactions, as
well as
other diversity generating techniques, including mutagenesis techniques and
standard
cloning methods as set forth in, e.g., Ausubel, Berger and Sambrook, supra,
i.e., to
produce additional interferon-alpha homologues with desired properties. Based
on the
screening or selection protocols employed, recombinant, e.g., shuffled,
interferon-alpha
homologue polypeptides can be generated and isolated that confer a variety of
desirable
characteristics, e.g., enhanced antiviral activity, enhanced antiproliferative
activity,
increased growth inhibitory, cytostatic and/or cytotoxic activities towards
particular target
cells, reduced immunogenicity, etc.
A variety of diversity generating protocols, including nucleic acid shuffling
protocols, are available and fully 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
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WO 01/25438 PCT/US00/27781
exclusive. Indeed, the various methods can be used singly or in combination,
in parallel or
in series, to access diverse sequence variants.
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 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.,
enhanced antiviral activity, enhanced antiproliferative activity, enhanced
anti-angiogenic
activity, increased growth inhibitory, cytostatic and/or cytotoxic activities
towards
particular target cells, reduced immunogenicity, etc. Methods for determining
nucleic
acids having enhanced antiviral, antiproliferative, growth inhibitory,
cytostatic, and/or
cytotoxic activity or reduced immunogenicity include those described herein.
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. A variety of related (or
even
unrelated) properties can be evaluated, in serial or in parallel, at the
discretion of the
practitioner.
The following publications describe a variety of diversity generating
procedures, including recursive recombination procedures, and/or methods for
generating
modified nucleic acid sequences for use in the procedures and methods of the
present
invention include the following publications and the references cited therein:
Soong, N.
W. et al. (2000) "Molecular Breeding of Viruses," Nature Genetics 25:436-439;
Stemmer,
W. 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 Biotechnolo~y 17:893-896; Chang et al. (1999)
"Evolution of a cytokine using DNA family shuffling," Nature Biotechnolo~y
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
Biotechnolo~y 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 BiotechnoloQV 15:436-438; Zhang et al. (1997) "Directed evolution of an
effective
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fucosidase from a galactosidase by DNA shuffling and screening," Proc. Nat'l
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 BiotechnoloQV 14:315-319;
Gates et al.
(1996) "Affinity selective isolation of ligands from peptide libraries through
display on a
lac repressor 'headpiece dimer,"' J. Mol. Biol. 255:373-386; Stemmer (1996)
"Sexual PCR
and Assembly PCR" In: The Encyclopedia of Molecular Biolo~~ 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," BiolTechnology 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. Nat'L Acad. Sci. USA 91:10747-10751.
Additional details regarding DNA shuffling and other diversity generating
methods can be found in the following U.S. patents, PCT publications, and EP
publications: USPN 5,605,793 to Stemmer (February 25, 1997), "Methods for In
vitro
Recombination;" USPN 5,811,238 to Stemmer et al. (September 22, 1998) "Methods
for
Generating Polynucleotides having Desired Characteristics by Iterative
Selection and
Recombination;" USPN 5,830,721 to Stemmer et al. (November 3, 1998), "DNA
Mutagenesis by Random Fragmentation and Reassembly;" USPN 5,834,252 to Stemmer
(November 10, 1998) "End-Complementary Polymerase Reaction;" USPN 5,837,458 to
Minshull (November 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
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WO 01/25438 PCT/US00/27781
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;" EP 0946755 by Patten and Stemmer, "Methods and
Compositions for Polypeptide Engineering;" and WO 98/13487 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."
Certain U.S. applications provide additional details regarding DNA
shuffling and related techniques, as well as other diversity generating
methods, including
"SHUFFLING OF CODON ALTERED GENES" by Patten et al. filed September 29,
1998 (USSN 60/102,362), January 29, 1999 (USSN 60/117,729), and September 28,
1999
(USSN 09/407,800); "EVOLUTION OF WHOLE CELLS AND ORGANISMS BY
RECURSIVE SEQUENCE RECOMBINATION", by Del Cardayre et al. filed July 15,
1998 (USSN 09/166,188), and July 15, 1999 (USSN 09/354,922);
"OLIGONUCLEOTIDE MEDIATED NUCLEIC ACID RECOMBINATION" by
Crameri et al., filed February 5, 1999 (USSN 60/118,813), June 24, 1999 (USSN
60/141,049), and September 28, 1999 (USSN 09/408,392); "USE OF CODON-BASED
CA 02385045 2002-03-14
WO 01/25438 PCT/US00/27781
OLIGONUCLEOTIDE SYNTHESIS FOR SYNTHETIC SHUFFLING" by Welch et al.,
filed September 28, 1999 (USSN 09/408,393); "METHODS FOR MAKING
CHARACTER STRINGS, POLYNUCLEOT>DES & POLYPEPT117ES HAVING
DESIRED CHARACTERISTICS" by Selifonov and Stemmer, filed February 5, 1999
(USSN 60/118854) and October 12, 1999 (USSN 09/416,375); RECOMBINATION OF
INSERTION MODIFIED NUCLEIC ACIDS by Patten et al., filed March 5, 1999 (USSN
60/122,943), July 2, 1999 (USSN 60/142,299), November 10, 1999 (USSN
60/164,618),
and November 10, 1999 (USSN 60/164,617); and "SINGLE-STRANDED NUCLEIC
ACID TEMPLATE-MEDIATED RECOMBINATION AND NUCLEIC ACID
FRAGMENT ISOLATION" by Affholter, USSN 60/186,482 filed March 2, 2000.
As a review of the foregoing publications, patents, published foreign
applications and U.S. patent applications reveals, diversity generation
methods, such as
shuffling (or "recursive recombination") of nucleic acids, to provide new
nucleic acids
with desired properties can be carned out by a number of established methods.
Any of
these methods can be adapted to the present invention to evolve the alpha
interferons
discussed herein to produce new alpha interferon homologues with new or
improved
properties. Both the methods of making such interferons and the interferons
(e.g., IFN
homologues) produced by these methods are a feature of the invention.In brief,
several
different general classes of sequence modification methods, such as
recombination, are
applicable to the present invention and set forth, e.g., in the references
above. First,
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. Second,
nucleic acids
can be recursively recombined in vivo or ex vivo, e.g., by allowing
recombination to occur
between nucleic acids in cells. Third, whole genome recombination methods can
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). Fourth,
synthetic
recombination methods can 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
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nucleotide addition methods, or can be made, e.g., by tri-nucleotide synthetic
approaches.
Fifth, 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. Any of the preceding general recombination formats can
be
practiced in a reiterative fashion to generate a more diverse set of
recombinant nucleic
acids. Sixth, 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
used. above references provide these and other basic recombination formats as
well as
many modifications of these formats. Regardless of the format which is used,
the nucleic
acids of the invention can be recombined (with each other, or with related (or
even
unrelated) nucleic acids to produce a diverse set of recombinant nucleic
acids, including
e.g., homologous nucleic acids. In general, the sequence recombination
techniques
described herein provide particular advantages in that they provide for
recombination
between the nucleic acids of SEQ m NO:1 to SEQ ID N0:35, and SEQ m N0:72 to
SEQ
>D N0:78, or fragments or variants thereof, in any available format, thereby
providing a
very fast way of exploring the manner in which different combinations of
sequences can
affect a desired result.
Following recombination, any nucleic acids which are produced can be
screened or selected for a desired activity. In the context of the present
invention, this can
include testing for and identifying any activity that can be detected, e.g.,
in an automatable
format, by any assay known in the art. In addition, useful properties such as
low
immunogenicity, increased half-life, improved solubility, oral availability,
or the like can
also be selected for. A varzety of alpha-interferon related (or even
unrelated) properties
can be assayed for, using any available assay.
DNA mutagenesis and shuffling provide a robust, widely applicable, means
of generating diversity useful for the engineering of proteins, pathways,
cells and
organisms with improved characteristics. In addition to the basic formats
described above,
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it is sometimes desirable to combine shuffling methodologies with other
techniques for
generating diversity. In conjunction with (or separately from) shuffling
methods, a variety
of diversity generation methods can be practiced and the results (i.e.,
diverse populations
of nucleic acids) screened for in the systems of the invention. Additional
diversity can be
introduced by methods which result in the alteration of individual nucleotides
or groups of
contiguous or non-contiguous nucleotides, i.e., mutagenesis methods. Many
mutagenesis
methods are found in the above-cited references; additional details regarding
mutagenesis
methods can be found in the references listed below.
Mutagenesis methods of generating diversity include, for example,
recombination (PCT/LTS98/05223; Publ. No. W098/42727); 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 Biolo~y
(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. Nat'1 Acad. Sci. USA 82:488-492; Kunkel et al.
(1987)
"Rapid and efficient site-specific mutagenesis without phenotypic selection,"
Results
Probl. Cell Differ. 154, 367-382; and Bass et al. (1988) "Mutant Trp
repressors with new
DNA-binding specificities," Science 242:240-245); oligonucleotide-directed
mutagenesis
(Results Probl. Cell Differ. 100:468-500 (1983); Results Probl. Cell Differ.
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,"
Results Probl. Cell Differ. 100:468-500; and Zoller & Smith (1987)
"Oligonucleotide-
directed mutagenesis: a simple method using two oligonucleotide primers and a
single-
stranded DNA template," Results Probl. Cell Differ. 154:329-350);
phosphorothioate-
modified DNA mutagenesis (Taylor et al. (1985) "The use of phosphorothioate-
modified
58
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WO 01/25438 PCT/US00/27781
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)
"Oligonucleotide-
directed construction of mutations via gapped duplex DNA," Results Probl. Cell
Differ.
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).
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," Results Probl. Cell
Differ.
154:382-403), deletion mutagenesis (Eghtedarzadeh & Henikoff (1986) "Use of
oligonucleotides to generate large deletions," Nucl. Acids Res. 14:5115),
restriction-
selection and 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
(transducing)," Nucl. Acids Res. 14:6361-6372; Wells et al. (1985) "Cassette
mutagenesis:
59
CA 02385045 2002-03-14
WO 01/25438 PCT/US00/27781
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. Nat'1 Acad.
Sci. USA,
83:7177-7181). Additional details on many of the above methods can be found in
Methods in Enzymolo~y, Vol. 154, which also describes useful controls for
trouble-
shooting problems with various mutagenesis methods.
Random or semi-random mutagenesis using doped or degenerate
oligonucleotides (Arkin and Youvan (1992) "Optimizing nucleotide mixtures to
encode
specific subsets of amino acids for semi-random mutagenesis," Biotechnolo~y
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); "Walk-Through Mutagenesis" (Crea, R.; US Patents 5,830,650 and
5,798,208, and EP Patent 0527809 B1) may also be employed to generate
diversity.
In one aspect of the present invention, 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 vial, with the products of one reaction priming the
products of another
reaction. Sexual PCR mutagenesis can be used in which homologous recombination
occurs between DNA molecules of different but related DNA sequence in vitro,
by
random fragmentation of the DNA molecule based on sequence homology, followed
by
fixation of the crossover by primer extension in a PCR reaction. This process
is described
in the references above, e.g., in Stemmer (1994) Proc. Nat'I Acad. Sci. USA
91:10747-
CA 02385045 2002-03-14
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10751. Recursive ensemble mutagenesis can be used in which an algorithm for
protein
mutagenesis is used to produce diverse populations of phenotypically related
mutants
whose members differ in amino acid sequence. This method uses a feedback
mechanism
to control successive rounds of combinatorial cassette mutagenesis. Examples
of this
approach are found in Arkin & Youvan (1992) Proc. Nat'1 Acad. Sci. USA 89:7811-
7815.
As noted, oligonucleotide directed mutagenesis can be used in a process
which allows for the generation of site-specific mutations in any 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 which 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).
In vivo (or ex vivo) mutagenesis can be used in a process of generating
random mutations in any cloned DNA of interest which involves the propagation
of 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.
Exponential ensemble mutagenesis can be used for generating
combinatorial libraries with a high percentage of unique and functional
mutants, where
small groups of residues are randomized in parallel to identify, at each
altered position,
amino acids which lead to functional proteins. Examples of such procedures are
found in
Delegrave & Youvan (1993) Biotechnolo~y Research 11:1548-1552. Similarly,
random
and site-directed mutagenesis can be used. Examples of such procedures are
found in
Arnold (1993) Current Opinion in Biotechnology 4:450-455.
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., QuickChangeTM site-directed mutagenesis kit; and Chameleons
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,
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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 Biotechnolo~y Ltd (e.g., using the
Carter/Winter
method above).
Any of the described shuffling or mutagenesis techniques can be used in
conjunction with procedures which introduce additional diversity into a
genome, e.g., a
bacterial, fungal, animal or plant genome. For example, in addition to the
methods above,
techniques have been proposed which produce chimeric nucleic acid multimers
suitable
for transformation into a variety of species (see, e.g., Schellenberger U.S.
Patent No.
5,756,316 and the references above). When such chimeric multimers consist 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), are transformed into a suitable host, this
provides a source
of nucleic acid diversity for DNA diversification.
Chimeric multimers transformed into host species are suitable as substrates
for in vivo (or ex vivo) shuffling protocols. Alternatively, a multiplicity of
polynucleotides
sharing regions of partial sequence similarity or homology can be transformed
into a host
species and recombined in vivo (or ex vivo) by the host cell. Subsequent
rounds of cell
division can be used to generate libraries, members of which, comprise a
single,
homogenous population of monomeric or pooled nucleic acid. Alternatively, the
monomeric nucleic acid can be recovered by standard techniques and recursively
recombined in any of the described shuffling formats.
Chain termination methods of diversity generation have also been proposed
(see, e.g., U.S. Patent No. 5,965,408 and the references above). In this
approach, double
stranded DNAs corresponding to one or more genes sharing regions of sequence
similarity
or homology are combined and denature, 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
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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 or homology and which are chimeric 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 diversity
generation methods
(e.g., RSR, DNA shuffling) according to any of the described formats.
Diversity can be further increased by using methods which are not
homology based with DNA shuffling (which, as set forth in the above
publications and
applications can be homology or non-homology based, depending on the precise
format).
For example, 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, can be used to generate
an
initial recombinant library which serves as a substrate for one or more rounds
of in vitro,
ex vivo, or in vivo diversity generation methods (e.g., RSR or shuffling
methods).
Methods for generating multispecies expression libraries have been
described (e.g., U.S. Patent Nos. 5,783,431; 5,824,485 and the references
above) and their
use to identify protein activities of interest has been proposed (U.S. Patent
5,958,672 and
the references above). Multispecies expression libraries are, 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 concatenated 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.
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 shuffling, or to otherwise bias the
substrates towards
nucleic acids that encode functional products (shuffling procedures can also,
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independently have these effects). For example, in the case of antibody
engineering, it is
possible to bias the shuffling process toward antibodies with functional
antigen binding
sites by taking advantage of in vivo (or ex vivo or in vitro) recombination
events prior to
diversity generation (e.g., DNA shuffling) by any described method. 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 diversity generation (e.g., DNA shuffling) according to any
of the
methods described herein.
Libraries can be biased towards nucleic acids which encode proteins with
desirable activities (e.g., binding affinities, enzymatic activities, anti-
viral activities,
ability to induce an immune response, antiproliferative activities, adjuvant
properties,
etc. ). 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, including, but not restricted to, DNA shuffling or another form
of recursive
sequence recombination or diversity generation. 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 U.S.
Patent No. 5,939,250. 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 using a fluorescent
analyzer, e.g., a flow
cytometry device, a CCD, a fluorometer, or a spectrophotometer.
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
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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 a shuffling-based gene reassembly process. In one such method the
fragment
population derived 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 the mediated by nuclease-
base removal
of non-hybridizing fragment ends, polymerization to fill gaps between such
fragments and
subsequent single stranded ligation. The parental strand can be removed by
digestion (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. As set
forth in
"Single-stranded nucleic acid template-mediated recombination and nucleic acid
fragment
isolation" by Affholter (USSN 60/186,482, filed March 2, 2000) and WO
98/27230,
"Methods and Compositions for Polypeptide Engineering" by Patten and Stemmer,
shuffling using single-stranded templates and nucleic acids of interest which
bind to a
portion of the template can also be performed.
In one 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 any of the shuffling reactions described
herein.
"Non-Stochastic" methods of generating nucleic acids and polypeptides are
alleged in Short, J. "Non-Stochastic Generation of Genetic Vaccines and
Enzymes," WO
CA 02385045 2002-03-14
WO 01/25438 PCT/US00/27781
00/46344. These methods, including the proposed non-stochastic polynucleotide
reassembly and gene site saturation mutagenesis and synthetic ligation
polynucleotide
reassembly methods outlined therein, can be applied to the present invention
as well.
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.
A recombinant nucleic acid produced by recursively recombining one or
more polynucleotides of the invention with one or more additional nucleic
acids 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 acids can include, e.g., a nucleic acid encoding a
naturally-
occurring interferon-alpha or a subsequence thereof, or any homologous
interferon-alpha
sequence or subsequence thereof, or an interferon-beta sequence or subsequence
thereof
(e.g., an interferon-alpha or interferon-beta sequence as found in GenBank or
other
available literature), or, e.g., any other homologous or non-homologous
nucleic acid
(certain recombination formats noted above, notably those performed
synthetically or in
silico, do not require homology for recombination).
The recombining steps may be performed in vivo, ex vivo, in vitro, or in
silico as described in more detail in the references above. Also included in
the invention
is a cell containing any resulting recombinant nucleic acid, nucleic acid
libraries produced
by diversity generation, recombination, or recursive recombination of the
nucleic acids set
forth herein, and populations of cells, vectors, viruses, plasmids or the like
comprising the
library or comprising any recombinant nucleic acid resulting from diversity
generation or
recombination (or recursive recombination) of a nucleic acid as set forth
herein with
another such nucleic acid, or an additional nucleic acid. Corresponding
sequence strings
in a database present in a computer system or computer readable medium are a
feature of
the invention.
OTHER POLYNUCLEOTIDE COMPOSITIONS
The invention also includes compositions comprising two or more
polynucleotides of the invention (e.g., as substrates for recombination). The
composition
can comprise a library of recombinant nucleic acids, where the library
contains at least 2,
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WO 01/25438 PCT/US00/27781
3, 5, 10, 20, or 50 or more nucleic acids. The nucleic acids are optionally
cloned into
expression vectors, providing expression libraries.
The invention also includes compositions produced by digesting one or
more polynucleotides 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 polynucleotides
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 above.
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.
Also included in the invention are compositions produced by incubating
one or more of the fragmented nucleic acid sets in the presence of
ribonucleotide- or
deoxyribonucelotide triphosphates and a nucleic acid polymerise. This
resulting
composition forms a recombination mixture for many of the recombination
formats noted
above. The nucleic acid polymerise may be an RNA polymerise, a DNA polymerise,
or
an RNA-directed DNA polymerise (e.g., a "reverse transcriptase"); the
polymerise can
be, e.g., a thermostable DNA polymerise (such as, VENT, TAQ, or the like).
INTERFERON HOMOLOGUE POLYPEPT>DES
The invention provides isolated or recombinant interferon-alpha homologue
polypeptides, also referred to herein as "interferon-alpha homologues," or
"interferon
homologues" or "IFN-alpha homologues" or "IFN homologues". An isolated or
recombinant interferon homologue polypeptide of the invention includes a
polypeptide
comprising a sequence selected from SEQ >D N0:36 to SEQ >D N0:70 and SEQ )D
N0:79 to SEQ >D N0:85, and conservatively modified variations thereof, and
fragments
thereof having an antiproliferative activity in, e.g., a human Daudi cell line-
based assay (or
other similar assay) and/or an antiviral activity in, e.g., a murine cell
lineBMCV-based
assay (or other similar assay). An alignment of exemplary interferon homologue
polypeptide sequences according to the invention is provided in Fig. 1.
Alignment of the
polypeptide sequences of the invention to each other or to sequences of known,
naturally-
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occurnng interferon-alphas is readily performed by one of ordinary skill in
the art using
publicly available databases and alignment programs.
The invention also provides a polypeptide comprising at least about 100,
120, 130, 140, 150, 155, 160, 163, 165, or 166 contiguous amino acids of any
one of SQ
D7 NOS:36-70 or SEQ ID N0:71. In one aspect, said amino acid sequence
comprises
amino acids Lys160 and G1u166, wherein the numbering of the amino acids in the
sequence corresponds to that of SEQ ID N0:36.
Several conclusions may be drawn from comparison of the exemplary
sequences of the invention (Fig. 1) to sequences of known, naturally-occurring
interferon-
alphas and other Type I interferons (including beta, delta, omega, and tau-
interferons)
from human and non-human sources. Such sequences are readily available from a
variety
of sources, such as GenBank, and the Pfam (Protein Families) database at
http://www.sanger.ac.uk/Software/Pfam/index.shtml.
Of particular note is the presence, in some interferon homologue
polypeptide sequences of the invention, of the following amino acid residues
(denoted
"Group I" residues) which do not appear in the equivalent position of known,
naturally-
occurring human or non-human Type 1 interferon sequences.
Group I: Aspll; Prol4; Arg50; Phe55; Asp75; Asn80; Prol l l; Leu124;
G1u134; Ser140, and A1a143; with residue numbering corresponding to the mature
interferon homologue sequence identified as SEQ ID N0:36.
Also of note is the presence, in some interferon homologue polypeptide
sequences of the invention, of the following amino acid residues (denoted
"Group II"
residues) which do not appear in the equivalent position of known, naturally-
occurring
human interferon-alpha subtype sequences.
Group II: Pro9; (Lys, Ser)12; (Thr, Val)24; G1n34; Arg40; Ser45; Arg47;
Leu56; I1e60; Phe67; A1a79, G1y88; His90; Arg9l; G1u95; Va1101; (Gly, Ala)104;
Va1112; Glyll4; Pro116; Lys133, and His136.
In other embodiments, the interferon homologue polypeptide comprises at
least 20, 50, 100, 150, 155, or 160 of more contiguous amino acids of any one
of SEQ 117
NOS:36-70 and/or one or more of amino acids A1a19, (Tyr or Gln)34, G1y37,
Phe38,
Lys7l, A1a76, Tyr90, I1e132, Arg134, Phe152, Lys160, and G1u166, wherein the
numbering of the amino acids corresponds to that of SEQ >D N0:36, or one or
more of
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amino acids Pro9, (Lys or Ser)12, (Thr or Val)24, G1n34, Arg40, Ser45, Arg47,
Leu56,
I1e60, Phe67, A1a79, G1y88, His90, Arg9l, G1u95, Va1101, (Gly, Ala)104,
Va1112,
G1y114, Pro116, Lys133, and His136, wherein the numbering of the amino acids
in said
polypeptide sequence corresponds to the numbering of individual amino acids in
the
amino acid sequence of SEQ ID N0:36. Thus, for example, in this embodiment, an
interferon polypeptide comprises an amino acid sequence comprising a proline
residue at
amino acid position 9 in the sequence, a lysine or serine residue at position
12, a threonine
or valine residue at position 24, a glutamine residue at position 34, an
arginine residue at
position 40, etc. Such polypeptides may exhibit antiproliferative activities
in a human
Daudi cell line-based proliferation assay (e.g., at least about 8.3x106
units/mg) and/or an
antiviral activities in a human WISH cell/EMCV-based assay (at least about
2.1x10'
units/mg). Some such polypeptides bind a human alpha interferon receptor. Some
such
polypeptides are 166 amino acids in length. In another aspect, such
polypeptides may
comprise a sequence selected from any of the group of SEQ >D N0:36 to SEQ ID
N0:54.
An antiproliferative activity of any polypeptide of the invention generally
relates to the capability or ability of a polypeptide to cause cells or parts
thereof to grow or
produce new cellular growth rapidly and often repeatedly.
The invention further includes a polypeptide (e.g., any of SEQ ID NOS:36-
71 or SEQ >D NOS:79-85) or a nucleic acid (e.g., any of SEQ >D NOS:1-35 or SEQ
ID
NOS:72-78)encoding a polypeptide, wherein said polypeptide having an anti-
angiogenic
activity as measured by an anti-angiogenesis assay well known to those of
ordinary skill in
the art.
The invention further includes:
(a) any interferon-alpha polypeptide comprising one or more Group I
amino acid residues above.
(b) any interferon-alpha polypeptide comprising one or more Group II
amino acid residues above in the context of a human like interferon sequence
(i.e., a
sequence which displays a high level of similarity or homology to a human
interferon), or
a sequence which is highly similar or homologous (i.e., having a percent
sequence
homology or sequence identity of at least about 80%, 90%, 95%, 96%, 97%, 98%
or
more) to any sequence listed in the attached sequence listing or fragment
thereof.
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(c) any interferon-alpha polypeptide containing a combination of the
following residues, which are localized in or near the regions of the
interferon-alpha
molecule known or proposed to interact with a Type I interferon receptor,
where such
sequence combinations (motifs) do not appear in the equivalent position of any
known
naturally-occurnng human or non-human Type 1 interferon:
(i) (Tyr or Gln)34; plus one or more of I1e132 or Arg134; or
(ii) Asp78, G1u79, or (Asp or Thr)80; plus one or more of I1e132 or
Arg134.
In another embodiment, the present invention provides an interferon alpha
homologue comprising the sequence show in SEQ 117 N0:71: CDLPQTHSLG-X11-Xlz-
R A-X 1 s-X 16-LL-X 19-QM-Xzz-R-Xz4-S-Xz6-FS CLKDR-X34-DFG-X38-P-X4o-EEFD-X4s-
X46-X47-FQ-X50-XS 1-QAI-Xss-Xs6-Xsr~-X6o-X61-QQTFN-X6~-FSTK-X~z-SS-X~s-X~~-
W-X~g-X~9-X8o-LL-Xg3-K-Xgs-Xg~-T-Xgg-L-X~o-QQLN-X9s-LEACV-Xlol-Q-Xlo3-V-Xlos-
Xlo6-XlorXloa-TPLMN-X114-D-X116-ILAV-X1 zl-KY-X1 z4-QRITLYL-Xl3z-E-X1 x4-
KYSPC-Xl4o-WEVVRAE>IVVIRSFSFSTNLQKRLRRKE, or a conservatively substituted
variation thereof, where X11 is N or D; Xlz is R, S, or K; Xls is L or M; X16
is I, M, or V;
X1~ is A or G; Xzz is G or R; Xz4 is I or T; Xz6 is P or H; X34 is H, Y or Q;
X3g is F or L;
X4o is Q or R; X4s is G or S; X46 is N or H; X4~ is Q or R; Xso is K or R; Xsl
is A or T; Xss
is S or F; Xs6 is V or A; Xs~ is L or F; X6o is M or I; X61 is I or M; X6~ is
L or F; X~z is D
or N; X~s is A or V; X~6 is A or T; X~g is E or D; X~9 is Q or E; Xgo is S, R,
T, or N; X83 is
E or D; Xgs is F or L; Xg6 is S or Y; X$$ is E or G; X~o is Y, H, N; X9s is D,
E, or N; Xlol is
I,M,orV;XlosisEorG;XlosisGorW;Xlo6isVorM;Xlo~isE,G,orK;XloBisEor
G; X114 is V, E, or G; X116 is S or P; Xlzl is K or R; Xlz4 is F or L; X13215
T, I, or M; X134
is K or R; and Xl4o is A or S; or a fragment of said SEQ ID N0:71. In another
aspect, the
interferon homologue polypeptide of SEQ ID N0:71, or a fragment thereof,
exhibits an
antiproliferative activity in a human Daudi cell line-based proliferation
assay (at least
about 8.3x106 units/mg) and/or an antiviral activity in a human WISH cell/EMCV-
based
assay (at least about 2.1x10' units/mg). Both such assays are discussed in
greater detail
below. Such polypeptide may comprise an amino acid sequence of the group of
from SEQ
>D N0:36 to SEQ >D N0:54 or may be encoded by a nucleotide sequence of the
group of
from SEQ ID NO:1 to SEQ ID N0:19.
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Fragments of the interferon homologue polypeptides described herein are
also a feature of the invention. An interferon alpha homologue fragment of the
invention
typically comprises an interferon homologue polypeptide comprising at least
about 20, 25,
or 30, and typically at least about 40, 50, 60, 70, 80, 90, or 100 contiguous
amino acids of
any one of SEQ ID NOS:36-71 or SEQ ID NOS:79-85. In other embodiments, the
fragment comprises usually at least about 100, 110, 120, 125, 130, 140, 150,
155, 158,
160, 162, 163, 164, or 165 contiguous amino acids of any one of SEQ )D NOS:36-
71 or
SEQ ID NOS:79-85. Such polypeptide fragments may have an antiproliferative
activity in
a human Daudi cell line-based assay and/or an antiviral activity in a human or
murine cell
lineBMCV-based assay.
In other embodiments, the invention provides polypeptides having a length
of 166 amino acids, and, in some such embodiments, such polypeptides have an
antiproliferative activity in a human Daudi cell line-based assay (or other
similar assay),
including, e.g., at least about 8.3x10 units/mg, and/or an antiviral activity
in a human
WISH cell line/EMCV-based assay (or other similar assay), including, e.g., at
least about
2.1x10' units/mg.
In other embodiments, the invention provides a polypeptide comprising at
least 100, 150, 155, or 160 contiguous amino acids of a protein encoded by a
coding
polynucleotide sequence comprising any of the following: (a) SEQ ID NO:1 to
SEQ ID
N0:35 or SEQ ID N0:72 to SEQ >D N0:78; (b) a coding polynucleotide sequence
that
encodes a first polypeptide selected from any of SEQ >D N0:36 to SEQ ID N0:70
or SEQ
ID N0:79 to SEQ ID N0:85; and (c) a complementary polynucleotide sequence that
hybridizes under at least highly stringent (or ultra-high stringent or ultra-
ultra- high
stringent conditions) hybridization conditions over substantially the entire
length of a
polynucleotide sequence of (a) or (b). Such polypeptides may have an
antiproliferative
activity in a human Daudi cell line-based assay (or other similar assay),
andlor an antiviral
activity in a human WISH cell line/EMCV-based assay (or other similar assay).
Some
such polypeptides of the invention specifically bind a human alpha interferon
receptor.
The polypeptides and nucleic acids of the subject invention need not be
identical, but can
be substantially identical, to the corresponding sequence of the target
molecule or related
molecule, including the polypeptides of any of SEQ >l.7 NOS:36-71 or fragments
thereof
(including those having antiviral or antiproliferative activities in the
assays described
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herein), or the nucleic acids of any of SEQ ID NOS:1-35 or fragments thereof
(including
those having antiviral or antiproliferative activities in the assays described
herein). The
polypeptides can be subject to various changes, such as insertions, deletions,
and
substitutions, either conservative or non-conservative, where such changes
might provide
for certain advantages in their use. The polypeptides of the invention can be
modified in a
number of ways so long as they comprise a sequence substantially identical (as
defined
below) or having a percent identity to a sequence in the naturally occurnng or
known
interferon polypeptide molecule.
Alignment and comparison of relatively short amino acid sequences (less
than about 30 residues) is typically straightforward. Comparison of longer
sequences can
require more sophisticated methods to achieve optimal alignment of two
sequences.
Optimal alignment of sequences for aligning a comparison window can be
conducted by
the local homology algorithm of Smith and Waterman (1981) Adv. Appl. Math.
2:482, by
the homology alignment algorithm of Needleman and Wunsch (1970) J. Mol. Biol.
48:443, by the search for similarity method of Pearson and Lipman (1988) Proc.
Nat'l
Acad. Sci. (USA) 85:2444, by computerized implementations of these algorithms
(GAP,
BESTFIT, FASTA, and TFASTA in the Wisconsin Genetics Software Package Release
7.0, Genetics Computer Group, 575 Science Dr., Madison, WI), or by inspection,
and the
best alignment (i.e., resulting in the highest percentage of sequence
similarity over the
comparison window) generated by the various methods is selected.
The term sequence identity means that two polynucleotide sequences are
identical (i.e., on a nucleotide-by-nucleotide basis) over a window of
comparison. The
term "percentage of sequence identity" or "percent sequence identity" is
calculated by
comparing two optimally aligned sequences over the window of comparison,
determining
the number of positions at which the identical residues occurs in both
sequences to yield
the number of matched positions, dividing the number of matched positions by
the total
number of positions in the window of comparison (i.e., the window size), and
multiplying
the result by 100 to yield the percentage of sequence identity. In one aspect,
the present
invention provides interferon homologue nucleic acids having at least about
80%, 85%,
90%, 95%, 96%, 97%, 98%, 99%, 99.5% or more percent sequence identity with the
nucleic acids of any of SEQ 117 NOS:1-35 or SEQ ID NOS:72-78 or fragments
thereof.
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As applied to polypeptides, the term substantial identity means that two
peptide sequences, when optimally aligned, such as by the programs GAP or
BESTFIT
using default gap weights (described in detail below), share at least about 80
percent
sequence identity, preferably at least about 90 percent sequence identity,
more preferably
at least about 95 percent sequence identity or more (e.g., 97, 98, or 99
percent sequence
identity). Preferably, residue positions which are not identical differ by
conservative
amino acid substitutions. Conservative amino acid substitutions refer to the
interchangeability of residues having similar side chains. For example, a
group of amino
acids having aliphatic side chains is glycine, alanine, valine, leucine, and
isoleucine; a
group of amino acids having aliphatic-hydroxyl side chains is serine and
threonine; a
group of amino acids having amide-containing side chains is asparagine and
glutamine; a
group of amino acids having aromatic side chains is phenylalanine, tyrosine,
and
tryptophan; a group of amino acids having basic side chains is lysine,
arginine, and
histidine; and a group of amino acids having sulfur-containing side chains is
cysteine and
methionine. Preferred conservative amino acids substitution groups are: valine-
leucine-
isoleucine, phenylalanine-tyrosine, lysine-arginine, alanine-valine, and
asparagine-
glutamine. In one aspect, the present invention provides interferon homologue
polypeptides having at least about 80%, 85%, 90%, 95%, 96%, 97%, 98% 99% 99.5%
or
more percent sequence identity with the polypeptides of any of SEQ )D NOS:36-
71 or
SEQ ID NOS:79-85 or fragments thereof.
A preferred example of an algorithm that is suitable for determining percent
sequence identity and sequence similarity is the FASTA algorithm, which is
described in
Pearson, W.R. & Lipman, D.J., 1988, Proc. Nat'l Acad. Sci. USA 85: 2444. See
also W.
R. Pearson, 1996, Methods Enzymol. 266: 227-258. Preferred parameters used in
a
FASTA alignment of DNA sequences to calculate percent identity are optimized,
BL50
Matrix 15: -5, k-tuple= 2; joining penalty= 40, optimization= 28; gap penalty -
12, gap
length penalty =-2; and width= 16.
Another preferred example of algorithm that is suitable for determining
percent sequence identity and sequence similarity are the BLAST and BLAST 2.0
algorithms, which are described in Altschul et al., 1977, Nuc. Acids Res. 25:
3389-3402
and Altschul et al., 1990, J. Mol. Biol. 215: 403-410, respectively. BLAST and
BLAST
2.0 are used, with the parameters described herein, to determine percent
sequence identity
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for the nucleic acids and proteins of the invention. Software for performing
BLAST
analyses is publicly available through the National Center for Biotechnology
Information
(http: //www.ncbi.nlm.nih.gov/). This algorithm involves first identifying
high scoring
sequence pairs (HSPs) by identifying short words of length W in the query
sequence,
which either match or satisfy some positive-valued threshold score T when
aligned with a
word of the same length in a database sequence. T is referred to as the
neighborhood word
score threshold (Altschul et al., supra). These initial neighborhood word hits
act as seeds
for initiating searches to find longer HSPs containing them. The word hits are
extended in
both directions along each sequence for as far as the cumulative alignment
score can be
increased. Cumulative scores are calculated using, for nucleotide sequences,
the
parameters M (reward score for a pair of matching residues; always > 0) and N
(penalty
score for mismatching residues; always < 0). For amino acid sequences, a
scoring matrix
is used to calculate the cumulative score. Extension of the word hits in each
direction are
halted when: the cumulative alignment score falls off by the quantity X from
its maximum
achieved value; the cumulative score goes to zero or below, due to the
accumulation of
one or more negative-scoring residue alignments; or the end of either sequence
is reached.
The BLAST algorithm parameters W, T, and X determine the sensitivity and speed
of the
alignment. The BLASTN program (for nucleotide sequences) uses as defaults a
wordlength (W) of 11, an expectation (E) of 10, M=5, N=-4 and a comparison of
both
strands. For amino acid sequences, the BLASTP program uses as defaults a
wordlength of
3, and expectation (E) of 10, and the BLOSUM62 scoring matrix (see Henikoff &
Henikoff (1989) Proc. Nat'l Acad. Sci. U.S.A. 89: 10915) alignments (B) of 50,
expectation (E) of 10, M=5, N=-4, and a comparison of both strands.
The BLAST algorithm also performs a statistical analysis of the similarity
between two sequences (see, e.g., Karlin & Altschul (1993) Proc. Nat'l Acad.
Sci. U.S.A.
90: 5873-5787). One measure of similarity provided by the BLAST algorithm is
the
smallest sum probability (P(N)), which provides an indication of the
probability by which
a match between two nucleotide or amino acid sequences would occur by chance.
For
example, a nucleic acid is considered similar to a reference sequence if the
smallest sum
probability in a comparison of the test nucleic acid to the reference nucleic
acid is less
than about 0.2, more preferably less than about 0.01, and most preferably less
than about
0.001.
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Another example of a useful algorithm is PILEUP. PILEUP creates a
multiple sequence alignment from a group of related sequences using
progressive,
pairwise alignments to show relationship and percent sequence identity. It
also plots a tree
or dendogram showing the clustering relationships used to create the
alignment. PILEUP
uses a simplification of the progressive alignment method of Feng & Doolittle
(1987) J.
Mol. Evol. 35: 351-360. The method used is similar to the method described by
Higgins &
Sharp (1989) CABIOS 5: 151-153. The program can align up to 300 sequences,
each of a
maximum length of 5,000 nucleotides or amino acids. The multiple alignment
procedure
begins with the pairwise alignment of the two most similar sequences,
producing a cluster
of two aligned sequences. This cluster is then aligned to the next most
related sequence or
cluster of aligned sequences. Two clusters of sequences are aligned by a
simple extension
of the pairwise alignment of two individual sequences. The final alignment is
achieved by
a series of progressive, pairwise alignments. The program is run by
designating specific
sequences and their amino acid or nucleotide coordinates for regions of
sequence
comparison and by designating the program parameters. Using PILEUP, a
reference
sequence is compared to other test sequences to determine the percent sequence
identity
relationship using the following parameters: default gap weight (3.00),
default gap length
weight (0.10), and weighted end gaps. PILEUP can be obtained from the GCG
sequence
analysis software package, e.g., version 7.0 (Devereaux et al. (1984) Nuc.
Acids Res. 12:
387-395.
Another preferred example of an algorithm that is suitable for multiple
DNA and amino acid sequence alignments is the CLUSTALW program (Thompson, J.
D.
et al. (1994) Nucl. Acids. Res. 22: 4673-4680). ClustalW performs multiple
pairwise
comparisons between groups of sequences and assembles them into a multiple
alignment
based on homology. Gap open and Gap extension penalties were 10 and 0.05,
respectively. For amino acid alignments, the BLOSUM algorithm can be used as a
protein
weight matrix (Henikoff and Henikoff (1992) Proc. Nat'l Acad. Sci. U.S.A. 89:
10915-
10919).
Making Polypeptides of the Invention
Recombinant methods for producing and isolating interferon homologue
polypeptides of the invention are described above. In addition to recombinant
production,
the polypeptides may be produced by direct peptide synthesis using solid-phase
techniques
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(cf. Stewart et al. (1969) Solid-Phase Peptide Synthesis, W.H. 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 interferon homologues.
Fragments of the interferon homologue polypeptides of the invention, as
discussed in
greater detail above, are also a feature of the invention and may be
synthesized by using
the procedures described above.
Polypeptides of the invention can be produced by introducing into a
population of cells a nucleic acid of the invention, wherein the nucleic acid
is operatively
linked to a regulatory sequence effective to produce the encoded polypeptide,
culturing the
cells in a culture medium to produce the polypeptide, and optionally isolating
the
polypeptide from the cells or from the culture medium.
In another aspect, polypeptides of the invention can be produced by
introducing into a population of cells a recombinant expression vector
comprising at least
one nucleic acid of the invention, wherein the at least one nucleic acid is
operatively
linked to a regulatory sequence effective to produce the encoded polypeptide,
culturing the
cells in a culture medium under suitable conditions to produce the polypeptide
encoded by
the expression vector, and optionally isolating the polypeptide from the cells
or from the
culture medium.
Using Polypeptides
Antibodies
In another aspect of the invention, an interferon homologue polypeptide of
the invention is used to produce antibodies which have, e.g., diagnostic,
prophylactic and
therapeutic uses, e.g., related to the activity, distribution, and expression
of interferon
homologues.
Antibodies to interferon homologues 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
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produced by an Fab expression library. Antibodies, i.e., those which block
receptor
binding, are especially preferred for therapeutic or prophylactic use.
Interferon homologue polypeptides for antibody induction do not require
biological activity; however, the polypeptide or oligopeptide must be
antigenic. Peptides
used to induce specific antibodies may have an amino acid sequence consisting
of at least
amino acids, preferably at least 15 or 20 amino acids. Short stretches of an
interferon
homologue polypeptide may be fused with another protein, such as keyhole
limpet
hemocyanin, and antibody produced against the chimeric molecule.
Methods of producing polyclonal and monoclonal antibodies are known to
10 those of skill in the art, and many antibodies are available. See, e.g.,
Coligan (1991)
Current Protocols in Immunology Wiley/Greene, NY; 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, CA,
and
references cited therein; Goding (1986) Monoclonal Antibodies: Principles and
Practice
(2d ed.) Academic Press, New York, NY; 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 KD of at least
about 0.1
~M, preferably at least about 0.01 ~M or better, and most typically and
preferably, 0.001
~M or better.
Detailed methods for preparation of chimeric (humanized) antibodies can
be found in U.S. Patent 5,482,856. Additional details on humanization and
other antibody
production and engineering techniques can be found in Borrebaeck (ed.) (1995)
Antibody
Engineering, 2°d 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, NJ (Paul).
In one useful embodiment, this invention provides for fully humanized
antibodies against the interferon homologues of the invention. Humanized
antibodies are
especially desirable in applications where the antibodies are used as
prophylactics and
therapeutics in vivo and ex vivo in human patients. Human antibodies consist
of
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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 embodiment, 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.
Ad'uI vants
In one aspect, the interferon homologue polypeptides of the present
invention or fragments thereof are useful as adjuvants to stimulate, enhance,
potentiate, or
augment an immune response related to an antigen when administered together
with the
antigen or after or before delivery of the antigen. In another aspect, the
invention provides
methods for administering one or more of the polypeptides invention described
herein to a
subject.
Therapeutic and Prophylactic Agents
As described in greater detail below, the interferon homologue
polypeptides of the present invention or fragments thereof are useful in the
prophylactic
and/or therapeutic treatment of a variety of diseases, disorders, or medical
conditions.
For example, the invention provides interferon-alpha homologue
polypeptides (and interferon-alpha homologue nucleic acids which encode such
polypeptides) that have both antiviral and antiproliferative activities in the
assays
described herein. In one aspect, the invention provides interferon-alpha
homologue
polypeptides (and interferon-alpha homologue nucleic acids which encode such
polypeptides) in which the ratio of antiviral activity to antiproliferative
activity is greater
than that of other known interferon-alphas such as those listed in GenBank as
noted
herein. Such polypeptides (and nucleic acids encoding them) are useful in the
therapeutic
and/or prophylactic treatment of various diseases and disorders, such as,
e.g., treatment
regimens for hepatitis B, hepatitis C, HIV, and HSV. In such treatment
regimens, some
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such polypeptides (and nucleic acids encoding them), such as interferon-alpha
homologue
2BA8, offer significant advantages over known interferon-alpha compounds,
since they
likely exhibit lower side effects upon administration than known interferon-
alpha
compounds, such as interferon-alpha 2a, are of higher potency, and thus may
require in
lower dosing and cause fewer immunogenicity effects.
SEQUENCE VARIATIONS
Conservatively Modified Variations
Interferon homologue polypeptides of the present invention include one or
more conservatively modified variations (or "conservative variations" or
conservative
substitutions") of the polypeptide sequences disclosed herein as SEQ 117 N0:36
to SEQ ID
N0:70 and SEQ >D N0:79 to SEQ 117 N0:85. Such conservatively modified
variations
comprise substitutions, additions or deletions which alter, add or delete a
single amino
acid or a small percentage of amino acids (typically less than about 5%, more
typically
less than about 4%, 2%, or 1 %) in any of SEQ ID N0:36 to SEQ ID N0:70 and SEQ
ID
N0:79 to SEQ 117 N0:85.
For example, a conservatively modified variation (e.g., deletion) of the 166
amino acid polypeptide identified herein as SEQ ID N0:36 has a length of at
least about
157 or 158 amino acids, preferably at least about 159 or 160 amino acids, more
preferably
at least about 162 or 163 amino acids, and still more preferably at least
about 164 or 165
amino acids, corresponding to a deletion of less than about 5%, 4%, 2% or 1%
of the
polypeptide sequence, respectively.
Another example of a conservatively modified variation (e.g., a
"conservatively substituted variation") of the polypeptide identified herein
as SEQ m
N0:36 will contain "conservative substitutions", according to the six
substitution groups
set forth in Table 2 (supra), in up to about 8 residues (i.e., less than about
5%) of the 166
amino acid polypeptide.
The interferon homologue polypeptide sequences of the invention,
including conservatively substituted sequences, can be present as part of
larger
polypeptide sequences such as which occur upon the addition of one or more
domains for
purification of the protein (e.g., poly His segments, FLAG epitope segments,
etc.), e.g.,
where the additional functional domains have little or no effect on the
activity of the
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interferon-alpha portion of the protein, or where the additional domains can
be removed
by post synthesis processing steps such as by treatment with a protease.
In another embodiment, interferon homologue polypeptides of the present
invention comprise the following sequence, identified herein as SEQ )D N0:71:
CDLPQTHSLG-X1 ~-Xlz-RA-Xls-X,6-LL-X»-QM-Xzz-R-Xz4-S-Xz6-FSCLKDR-X34-
DFG-X3$-P-X4o-EEFD-X4s-X46-X4~-FQ-Xso-Xs 1-QAI-Xss-Xs6-Xs7-~-X6o-X61-QQT~-
X6~-FSTK-X~z-SS-X~s-X~6-W-X~g-X~9-X8o-LL-X83-K-X$s-Xg~-T-Xg$-L-X9o-QQLN-X9s-
LEAC V -X i o 1-Q-X 1 o3-V-X 1 os-X ~ o6-X ~ orX 1 os-TPLMN-X, , 4-D-X 116-ILA
V-X ~ z ~ -KY-X 1 z4-
QRTTLYL-Xl3z-E-Xls4-KYSPC-Xl4o-WEVVRAEIMRSFSFSTNLQKRLRRKE, or a
conservatively substituted variation thereof, where Xl > is N or D; Xlz is R,
S, or K; Xls is
LorM;Xl6isI,M,orV;Xl9isAorG;XzzisGorR;Xz4isIorT;Xz6isPorH;X34is
H, Y or Q; X3g is F or L; X4o is Q or R; X4s is G or S; X46 is N or H; X4~ is
Q or R; Xso is
K or R; Xsl is A or T; Xss is S or F; Xs6 is V or A; Xs~ is L or F; X6o is M
or I; X61 is I or
M;X6~isLorF;X~zisDorN;X~sisAorV;X~6isAorT;X~BisEorD;X~9isQorE;
Xgo is S, R, T, or N; X83 is E or D; Xgs is F or L; X86 is S or Y; X8g is E or
G; X9o is Y, H,
N;X9sisD,E,orN;Xlo1 isI,M,orV;XlosisEorG;XlosisGorW;Xlo6isVorM;
Xlo~isE,G,orK;XloBisEorG;X114isV,E,orG;X116isSorP;XlzlisKorR;Xlz4is
F or L; Xl3z is T, I, or M; X134 is K or R; and Xl4o is A or S; or a fragment
of said SEQ >D
N0:71. As defined above, a conservatively modified variation of the sequence
of SEQ ID
N0:71 can include up to a total of about 8 amino acid deletions, insertions,
or conservative
substitutions in the 166 amino acid polypeptide, excluding the positions
designated X in
SEQ ID N0:71, which correspond to the amino acid explicitly defined.
As an example, if four conservative substitutions were localized in the
subsequence corresponding to amino acids 141-166 of SEQ 117 N0:71, examples of
conservatively substituted variations of this subsequence,
WEVVR AEIMR SFSFS TNLQK RLRRKE, include:
WEVVR SEIMR SFSYS TNLQR RLRRKD and
WELVR AEIVR SFSFS TNLNK RLRKKE, and the like, where the conservative
substitutions are underlined.
A feature of the invention is an interferon homologue polypeptide
comprising at least about 20, usually at least about 25, typically at least
about 30, 40, 50,
60, 70, 80, 90, or 100 contiguous amino acids of any one of SEQ ID NOS:36-71
or SEQ
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ID NOS:79-85. In other embodiments, the polypeptide typically comprises at
least about
100, 110, 120, 125, 130, 140, 150, 155, 158, 160, 163, 164, or 165 contiguous
amino acids
of any one of SEQ ID NOS:36-70 or SEQ ID NOS:79-85.
In other embodiments, the interferon homologue polypeptide of the
invention comprises an amino acid sequence comprising one or more of amino
acid
residues (Tyr or Gln)34, G1y37, Phe38, Lys7l, A1a76, Tyr90, I1e132, Arg134,
Phe152,
Lys160, and G1u166, wherein the numbering of the amino acids corresponds to
the
numbering of amino acids in the amino acid sequence of SEQ ID N0:36. In a
preferred
embodiment, the interferon homologue polypeptide comprises an amino acid
sequence
comprising at least 150, 155, or 166 contiguous amino acid residues of any one
of SEQ ID
NOS:36-70, further comprising Lys160 and Glul66, wherein the numbering of the
amino
acids corresponds to the numbering of amino acids in the amino acid sequence
of SEQ ID
N0:36. Some such polypeptides also exhibit an antiproliferative activity of at
least about
8.3x106 units/milligram in the human Daudi cell line - based assay, or an
antiviral activity
of at about least 2.1x10' units/milligram (mg) in the human WISH cell/EMCV-
based
assay.
DEFINING POLYPEPT117ES BY IMMUNOREACTIVITY
Because the polypeptides of the invention provide a variety of new
polypeptide sequences as compared to other alpha interferon homologues, the
polypeptides also provide a new structural features which can be recognized,
e.g., in
immunological assays. The generation of antisera which specifically binds the
polypeptides of the invention, as well as the polypeptides which are bound by
such
antisera, are features of the invention.
The invention includes interferon-alpha homologue polypeptides that
specifically bind to or that are specifically immunoreactive with an antibody
or antisera
generated against an immunogen comprising an amino acid sequence selected from
one or
more of SEQ >D N0:36 to SEQ )D N0:70, SEQ ID N0:71, and SEQ ID N0:79 to SEQ
ID N0:85. To eliminate cross-reactivity with other interferon-alpha
polypeptides, e.g.,
known interferon-alpha polypeptides, the antibody or antisera (or antiserum)
is subtracted
with available known alpha interferons, such as those polypeptides encoded by
nucleic
acids represented by GenBank accession numbers J00210 (alpha-D), J00207 (Alpha-
A),
X02958 (Alpha-6), X02956 (Alpha-5), V00533 (alpha-H), V00542 (alpha-14),
V00545
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(IFN-1B), X03125 (alpha-8), X02957 (alpha-16), V00540 (alpha-21), X02955
(alpha-4b),
V00532 (alpha-C), X02960 (alpha-7), X02961 (alpha-10 pseudogene), 80067 (Gx-
1),
I01614, I01787, I07821, M12350 (alpha-F), and M38289, V00549 (alpha-2a), and
I08313
(alpha-Conl), or any other known interferon-alpha polypeptides (typically
referred to as
the "control alpha interferon polypeptides"). Where the accession number
corresponds to
a nucleic acid, a polypeptide encoded by the nucleic acid is generated and
used for
antibody/antisera subtraction purposes. Where the nucleic acid corresponds to
a non-
coding sequence, e.g., a pseudo gene, an amino acid which corresponds to the
reading
frame of the nucleic acid is generated (e.g., synthetically), or is minimally
modified to
include a start codon for recombinant production.
In one typical format, the immunoassay uses a polyclonal antiserum which
was raised against one or more polypeptides comprising one or more of the
amino acid
sequences corresponding to one or more of: SEQ ID N0:36 to SEQ ID N0:70, SEQ m
N0:71, and SEQ ID N0:79 to SEQ ll7 N0:85, or a substantial subsequence thereof
(i.e.,
at least about 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, or 98% or more of the
full
length sequence provided). The full set of potential polypeptide immunogens
derived
from one or more of SEQ ID N0:36 to SEQ >D N0:70, SEQ ID N0:7, and SEQ 117
N0:79 to SEQ >D N0:85 are collectively referred to below as "the immunogenic
polypeptides." The resulting antisera is optionally selected to have low cross-
reactivity
against the control alpha interferon polypeptides and/or other known
interferon
polypeptides and any such cross-reactivity is removed by immunoabsorption with
one or
more of the control alpha interferon polypeptides, prior to use of the
polyclonal antiserum
in the immunoassay.
In order to produce antisera for use in an immunoassay, one or more of the
immunogenic polypeptides is produced and purified as described herein. For
example,
recombinant protein may be produced in a mammalian cell line. An inbred strain
of mice
(used in this assay because results are more reproducible due to the virtual
genetic identity
of the mice) is immunized with the immunogenic polypeptide(s) in combination
with a
standard adjuvant, such as Freund's adjuvant, and a standard mouse
immunization protocol
(see Harlow and Lane (1988) Antibodies, A Laboratory Manual, Cold Spring
Harbor
Publications, New York, for a standard description of antibody generation,
immunoassay
formats and conditions that can be used to determine specific
immunoreactivity).
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Alternatively, one or more synthetic or recombinant polypeptides derived from
the
sequences disclosed herein is conjugated to a carrier protein and used as an
immunogen.
Polyclonal sera are collected and titered against the immunogenic
polypeptide(s) in an immunoassay, for example, a solid phase immunoassay with
one or
more of the immunogenic polypeptides immobilized on a solid support.
Polyclonal
antisera with a titer of 10~ or greater are selected, pooled and subtracted
with the control
alpha interferon polypeptides to produce subtracted pooled titered polyclonal
antisera.
The subtracted pooled titered polyclonal antisera are tested for cross
reactivity against the control alpha interferon polypeptides. Preferably at
least two of the
immunogenic alpha interferon polypeptides are used in this determination,
preferably in
conjunction with at least two of the control alpha interferon polypeptides, to
identify
antibodies which are specificallybound by the immunogenic polypeptides(s).
In this comparative assay, discriminatory binding conditions are determined
for the subtracted titered polyclonal antisera which result in at least about
a 5-10 fold
higher signal to noise ratio for binding of the titered polyclonal antisera to
the
immunogenic alpha interferons as compared to binding to the control alpha
interferons .
That is, the stringency of the binding reaction is adjusted by the addition of
non-specific
competitors such as albumin or non-fat dry milk, or by adjusting salt
conditions,
temperature, or the like. These binding conditions are used in subsequent
assays for
determining whether a test polypeptide is specifically bound by the pooled
subtracted
polyclonal antisera. In particular, test polypeptides which show at least a 2-
5x higher
signal to noise ratio than the control polypeptides under discriminatory
binding conditions,
and at least about a'h signal to noise ratio as compared to the immunogenic
polypeptide(s), shares substantial structural similarity or homology with the
immunogenic
polypeptide as compared to known alpha interferons, and is, therefore a
polypeptide of the
invention.
In another example, immunoassays in the competitive binding format are
used for detection of a test polypeptide. For example, as noted, cross-
reacting antibodies
are removed from the pooled antisera mixture by immunoabsorption with the
control alpha
interferon polypeptides. The immunogenic polypeptide(s) are then immobilized
to a solid
support which is exposed to the subtracted pooled antisera. Test proteins are
added to the
assay to compete for binding to the pooled subtracted antisera. The ability of
the test
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proteins) to compete for binding to the pooled subtracted antisera as compared
to the
immobilized proteins) is compared to the ability of the immunogenic
polypeptide(s)
added to the assay to compete for binding (the immunogenic polypeptides
compete
effectively with the immobilized immunogenic polypeptides for binding to the
pooled
antisera). The percent cross-reactivity for the test proteins is calculated,
using standard
calculations.
In a parallel assay, the ability of the control proteins to compete for
binding
to the pooled subtracted antisera is determined as compared to the ability of
the
immunogenic polypeptide(s) to compete for binding to the antisera. Again, the
percent
cross-reactivity for the control polypeptides is calculated, using standard
calculations.
Where the percent cross-reactivity is at least 5-lOx as high for the test
polypeptides, the
test polypeptides are said to specifically bind the pooled subtracted
antisera.
In general, the immunoabsorbed and pooled antisera can be used in a
competitive binding immunoassay as described herein to compare any test
polypeptide to
the immunogenic polypeptide(s). In order to make this comparison, the two
polypeptides
are each assayed at a wide range of concentrations and the amount of each
polypeptide
required to inhibit 50% of the binding of the subtracted antisera to the
immobilized protein
is determined using standard techniques. If the amount of the test polypeptide
required is
less than twice the amount of the immunogenic polypeptide that is required,
then the test
polypeptide is said to specifically bind to an antibody generated to the
immunogenic
polypeptide, provided the amount is at least about 5-lOx as high as for a
control
polypeptide.
As a final determination of specificity, the pooled antisera is optionally
fully immunosorbed with the immunogenic polypeptide(s) (rather than the
control
polypeptides) until little or no binding of the resulting immunogenic
polypeptide
subtracted pooled antisera to the immunogenic polypeptide(s) used in the
immunoabsorption is detectable. This fully immunosorbed antisera is then
tested for
reactivity with the test polypeptide. If little or no reactivity is observed
(i.e., no more than
2x the signal to noise ratio observed for binding of the fully immunosorbed
antisera to the
immunogenic polypeptide), then the test polypeptide is specifically bound by
the antisera
elicited by the immunogenic protein.
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ANTIPROLIFERATIVE PROPERTIES OF INTERFERON HOMOLOGUES
The effect of interferon homologues on cellular growth was examined in a
human Daudi cell line - based assay as described in Example 1. Fig. 2 shows
the
antiproliferative activity of exemplary interferon homologues of the invention
comprising
amino acid sequences SEQ ID N0:36 to SEQ ID N0:54, in comparison to control
interferons, human IFN-alpha 2a and consensus human IFN-alpha (Conl). The
graph
shows the number of Units of activity per milligram (mg) of interferon test
sample (Y
axis) for a set of exemplary interferon alpha homologues, each of which is
designated with
a name (clone name) on the X axis, compared with that of human IFN-alpha 2a
and
consensus human IFN-alpha. These results indicate that compositions comprising
an
interferon-alpha homologue of the present invention can be used in methods to
inhibit or
reduce proliferation of tumor cells, including, but not limited to: human
carcinoma cells,
hematopoietic cancer cells, human leukemia cells, human lymphoma cells, and
human
melanoma cells. Inhibition can be performed in vitro (useful, e.g., in a
variety of
proliferation assays), ex vivo or in vivo (useful, e.g., as a therapeutic or
prophylactic
agent).
Interferon-alpha homologues of the present invention show diverse activity
patterns against a variety of cancer cell lines (see, e.g., Example 2). An in
vitro cell line
screen (as described in, e.g., Monks, A. et al. (1991) J. Nat'1 Cancer Inst.
83:757-766) was
used to assay interferon-alpha homologues of the invention for selective
growth inhibition
and/or cell killing of particular cancer cell lines. The human cancer cell
lines screened
(see, e.g., Example 2, Table 3) include leukemias, melanomas, and cancers of
the lung,
colon, brain, central nervous system, ovary, breast, prostate, and kidney.
Three activity parameters were determined in the cancer cell line screen: 1)
GI50 ("growth inhibition at 50%"), a measure of growth inhibition activity, is
the
concentration of interferon test sample (IFN alpha homologue or control IFN
alpha) at
which cell growth is inhibited by 50%, as measured by a 50% reduction in the
net
protein/polypeptide increase in the interferon test sample as compared to that
observed in
the control cells (no test sample) at the end of the incubation period; 2) TGI
("total growth
inhibition") a measure of cytostatic activity, is the concentration of
interferon test sample
at which cell growth of a particular cell line is totally inhibited, wherein
the amount of
cellular protein at the end of the incubation period equals the amount of
cellular protein at
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the beginning of the incubation period ; and 3) LC50, a measure of cytotoxic
activity, is
the concentration of interferon test sample at which a 50% reduction in the
measured
amount of cellular protein at the end of the incubation as compared to that at
the beginning
of the incubation period is observed, indicating a net loss of cells following
interferon test
sample addition. Further details of the assay and data analysis procedures are
provided in
Example 2.
The activity parameters of exemplary interferon-alpha homologue 3DA11
(SEQ ID N0:40) against a variety of cancer cell lines are shown in Figs. 3A,
3B, and 3C,
in comparison with the interferon-alpha Conl and human interferon-alpha 2a
controls.
With respect to growth inhibition activity, in particular, homologue 3DA11
and control interferon-alpha Conl showed significant activity against most of
the cell lines
tested, with the interferon-alpha Con 1 exhibiting generally higher activity,
and interferon-
alpha 2a generally exhibiting lower overall activity and in only a subset of
the cell lines
(Fig. 3A).
In contrast, in particular, a pronounced difference was observed in the
cytotoxic and cytostatic activities of homologue 3DA11 in comparison to both
interferon-
Con 1 and human interferon-alpha 2a controls. In the concentration range
tested,
homologue 3DA11 showed significant cytostatic activity against a population of
cells of
eleven of the cell lines, while interferon-Conl showed activity against only a
population
of cells of one of the cell lines, against which homologue 3DA11 was also
active (Fig.
3B). IFN-alpha 2a, on the other hand, was not active in this assay against any
of the
tested cell lines. Homologue 3DA11 thus has a broader cytostatic activity
profile than
consensus human interferon-alpha (Conl) and human interferon-alpha 2a.
Homologue 3DA11 also showed significant cytotoxic activity in
comparison to the interferon-Conl and human interferon-alpha 2a controls (Fig.
3C).
Surprisingly, homologue 3DA11 displayed cytotoxic activity against a
population of cells
of 8 of the cell lines, whereas neither the interferon-Conl nor the interferon-
alpha 2a
controls exhibited measurable activity against a population of cells of any of
the cell lines
at the concentration range employed in the assay. Thus, homologue 3DA11 also
has a
broader cytotoxic activity profile than interferon-Conl and human interferon-
alpha 2a.
Figs. 4A-4D illustrate the cytostatic activity (as reflected by the TGI value)
of exemplary interferon-alpha homologues of the invention. In each figure, the
relative
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cytostatic activity (expressed as -log TGI) against a population of cells of
particular cancer
cell line is plotted for various interferon-alpha homologues and for the two
control
interferons (interferon-Conl and human interferon-alpha 2a).
Of the exemplary homologues tested, homologues 1D3 (SEQ >D N0:54)
and 3DA11 (SEQ >D N0:40), but neither of the control interferons, exhibited
significant
cytostatic activity against a population of cells of leukemia cell line RPMI-
8226 over the
concentration range of the assay (Fig. 4A). In this example, the 1D3 and 3DA11
homologues showed at least about 25-fold higher cytostatic activity against a
population
of the cells (corresponding to a difference in TGI of at least about 1.4 log
units) than did
either of the controls (interferon-Conl or interferon-alpha 2a) against a
population of cells
of the leukemia cell line.
Homologues 1D3, 2G5 (SEQ ll~ N0:45), 6CG3 (SEQ )D N0:52) and
3DA11, but neither of the control interferons, exhibited significant
cytostatic activity
against lung cancer cell line NCI-H23 (Fig. 4B). In this example, the 1D3,
2G5, 6CG3,
and 3DAl l homologues showed at least about 12-fold higher cytostatic activity
a
population of cells of a lung cancer cell line (corresponding to a difference
in TGI of at
least about 1.1 log units) than either interferon-Conl or interferon-alpha 2a
against a
population of cells of the lung cancer cell line.
Homologues 1D3, 2G5, and 3DA11, but neither of the control interferons,
showed significant cytostatic activity against a population of cells of renal
cancer cell line
ACHN (Fig. 4C). In this example, the 1D3, 2G5, and 3DA11 homologues showed at
least
about 35-fold higher cytostatic activity a population of cells of said renal
cancer cell line
(corresponding to a difference in TGI of at least about 1.55 log units) than
either
interferon-Conl or interferon-alpha 2a against a population of cells of renal
cancer cell
line.
Homologues 1D3, 2G5, 3DAll, 2CA5 (SEQ >D N0:42) and 2DB11 (SEQ
>D N0:41), and the interferon-Conl control, but not the interferon alpha-2a
control,
exhibited significant cytostatic activity against a population of cells of an
ovarian cancer
cell line OVCAR-3 (Fig. 4D). In this example, homologue 1D3 showed at least
about 2-
fold higher cytostatic activity (corresponding to a difference in TGI of at
least about 0.3
log units) than interferon-Conl, and the 1D3, 2G5, 3DA11, 2CA5, and 2DB11
homologues showed at least about 40-fold higher cytostatic activity
(corresponding to a
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difference in TGI of at least about 1.6 log units) than interferon-alpha 2a,
against
respective populations of cells of the ovarian cancer cell line.
From the exemplary data provided herein, it is apparent that interferon-
alpha homologues of the invention showed a variety of cytostatic activity
profiles, which
differed significantly from those of the interferon-alpha Conl and interferon
alpha-2a.
The present invention includes an interferon-alpha homologue having
increased cytostatic activity relative to human interferon-alpha 2a or to
consensus human
interferon-alpha, Conl. In various embodiments, the interferon-alpha homologue
has at
least about 2-fold higher cytostatic activity a population of cells of a
cancer cell line (i.e.,
has a TGI value at least about 2-fold lower) than does human interferon-alpha
2a, or has at
least 2-fold higher cytostatic activity than interferon-Conl, against a
population of cells of
one or more cancer cell lines selected from the following: a leukemia cell
line; a
melanoma cell line; a lung cancer cell line; a colon cancer cell line; a
central nervous
system (CNS) cancer cell line; an ovarian cancer cell line; a breast cancer
cell line; a
prostate cancer cell line; and a renal cancer cell line.
In other embodiments, the interferon-alpha homologue has at least about 5-
fold higher cytostatic activity a population of cells of a cancer cell line
(i.e., has a TGI
value at least about 5-fold lower) than does human interferon-alpha 2a, or has
at least
about 5-fold higher cytostatic activity than interferon-Conl, against a
population of cells
of one or more cancer cell lines selected from the following: a leukemia cell
line; a
melanoma cell line; a lung cancer cell line; a colon cancer cell line; a
central nervous
system (CNS) cancer cell line; an ovarian cancer cell line; a breast cancer
cell line; a
prostate cancer cell line; and a renal cancer cell line. In other embodiments,
the
interferon-alpha homologue has at least about 10-fold higher cytostatic
activity a
population of cells of a cancer cell line (i.e., has a TGI value at least
about 10-fold lower)
than does human interferon-alpha 2a, or has at least about 10-fold higher
cytostatic
activity than interferon-Conl, against a population of cells of one or more
cancer cell lines
selected from the following: a leukemia cell line; a melanoma cell line; a
lung cancer cell
line; a colon cancer cell line; a CNS cancer cell line; an ovarian cancer cell
line; a breast
cancer cell line; a prostate cancer cell line; and a renal cancer cell line.
The invention includes an interferon-alpha homologue having increased
cytotoxic activity relative to human interferon-alpha 2a or relative to
interferon-Conl. In
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various embodiments, the interferon-alpha homologue has at least about 2-fold
higher
cytotoxic activity (i.e., has an LC50 value at least about 2-fold lower), at
least 5-fold
higher cytotoxic activity, or at least 10-fold higher cytotoxic activity, than
human
interferon-alpha 2a against a population of cells of one or more cancer cell
lines selected
from the following: a leukemia cell line; a melanoma cell line; a lung cancer
cell line; a
colon cancer cell line; a CNS cancer cell line; an ovarian cancer cell line; a
breast cancer
cell line; a prostate cancer cell line; and a renal cancer cell line. In other
embodiments, the
interferon-alpha homologue has at least about 2-fold higher cytotoxic activity
(i.e., has an
LC50 value at least about 2-fold lower), at least about 5-fold higher
cytotoxic activity, or
at least about 10-fold higher cytotoxic activity, than interferon-Conl,
against a population
of cells of at least one cancer cell line selected from: a leukemia cell line;
a melanoma cell
line; a lung cancer cell line; a colon cancer cell line; a CNS cancer cell
line; an ovarian
cancer cell line; a breast cancer cell line; a prostate cancer cell line; and
a renal cancer cell
line.
The invention includes an interferon-alpha homologue having increased
growth inhibition activity relative to human interferon-alpha 2a or to
interferon-Conl. In
various embodiments, the interferon-alpha homologue has at least about 2-fold
higher
growth inhibition activity (i.e., has a GI50 value at least about 2-fold
lower), at least about
5-fold higher growth inhibition activity, or at least about 10-fold higher
growth inhibition
activity, than human interferon-alpha 2a, against a population of cells of one
or more
cancer cell lines selected from: a leukemia cell line; a melanoma cell line; a
lung cancer
cell line; a colon cancer cell line; a CNS cancer cell line; an ovarian cancer
cell line; a
breast cancer cell line; a prostate cancer cell line; and a renal cancer cell
line. In other
embodiments, the interferon-alpha homologue has at least about 2-fold higher
growth
inhibition activity (i.e., has a GI50 value at least about 2-fold lower), at
least about 5-fold
higher growth inhibition activity, or at least about 10-fold higher growth
inhibition
activity, than interferon-Con 1, against at least one cancer cell line
selected from the
following: a leukemia cell line; a melanoma cell line; a lung cancer cell
line; a colon
cancer cell line; a CNS cancer cell line; an ovarian cancer cell line; a
breast cancer cell
line; a prostate cancer cell line; and a renal cancer cell line.
The discovery set forth herein that interferons (such as the interferon-alpha
homologues described herein) can be evolved, modified, or recombined to
display a
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variety of activity profiles provides an opportunity for evolving and creating
customized
and specific interferon homologues for the treatment of a variety of specific
diseases or
disease conditions, including, e.g., a variety of cancers or related
conditions. For example,
an interferon homologue of the invention optimized to have increased potency
against a
particular target cancer cell type may also be optimized to have
(advantageously) reduced
toxicity towards a non-target cell(s), and thus may produce lower side effects
in the
subject to which the homologue is administered (e.g., patient).
The present invention further provides an opportunity to optimize
interferon homologues against tumor cells taken from a subpopulation of
subjects (e.g.,
mammals or human patients), or even from an individual subject (e.g., mammal
or human
patient), providing therapeutic or prophylactic treatment tailored to the
individual subject.
Optimized interferon homologues of the invention may provide therapeutic or
prophylactic benefit against cancers or related conditions or other interferon-
treatable
disorders or conditions which are otherwise unresponsive to currently-
available interferons
or to other treatment regimes.
ANTIVIRAL PROPERTIES OF INTERFERON HOMOLOGUES
The antiviral activity of interferon homologues of the present invention was
evaluated in a human WISH cell/EMCV assay as described in Example 1. Fig. 2
shows
the antiviral activity of exemplary interferon homologues of the invention
comprising
amino acid sequences SEQ ID N0:36 to SEQ ID N0:54.
Improved in vitro antiviral activity of exemplary IFN-alpha homologues of
the invention has been shown to be maintained in vivo in a murine model
system. Two
IFN-alpha homologues of the invention, designated CH2.2 and CH2.3 (SEQ ID
NOS:84
and 85, respectively), were previously shown to have about 206,000-fold and
138,000-fold
improved antiviral activity, respectively, compared to human IFN-alpha 2a in a
murine
cell-based assay, as well as significantly higher activity in the same assay
as compared to
native murine interferons (Chang et al. (1999) Nature Biotechnol. 17:793-797).
As
described in Example 3 below, Balb/c mice challenged with a lethal dose of
vesicular
stomatitis virus (VSV) were administered varying doses of IFN-alpha
homologues,
designated CH2.2 and CH2.3, native murine interferon Mu-IFN alpha 4, and human
IFN-
alpha 2a. The high in vitro activity correlated well with the observed in vivo
activity (Fig.
S). The CH2.2 and CH2.3 homologues were fully effective in protecting mice
from the
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lethal viral challenge, while the same dosage of the native murine interferon
was partially
effective and the human IFN-alpha 2a was completely ineffective. These results
indicate
that compositions comprising interferon homologues of the present invention
can be used
in methods to inhibit viral replication in subjects infected with viruses
including, but not
limited to: human immunodeficiency virus (HIV), hepatitis C virus (HCV),
herpes simplex
virus (HSV), and hepatitis B virus (HBV). Inhibition can be performed in vitro
( useful,
e.g., in a variety of antiviral assays), ex vivo (useful e.g., as a
therapeutic or prophylactic
agent in ex vivo methods discussed herein), or in vivo ( useful, e.g., as a
therapeutic or
prophylactic agent in in vivo methods discussed herein).
INTERFERON HOMOLOGUES IN THE TREATMENT OF AUTOIMMUNE AND
OTHER IMMUNE-RELATED DISORDERS
Compositions of the present invention can be used to therapeutically or
prophylactically treat and thereby alleviate a variety of immune system-
related disorders
characterized by hyper- or hypo-active immune system function or other
features. Such
disorders include hyperallergenicity and autoimmune disorders, such as
multiple sclerosis,
type I (insulin dependent) diabetes mellitus, lupus erythematosus, amyotrophic
lateral
sclerosis, Crohn's disease, rheumatoid arthritis, stomatitis, asthma,
allergies, psoriasis and
the like.
THERAPEUTIC AND PROPHYLACTIC COMPOSITIONS
Therapeutic or prophylactic compositions comprising one or more
interferon homologue polypeptides or nucleic acids of the invention are tested
in
appropriate in vitro, ex vivo, and in vivo animal models of disease, to
confirm efficacy,
tissue metabolism, and to estimate dosages, according to methods well known in
the art.
In particular, dosages can be determined by activity comparison of the alpha
interferon
homologues to existing alpha interferon therapeutics or prophylactics, i.e.,
in a relevant
assay. In one aspect, the invention provides methods comprising administering
one or
more interferon homologue nucleotides or polypeptides of the invention (or
fragments
thereof) described above to a mammal, including, e.g., a human, primate,
mouse, pig, cow,
goat, rabbit, rat, guinea pig, hamster, horse, sheep; or a non-mammalian
vertebrate such as
a bird (e.g., a chicken or duck) or a fish, or invertebrate, as described in
greater detail
below. Such compositions typically comprise one or more interferon homologue
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nucleotides or polypeptides of the invention (or fragments thereof) and an
excipient,
including, e.g., a pharmaceutically acceptable excipient.
In one aspect, a composition of the invention is produced by digesting one
or more nucleic acids of the invention (or fragments thereof) with a
restriction
endonuclease, an RNase, or a DNase.
In another aspect of the invention, compositions produced by incubating
one or more nucleic acids described above in the presence of
deoxyribonucelotide
triphosphates and a nucleic acid polymerase, e.g., a thermostable polymerase,
are
provided.
The invention also includes compositions comprising two or more nucleic
acids described above. The composition may comprise a library of nucleic
acids, where
the library contains at least about 5, 10, 20, 50, 100, 150, or 200 or more
such nucleic
acids.
Administration is by any of the routes normally used for introducing a
molecule into ultimate contact with blood or tissue cells. The interferon-
alpha
homologues of the invention are administered in any suitable manner,
preferably with
pharmaceutically acceptable carriers. Suitable methods of administering such
interferon
homologues in the context of the present invention to a patient are available,
and, although
more than one route can be used to administer a particular composition, a
particular route
can often provide a more immediate and more effective reaction than another
route.
Pharmaceutically acceptable carriers are determined in part by the
particular composition being administered, as well as by the particular method
used to
administer the composition. Accordingly, there is a wide variety of suitable
formulations
of pharmaceutical compositions of the present invention.
Polypeptide compositions can be administered for any of the prophylactic,
therapeutic, and diagnostic methods described herein by a number of routes
including, but
not limited to oral, intravenous, intraperitoneal, intramuscular, transdermal,
subcutaneous,
topical, sublingual, vaginal, or rectal means, or by inhalation. Interferon
homologue
polypeptide compositions can also be administered via liposomes. Such
administration
routes and appropriate formulations are generally known to those of skill in
the art.
The interferon homologue polypeptide or nucleic acid, alone or in
combination with other suitable components, can also be made into aerosol
formulations
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(i.e., they can be "nebulized") to be administered via inhalation. Aerosol
formulations can
be placed into pressurized acceptable propellants, such as
dichlorodifluoromethane,
propane, nitrogen, and the like.
Formulations suitable for parenteral administration, such as, for example,
by intraarticular (in the joints), intravenous, intramuscular, intradermal,
intraperitoneal,
and subcutaneous routes, include aqueous and non-aqueous, isotonic sterile
injection
solutions, which can contain antioxidants, buffers, bacteriostats, and solutes
that render the
formulation isotonic with the blood of the intended recipient, and aqueous and
non-
aqueous sterile suspensions that can include suspending agents, solubilizers,
thickening
agents, stabilizers, and preservatives. The formulations of packaged nucleic
acid can be
presented in unit-dose or mufti-dose sealed containers, such as ampules and
vials.
Parenteral administration and intravenous administration are preferred
methods of administration. In particular, the routes of administration already
in use for
existing alpha interferon therapeutics or prophylactics, along with
formulations in current
use, are preferred routes of administration and formulation for the alpha
interferon
homologue polypeptide and nucleic acids of the invention.
Cells transduced with the interferon homologue nucleic acids as described
above in the context of ex vivo or in vivo therapy can also be administered
intravenously or
parenterally as described above. It will be appreciated that the delivery of
cells to subjects
(e.g., human patients) is routine, e.g., delivery of cells to the blood via
intravenous or
intraperitoneal administration.
The dose of interferon homologue polypeptide or nucleic acid of the
invention administered to a subject (e.g., patient), in the context of the
present invention is
sufficient to effect a beneficial therapeutic or prophylactic response in the
subject (e.g.,
patient) over time, or to inhibit infection by a pathogen, depending on the
application. The
dose will be determined by the efficacy of the particular vector, or
formulation, and the
activity interferon homologue employed and the condition of the patient, as
well as the
body weight or surface area of the patient to be treated. The size of the dose
also will be
determined by the existence, nature, and extent of any adverse side-effects
that accompany
the administration of a particular vector, formulation, transduced cell type
or the like in a
particular patient.
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In the therapeutic and prophylactic treatment methods of the invention
described herein, an effective amount of an interferon-alpha nucleic acid
(e.g., DNA or
mRNA) of the invention (e.g., nucleic acid dosage) will generally be in the
range of, e.g.,
from about 0.05 microgram/kilogram (kg) to about 50 mg/kg, usually about 0.005-
5
S mg/kg. However, as will be understood, the effective amount of the nucleic
acid (e.g.,
nucleic acid dosage) and/or polpeptide (e.g., polypeptide dosage) will vary in
a manner
apparent to those of ordinary skill in the art according to a number of
factors, including the
activity or potency of the polypeptide, the activity or potency of any nucleic
acid construct
(e.g., vector, promoter, expression system) to be administered, the disease or
condition
(e.g., particular cancer) to be treated, and the subject to which or whom the
nucleic acid is
delivered.
For delivery of some polypeptides, e.g., by delivering nucleic acids
encoding such polypeptides, for example, adequate levels of translation and/or
expression
are achieved with a nucleic acid dosage of, e.g., about O.OOSmglkg to about 5
mg/kg.
Dosages for other polypeptides (and nucleic acids encoding them) having a
known
biological activity can be readily determined by those of skill in the art
according to the
factors noted above. Dosages used for other known interferon-alphas for
particular
diseases provide guidelines for determining dosage and treatment regimen for a
nucleic
acid or polypeptide of the invention. An effective amount of an interferon-
alpha
homologue polypeptide may be in the range of from about 1 microgram to about 1
milligram, and more typically from about 1 microgram to about 100 micrograms.
A composition for use in therapeutic and prophylactic treatment methods of
the invention described herein may comprise, e.g., a concentration of an
interferon-alpha
homologue nucleic acid (e.g., DNA or mRNA) of the invention of from about 0.1
microgram/milliliter (ml) to about 20 mg/ml and a pharmaceutically acceptable
Garner
(e.g., aqueous carrier).
A composition for use in therapeutic and prophylactic treatment methods of
the invention described herein may comprise, e.g., a concentration of an
interferon-alpha
homologue polypeptide of the invention in an amount as described above and
herein and a
pharmaceutically acceptable Garner (e.g., aqueous Garner).
In determining the effective amount of the vector, cell type, or formulation
to be administered in the treatment or prophylaxis of cancers or viral
diseases, the
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physician evaluates circulating plasma levels, vector/cell/formulation/
interferon
homologue toxicities, progression of the disease, and the production of anti-
vector/interferon homologue antibodies.
The dose administered, e.g., to a 70 kilogram patient will be in the range
equivalent to dosages of currently-used interferon-alpha therapeutic or
prophylactic
proteins, and doses of vectors or cells which produce interferon homologue
sequences are
calculated to yield an equivalent amount of interferon homologue nucleic acid
or
expressed protein. The vectors of this invention can supplement treatment of
cancers and
virally-mediated conditions by any known conventional therapy, including
cytotoxic
agents, nucleotide analogues (e.g., when used for treatment of HIV infection),
biologic
response modifiers, and the like.
For administration, interferon homologues and transduced cells of the
present invention can be administered at a rate determined by the LD-50 of the
interferon
homologue polypeptide or nucleic acid, vector, or transduced cell type, and
the side-
effects of the interferon homologue polypeptides or nucleic acids, vector or
cell type at
various concentrations, as applied to the mass and overall health of the
patient.
Administration can be accomplished via single or divided doses.
For introduction of recombinant alpha-interferon nucleic acid transduced
cells into a subject (e.g., patient), blood samples are obtained prior to
infusion, and saved
for analysis. Between 1 X 106 and 1 X 1012 transduced cells are infused
intravenously
over 60- 200 minutes. Vital signs and oxygen saturation by pulse oximetry are
closely
monitored. Blood samples are obtained 5 minutes and 1 hour following infusion
and
saved for subsequent analysis. Leukopheresis, transduction and reinfusion are
optionally
repeated every 2 to 3 months for a total of 4 to 6 treatments in a one year
period. After the
first treatment, infusions can be performed on a outpatient basis at the
discretion of the
clinician. If the reinfusion is given as an outpatient, the participant is
monitored for at
least 4, and preferably 8 hours following the therapy. Transduced cells are
prepared for
reinfusion according to established methods. See Abrahamsen et al. (1991) J.
Clin.
Apheresis 6:48-53; Carter et al. (1988) J. Clin. Arpheresis 4:113-117;
Aebersold et al.
(1988), J. Immunol. Methods 112:1-7; Muul et al. (1987) J. Immunol. Methods
101:171-
181 and Carter et al. (1987) Transfusion 27:362-365. After a period of about 2-
4 weeks in
culture, the cells should number between 1 X 106 and 1 X 1012. In this regard,
the growth
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characteristics of cells vary from patient to patient and from cell type to
cell type. About
72 hours prior to reinfusion of the transduced cells, an aliquot is taken for
analysis of
phenotype, and percentage of cells expressing the therapeutic or prophylactic
agent.
If a subject (e.g., patient) undergoing infusion of a vector or transduced
cell
or protein formulation develops fevers, chills, or muscle aches, he/she
receives the
appropriate dose of aspirin, ibuprofen, acetaminophen or other pain/fever
controlling drug.
Subjects (e.g., patients) who experience reactions to the infusion such as
fever, muscle
aches, and chills are premedicated 30 minutes prior to the future infusions
with either
aspirin, acetaminophen, or, e.g., diphenhydramine. Meperidine is used for more
severe
chills and muscle aches that do not quickly respond to antipyretics and
antihistamines.
Cell infusion is slowed or discontinued depending upon the severity of the
reaction.
THERAPEUTIC AND PROPHYLACTIC TREATMENT METHODS
The present invention also includes methods of therapeutically or
prophylactically treating a disease or disorder by administering in vivo or ex
vivo one or
more nucleic acids or polypeptides of the invention described above (or
compositions
comprising a pharmaceutically acceptable excipient and one or more such
nucleic acids or
polypeptides) to a subject, including, e.g., a mammal, including, e.g., a
human, primate,
mouse, pig, cow, goat, rabbit, rat, guinea pig, hamster, horse, sheep; or a
non-mammalian
vertebrate such as a bird (e.g., a chicken or duck) or a fish, or
invertebrate.
In one aspect of the invention, in ex vivo methods, one or more cells or a
population of cells of interest of the subject (e.g., tumor cells, tumor
tissue sample, organ
cells, blood cells, cells of the skin, lung, heart, muscle, brain, mucosae,
liver, intestine,
spleen, stomach, lymphatic system, cervix, vagina, prostate, mouth, tongue,
etc.) are
obtained or removed from the subject and contacted with an amount of a
polypeptide of
the invention that is effective in prophylactically or therapeutically
treating the disease,
disorder, or other condition. The contacted cells are then returned or
delivered to the
subject to the site from which they were obtained o~ to another site (e.g.,
including those
defined above) of interest in the subject to be treated. If desired, the
contacted cells may
be grafted onto a tissue, organ, or system site (including all described
above) of interest in
the subject using standard and well-known grafting techniques or, e.g.,
delivered to the
blood or lymph system using standard delivery or transfusion techniques.
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The invention also provides in vivo methods in which one or more cells or a
population of cells of interest of the subject are contacted directly or
indirectly with an
amount of a polypeptide of the invention effective in prophylactically or
therapeutically
treating the disease, disorder, or other condition. In direct
contact/administration formats,
the polypeptide is typically administered or transferred directly to the cells
to be treated or
to the tissue site of interest (e.g., tumor cells, tumor tissue sample, organ
cells, blood cells,
cells of the skin, lung, heart, muscle, brain, mucosae, liver, intestine,
spleen, stomach,
lymphatic system, cervix, vagina, prostate, mouth, tongue, etc.) by any of a
variety of
formats, including topical administration, injection (e.g., by using a needle
or syringe), or
vaccine or gene gun delivery, pushing into a tissue, organ, or skin site. The
polypeptide
can be delivered, for example, intramuscularly, intradermally, subdermally,
subcutaneously, orally, intraperitoneally, intrathecally, intravenously, or
placed within a
cavity of the body (including, e.g., during surgery), or by inhalation or
vaginal or rectal
administration.
In in vivo indirect contact/administration formats, the polypeptide is
typically administered or transferred indirectly to the cells to be treated or
to the tissue site
of interest, including those described above (such as, e.g., skin cells, organ
systems,
lymphatic system, or blood cell system, etc.), by contacting or administering
the
polypeptide of the invention directly to one or more cells or populatiowof
cells from
which treatment can be facilitated. For example, tumor cells within the body
of the
subject can be treated by contacting cells of the blood or lymphatic system,
skin, or an
organ with a sufficient amount of the polypeptide such that delivery of the
polypeptide to
the site of interest (e.g., tissue, organ, or cells of interest or blood or
lymphatic system
within the body) occurs and effective prophylactic or therapeutic treatment
results. Such
contact, administration, or transfer is typically made by using one or more of
the routes or
modes of administration described above.
In another aspect, the invention provides ex vivo methods in which one or
more cells of interest or a population of cells of interest of the subject
(e.g., tumor cells,
tumor tissue sample, organ cells, blood cells, cells of the skin, lung, heart,
muscle, brain,
mucosae, liver, intestine, spleen, stomach, lymphatic system, cervix, vagina,
prostate,
mouth, tongue, etc.) are obtained or removed from the subject and transformed
by
contacting said one or more cells or population of cells with a polynucleotide
construct
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comprising a target nucleic acid sequence of the invention that encodes a
biologically
active polypeptide of interest (e.g., a polypeptide of the invention) that is
effective in
prophylactically or therapeutically treating the disease, disorder, or other
condition. The
one or more cells or population of cells is contacted with a sufficient amount
of the
polynucleotide construct and a promoter controlling expression of said nucleic
acid
sequence such that uptake of the polynucleotide construct (and promoter) into
the cells)
occurs and sufficient expression of the target nucleic acid sequence of the
invention results
to produce an amount of the biologically active polypeptide effective to
prophylactically
or therapeutically treat the disease, disorder, or condition. The
polynucleotide construct
may include a promoter sequence (e.g., CMV promoter sequence) that controls
expression
of the nucleic acid sequence of the invention and/or, if desired, one or more
additional
nucleotide sequences encoding at least one or more of another polypeptide of
the
invention, a cytokine, adjuvant, or co-stimulatory molecule, or other
polypeptide of
interest.
Following transfection, the transformed cells are returned, delivered, or
transferred to the subject to the tissue site or system from which they were
obtained or to
another site (e.g., tumor cells, tumor tissue sample, organ cells, blood
cells, cells of the
skin, lung, heart, muscle, brain, mucosae, liver, intestine, spleen, stomach,
lymphatic
system, cervix, vagina, prostate, mouth, tongue, etc.) to be treated in the
subject. If
desired, the cells may be grafted onto a tissue, skin, organ, or body system
of interest in
the subject using standard and well-known grafting techniques or delivered to
the blood or
lymphatic system using standard delivery or transfusion techniques. Such
delivery,
administration, or transfer of transformed cells is typically made by using
one or more of
the routes or modes of administration described above. Expression of the
target nucleic
acid occurs naturally or can be induced (as described in greater detail below)
and an
amount of the encoded polypeptide is expressed sufficient and effective to
treat the disease
or condition at the site or tissue system.
In another aspect, the invention provides in vivo methods in which one or
more cells of interest or a population of cells of the subject (e.g.,
including those cells and
cells systems and subjects described above) are transformed in the body of the
subject by
contacting the cells) or population of cells with (or administering or
transferring to the
cells) or population of cells using one or more of the routes or modes of
administration
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described above) a polynucleotide construct comprising a nucleic acid sequence
of the
invention that encodes a biologically active polypeptide of interest (e.g., a
polypeptide of
the invention) that is effective in prophylactically or therapeutically
treating the disease,
disorder, or other condition.
The polynucleotide construct can be directly administered or transferred to
cells) suffering from the disease or disorder (e.g., by direct contact using
one or more of
the routes or modes of administration described above). Alternatively, the
polynucleotide
construct can be indirectly administered or transferred to cells) suffering
from the disease
or disorder by first directly contacting non-diseased cells) or other diseased
cells using
one or more of the routes or modes of administration described above with a
sufficient
amount of the polynucleotide construct comprising the nucleic acid sequence
encoding the
biologically active polypeptide, and a promoter controlling expression of the
nucleic acid
sequence, such that uptake of the polynucleotide construct (and promoter) into
the cells)
occurs and sufficient expression of the nucleic acid sequence of the invention
results to
produce an amount of the biologically active polypeptide effective to
prophylactically or
therapeutically treat the disease or disorder, and whereby the polynucleotide
construct or
the resulting expressed polypeptide is transferred naturally or automatically
from the
initial delivery site, system, tissue or organ of the subject's body to the
diseased site,
tissue, organ or system of the subject's body (e.g., via the blood or
lymphatic system).
Expression of the target nucleic acid occurs naturally or can be induced (as
described in
greater detail below) such that an amount of the encoded polypeptide is
expressed
sufficient and effective to treat the disease or condition at the site or
tissue system. The
polynucleotide construct may include a promoter sequence (e.g., CMV promoter
sequence) that controls expression of the nucleic acid sequence and/or, if
desired, one or
more additional nucleotide sequences encoding at least one or more of another
polypeptide
of the invention, a cytokine, adjuvant, or co-stimulatory molecule, or other
polypeptide of
interest.
In each of the in vivo and ex vivo treatment methods as described above, a
composition comprising an excipient and the polypeptide or nucleic acid of the
invention
can be administered or delivered. In one aspect, a composition comprising a
pharmaceutically acceptable excipient and a polypeptide or nucleic acid of the
invention is
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administered or delivered to the subject as described above in an amount
effective to treat
the disease or disorder.
In another aspect, in each in vivo and ex vivo treatment method described
above, the amount of polynucleotide administered to the cells) or subject can
be an
amount sufficient that uptake of said polynucleotide into one or more cells of
the subject
occurs and sufficient expression of said nucleic acid sequence results to
produce an
amount of a biologically active polypeptide effective to enhance an immune
response in
the subject, including an immune response induced by an immunogen (e.g.,
antigen). In
another aspect, for each such method, the amount of polypeptide administered
to cells) or
subject can be an amount sufficient to enhance an immune response in the
subject,
including that induced by an immunogen (e.g., antigen).
In yet another aspect, in an in vivo or in vivo treatment method in which a
polynucleotide construct (or composition comprising a polynucleotide
construct) is used to
deliver a physiologically active polypeptide to a subject, the expression of
the
polynucleotide construct can be induced by using an inducible on- and off-gene
expression
system. Examples of such on- and off-gene expression systems include the Tet-
OnTM
Gene Expression System and Tet-OfITM Gene Expression System (see, e.g.,
Clontech
Catalog 2000, pg. 110-111 for a detailed description of each such system),
respectively.
Other controllable or inducible on- and off-gene expression systems are known
to those of
ordinary skill in the art. With such system, expression of the target nucleic
of the
polynucleotide construct can be regulated in a precise, reversible, and
quantitative manner.
Gene expression of the target nucleic acid can be induced, for example, after
the stable
transfected cells containing the polynucleotide construct comprising the
target nucleic acid
are delivered or transferred to or made to contact the tissue site, organ or
system of
interest. Such systems are of particular benefit in treatment methods and
formats in which
it is advantageous to delay or precisely control expression of the target
nucleic acid (e.g.,
to allow time for completion of surgery and/or healing following surgery; to
allow time for
the polynucleotide construct comprising the target nucleic acid to reach the
site, cells,
system, or tissue to be treated; to allow time for the graft containing cells
transformed with
the construct to become incorporated into the tissue or organ onto or into
which it has been
spliced or attached, etc. )
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INTEGRATED SYSTEMS
The present invention provides computers, computer readable media and
integrated systems comprising character strings corresponding to the sequence
information
herein for the polypeptides and nucleic acids herein, including, e.g., those
sequences listed
herein and the various silent substitutions and conservative substitutions
thereof.
Various methods and genetic algorithms (GOs) known in the art can be
used to detect homology or similarity between different character strings, or
can be used to
perform other desirable functions such as to control output files, provide the
basis for
making presentations of information including the sequences and the like.
Examples
include BLAST, discussed supra.
Thus, different types of homology and similarity of various stringency and
length can be detected and recognized in the integrated systems herein. For
example,
many homology determination methods have been designed for comparative
analysis of
sequences of biopolymers, for spell-checking in word processing, and for data
retrieval
from various databases. With an understanding of double-helix pair-wise
complement
interactions among 4 principal nucleobases in natural polynucleotides, models
that
simulate annealing of complementary homologous polynucleotide strings can also
be used
as a foundation of sequence alignment or other operations typically performed
on the
character strings corresponding to the sequences herein (e.g., word-processing
manipulations, construction of figures comprising sequence or subsequence
character
strings, output tables, etc.). An example of a software package with GOs for
calculating
sequence similarity or homology is BLAST, which can be adapted to the present
invention
by inputting character strings corresponding to the sequences herein.
Similarly, standard desktop applications such as word processing software
(e.g., Microsoft WordTM or Corel WordPerfectTM) and database software (e.g.,
spreadsheet
software such as Microsoft ExcelTM, Corel Quattro ProTM, or database programs
such as
Microsoft AccessTM or ParadoxTM) can be adapted to the present invention by
inputting a
character string corresponding to the interferon alpha homologues of the
invention (either
nucleic acids or proteins, or both). For example, the integrated systems can
include the
foregoing software having the appropriate character string information, e.g.,
used in
conjunction with a user interface (e.g., a GUI in a standard operating system
such as a
Windows, Macintosh or LINUX system) to manipulate strings of characters. As
noted,
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specialized alignment programs such as BLAST can also be incorporated into the
systems
of the invention for alignment of nucleic acids or proteins (or corresponding
character
strings).
Integrated systems for analysis in the present invention typically include a
digital computer with GO software for aligning sequences, as well as data sets
entered into
the software system comprising any of the sequences herein. The computer can
be, e.g., a
PC (Intel x86 or Pentium chip- compatible DOSTM, OS2TM WINDOWSTM WINDOWS
NTTM, WINDOWS95TM, WINDOWS98TM LINUX based machine, a MACINTOSHTM,
Power PC, or a UNIX based (e.g., SUNTM work station) machine) or other
commercially
common computer which is known to one of skill. Software for aligning or
otherwise
manipulating sequences is available, or can easily be constructed by one of
skill using a
standard programming language such as Visualbasic, Fortran, Basic, Java, or
the like.
Any controller or computer optionally includes a monitor which is often a
cathode ray tube ("CRT") display, a flat panel display (e.g., active matrix
liquid crystal
display, liquid crystal display), or others. Computer circuitry is often
placed in a box
which includes numerous integrated circuit chips, such as a microprocessor,
memory,
interface circuits, and others. The box also optionally includes a hard disk
drive, a floppy
disk drive, a high capacity removable drive such as a writeable CD-ROM, and
other
common peripheral elements. Inputting devices such as a keyboard or mouse
optionally
provide for input from a user and for user selection of sequences to be
compared or
otherwise manipulated in the relevant computer system.
The computer typically includes appropriate software for receiving user
instructions, either in the form of user input into a set parameter fields,
e.g., in a GUI, or in
the form of preprogrammed instructions, e.g., preprogrammed for a variety of
different
specific operations. The software then converts these instructions to
appropriate language
for instructing the operation of the fluid direction and transport controller
to carry out the
desired operation.
The software can also include output elements for controlling nucleic acid
synthesis (e.g., based upon a sequence or an alignment of a sequences herein)
or other
operations which occur downstream from an alignment or other operation
performed using
a character string corresponding to a sequence herein.
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In one embodiment, the invention provides an integrated system
comprising a computer or computer readable medium comprising a database having
one or
more sequence records. Each of the sequence records comprises one or more
character
strings corresponding to a nucleic acid or polypeptide or protein sequence
selected from
SEQ ll~ NO:1 to SEQ ID N0:85. The integrated system further comprises a use
input
interface allowing a use to selectively view the one or more sequence records.
In one such
integrated system, the computer or computer readable medium comprises an
alignment
instruction set that aligns the character strings with one or more additional
character
strings corresponding to a nucleic acid or polypeptide or protein sequence.
One such integrated system includes an instruction set that comprises at
least one of the following: a local homology comparison determination, a
homology
alignment determination, a search for similarity determination, and a BLAST
determination. In some embodiments, the system further comprises a readable
output
element that displays an alignment produced by the alignment instruction set.
In another
embodiment, the computer or computer readable medium further comprises an
instruction
set that translates at least one nucleic acid sequence which comprises a
sequence selected
from SEQ 1T7 NO:1 to SEQ ID N0:35 or SEQ ID N0:72 to SEQ ID N0:78 into an
amino
acid sequence. The instruction set may select the nucleic acid by applying a
codon usage
instruction set or an instruction set which determines sequence identity to a
test nucleic
acid sequence.
Methods of using a computer system to present information pertaining to at
least one of a plurality of sequence records stored in a database are also
provided. Each of
the sequence records comprises at least one character string corresponding to
SEQ ID
NO:I to SEQ ID N0:85. The method comprises determining at least one character
string
corresponding to one or more of SEQ >D NO:I to SEQ ID N0:85 or a subsequence
thereof; determining which of the at least one character string of the list
are selected by a
user; and displaying each of the selected character strings, or aligning each
of the selected
character strings with an additional character string. The method may further
comprise
displaying an alignment of each of the selected character strings with an
additional
character string and/or displaying the list.
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KITS
In an additional aspect, the present invention provides kits embodying the
methods, composition, systems and apparatus herein. Kits of the invention
optionally
comprise one or more of the following: (1) an apparatus, system, system
component or
apparatus component as described herein; (2) instructions for practicing the
methods
described herein, and/or for operating the apparatus or apparatus components
herein
and/or for using the compositions herein; (3) one or more alpha interferon
homologue
compositions (such as e.g., compositions comprising at least one interferon
alpha
homologue nucleic acid or polypeptide or fragment thereof, cell, vector, etc.,
of the
invention) or components (interferon alpha homologue nucleic acid or
polypeptide or
fragment thereof, cell, vector, etc., of the invention); (4) a container for
holding one or
more aspects of the invention, including such components or compositions, and
(5)
packaging materials.
In a further aspect, the present invention provides for the use of any
apparatus, apparatus component, composition or kit herein, for the practice of
any method
or assay herein, and/or for the use of any apparatus or kit to practice any
assay or method
herein.
EXAMPLES
EXAMPLE I: PREPARATION AND SCREENING OF SHUFFLED INTERFERON-
ALPHA LIBRARIES
Fragments (25-60 base pairs (bp) in length) of about 20 human interferon-
alpha subspecies genes were prepared by PCR amplification and DNAse treatment,
and
recombined essentially as described in Crameri A. et al. (1998; Nature 15:288-
291), to
produce shuffled interferon-alpha mature coding sequences. Expression
libraries were
prepared by subcloning shuffled interferon-alpha mature coding sequences into
an E. coli
secretion vector. Shuffled interferon polypeptides were expressed as mature
proteins
fused at the C-termini to an E tag (Amersham-Pharmacia) to facilitate
quantitation and
purification from the periplasmic space. E. coli transformants were picked
using a robotic
colony picker (Q-Bot, Genetix Pharmaceuticals) into microtiter plates, and
periplasmic
extracts were prepared.
Periplasmic extracts were assayed for antiproliferative activity on a human
Daudi cell line as described by Scarozza, A.M. et al. (1992) J. Interferon
ReS. 12:35-42.
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Clones exhibiting antiproliferative activity in the Daudi assay were re-
screened and expression levels determined by Western blot using an anti-E tag
antibody
(Amersham-Pharmacia). Clones exhibiting highest activity normalized to
expression
levels were selected for sequencing and were also utilized as substrates for
additional
rounds of shuffling and screening as described above.
Clones from the first and second rounds of shuffling having relatively high
antiproliferative activity by the Daudi assay were subcloned into a CHO
expression vector
(pDEI-1011) in which the E-tag/6-His tag (Amersham-Pharmacia) is fused to the
C-
terminus of the shuffled interferons. Clones were transfected into CHO cells
and stable
cell lines were selected with 1 mg/ml 6418. CHO-expressed mature interferons
were
purified on anti-E tag Sepharose column (Amersham-Pharmacia) and quantitated
by a
Bradford assay (Biorad). CHO-purified shuffled interferons were assayed for
antiproliferative activity by the Daudi assay and for antiviral activity using
a human WISH
cell/EMCV assay as described below.
Human WISH cell / EMCV antiviral assay
WISH cells were seeded to a density of 6 x 104 cells/well in 96-well plates
in 100 u1 RPMI medium (Gibco-BRL) supplemented with 10% fetal calf serum,
penicillin
(100 p.g/ml), and streptomycin (100 pg/ml), and incubated for 24 hours at
37°C. Samples
of interferon-alpha polypeptides in medium (100 ~1 total volume) were added to
wells and
incubated for 3 hours at 37°C under a 5% COZ atmosphere. Dilutions of
EMCV
(encephalomyocarditis virus) were added to wells in 50 p,1 volumes, and
incubated for 24
hours as above. Medium was carefully removed and wells were rinsed 2x with
warm
phosphate-buffered saline (PBS). Neutral red (100 pl/well of 1:50 dilution in
medium)
was added to the wells and incubated for 2 hours as above. Glutaraldehyde (50
p,l/well of
0.5% in PBS) was added and incubated for 30 minutes as above. Wells were
washed 2x in
PBS, and 100 pl/well of a solution of 50% methanol, 1% acetic acid was added.
Absorbance at 540 nanometers (nm) was measured using a microplate reader.
Fig. 2 shows the antiproliferative activity and the antiviral activity of
exemplary
interferon homologues of the invention, in comparison with interferon alpha-2a
and
interferon-alpha Conl. The graph shows the number of Units activity per
milligram of
homologue (Y axis) for a set of exemplary interferon alpha homologues, each of
which is
designated with a "name" on the X axis.
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EXAMPLE 2: IN VITRO CANCER CELL LINE SCREEN
An in vitro cell line screen (as described in, e.g., Monks, A. et al. (1991)
J. Nat'l
Cancer Inst. 83:757-766 (hereinafter "Monks") and
http://dtp.nci.~ov./branches/btb/ivclsp.html, each of which is incorporated
herein by
reference in its entirety for all purposes) was used to assay interferon-alpha
homologues of
the invention for selective growth inhibition and/or cell killing of
particular cancer cell
lines. The 60 human cancer cell lines used (Table 3) include leukemias,
melanomas, and
cancers of the lung, colon, brain, ovary, breast, prostate, central nervous
system, renal
system, and kidney. Human tumor cell lines were grown according to procedures
outlined
in Monks") and http://dtp.nci.~ov./branches/btb/ivclsp.html.
Table 3
Human cancer cell lines screened
Cancer t Cell lines
a
Leukemia CCRF-CEM, HL-60 (TB), K-562, MOLT-4, RPMI-8226,
SR
Colon cancerCOLO 205, HCC-2998, HCT-15, HCT-116, HT29, KM12,
SW-620
CNS cancer SF-268, SF-295, SF-539, SNB-19, SNB-75, U251
Lung cancer A549/ATCC, EKVX, HOP-62, HOP-92, NCI-H23, NCI-H226,
NCI-
H322M, NCI-H460, NCI-H522
Breast cancerMCF-7, NCI/ADR HS578T, MDA-MB-231/ATCC, MDA-MB-435,
MDA-N, BT-549, T-47D
Melanoma LOX IMVI, M14, MALME-3M, SK-MEL-2, SK-MEL-5, SK-MEL-
28, UACC-62, UACC-257
Ovarian cancerIGROV1, OVCAR-3, OVCAR-4, OVCAR-5, OVCAR-8, SK-OV-3
Prostate DU-145, PC-3
cancer
Renal cancer786-0, A498, ACHN, CAKI-1, RXF 393, SN12C, TK-10,
~ UO-31
Briefly, cells were inoculated into 96 well microtiter plates at densities
ranging from about 5,000 to about 40,000 cells/well, depending on the growth
properties
of the particular cell line. After inoculation, the microtiter plates were
incubated for 24
hours (h) at 37 degrees C prior to addition of test samples (e.g., interferon
homologues of
the invention or control interferons). After 24 h, two plates of each cell
line were fixed in
situ with trichloroacetic acid (TCA), to provide a measurement of the cell
population for
each cell line at the time of test sample addition (T°). To the
remaining plates, interferon
samples (affinity-purified from CHO cell supernatants) were added in five 10-
fold serial
dilutions ranging from 10-°'8 to 10-4'g ~,g/ml.
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Following sample addition, the plates were incubated for an additional 6
days. The assay was terminated by addition of TCA.
Cell population was determined by measuring cellular protein in a
quantitative protein dye-binding assay. Sulforhodamine B solution (100 ~,l) at
0.4 % (w/v)
in 1% acetic acid was added to each well, followed by incubation for 10
minutes at room
temperature. Unbound dye was removed by washing five times with 1% acetic acid
and
the plates air- dried. Protein-bound dye was solubilized with 10 milliMolar
(mM) Tris,
and the absorbance read at 515 nanometer (nm) on an automated plate reader .
Seven absorbance measurements were taken for each dose-response assay,
corresponding to: the amount of cellular protein prior to sample addition
(time zero; To),
the amount of cellular protein at the end of the incubation period in the
absence of test
sample (control growth, C), and five measurements corresponding to the amount
of
cellular protein at the end of the incubation period in the presence of each
of the five
concentrations of interferon test sample (test growth in presence of
interferon test sample
at the five concentration levels, T;). These measurements were used to
calculate the
following three parameters for each test sample:
GI50, or "growth inhibition of 50%," is the concentration of interferon test
sample at which cell growth is inhibited by 50%, as measured by a 50%
reduction in the
net protein/polypeptide increase in the interferon test sample as compared to
that observed
in the control cells (no test sample) at the end of the incubation period.
GI50 is calculated
as the concentration of test sample where [(T;-To)/(C-To)] x 100 = 50. See
Fig. 3A.
TGI, or "total growth inhibition," is the concentration of interferon test
sample at which cell growth is totally inhibited, wherein the amount of
cellular protein at
the end of the incubation period equals the amount of cellular protein at the
beginning of
the incubation period. The concentration of interferon test sample that
produces total
growth inhibition (TGI) is calculated as the concentration of test sample
where T; = To.
LC50 is the concentration of interferon test sample at which a 50%
reduction in the measured amount of cellular protein at the end of the
incubation as
compared to that at the beginning of the incubation period is observed,
indicating a net
loss of cells following interferon test sample addition. LC50 is calculated as
the
concentration of test sample where [(T;-To)/To] x 100 = -50.
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If, for a particular test sample, an effect was not achieved or was exceeded
at the concentration range tested, the value for that parameter was expressed
as greater or
less than the maximum or minimum concentration tested.
S EXAMPLE 3: IN VITRO ACTIVITY OF IFN-ALPHA HOMOLOGUES CORRELATES
WITH IN VIVO EFFICACY.
Fragments of human interferon-alpha genes were shuffled and screened for
activity in a murine cell-based antiviral assay as described by Chang et al.
(1999) Nature
Biotechnol. 17:793-797. Interferon-alpha homologues that exhibited over 105-
fold higher
antiviral activity than human interferon-alpha 2a against mouse cells were
isolated. The
antiviral activities of a number of the interferon-alpha homologues even
significantly
exceeded the antiviral activity of native mouse interferons, including Mu-IFN-
alpha 4
(Chang et al., supra). Recursive sequence recombination (e.g., DNA shuffling)
of human
interferon-alpha gene fragments to produce novel interferon alpha homologues
and
subsequent screening of such homologues against murine interferon receptors
resulted in
the identification and isolation of interferon-alpha homologues with activity
optimized for
the distantly related murine species.
A dose-response study in mice was performed to determine if the high
antiviral activity observed in vitro is sustained in vivo. Two of the mouse-
optimized
interferon-alpha homologues, designated herein as CH2.2 and CH2.3 (SEQ ID
NOS:84
and 85, respectively), were used in this study. CH2.2 and CH2.3 were shown to
have
about 138,000-fold and about 206,00-fold higher activity, respectively, than
human
interferon-alpha 2a, and about 2.5-fold and about 1.6-fold higher activity
than native
mouse interferon-alpha 4, in the in vitro mouse cell antiviral assay (Chang et
al., supra).
Groups of Balb/c mice received subcutaneous doses of either phosphate
buffered saline (PBS), interferon-alpha homologue CH2.2, interferon-alpha
homologue
CH2.3, murine IFN-alpha 4, or human interferon-alpha 2a, in daily subcutaneous
doses of
2, 10, or 50 pg (total volume of 50 w1) for four consecutive days. On day 2,
the mice were
exposed to a lethal intranasal dose (ten times the LCSO) of vesicular
stomatitis virus
(VSV). Data is expressed as the number of mice which survive to day 21.
Fig. 5 shows that both of the mouse-optimized interferon-alpha
homologues, CH2.2 and CH2.3, were as effective or more effective than native
murine
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interferon Mu-IFN alpha 4 in protecting mice from VSV. At the concentrations
tested,
human IFN-alpha 2a was nearly completely ineffective in protecting mice from
the virus.
Thus, the in vivo efficacy of the interferon-alpha homologues of the invention
correlates
remarkably well with the antiviral activities observed in the in vitro assays.
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. For example, all the
techniques, methods,
compositions, apparatus and systems described above may be used in various
combinations. All publications, patents, patent applications, 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,
or other
document were individually indicated to be incorporated by reference for all
purposes.
SEQUENCES
SEQ ID Clone Sequence
ID
SEQ)D NO:1 2DH12 TGTGATCTGCCTCAGACCCACAGCCTTGGCAACAGGAGGGCCTTGATG
CTCCTGGCACAAATGGGACGAATCTCTCCTTTCTCCTGCCTGAAGGAC
AGACAAGACTTTGGATTCCCCCAGGAGGAGTTTGATGGCAACCAGTTC
CAGAAGGCTCAAGCCATCTCTGTCCTCCATGAGATGATCCAGCAGACC
TTCAATCTCTTCAGCACAAAGGATTCATCTGCTGCTTGGGAACAGACC
CTCCTAGAA.A.A.ATTTTCCACTGAACTCTACCAGCAGCTGAATGACCTG
GAAGCCTGCGTGATACAGGAGGTAGGGGTGAAAGAGACTCCCCTGATG
AATGTGGACTCCATCCTGGCTGTGAGGAAGTACTTCCAAAGAATCACT
CTTTATCTAATAGAGAGGAAATACAGCCCTTGTGCATGGGAGGTTGTC
AGAGCAGAAATCATGAGATCTTTCTCTTTTTCAACAAACTTGCAA.AAA
AGATTAAGGAGGAAGGAA
SEQ)D N0:2 2CA3 TGTGATCTGCCTCAGACCCACAGCCTTGGTGACAGGAGGGCCATGATA
CTCCTGGCACAAATGGGACGAATCTCTCCTTTCTCCTGCCTGAAGGAC
AGATATGATTTCGGATTCCCCCAGGAGGAGTTTGATGGCAACCAGTTC
CAGAAGGCTCAAGCCATCTCTGTCCTCCATGAGATGATCCAGCAGACC
TTCAATCTCTTCAGCACAAAGGATTCATCTGCTGCTTGGGAACAGAGC
CTCCTAGAA.AP~ATTTTCCACTGAACTTTACCAGCAGCTGAATGAACTG
GAAGCATGTGTGATACAGGAGGTTGGGGTGGGAGAGACTCCCCTGATG
AATGGGGACTCCATCCTGGCTGTGAAGAAGTACTTCCAAAGAATCACT
CTTTATCTAATAGAGAGGAAATACAGCCCTTGTGCATGGGAGGTTGTC
AGAGCAGAAATCATGAGATCTTTCTCTTTTTCAACAAACTTGCAA.A.AA
AGATTAAGGAGGAAGGAA
SEQ)D N0:3 4AB9 TGTGATCTGCCTCAGACCCACAGCCTTGGCAACAGGAGGGCCTTGATA
CTCCTGGCACAAATGGGACGAATCTCTCCTTTCTCCTGCCTGAAGGAC
AGACATGACTTTGGATTCCCCCGGGAGGAGTTTGATGGCAACCAGTTC
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CTCCTGGCACAAATGGGACGAATCTCTCCTTTCTCCTGCCTGAAGGAC
AGACATGACTTTGGATTCCCCCGGGAGGAGTTTGATGGCAACCAGTTC
CAGAAGGCTCAAGCCATCTCTGTCCTCCATGAGATGATGCAGCAGACC
TTCAATCTCTTCAGCACAAAGAACTCATCTGCTGCTTGGGATGAGACC
CTCCTAGAAAAATTTTCCACTGAACTTTACCAGCAACTGAATGAACTG
GAAGCATGTGTGATACAGGAGGTTGGGGTGGAAGAGACTCCCCTGATG
AATGAGGACTCCATCCTGGCTGTGAAGAAATACTTCCAAAGAATCACT
CTTTATCTGACAGAGAAGAAGTATAGCCCTTGTTCCTGGGAGGTTGTC
AGAGCAGAAATCATGAGATCTTTCTCTTTTTCAACAAACTTGCAAAAA
AGATTAAGGAGGAAGGAA
SEQ ~ N0:4 2DA4 TGTGATCTGCCTCAGACCCACAGCCTTGGTAACAGGAGGGCCTTGATG
CTCCTGGCACAAATGGGAAGAATCTCTCCTTTCTCCTGCCTGAAGGAC
AGACAAGACTTTGGATTCCCCCAGGAGGAGTTTGATAGCAACCAGTTC
CAGAAGGCTCAAGCCATCTCTGTCCTCCATGAGATGATGCAGCAGACC
TTCAATCTCTTCAGCACAAAGGACTCATCTGCTGCTTGGGATGAGACC
CTCCTAGAAP~AATTTTCCACTGAACTCTACCAGCAGCTGAATGACCTG
GAAGCCTGCGTGATACAGGAGGTTGGGGTGGAAGAGACCCCCCTGATG
AATGTGGACTCCATCCTGGCTGTGAGGAAGTACTTCCAAAGAATCACT
CTTTATCTAATAGAGAGGAAATACAGCCCTTGTGCATGGGAGGTTGTC
AGAGCAGAAATCATGAGATCTTTCTCTTTTTCAACAAACTTGCAAAA.A
AGATTAAGGAGGAAGGAA
SEQ ~ N0:5 3DA11 TGTGATCTGCCTCAGACCCACAGCCTTGGTAACAGGAGGGCCTTGGTA
CTCCTGGCACAAATGGGAAGAATCTCTCCTTTCTCCTGCCTGAAGGAC
AGATATGATTTCGGATTCCCCCAGGAGGAGTTTGATGGCAACCAGTTC
CAGAAGGCTCAAGCCATCTCTGTCCTCCATGAGATGATCCAGCAGACC
TTCAATCTCTTCAGCACAAAGGATTCATCTGCTGCTTGGGATGAGACC
CTCCTAGAAAAATTTTCCACTGAACTTTACCAGCAGCTGAATGACCTG
GAAGCCTGCGTGATACAGGAGGTTGGGGTGGAAGAGACCCCCCTGATG
AATGAGGACTCCATCCTGGCTGTGAAGAAATACTTCCAAAGAATCACT
CTTTATCTAATAGAGAGGAAATACAGCCCTTGTGCATGGGAGGTTGTC
AGAGCAGAAATCATGAGATCTTTCTCTTTTTCAACAAACTTGCAAAAA
AGATTAAGGAGGAAGGAA
SEQ ~ N0:6 2D811 TGTGATCTGCCTCAGACCCACAGCCTTGGTAACAGGAGGGCCTTGATG
CTCCTGGCACAAATGGGAAGAATCTCTCCTTTCTCCTGCCTGAAGGAC
AGATATGATTTCGGATTCCCCCAGGAGGAGTTTGATGGCAACCAGTTC
CAGAAGGCTCAAGCCATCTCTGTCCTCCATGAGATGATCCAGCAGACC
TTCAATCTCTTCAGCACAAAGGATTCATCTGCTGCTTGGGATGAGACC
CTCCTAGAAAAATTTTCCACTGAACTTTACCAGCAGCTGAATGACTTG
GAAGCCTGTGTGATACAGGAGGTTGGGGTGGAAGAGACTCCCCTGATG
AATGTGGACTCCATCCTGGCTGTGAGGAAGTACTTCCAAAGAATCACT
CTTTATCTAATAGAGAGGAAATACAGCCCTTGTGCATGGGAGGTTGTC
AGAGCAGAAATCATGAGATCTTTCTCTTTTTCAACAAACTTGCAAAA.A
AGATTAAGGAGGAAGGAA
SEQ m N0:7 2CA5 TGTGATCTGCCTCAGACCCACAGCCTTGGTAACAGGAGGGCCTTGATA
CTCCTGGCACAAATGGGACGAATCTCTCCTTTCTCCTGCCTGAAGGAC
AGACAAGACTTTGGATTCCCCCAGGAGGAGTTTGATGGCAACCGGTTC
CAGAAGGCTCAAGCCATCTCTGTCCTCCATGAGATGATCCAGCAGACC
TTCAATCTCTTCAGCACAAAGAACTCATCTGCTGCTTGGGAACAGAGC
CTCCTAGAA.A.A.ATTTTCCACTGAACTCTACCAGCAGCTGAATGACCTG
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GAAGCCTGCGTGATACAGGAGGTTGGGGTGGAAGAGACCCCCCTGATG
AATGAGGACTCCATCCTGGCTGTGAAGAAATACTTCCAAAGAATCACT
CTTTATCTAATAGAGAGGAAATACAGCCCTTGTGCATGGGAGGTTGTC
AGAGCAGAAATCATGAGATCTTTCTCTTTTTCAACAAACTTGCAAAAA
AGATTAAGGAGGAAGGAA
SEQ B7N0:8 2G6 TGTGATCTGCCTCAGACCCACAGCCTTGGTAACAGGAGGGCCTTGATA
CTCCTGGCACAAATGGGAAGAATCTCTCCTTTCTCCTGCCTGAAGGAC
AGACATGACTTTGGATTCCCCCAGGAGGAGTTTGATGGCAACCAGTTC
CAGAAGGCTCAAGCCATCTCTGTCCTCCATGAGATGATCCAGCAGACC
TTCAATCTCTTCAGCACAAAGGACTCATCTGCTACTTGGGAACAGAGC
CTCCTAGAAA.A.ATTTTCCACTGAACTTAACCAGCAGCTGAATGACCTG
GAAGCCTGCGTGATACAGGAGGTTGGGGTGGAAGAGACTCCCCTGATG
AATGTGGACCCCATCCTGGCTGTGAAGAAATACTTCCAAAGAATCACT
CTCTATCTGACAGAGAAGAAATACAGCCCTTGTGCCTGGGAGGTTGTC
AGAGCAGAAATCATGAGATCTTTCTCTTTTTCAACAAACTTGCAA.AAA
AGATTAAGGAGGAAGGAA
SEQ m N0:9 3AH7 TGTGATCTGCCTCAGACCCACAGCCTTGGTAACAGGAGGGCCTTGATA
CTCCTGGCACAAATGCGAAGAATCTCTCCTTTCTCCTGCCTGAAGGAC
AGACATGACTTTGGATTCCCCCAGGAGGAGTTTGATAGCAACCAGTTC
CAGAAGGCTCAAGCCATCTCTGTCCTCCATGAGATGATCCAGCAGACC
TTCAATCTCTTCAGCACAAAGGATTCATCTGCTGCTTGGGAACAGAGC
CTCCTAGAAA.AATTTTCCACTGAACTTCACCAGCAACTGAATGAACTG
GAAGCATGTGTAGTACAGGAGGTTGGGGTGGAAGAGACTCCCCTGATG
AATGAGGACTCCATCCTGGCTGTGAAGAAATACCTCCAAAGAATCACT
CTTTATCTGACAGAGAAGAAGTATAGCCCTTGTGCATGGGAGGTTGTC
AGAGCAGAAATCATGAGATCTTTCTCTTTTTCAACAA.ACTTGCAAAAA
AGATTAAGGAGGAAGGAA
SEQ ~ NO:10 2G5 TGTGATCTGCCTCAGACCCACAGCCTTGGTAACAGGAGGGCCTTGATG
CTCCTGGCACAAATGGGAAGAATCTCTCCTTTCTCCTGCCTGAAGGAC
AGACAAGACTTTGGATTCCCCCAGGAGGAGTTTGATGGCAACCAGTTC
CAGAAGGCTCAAGCCATCTCTGTCCTCCATGAGATGATCCAGCAGACC
TTCAATCTCTTCAGCACAAAGGATTCATCTGCTGCTTGGGAACAGAGC
CTCCTAGAAAAATTTTCCACTGAACTCTACCAGCAGCTGAATGACCTG
GAAGCCTGCGTGATACAGGAGGTTGGGGTGGAAGAGACCCCCCTGATG
AATGTGGACTCCATCCTGGCTGTGAGGAAGTACTTCCAAAGAATCACT
CTTTATCTAATAGAGAGGAAATACAGCCCTTGTGCATGGGAGGTTGTC
AGAGCAGAAATCATGAGATCTTTCTCTTTTTCAACAAACTTGCAAAA.A
AGATTAAGGAGGAAGGAA
SEQ ~ NO:11 2BA8 TGTGATCTGCCTCAGACCCACAGCCTTGGTAACAGGAGGGCCCTGATA
CTCCTGGCACAAATGGGACGAATCTCTCCTTTCTCCTGCCTGAAGGAC
AGATATGATTTCGGATTCCCCCAGGAGGAGTTTGATGGCAACCAGTTC
CAGAAGGCTCAAGCCATCTCTGTCCTCCATGAGATGATCCAGCAGACC
TTCAATCTCTTCAGCACAAAGGATTCATCTGCTGCTTGGGAACAGAGC
CTCCTAGAAAAATTTTCCACTGAACTTTACCAGCAGCTGAATGACCTG
GAAGCCTGCGTGATACAGGAGGTTGGGGTGGAAGAGACCCCCCTAATG
AATGTGGACTCCATCCTGGCTGTGAGGAAGTACTTCCAAAGAATCACT
CTTTATCTAATAGAGAGGAAATACAGCCCTTGTGCATGGGAGGTTGTC
AGAGCAGAAATCATGAGATCTTTCTCTTTTTCAACAAACTTGCAAAAA
AGATTAAGGAGGAAGGAA
111
CA 02385045 2002-03-14
WO 01/25438 PCT/US00/27781
SEQ m N0:12 1F3 TGTGATCTGCCTCAGACCCACAGCCTTGGTAACAGGAGGGCCTTGATA
CTCCTGGGACAAATGGGAAGAATCTCTCATTTCTCCTGCCTGAAGGAC
AGACATGACTTTGGATTCCCCCAGGAGGAGTTTGATGGCAACCAGTTC
CAGAAGGCTCAAGCCATCTCTGTCCTCCATGAGATGATCCAGCAGACC
TTCAACCTCTTCAGCACAAAGGACTCATCTGTTGCTTGGGATGAGAGG
CTTCTAGACAAACTCTATACTGAACTTTACCAGCAGCTGAATGACCTG
GAAGCCTGTGTGATGCAGGAGGTGTGGGTGGGAGGGACTCCCCTGATG
AATGAGGACTCCATCCTGGCTGTGAGAAAATACTTCCAAAGAATCACT
CTCTATCTGACAGAGAAGAAATACAGCCCTTGTGCCTGGGAGGTTGTC
AGAGCAGAAATCATGAGATCTTTCTCTTTTTCAACAAACTTGCAAAA.A
AGATTAAGGAGGAAGGAA
SEQ ~ N0:13 4BE10 TGTGATCTGCCTCAGACCCACAGCCTTGGTAACAGGAGGGCCTTGATA
CTCCTGGCACAGATGGGACGAATCTCTCCTTTCTCCTGCCTGAAGGAC
AGATATGATTTCGGATTCCCCCAGGAGGAGTTTGATGGCAACCAGTTC
CAGAAGGCTCAAGCCATCTCTGTCCTCCATGAGATAATGCAGCAGACC
TTCAATCTCTTCAGCACAAAGAACTCATCTGCTGCTTGGGATGAGACC
CTCCTAGAAP~AATTTTCCACTGAACTTTACCAGCAACTGAATGAACTG
GAAGCATGTGTGATACAGGGGGTTGGGGTGGAAGAGACTCCCCTGATG
AATGAGGACTCCATCTTGGCTGTGAGGAAATACTTCCAAAGAATCACT
CTTTATCTGACAGAGAAGAAGTATAGCCCTTGTTCCTGGGAGGTTGTC
AGAGCAGAAATCATGAGATCTTTCTCTTTTTCAACAAACTTGCAA.A.AA
AGATTAAGGAGGAAGGAA
SEQ ~ N0:14 2DD9 TGTGATCTGCCTCAGACCCACAGCCTTGGTAACAGGAGGGCCTTGATG
CTCCTGGCACAAATGGGAAGAATCTCCCCTTTCTCCTGCCTGAAGGAC
AGATATGATTTCGGATTCCCCCAGGAGGAGTTTGATGGCAACCAGTTC
CAGAAGGCTCAAGCCATCTCTGTCCTCCATGAGATGATCCAGCAGACC
TTCAATCTCTTCAGCACAAAGGATTCATCTGCTGCTTGGGAACAGAGC
CTCCTAGAAAAATTTTCCACTGGACTCTACCAGCAGCTGAATGACCTG
GAAGCCTGCGTGATACAGGAGGTTGGGGTGGAAGAGACCCCCCTGATG
AATGAGGACTCCATCCTGGCTGTGAAGAAATACTTCCAAAGAATCACT
CTTTATCTGACAGAGAAGAAGTATAGCCCTTGTTCCTGGGAGGTTGTC
AGAGCAGAAATCATGAGATCTTTCTCTTTTTCAACAAACTTGCAAA.A.A
AGATTAAGGAGGAAGGAA
SEQ m NO:15 3CA1 TGTGATCTGCCTCAGACCCACAGCCTTGGCAACAGGAGGGCCTTGATA
CTCCTGGCACAAATGGGAAGAATCTCTCCTTTCTCCTGCCTGAAGGAC
AGACATGACTTTGGATTACCCCAGGAGGAGTTTGATGGCAACCAGTTC
CAGAAGGCTCAAGCCATCTCTGTCCTCCATGAGATGATCCAGCAGACC
TTCAATCTCTTCAGCACAAAGAACTCATCTGCTGCTTGGGATGAGACC
CTCCTAGAA.A.AATTTTCCACTGAACTTTACCAGCAACTGAATAACCTG
GAAGCATGTGTGATACAGGAGGTTGGGATGGAAGAGACTCCCCTGATG
AATGTGGACTCCATCCTGGCTGTGAAGAAATACTTCCAAAGAATCACT
CTTTATCTGACAGAGAAGAAGTATAGCCCTTGTGCCTGGGAGGTTGTC
AGAGCAGAAATCATGAGATCTTTCTCTTTTTCAACAAACTTGCAA.AAA
AGATTAAGGAGGAAGGAA
SEQ ~ N0:16 2F8 TGTGATCTGCCTCAGACCCACAGCCTTGGTAACAGGAGGGCCTTGATA
CTCCTGGCACAAATGGGACGAATCTCTCCTTTCTCCTGCCTGAAGGAC
AGATATGATTTCGGATTCCCCCAGGAGGAGTTTGATGGCAACCAGTTC
CAGAAGGCTCAAGCCATCTCTGTCCTCCATGAGATGATGCAGCAGACC
TTCAATCTCTTCAGCACAAAGAACTCATCTGCTGCTTGGGATGAGACC
112
CA 02385045 2002-03-14
WO 01/25438 PCT/US00/27781
CTCCTAGAAAAATTTTCCACTGAACTTTACCAGCAACTGAATGAACTG
GAAGCATGTGTGATACAGGAGGTTGGGGTGGAAGAGACTCCCCTGATG
AATGAGGACTCCATCCTGGCTGTGAAGAAATACTTCCAAAGAATCACT
CTTTATCTGACAGAGAAGAAGTATAGCCCTTGTTCCTGGGAGGTTGTC
AGAGCAGAAATCATGAGATCTTTCTCTTTTTCAACAAACTTGCAAAA.A
AGATTAAGGAGGAAGGAA
SEQ m N0:17 6CG3 TGTGATCTGCCTCAGACCCACAGCCTTGGTAACAAGAGGGCCATGATG
CTCCTGGCACAAATGGGAAGAACCTCTCCTTTCTCCTGTCTGAAGGAC
AGACATGACTTTGGATTCCCCCAGGAGGAGTTTGATGGCAACCAGTTC
CAGAGGGCTCAAGCCATCTTTGTCCTCCATGAGATGATCCAGCAGACC
TTCAATTTCTTCAGCACAAAGGACTCATCTGCTGCTTGGGAACAGAGC
CTCCTAGAAAAATTTTCCACTGAACTTAACCAGCAGCTGAATGACCTG
GAAGCCTGCGTGATACAGGAAGTTGGGGTGGAAGAGACTCCCCTGATG
AATGAGGACTCCATCCTGGCTGTGAAGAAATACTTCCAAAGAATCACT
CTTTATCTGACAGAGAAGAAATACAGCCCTTGTGCCTGGGAGGTTGTC
AGAGCAGAAATCATGAGATCTTTCTCTTTTTCAACAAACTTGCAAA.A.A
AGATTAAGGAGGAAGGAA
SEQI17 N0:183CG7 TGTGATCTGCCTCAGACCCACAGCCTTGGTAACAGTAGGGCCTTGATG
CTCCTGGCACAAATGGGAAGAATCTCCCCTTTCTCCTGCCTGAAGGAC
AGACATGATTTCGGATTCCCCCAGGAGGAGTTTGATGGCAACCAGTTC
CAGAAGGCTCAAGCCATCTCTGCCTTCCATGAGATGATCCAGCAGACC
TTCAATCTCTTCAGCACAAAGGATTCATCTGCTGCTTGGGAACAGAAC
CTCCTAGAAA.A.ATTTTCCACTGAACTTTACCAGCAACTGAATAACCTG
GAAGCATGTGTGATACAGGAGGTTGGGATGGAAGAGACTCCCCTGATG
AATGTGGACTCCATCCTGGCTGTGAGGAAGTACTTCCAAAGAATCACT
CTTTATCTAATAGAGAGGAAATACAGCCCTTGTGCCTGGGAGGTTGTC
AGAGCAGAAATCATGAGATCTTTCTCTTTTTCAACAAACTTGCAA.AA.A
AGATTAAGGAGGAAGGAA
SEQ ~ N0:19 1D3 TGTGATCTGCCTCAGACCCACAGCCTTGGTAACAGGAGGGCCTTGATA
CTCCTGGCACAAATGGGAAGAATCTCTCATTTCTCCTGCCTGAAGGAC
AGACATGATTTCGGATTCCCCCAGGAGGAGTTTGATGGCCACCAGTTC
CAGAAGACTCAAGCCATCTCTGTCCTCCATGAGATGATCCAGCAGACC
TTCAATCTCTTCAGCACAAAGGACTCATCTGCTGCTTGGGAACAGAGC
CTCCTAGAA.AAATTTTCCACTGAACTTTACCAGCAACTGAATGACCTG
GAAGCATGTGTGATACAGGAGGTTGGGGTGGAAGAGACTCCCCTGATG
AATGAGGACTCCATCCTGGCTGTGAAGAAATACTTCCAAAGAATCACT
CTTTATCTGATGGAGAAGAAATACAGCCCTTGTGCCTGGGAGGTTGTC
AGAGCAGAAATCATGAGATCTTTCTCTTTTTCAACAAACTTGCAAAAA
AGATTAAGGAGGAAGGAA
SEQ ~ N0:20 2G4 TGTGATCTGCCTCAGACCCACAGCCTTGGTAACAGGAGGGCCATGATG
CTCCTGGCACAAATGAGCAGAATCTCTCCTTCCTCCTGTCTGATGGAC
AGACATGACTTTGAATTTCCCCAGGAGGAATTTGATGATAAACAGTTC
CAGAAGGCTCCAGCCATCTCTGTCCTCCATGAGGTGATTCAGCAGACC
TTCAATCTCTTCAGCACAGAGGACTCATCTGCTGCTTGGGAACAGACC
CTCCTAGAAAAATTTTCCACTGAACTTTACCAGCAACTGAATGACCTG
GAAGCATGTGTGATGCAGGAGGAGAGGGTGGGAGAAACTCCCCTGATG
AATGCGGACTCCATCTTGGCTGTGAGGAAATACTTCCAAAGAATCACT
CTTTATCTGACAAAGAAGAAGTATAGCCCTTGTTCCTGGGAGGTTGTC
AGAGCAGAAATCATGAGATCTTTCTCTTTTTCAACAAACTTGCAA.A.AA
113
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AGATTAAGGAGGAAGGAA
SEQ ~ N0:21 lAl TGTGATCTGCCTCAGACCCACAGCCTTGGTAACAGGAGGGCCTTGATA
CTCCTGGCACAAATGGGAAGAATCTCTCATTTCTCCTGCCTGAAGGAC
AGATATGATTTCGGATTCCCCCAGGAGGTGTTTGATGGCAACCAGTTC
CAGAAGGCCCAAGCCATCTCTGCCTTCCATGAGATGATGCAGCAGACC
TTCAATCTCTTCAGCACAGAGGACTCATCTGCTGCTTGGGAACAGAGC
CTCCTAGAAAA.ATTTTCCACTGAACTTCACCAGCAACTGAATGACCTG
GAAGCCTGTGTGATACAGGAGGTTGGGGTGGAAGAGACTCCCCTGATG
AATGAGGACTCCATCCTGGCTGTGAGGAAATACTTTCAAAGAATCACT
CTTTATCTAATGGAGAAGAAATACAGCCCTTGTGCCTGGGAGGTTGTC
AGAGCAGAAATCATGAGATCTTTCTCTTTTTCAACAAACTTGCAAAAA
AGATTAAGGAGGAAGGAA
SEQ ~ N0:22 1D10 TGTGATCTGCCTCAGACCCACAGCCTTGGTAACAGGAGGGCCTTGATA
CTCCTGGCACAAATGGGAAGAATCTCTCATTTCTCCTGCCTGAAGGAC
AGACATGATTTCGGATTCCCCCAGGAGGAGTTTGATGGCCACCAGTTC
CAGAAGACTCAAGCCATCTCTGTCCTCCATGAGATGATCCAGCAGACC
TTCAATCTCTTCAGCACAAAGGACTCATCTGCTGCTTGGGAACAGAGC
CTCCTAGAAA.AATTTTCCACTGAACTTTACCAGCAACTGAATGACCTG
GAAGCATGTGTGATACAGGAGGTTGGGGTGGAAGAGACTCCCCTGATG
AATGAGGACTCCATCCTGGCTGTGAAGAAATACTTCCAAAGAATCACT
CTTTATCTGATGGAGAAGAAATACAGCCCTTGTGCCTGGGAGGTTGTC
AGAGCAGAAATCATGAGATCTTTCTCTTTTTCAACAAACTTGCAAAA.A
AGATTAAGGAGGAAGGAA
SEQ ~ N0:23 1F6 TGTGATCTGCCTCAGACCCACAGCCTTGGTAACAGGAGGACTTTGATG
ATAATGGCACAAATGGGAAGAATCTCTCCTTTCTCCTGCCTGAAGGAC
AGACATGACTTTGGATTTCCCCAGGAGGAGTTTGATGGCAACCAGTTC
CAGAAGGCTCAAGCCATCTCTGTCCTCCATGAGATGATCCAGCAGACC
TTCAATCTCTTCAGCACAAAGGACTCATCTGCTACTTGGGAACAGAGC
CTCCTAGAAAAATTTTCCACTGAACTTAACCAGCAGCTGAATGACCTG
GAAGCCTGCGTGATACAGGAGGCTGGGGTGGAAGAGACTCCCCTGATG
AATGTGGACTCCATCCTGGCTGTGAAGAAATACTTCCAAAGAATCACT
CTTTATCTAACAGAGAAGAAATACAGCCCTTGTGCCTGGGAGGTTGTC
AGAGCAGAAATCATGAGATCTTTCTCTTTTTCAACAAACTTGCAA.AAA
AGATTAAGGAGGAAGGAA
SEQ m N0:24 2A10 TGTGATCTGCCTCAGACCCACAGCCTTGGTAACAGGAGGGCCTTGATA
CTCCTGGCACAAATGGGAAGAATCTCTCATTTCTCCTGCCTGAAGGAC
AGATATGATTTCGGATTCCCCCAGGAGGTGTTTGATGGCAACCAGTTC
CAGAAGGCTCAAGCCATCTCTGCCTTCCATGAGATGATCCAGCAGACC
TTCAATCTCTTCAGCACAAAGGACTCATCTGCTACTTGGGAACAGAGC
CTCCTAGAAAAATTTTCCACTGAACTTTACCAGCAACTGAATAACCTG
GAAGCATGTGTGATACAGGAGGTTGGGGTGGAAGAGACTCCCCTGATG
AATGAGGACTCCATCCTGGCTGTGAGGAAATACTTTCAAAGAATCACT
CTTTATCTGATGGAGAAGAAATACAGCCCTTGTGCCTGGGAGGTTGTC
AGAGCAGAAATCATGAGATCTTTCTCTTTTTCAACAAACTTGCAAAAA
AGATTAAGGAGGAAGGAA
SEQ m N0:25 2C3 TGTGATCTGCCTCAGACCCACAGCCTTGGTAACAGGAGGGCCTTGATA
CTCCTGGCACAAATGGGAAGAATCTCTCCTTTCTCCTGCCTGAAGGAC
AGACATGACTTTGGATTTCCTCAGGAGGAGTTTGATGGCAACCAGTCC
114
CA 02385045 2002-03-14
WO 01/25438 PCT/US00/27781
CAGAAGGCTCAAGCCATCTCTGTCCTCCATGAGATGATCCAGCAGACC
TTCAATCTCTTCAGCACAAAGGACTCATCTGATACTTGGGATGCGACC
CTTTTAGAAAAATTTTCCACTGAACTTAACCAGCAGCTGAATGACCTG
GAAGCCTGCGTGATACAGGAGGTTGGGGTGGAAGAGACCCCCCTGATG
AATGTGGACTCCATCCTGGCTGTGAAGAAATACTTCCAAAGAATCACT
CTTTATCTGACAGAGAAGAAATACAGCCCTTGTGCCTGGGAGGTTGTC
AGAGCAGAAATCATGAGATCTTTCTCTTTTTCAACAAACTTGCAAAAA
AGATTAAGGAGGAAGGAA
SEQ ~ N0:26 2D1 TGTGATCTGCCTCAGACCCACAGCCTTGGTAACAGGAGGGCCTTGATA
CTCCTGGCACAAATGGGACGAATCTCTCCTTTCTCCTGCCTGAAGGAC
AGACAAGACTTTGGATTCCCCCAGGAGGAGTTTGATGGCAACCGGTTC
CAGAAGGCTCAAGCCATCTCTGTCCTCCATGAGATGATCCAGCAGACC
TTCAATCTCTTCAGCACAAAGAACTCATCTGCTGCTTGGGAACAGAGC
CTCCTAGAAAAATTTTCCACTGAACTCTACCAGCAGCTGAATGACCTG
GAAGCCTGCGTGATACAGGAGGTTGGGGTGGAAGAGACCCCCCTGATG
AATGAGGACTCCATCCTGGCTGTGAAGAAATACTTCCAAAGAATCACT
CTTTATCTAATAGAGAGGAAATACAGCCCTTGTGCATGGGAGGTTGTC
AGAGCAGAAATCATGAGATCTTTCTCTTTTTCAACAAACTTGCAA.A.A.A
AGATTAAGGAGGAAGGAA
SEQ ~ N0:27 2D10 TGTGATCTGCCTCAGACCCACAGCCTTGGTAACAGGAGGGCCTTGATA
CTCCTGGCACAAATGGGAAGAGTCTCTCCTTTCTCCTGCCTGAAGGAC
AGACATGACTTTGGATTCCCCCAGGAGGAGTTTGATGGCAACCAGTTC
CAGAAGGCTCAAGCCATCTCTGCCTTCCATGAGATGATCCAGCAGACC
TTCAATCTCTTCAGCACAAAGGACTCATCTGCTACTTGGGAACAGAGC
CTCCTAGAA.AAATTTTCCACTGAACTTTACCAGCAACTGAATAACCTG
GAAGCCTGCGTGATACAGGAGGTTGGGGTGGAAGAGACTCCCCTGATG
AATGTGGACTCCATCCTGGCTGTGAAGAAATACTTCCGAAGAATCACT
CTCTATCTGACAGAGAAGAAATACAGCCCTTGTGCCTGGGAGGTTGTC
AGAGCAGAAATCATGAGATCTTTCTCTTTTTCAACAAACTTGCAAAAA
AGATTAAGGAGGAAGGAA
SEQ ~ N0:28 2D7 TGTGATCTGCCTCAGACCCACAGCCTTGGTAACAGGCGGGCCTTGATA
CTCCTGGCACAAATGGGAAGAATCTCTCCTTTCTCCTGTCTGAAGGAC
AGACATGACTTCAGATTTCCCCAGGAGGAGTTTGATGGCAACCAGTTC
CAGAAGGCTCAAGCCATCTCTGTCCTCCATGAGATGATCCAGCAGACC
TTCAATCTCTTCAGCACAAAGGACTCATCTGCTACTTGGGAACAGAGC
CTCCTAGAAAAATTTTCCACTGAACTTTACCAGCAACTGAATAACCTG
GAAGCTTGCGTGATACAGGAGGTTGGGGTGGAAGAGACTCCCCTGATG
AATGTGGACTCTATCCTGGCTGTGAAGAAATACTTCCAAAGAATCACT
CTTTATCTGACAGAGAGGAAATACAGCCCTTGTGCCTGGGAGGTTGTC
AGAGCAGAAATCATGAGATCTTTCTCTTTTTCAACAAACTTGCAAAAA
AGATTAAGGAGGAAGGAA
SEQ ~ N0:29 2D9 TGTGATCTGCCTCAGACCCACAGCCTTGGTAACAGGAGGGCCTTGATA
CTCCTGGCACAAATGGGAAGAATCTCTCCTTTCTCCTGCCTGAAGGAC
AGACATGACTTTGGATTCCCCCAGGAGGAGTTTGATGGCAACCAGTTC
CAGAAGGCTCAAGCCATCTCTGTCCTCCATGAGATGATCCAGCAGACT
TTCAATCTCTTCAGCACAAAGGACTCATCTGCTACTTGGGAACAGAGC
CTCCTAGAAAAATTTTCCACTGAACTTAACCAGCAGCTGAATGACCTG
GAAGCCTGCGTGATACAGGAGGTTGGGGTGGAAGAGACTCCCCTGGTG
AATGTGGACTCCATCCTGGCTGTGAAGAAATACTTCCAAAGAATCACT
115
CA 02385045 2002-03-14
WO 01/25438 PCT/US00/27781
CTTTATCTGACAGAGAAGAAATACAGCCCTTGTGCCTGGGAGGTTGTC
AGAGCAGAAATCATGAGATCTTTCTCTTTTTCAACAAACTTGCAAA.A.A
AGATTAAGGAGGAAGGAA
SEQ m N0:30 2DA2 TGTGATCTGCCTCAGACCCACAGCCTTGGTAACAGGAGGCCCTTGATA
CTCCTGGCACAAATGGGAAGAATCTCTCCTTTCTCCTGCCTGAAGGAC
AGACAGGACTTCGGATTCCCCCAGGAGGAGTTTGATGGCAACCAGTTC
CAGAAGGCTCAAGCCATCTCTGTCCTCCATGAGATGATGCAGCAGACC
TTCAATCTCTTCAGCACAAAGAACTCATCTGCTGCTTGGGAACAGAGC
CTCCTAGAA.A.A.ATTTTCCACTGAACTCCACCAGCAACTGAATGAACTG
GAAGCATGTGTGATACAGGAGGTTGGGGTGGAAGAGACTCCCCTGATG
AATGTGGACTCCATCCTGGCTGTGAAGAAATACTTCCAAAGAATCACT
CTTTATCTAATAGAGAGGAAATACAGCCCTTGTGCATGGGAGGTTGTC
AGAGCAGAAATCATGAGATCTTTCTCTTTTTCAACAAACTTGCAAAAA
AGATTAAGGAGGAAGGAA
SEQ ll~N0:312DH9 TGTGATCTGCCTCAGACCCACAGCCCTGGTAACAGGAGGGCCTTGATG
CTCCTGGCACAAATGGGACGAATCTCTCCTTTCTCCTGCCTGAAGGAC
AGATATGATTTCGGATTCCCCCAGGGGGAGTTTGATGGCAACCAGTTC
CAGAAGGCTCAAGCCATCTCTGTCCTCCATGAGATGATGCAGCAGACC
TTCAATCTCTTCAGCACAAAGGATTCATCTGCTGCTTGGGAACAGAGC
CTCCTAGAAA.AATTTTCCACTGAACTCTACCGGCAGCTGAATGACCTG
GAAGCCTGTGTGATACAGGAGGTTGGGGTGGAAGAGACCCCCCTGATG
AATGTGGACTCCATCCTGGCTGTGAGGAAGTACTTCCAAAGAATCACT
CTTTATCTGACAGAGAAGAAGCATAGCCCTTGTTCCTGGGAGGTTGTC
AGAGCAGAAATCATGAGATCTTTCTCTTTTTCAACAAACTTGCAAAAA
AGATTAAGGAGGAAGGAA
SEQ ~ N0:32 2611 TGTGATCTGCCTCAGACCCACAGCCTTGGTAACAGGAGGGCCTTGATA
CTCCTGGCACAAATGGGAAGAATCTCTCCTTTCTCCTGCCTGAAGGAC
AGACATGACTTTGGACTTCCCCAGGAGGAGTTTGATGGCAACCAGTTC
CAGAAGACTCAAGCCATCTCTGTCCTCCATGAGATGATCCAGCAGACC
TTCAATCTCTTCAGCACAAAGGACTCATCTGATACTTGGGAACAGAGC
CTCCTAGAAAAATTCTACATTGAACTTTTCCAGCAGCTGAATGACCTG
GAAGCCTGCGTGATACAGGAGGTTGGGGTGGAAGAGACTCCCCTGATG
AATGTGGACTCCATCCTGGCTGTGAGAAAATACTTCCAAAGAATCACT
CTTTATCTGACAGAGGAGAAATACAGCCCTTGTGCCTGGGAGGTTGTC
AGAGCAGAAATCATGAGATCTTTCTCTTTTTCAACAAACTTGCAAAAA
AGATTAAGGAGGAAGGAA
SEQ ~ N0:33 2612 TGTGATCTGCCTCAGACCCACAGCCTTGGTAACAGGAGGACTTTGATG
CTCATGGCACAAATGAGGAGAATCTCTCCTTTCCCCCGCCTGAAGGAC
AGATATGATTTCGGATTCCCCCAGGAGGTGTTTGATGGCAACCAGTTC
CAGAAGGCTCAAGCTATCTTCCTTTTCCATGAGATGATGCAGCAGACC
TTCAATCTCTTCAGCACAAAGAACTCATCTGCTGCTTGGGATGAGACC
CTCCTAGACAAATTCTACACTGAACTCTACCAGCAGCTGAATGACTTG
GAAGCCTGTGTGATGCAGGAGGGGAGGGTGGGAGAAACTCCCCTGATG
AATGCGGACTCCATCTTGGCTGTGAAGAAATACTTCCGAAGAATCACT
CTCTATCTGACAGAGAAGAAATACAGCCCTTGTGCCTGGGAGGCTGTC
AGAGCAGAAATCATGAGATCTTTCTCTTTTTCAACAAACTTGCAAA.A.A
AGATTAAGGAGGAAGGAA
116
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WO 01/25438 PCT/US00/27781
SEQ m N0:34 2H9 TGTGATCTGCCTCAGACCCACAGCCTTGGTAACAGGAGGGCCTTGATA
CTCCTGGCACAAATGGGAAGAATCTCTCCTTTCTCCTGCCTGAAGGAC
AGACATGACTTTGGATTCCCCCAGGAGGAGTTTGATGGCAACCAGTTC
CAGAAGGCTCAAGCCATCTCTGTCCTCCATGAGATGATCCAGCAGACC
TTCAATCTCTTCAGCACAAAGGACTCATCTGCTACTTGGGAACAGAGC
CTCCTAGAAAP~ATTTTCCACTGAACTTAACCAGCAGCTGAATGACCTA
GAAGCCTGTGTGACACAGGAGGTTGGGGTGGAAGAGACTCCCCTGATG
AATGAGGACTCTATCCTGGCTGTGAAGAAATACTTCCAAAGAATCACT
CTTTATCTGACAGAGAAGAAATACAGCCCTTGTGCCTGGGAGGTTGTC
AGAGCAGAAATCATGAGATCTTTCTCTTTTTCAACAAACTTGCAAA.A.A
AGATTAAGGAGGAAGGAA
SEQ ll~ N0:356BC11 TGTGATCTGCCTCAGACCCACAGCCTTGGTAACAGGAGGGCCTTGATA
CTCCTGGCACAAATGGGAAGAATCTCTCCTTTCTCCTGCCTGAAGGAC
AGATATGATTTCGGATTCCCCCAGGAGGAGTTTGATGGCAACCAGCTC
CAGAAGGCTCAAGCCATCTCTGTCCTCCATGAGATGATCCAGCAGACC
TTCAATCTCTTCAGCACAAAGGATTCATCTGCTGCTTGGGAACAGAGC
CTCCTAGAAAAATTTTCCACTGAACTTAACCAGCAGCTGAATGACCTG
GAAGCCTGCGTGATACAGGAGGTTGGAGTGGAAGAGACTCCCCTGATG
AATGTGGACTCCATCCTGGCTGTGAAGAAATACTTCCAAAGAATCACT
CTTTATCTGACAGAGAGGAAATACAGCCCTTGTGCCTGGGAGGTTGTC
AGAGCAGAAATCATGAGATCTTTCTCTTTTTCAACAAACTTGCAA.A.A.A
AGATTAAGGAGGAAGGAA
SEQ ~ N0:36 2DH12 CDLPQTHSLGNRR.ALMLLAQMGRISPFSCLKDRQDFGFPQEEFDGNQF
QKAQAISVLHEMIQQTFNLFSTKDSSAAWEQTLLEKFSTELYQQLNDL
EACVIQEVGVKETPLMNVDSILAVRKYFQRITLYLIERKYSPCAWEW
RAEIMRSFSFSTNLQKRLRRKE
SEQ ~ N0:37 2CA3 CDLPQTHSLGDRRAMILLAQMGRISPFSCLKDRYDFGFPQEEFDGNQF
QKAQAISVLHEMIQQTFNLFSTKDSSAAWEQSLLEKFSTELYQQLNEL
EACVIQEVGVGETPLMNGDSILAVKKYFQRITLYLIERKYSPCAWEW
RAEIMRSFSFSTNLQKRLRRKE
SEQ ~ N0:38 4AB9 CDLPQTHSLGNRRALILLAQMGRISPFSCLKDRHDFGFPREEFDGNQF
QKAQAISVLHEMMQQTFNLFSTKNSSAAWDETLLEKFSTELYQQLNEL
EACVIQEVGVEETPLMNEDSILAVKKYFQRITLYLTEKKYSPCSWEW
RAEIMRSFSFSTNLQKRLRRKE
SEQ ~ N0:39 2DA4 CDLPQTHSLGNRRALMLLAQMGRISPFSCLKDRQDFGFPQEEFDSNQF
QKAQAISVLHEMMQQTFNLFSTKDSSAAWDETLLEKFSTELYQQLNDL
EACVIQEVGVEETPLMNVDSILAVRKYFQRITLYLIERKYSPCAWEW
RAEIMRSFSFSTNLQKRLRRKE
SEQ ~ N0:40 3DA11 CDLPQTHSLGNRRALVLLAQMGRISPFSCLKDRYDFGFPQEEFDGNQF
QKAQAISVLHEMIQQTFNLFSTKDSSAAWDETLLEKFSTELYQQLNDL
EACVIQEVGVEETPLMNEDSILAVKKYFQRITLYLIERKYSPCAWEW
RAEIMRSFSFSTNLQKRLRRKE
SEQ B7 N0:412DB11 CDLPQTHSLGNRRALMLLAQMGRISPFSCLKDRYDFGFPQEEFDGNQF
QKAQAISVLHEMIQQTFNLFSTKDSSAAWDETLLEKFSTELYQQLNDL
EACVIQEVGVEETPLMNVDSILAVRKYFQRITLYLIERKYSPCAWEW
RAEIMRSFSFSTNLQKRLRRKE
SEQ ~ N0:42 2CA5 CDLPQTHSLGNRRALILLAQMGRISPFSCLKDRQDFGFPQEEFDGNRF
QKAQAISVLHEMIQQTFNLFSTKNSSAAWEQSLLEKFSTELYQQLNDL
117
CA 02385045 2002-03-14
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EACVIQEVGVEETPLMNEDSILAVKKYFQRITLYLIERKYSPCAWEW
RAEIMRSFSFSTNLQKRLRRKE
SEQ ~ N0:43 2G6 CDLPQTHSLGNRRALILLAQMGRISPFSCLKDRHDFGFPQEEFDGNQF
QKAQAISVLHEMIQQTFNLFSTKDSSATWEQSLLEKFSTELNQQLNDL
EACVIQEVGVEETPLMNVDPILAVKKYFQRITLYLTEKKYSPCAWEW
RAEIMRSFSFSTNLQKRLRRKE
SEQ B7N0:44 3AH7 CDLPQTHSLGNRRALILLAQMRRISPFSCLKDRHDFGFPQEEFDSNQF
QKAQAISVLHEMIQQTFNLFSTKDSSAAWEQSLLEKFSTELHQQLNEL
EACWQEVGVEETPLMNEDSILAVKKYLQRITLYLTEKKYSPCAWEW
RAEIMRSFSFSTNLQKRLRRKE
SEQ B7 N0:452G5 CDLPQTHSLGNRRALMLLAQMGRISPFSCLKDRQDFGFPQEEFDGNQF
QKAQAISVLHEMIQQTFNLFSTKDSSAAWEQSLLEKFSTELYQQLNDL
EACVIQEVGVEETPLMNVDSILAVRKYFQRITLYLIERKYSPCAWEW
RAEIMRSFSFSTNLQKRLRRKE
SEQ ~ N0:46 2BA8 CDLPQTHSLGNRRALILLAQMGRISPFSCLKDRYDFGFPQEEFDGNQF
QKAQAISVLHEMIQQTFNLFSTKDSSAAWEQSLLEKFSTELYQQLNDL
EACVIQEVGVEETPLMNVDSILAVRKYFQRITLYLIERKYSPCAWEW
RAEIMRSFSFSTNLQKRLRRKE
SEQ m N0:47 1F3 CDLPQTHSLGNRRALILLGQMGRISHFSCLKDRHDFGFPQEEFDGNQF
QKAQAISVLHEMIQQTFNLFSTKDSSVAWDERLLDKLYTELYQQLNDL
EACVMQEVWVGGTPLMNEDSILAVRKYFQRITLYLTEKKYSPCAWEW
RAEIMRSFSFSTNLQKRLRRKE
SEQ ~ N0:48 4BE10 CDLPQTHSLGNRRALILLAQMGRISPFSCLKDRYDFGFPQEEFDGNQF
QKAQAISVLHEIMQQTFNLFSTKNSSAAWDETLLEKFSTELYQQLNEL
EACVIQGVGVEETPLMNEDSILAVRKYFQRITLYLTEKKYSPCSWEW
RAEIMRSFSFSTNLQKRLRRKE
SEQ ~ N0:49 2DD9 CDLPQTHSLGNRRALMLLAQMGRISPFSCLKDRYDFGFPQEEFDGNQF
QKAQAISVLHEMIQQTFNLFSTKDSSAAWEQSLLEKFSTGLYQQLNDL
EACVIQEVGVEETPLMNEDSILAVKKYFQRITLYLTEKKYSPCSWEW
RAEIMRSFSFSTNLQKRLRRKE
SEQ B7N0:50 3CA1 CDLPQTHSLGNRRALILLAQMGRISPFSCLKDRHDFGLPQEEFDGNQF
QKAQAISVLHEMIQQTFNLFSTKNSSAAWDETLLEKFSTELYQQLNNL
EACVIQEVGMEETPLMNVDSILAVKKYFQRITLYLTEKKYSPCAWEW
RAEIMRSFSFSTNLQKRLRRKE
SEQ ~ N0:51 2F8 CDLPQTHSLGNRRALILLAQMGRISPFSCLKDRYDFGFPQEEFDGNQF
QKAQAISVLHEMMQQTFNLFSTKNSSAAWDETLLEKFSTELYQQLNEL
EACVIQEVGVEETPLMNEDSILAVKKYFQRITLYLTEKKYSPCSWEW
RAEIMRSFSFSTNLQKRLRRKE
SEQ ~ N0:52 6CG3 CDLPQTHSLGNKRAMMLLAQMGRTSPFSCLKDRHDFGFPQEEFDGNQF
QRAQAIFVLHEMIQQTFNFFSTKDSSAAWEQSLLEKFSTELNQQLNDL
EACVIQEVGVEETPLMNEDSILAVKKYFQRITLYLTEKKYSPCAWEW
RAEIMRSFSFSTNLQKRLRRKE
SEQ ~ N0:53 3CG7 CDLPQTHSLGNSRALMLLAQMGRISPFSCLKDRHDFGFPQEEFDGNQF
QKAQAISAFHEMIQQTFNLFSTKDSSAAWEQNLLEKFSTELYQQLNNL
EACVIQEVGMEETPLMNVDSILAVRKYFQRITLYLIERKYSPCAWEW
RAEIMRSFSFSTNLQKRLRRKE
SEQ ~ N0:54 1D3 CDLPQTHSLGNRRALILLAQMGRISHFSCLKDRHDFGFPQEEFDGHQF
QKTQAISVLHEMIQQTFNLFSTKDSSAAWEQSLLEKFSTELYQQLNDL
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EACVIQEVGVEETPLMNEDSILAVKKYFQRITLYLMEKKYSPCAWEW
RAEIMRSFSFSTNLQKRLRRKE
SEQ ~ NO:55 2G4 CDLPQTHSLGNRRAMMLLAQMSRISPSSCLMDRHDFEFPQEEFDDKQF
QKAPAISVLHEVIQQTFNLFSTEDSSAAWEQTLLEKFSTELYQQLNDL
EACVMQEERVGETPLMNADSILAVRKYFQRITLYLTKKKYSPCSWEW
RAEIMRSFSFSTNLQKRLRRKE
SEQ ~ N0:56 lAl CDLPQTHSLGNRRALILLAQMGRISHFSCLKDRYDFGFPQEVFDGNQF
QKAQAISAFHEMMQQTFNLFSTEDSSAAWEQSLLEKFSTELHQQLNDL
EACVIQEVGVEETPLMNEDSILAVRKYFQRITLYLMEKKYSPCAWEW
RAEIMRSFSFSTNLQKRLRRKE
SEQ ~ N0:57 1D10 CDLPQTHSLGNRRALILLAQMGRISPFSCLKDRHDFRFPQEEFDGNQL
QKTQAISVLHEMIQQTFNLFSTKDSSATWEQSLLEKFSTELNQQLNDL
EACVIQGVGVEETPPMNVDSILAVKKYFQRITLYLTEKKYSPCAWEW
RAEIMRSFSFSTNLQKRLRRKE
SEQ ~ NO:58 1F6 CDLPQTHSLGNRRTLMIMAQMGRISPFSCLKDRHDFGFPQEEFDGNQF
QKAQAISVLHEMIQQTFNLFSTKDSSATWEQSLLEKFSTELNQQLNDL
EACVIQEAGVEETPLMNVDSILAVKKYFQRITLYLTEKKYSPCAWEW
RAEIMRSFSFSTNLQKRLRRKE
SEQ ~ N0:59 2A10 CDLPQTHSLGNRRALILLAQMGRISHFSCLKDRYDFGFPQEVFDGNQF
QKAQAISAFHEMIQQTFNLFSTKDSSATWEQSLLEKFSTELYQQLNNL
EACVIQEVGVEETPLMNEDSILAVRKYFQRITLYLMEKKYSPCAWEW
RAEIMRSFSFSTNLQKRLRRKE
SEQ ~ N0:60 2C3 CDLPQTHSLGNRRALILLAQMGRISPFSCLKDRHDFGFPQEEFDGNQS
QKAQAISVLHEMIQQTFNLFSTKDSSDTWDATLLEKFSTELNQQLNDL
EACVIQEVGVEETPLMNVDSILAVKKYFQRITLYLTEKKYSPCAWEW
RAEIMRSFSFSTNLQKRLRRKE
SEQ ll~ N0:612D1 CDLPQTHSLGNRRALILLAQMRRISPFSCLKDRHDFGFPQEEFDGNQF
QKAQAISAFHEMIQQTFNLFSTKDSSAAWEQSLLEKFSTELYQQLNNL
EACVIQEVGMEETPLMNEDSILAVKKYFQRITLYLTEKKYSPCAWEW
RAEIMRSFSFSTNLQKRLRRKE
SEQ ~ N0:62 2D10 CDLPQTHSLGNRRALILLAQMGRVSPFSCLKDRHDFGFPQEEFDGNQF
QKAQAISAFHEMIQQTFNLFSTKDSSATWEQSLLEKFSTELYQQLNNL
EACVIQEVGVEETPLMNVDSILAVKKYFRRITLYLTEKKYSPCAWEW
RAEIMRSFSFSTNLQKRLRRKE
SEQ ~ N0:63 2D7 CDLPQTHSLGNRRALILLAQMGRISPFSCLKDRHDFRFPQEEFDGNQF
QKAQAISVLHEMIQQTFNLFSTKDSSATWEQSLLEKFSTELYQQLNNL
EACVIQEVGVEETPLMNVDSILAVKKYFQRITLYLTERKYSPCAWEW
RAEIMRSFSFSTNLQKRLRRKE
SEQ ~ N0:64 2D9 CDLPQTHSLGNRRALILLAQMGRISPFSCLKDRHDFGFPQEEFDGNQF
QKAQAISVLHEMIQQTFNLFSTKDSSATWEQSLLEKFSTELNQQLNDL
EACVIQEVGVEETPLVNVDSILAVKKYFQRITLYLTEKKYSPCAWEW
RAEIMRSFSFSTNLQKRLRRKE
SEQ ~ N0:65 2DA2 CDLPQTHSLGNRRPLILLAQMGRISPFSCLKDRQDFGFPQEEFDGNQF
QKAQAISVLHEMMQQTFNLFSTKNSSAAWEQSLLEKFSTELHQQLNEL
EACVIQEVGVEETPLMNVDSILAVKKYFQRITLYLIERKYSPCAWEW
RAEIMRSFSFSTNLQKRLRRKE
SEQ ~ N0:66 2DH9 CDLPQTHSPGNRRALMLLAQMGRISPFSCLKDRYDFGFPQGEFDGNQF
QKAQAISVLHEMMQQTFNLFSTKDSSAAWEQSLLEKFSTELYRQLNDL
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EACVIQEVGVEETPLMNVDSILAVRKYFQRITLYLTEKKHSPCSWEW
RAEIMRSFSFSTNLQKRLRRKE
SEQ ~ N0:67 2611 CDLPQTHSLGNRRALILLAQMGRISPFSCLKDRHDFGLPQEEFDGNQF
QKTQAISVLHEMIQQTFNLFSTKDSSDTWEQSLLEKFYIELFQQLNDL
EACVIQEVGVEETPLMNVDSILAVRKYFQRITLYLTEEKYSPCAWEW
RAEIMRSFSFSTNLQKRLRRKE
SEQ ~ N0:68 2612 CDLPQTHSLGNRRTLMLMAQMRRISPFPRLKDRYDFGFPQEVFDGNQF
QKAQAIFLFHEMMQQTFNLFSTKNSSAAWDETLLDKFYTELYQQLNDL
EACVMQEGRVGETPLMNADSILAVKKYFRRITLYLTEKKYSPCAWEAV
RAEIMRSFSFSTNLQKRLRRKE
SEQ ~ N0:69 2H9 CDLPQTHSLGNRRALILLAQMGRISPFSCLKDRHDFGFPQEEFDGNQF
QKAQAISVLHEMIQQTFNLFSTKDSSATWEQSLLEKFSTELNQQLNDL
EACVTQEVGVEETPLMNEDSILAVKKYFQRITLYLTEKKYSPCAWEW
RAEIMRSFSFSTNLQKRLRRKE
SEQ ~ N0:70 6BC11 CDLPQTHSLGNRRALILLAQMGRISPFSCLKDRYDFGFPQEEFDGNQL
QKAQAISVLHEMIQQTFNLFSTKDSSAAWEQSLLEKFSTELNQQLNDL
EACVIQEVGVEETPLMNVDSILAVKKYFQRITLYLTERKYSPCAWEW
RAEIMRSFSFSTNLQKRLRRKE
SEQ ~ N0:71 tl9bb CDLPQTHSLGXXRAXXLLXQMXRXSXFSCLKDRXDFGXPXEEFDXXXF
QXXQAIXXXHEXXQQTFNXFSTKXSSXXWXXXLLXKXXTXLXQQLNXL
EACVXQXVXXXXTPLMNXDXILAVXKYXQRITLYLXEXKYSPCXWEW
RAEIMRSFSFSTNLQKRLRRKE
SEQ ~ N0:72 CH1.1 TGTGATCTGCCTCAGACCCACAGCCTTGGTAACAGGAGGGCCTTGATA
CTCCTGGCACAAATGGGAAGAATCTCTCCTTTCTCCTGTCTGATGGAC
AGACATGACTTTGGATTTCCCCAGGAGGAGTTTGATGACAACCAGTTC
CAGAAGGCTCAAGCCATCTCTGTCCTCCATGAGATGATCCAACAGACC
TTCAATCTCTTCAGCACAAAGGACTCATCTGCTACTTGGGATGAGACA
CTTCTAGACAAATTCTACACTGAACTTTACCAGCAGCTGAATGACCTG
GAAGCCTGCGTGATACAGGAGGTTGGGGTGGAAGAGACTCCCCTGATG
AATGAGGACTCCATCTTGGCTGTGAAGAAATACTTCCGAAGAATCACT
CTCTATCTGACAGAGAAGAAATACAGCCCTTGTGCCTGGGAGGTTGTC
AGAGCAGAAATCATGAGATCTTTCTCTTTTTCAACAAACTTGCAAAA.A
AGATTAAGGAGGAAGGAA
SEQ ~ N0:73 CH1.2 TGTGATCTGCCTCAGACCCACAGCCTTGGTAACAGGAGGGCCTTGATA
CTCCTGGCACAAATGGGAAGAATCTCTCCTTTCTCCTGCCTGAAGGAC
AGACATGACTTTGGATTCCCCCAGGAGGAGTTTGATGGCAACCAGTTC
CAGAAGGCTCAAGGCATCTCTGTCCTCCATGAGATGATCCAGCAGACC
TTCCATCTCTTCAGCACAAAGGACTCATCTGCTACTTGGGAACAGAGC
CTCCTAGAAAAATTTTCCACTGAACTTAACCAGCAGCTGAATGACCTG
GAAGCCTGCGTGATACAGGAGGTTGGGGTGGAAGAGACTCCCCTGATG
AATGTGGACTCCATCCTGGCTGTGAAGAAATACTTCCGAAGAATCACT
CTTTATCTGACAGAGAAGAAATACAGCCCTTGTGCCTGGGAGGTTGTC
AGAGCAGAAATCATGAGATCTTTCTCTTTTTCAACAAACTTGCAAAAA
AGATTAAGGAGGAAGGAA
SEQ m N0:74 CH1.3 TGTGATCTGCCTCAGACCCACAGCCTTGGTAACAGGAGGACTTTGATG
ATAATGGCACAAATGGGAAGAATCTCTCCTTTCTCCTGCCTGAAGGAC
AGACATGACTTTGGATTTCCTCAGGAGGAGTTTGATGGCAACCAGTTC
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CAGAAGGCTCAAGCCATCTCTGTCCTCCATGAGATGATCCAGCAGACC
TTCAATCTCTTCAGCACAAAGGACTCATCTGCTACTTGGGATGAGACA
CTTCTAGACAAATTCTACACTGAACTTTACCAGCAGCTGAATGACCTG
GAAGCCTGTATGATGCAGGAGGTTGGAGTGGAAGACACTCCTCTGATG
AATGTGGACTCTATCCTGACTGTGAGA.A.AATACTTTCGAAGAATCACT
CTTTATCTGACAGAGAAGAAATACAGCCCTTGTGCCTGGGAGGTTGTC
AGAGCAGAAATCATGAGATCTTTCTCTTTTTCAACAAACTTGCAAAA.A
AGATTAAGGAGGAAGGAA
SEQ ~ N0:75 CH1.4 TGTGATCTGCCTCAGACCCACAGCCTGGGTAATAGGAGGGCCTTGATA
CTCCTGGCACAAATGGGAAGAATCTCTCCTTTCTCCTGCCTGAAGGAC
AGACATGACTTTGGATTCCCCCAGGAGGAGTTTGGTGGCAACCAGTTC
CAGAAGGCTCAAGCCATCTCTGTCCTCCATGAGATGATCCAGCAGACC
TTCAATCTCTTCAGCACAGAGGACTCATCTGCTGCTTGGGATGAGACC
CTCCTAGACAAATTCTACATTGAACTTTTCCAGCAACTGAATGACCTG
GAAGCCTGTGTGATGCAGGAGGAGAGGGTGGGAGAAACTCCCCTGATG
AATGCGGACTCCATCTTGGCTGTGAAGAAATACTTCCAAAGAATCACT
CTTTATCTGACAGAGAAGAAATACAGCCCTTGTGCCTGGGAGGTTGTC
AGAGCAGAAATCATGAGATCTTTCTCTTTTTCAACAAACTTGCAAAAA
AGATTAAGGAGGAAGGAA
SEQ ~ N0:76 CH2.1 TGTGATCTGCCTCAGACCCACAGCCTTGGTAACAGGAGGACTTTGATG
ATAATGGCACAAATGGGAAGAATCTCTCCTTTCTCCTGCCTGAAGGAC
AGACATGACTTTGGATTTCCTCAGGAGGAGTTTGATGGCAACCAGTTC
CAGAAGGCTCAAGCCATCTCTGTCCTCCATGAGATGATCCAGCAGACC
TTCAATCTCTTCAGCACAAAGGACTCATCTGCTACTTGGGATGAGACA
CTTCTAGACAAATTCTACACTGAACTTTACCAGCAGCTGAATGACCTG
GAAGCCTGTATGATACAGGAGGTTGGGGTGGAAGAGACTCCCCTGATG
AATGAGGACTCCATCTTGGCTGTGAAGAAATACTTCCGAAGAATCACT
CTCTATCTGACAGAGAAGAAATACAGCCCTTGTGCCTGGGAGGTTGTC
AGAGCAGAAATCATGAGATCTTTCTCTTTTTCAACAAACTTGCAAAAA
AGATTAAGGAGGAAGGAA
SEQ B7 N0:77CH2.2 TGTGATCTGCCTCAGACCCACAGCCTTGGTAACAGGAGGGCCTTGATA
CTCCTGGCACAAATGGGAAGAATCTCTCCTTTCTCCTGTCTGATGGAC
AGACATGACTTTGGATTTCCCCAGGAGGAGTTTGATGACAACCAGTTC
CAGAAGGCTCAAGCCATCTCTGTCCTCCATGAGATGATCCAACAGACC
TTCAATCTCTTCAGCACAAAGGACTCATCTGCTACTTGGGATGAGACA
CTTCTAGACAAATTCTACACTGAACTTTACCAGCAGCTGAATGACCTG
GAAGCCTGTATGATGCAGGAGGTTGGAGTGGAAGACACTCCTCTGATG
AATGTGGACTCTATCCTGACTGTGAAGAAATACTTCCGAAGAATCACT
CTTTATCTGACAGAGAAGAAATACAGCCCTTGTGCCTGGGAGGTTGTC
AGAGCAGAAATCATGAGATCTTTCTCTTTTTCAACAAACTTGCAA.A.A.A
AGATTAAGGAGGAAGGAA
SEQ ~ N0:78 CH2.3 TGTGATCTGCCTCAGACCCACAGCCTTGGTAACAGGAGGACTTTGATG
ATAATGGCACAAATGGGAAGAATCTCTCCTTTCTCCTGCCTGAAGGAC
AGACATGACTTTGGATTTCCTCAGGAGGAGTTTGATGGCAACCAGTTC
CAGAAGGCTCAAGCCATCTCTGTCCTCCATGAGATGATCCAGCAGACC
TTCAATCTCTTCAGCACAAAGGACTCATCTGCTACTTGGGATGAGACA
CTTCTAGACAAATTCTACACTGAACTTTACCAGCAGCTGAATGACCTG
GAAGCCTGTATGATGCAGGAGGTTGGAGTGGAAGACACTCCTCTGATG
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AATGAGGACTCCATCTTGGCTGTGAAGAAATACTTCCGAAGAATCACT
CTCTATCTGACAGAGAAGAAATACAGCCCTTGTGCCTGGGAGGTTGTC
AGAGCAGAAATCATGAGATCTTTCTCTTTCTCAACAAACTTGC~~AAA.A
AGATTAAGGAGGAAGGAA
SEQ m N0:79 CH1.1 CDLPQTHSLGNRRALILLAQMGRISPFSCLMDRHDFGFPQEEFDDNQF
QKAQAISVLHEMIQQTFNLFSTKDSSATWDETLLDKFYTELYQQLNDL
EACVIQEVGVEETPLMNEDSILAVKKYFRRITLYLTEKKYSPCAWEW
RAEIMRSFSFSTNLQKRLRRKE
SEQ ~ N0:80 CH1.2 CDLPQTHSLGNRRALILLAQMGRISPFSCLKDRHDFGFPQEEFDGNQF
QKAQGISVLHEMIQQTFHLFSTKDSSATWEQSLLEKFSTELNQQLNDL
EACVIQEVGVEETPLMNVDSILAVKKYFRRITLYLTEKKYSPCAWEW
RAEIMRSFSFSTNLQKRLRRKE
SEQ ~ N0:81 CH1.3 CDLPQTHSLGNRRTLMIMAQMGRISPFSCLKDRHDFGFPQEEFDGNQF
QKAQAISVLHEMIQQTFNLFSTKDSSATWDETLLDKFYTELYQQLNDL
EACMMQEVGVEDTPLMNVDSILTVRKYFRRITLYLTEKKYSPCAWEW
RAEIMRSFSFSTNLQKRLRRKE
SEQ ~ N0:82 CH1.4 CDLPQTHSLGNRRALILLAQMGRISPFSCLKDRHDFGFPQEEFGGNQF
QKAQAISVLHEMIQQTFNLFSTEDSSAAWDETLLDKFYIELFQQLNDL
EACVMQEERVGETPLMNADSILAVKKYFQRITLYLTEKKYSPCAWEW
RAEIMRSFSFSTNLQKRLRRKE
SEQ ~ N0:83 CH2.1 CDLPQTHSLGNRRTLMIMAQMGRISPFSCLKDRHDFGFPQEEFDGNQF
QKAQAISVLHEMIQQTFNLFSTKDSSATWDETLLDKFYTELYQQLNDL
EACMIQEVGVEETPLMNEDSILAVKKYFRRITLYLTEKKYSPCAWEW
RAEIMRSFSFSTNLQKRLRRKE
SEQ ~ N0:84 CH2.2 CDLPQTHSLGNRRALILLAQMGRISPFSCLMDRHDFGFPQEEFDDNQF
QKAQAISVLHEMIQQTFNLFSTKDSSATWDETLLDKFYTELYQQLNDL
EACMMQEVGVEETPLMNVDSILTVKKYFRRITLYLTEKKYSPCAWEW
RAEIMRSFSFSTNLQKRLRRKE
SEQ ~ N0:85 CH2.3 CDLPQTHSLGNRRTLMIMAQMGRISPFSCLKDRHDFGFPQEEFDGNQF
QKAQAISVLHEMIQQTFNLFSTKDSSATWDETLLDKFYTELYQQLNDL
EACMMQEVGVEETPLMNEDSILAVKKYFRRITLYLTEKKYSPCAWEW
RAEIMRSFSFSTNLQKRLRRKE
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SEQUENCE LISTING
<110> HEINRICHS, VOLKER
S CHEN, TEDDY
PATTEN, PHILLIP A.
<120> IFN-ALPHA HOMOLOGUES
<130> 02-101510/0140.002
<140>
<141>
1S <150> 09/415,183
<151> 1999-10-07
<160> 88
<170> PatentIn Ver. 2.0
<210> 1
<211> 498
<212> DNA
2S <213> Artificial Sequence
<220>
<223> Description of Artificial Sequence: Synthetic DNA
<220>
<223> Clone ID 2DH12
<400> 1
tgtgatctgc ctcagaccca cagccttggc aacaggaggg ccttgatgct cctggcacaa 60
3S atgggacgaa tctctccttt ctcctgcctg aaggacagac aagactttgg attcccccag 120
gaggagtttg atggcaacca gttccagaag gctcaagcca tctctgtcct ccatgagatg 180
atccagcaga ccttcaatct cttcagcaca aaggattcat ctgctgcttg ggaacagacc 240
ctcctagaaa aattttccac tgaactctac cagcagctga atgacctgga agcctgcgtg 300
atacaggagg taggggtgaa agagactccc ctgatgaatg tggactccat cctggctgtg 360
aggaagtact tccaaagaat cactctttat ctaatagaga ggaaatacag cccttgtgca 420
tgggaggttg tcagagcaga aatcatgaga tctttctctt tttcaacaaa cttgcaaaaa 480
agattaagga ggaaggaa 498
<210> 2
4S <211> 498
<212> DNA
<213> Artificial Sequence
<220>
S0 <223> Description of Artificial Sequence: Synthetic DNA
<220>
<223> Clone ID 2CA3
$$ <400> 2
tgtgatctgc ctcagaccca cagccttggt gacaggaggg ccatgatact cctggcacaa 60
atgggacgaa tctctccttt ctcctgcctg aaggacagat atgatttcgg attcccccag 120
gaggagtttg atggcaacca gttccagaag gctcaagcca tctctgtcct ccatgagatg 180
1
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atccagcaga ccttcaatct cttcagcaca aaggattcat ctgctgcttg ggaacagagc 240
ctcctagaaa aattttccac tgaactttac cagcagctga atgaactgga agcatgtgtg 300
atacaggagg ttggggtggg agagactccc ctgatgaatg gggactccat cctggctgtg 360
aagaagtact tccaaagaat cactctttat ctaatagaga ggaaatacag cccttgtgca 420
tgggaggttg tcagagcaga aatcatgaga tctttctctt tttcaacaaa cttgcaaaaa 480
498
agattaagga ggaaggaa
<210> 3
<211> 498
<212> DNA
<213> Artificial Sequence
<220>
<223> Description of Artificial Sequence: Synthetic DNA
<220>
<223> Clone ID 4AB9
<400> 3
tgtgatctgc ctcagaccca cagccttggc aacaggaggg ccttgatact cctggcacaa 60
atgggacgaa tctctccttt ctcctgcctg aaggacagac atgactttgg attcccccgg 120
gaggagtttg atggcaacca gttccagaag gctcaagcca tctctgtcct ccatgagatg 180
atgcagcaga ccttcaatct cttcagcaca aagaactcat ctgctgcttg ggatgagacc 240
ctcctagaaa aattttccac tgaactttac cagcaactga atgaactgga agcatgtgtg 300
2$ atacaggagg ttggggtgga agagactccc ctgatgaatg aggactccat cctggctgtg 360
aagaaatact tccaaagaat cactctttat ctgacagaga agaagtatag cccttgttcc 420
tgggaggttg tcagagcaga aatcatgaga tctttctctt tttcaacaaa cttgcaaaaa 480
agattaagga ggaaggaa 498
<210> 4
<211> 498
<212> DNA
<213> Artificial Sequence
<z2o>
<223> Description of Artificial Sequence: Synthetic DNA
<220>
<223> Clone ID 2DA4
<400> 4
tgtgatctgc ctcagaccca cagccttggt aacaggaggg ccttgatgct cctggcacaa 60
atgggaagaa tctctccttt ctcctgcctg aaggacagac aagactttgg attcccccag 120
gaggagtttg atagcaacca gttccagaag gctcaagcca tctctgtcct ccatgagatg 180
atgcagcaga ccttcaatct cttcagcaca aaggactcat ctgctgcttg ggatgagacc 240
ctcctagaaa aattttccac tgaactctac cagcagctga atgacctgga agcctgcgtg 300
atacaggagg ttggggtgga agagaccccc ctgatgaatg tggactccat cctggctgtg 360
aggaagtact tccaaagaat cactctttat ctaatagaga ggaaatacag cccttgtgca 420
tgggaggttg tcagagcaga aatcatgaga tctttctctt tttcaacaaa cttgcaaaaa 480
agattaagga ggaaggaa 498
<210> 5
<211> 498
<212> DNA
$5 <213> Artificial Sequence
<220>
<223> Description of Artificial Sequence: Synthetic DNA
2
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<220>
<223> Clone ID 3DA11
<400> 5
$ tgtgatctgc ctcagaccca cagccttggt aacaggaggg ccttggtact cctggcacaa 60
atgggaagaa tctctccttt ctcctgcctg aaggacagat atgatttcgg attcccccag 120
gaggagtttg atggcaacca gttccagaag gctcaagcca tctctgtcct ccatgagatg 180
atccagcaga ccttcaatct cttcagcaca aaggattcat ctgctgcttg ggatgagacc 240
ctcctagaaa aattttccac tgaactttac cagcagctga atgacctgga agcctgcgtg 300
atacaggagg ttggggtgga agagaccccc ctgatgaatg aggactccat cctggctgtg 360
aagaaatact tccaaagaat cactctttat ctaatagaga ggaaatacag cccttgtgca 420
tgggaggttg tcagagcaga aatcatgaga tctttctctt tttcaacaaa cttgcaaaaa 480
agattaagga ggaaggaa 498
1$ <210> 6
<211> 498
<212> DNA
<213> Artificial Sequence
<220>
<223> Description of Artificial Sequence: Synthetic DNA
<220>
<223> Clone ID 2DB11
2$
<400> 6
tgtgatctgc ctcagaccca cagccttggt aacaggaggg ccttgatgct cctggcacaa 60
atgggaagaa tctctccttt ctcctgcctg aaggacagat atgatttcgg attcccccag 120
gaggagtttg atggcaacca gttccagaag gctcaagcca tctctgtcct ccatgagatg 180
~ atccagcaga ccttcaatct cttcagcaca aaggattcat ctgctgcttg ggatgagacc 240
ctcctagaaa aattttccac tgaactttac cagcagctga atgacttgga agcctgtgtg 300
atacaggagg ttggggtgga agagactccc ctgatgaatg tggactccat cctggctgtg 360
aggaagtact tccaaagaat cactctttat ctaatagaga ggaaatacag cccttgtgca 420
tgggaggttg tcagagcaga aatcatgaga tctttctctt tttcaacaaa cttgcaaaaa 480
3$ agattaagga ggaaggaa 498
<210> 7
<211> 498
<212> DNA
40 <213> Artificial Sequence
<220>
<223> Description of Artificial Sequence: Synthetic DNA
4$ <220>
<223> Clone ID 2CA5
<400> 7
tgtgatctgc ctcagaccca cagccttggt aacaggaggg ccttgatact cctggcacaa 60
$0 atgggacgaa tctctccttt ctcctgcctg aaggacagac aagactttgg attcccccag 120
gaggagtttg atggcaaccg gttccagaag gctcaagcca tctctgtcct ccatgagatg 180
atccagcaga ccttcaatct cttcagcaca aagaactcat ctgctgcttg ggaacagagc 240
ctcctagaaa aattttccac tgaactctac cagcagctga atgacctgga agcctgcgtg 300
atacaggagg ttggggtgga agagaccccc ctgatgaatg aggactccat cctggctgtg 360
$$ aagaaatact tccaaagaat cactctttat ctaatagaga ggaaatacag cccttgtgca 420
tgggaggttg tcagagcaga aatcatgaga tctttctctt tttcaacaaa cttgcaaaaa 480
agattaagga ggaaggaa 498
<210> 8
3
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<211> 498
<212> DNA
<213> Artificial Sequence
$ <220>
<223> Description of Artificial Sequence: Synthetic DNA
<220>
<223> Clone ID 2G6
1~
<400> 8
tgtgatctgc ctcagaccca cagccttggt aacaggaggg ccttgatact cctggcacaa 60
atgggaagaa tctctccttt ctcctgcctg aaggacagac atgactttgg attcccccag 120
gaggagtttg atggcaacca gttccagaag gctcaagcca tctctgtcct ccatgagatg 180
1$ atccagcaga ccttcaatct cttcagcaca aaggactcat ctgctacttg ggaacagagc 240
ctcctagaaa aattttccac tgaacttaac cagcagctga atgacctgga agcctgcgtg 300
atacaggagg ttggggtgga agagactccc ctgatgaatg tggaccccat cctggctgtg 360
aagaaatact tccaaagaat cactctctat ctgacagaga agaaatacag cccttgtgcc 420
tgggaggttg tcagagcaga aatcatgaga tctttctctt tttcaacaaa cttgcaaaaa 480
20 agattaagga ggaaggaa 498
<210> 9
<211> 498
<212> DNA
2$ <213> Artificial Sequence
<220>
<223> Description of Artificial Sequence: Synthetic DNA
30 <220>
<223> Clone ID 3AH7
<400> 9
tgtgatctgc ctcagaccca cagccttggt aacaggaggg ccttgatact cctggcacaa 60
3$ atgcgaagaa tctctccttt ctcctgcctg aaggacagac atgactttgg attcccccag 120
gaggagtttg atagcaacca gttccagaag gctcaagcca tctctgtcct ccatgagatg 180
atccagcaga ccttcaatct cttcagcaca aaggattcat ctgctgcttg ggaacagagc 240
ctcctagaaa aattttccac tgaacttcac cagcaactga atgaactgga agcatgtgta 300
gtacaggagg ttggggtgga agagactccc ctgatgaatg aggactccat cctggctgtg 360
40 aagaaatacc tccaaagaat cactctttat ctgacagaga agaagtatag cccttgtgca 420
tgggaggttg tcagagcaga aatcatgaga tctttctctt tttcaacaaa cttgcaaaaa 480
agattaagga ggaaggaa 498
<210> 10
4$ <211> 49s
<212> DNA
<213> Artificial Sequence
<220>
$~ <223> Description of Artificial Sequence: Synthetic DNA
<220>
<223> Clone ID 2G5
$$ <400> to
tgtgatctgc ctcagaccca cagccttggt aacaggaggg ccttgatgct cctggcacaa 60
atgggaagaa tctctccttt ctcctgcctg aaggacagac aagactttgg attcccccag 120
gaggagtttg atggcaacca gttccagaag gctcaagcca tctctgtcct ccatgagatg 180
atccagcaga ccttcaatct cttcagcaca aaggattcat ctgctgcttg ggaacagagc 240
4
CA 02385045 2002-03-14
WO 01/25438 PCT/US00/27781
ctcctagaaa aattttccac tgaactctac eagcagctga atgacctgga agcctgcgtg 300
atacaggagg ttggggtgga agagaccccc ctgatgaatg tggactccat cctggctgtg 360
aggaagtact tccaaagaat cactctt.tat ctaatagaga ggaaatacag cccttgtgca 420
tgggaggttg tcagagcaga aatcatgaga tctttctctt tttcaacaaa cttgcaaaaa 480
$ agattaagga ggaaggaa 498
<210> 11
<211> 498
<212> DNA
<213> Artificial Sequence
<220>
<223> Description of Artificial Sequence: Synthetic DNA
1$ <220>
<223> Clone ID 2BA8
<400> 11
tgtgatctgc ctcagaccca cagccttggt aacaggaggg ccctgatact cctggcacaa 60
atgggacgaa tctctccttt ctcctgcctg aaggacagat atgatttcgg attcccccag 120
gaggagtttg atggcaacca gttccagaag gctcaagcca tctctgtcct ccatgagatg 180
atccagcaga ccttcaatct cttcagcaca aaggattcat ctgctgcttg ggaacagagc 240
ctcctagaaa aattttccac tgaactttac cagcagctga atgacctgga agcctgcgtg 300
atacaggagg ttggggtgga agagaccccc ctaatgaatg tggactccat cctggctgtg 360
2$ aggaagtact tccaaagaat cactctttat ctaatagaga ggaaatacag cccttgtgca 420
tgggaggttg tcagagcaga aatcatgaga tctttctctt tttcaacaaa cttgcaaaaa 480
agattaagga ggaaggaa 498
<210> 12
<211> 498
<212> DNA
<213> Artificial Sequence
<220>
3$ <223> Description of Artificial Sequence: Synthetic DNA
<220>
<223> Clone ID 1F3
<400> 12
tgtgatctgc ctcagaccca cagccttggt aacaggaggg ccttgatact cctgggacaa 60
atgggaagaa tctctcattt ctcctgcctg aaggacagac atgactttgg attcccccag 120
gaggagtttg atggcaacca gttccagaag gctcaagcca tctctgtcct ccatgagatg 180
atccagcaga ccttcaacct cttcagcaca aaggactcat ctgttgcttg ggatgagagg 240
4$ cttctagaca aactctatac tgaactttac cagcagctga atgacctgga agcctgtgtg 300
atgcaggagg tgtgggtggg agggactccc ctgatgaatg aggactccat cctggctgtg 360
agaaaatact tccaaagaat cactctctat ctgacagaga agaaatacag cccttgtgcc 420
tgggaggttg tcagagcaga aatcatgaga tctttctctt tttcaacaaa cttgcaaaaa 480
agattaagga ggaaggaa 498
$0
<210> 13
<211> 498
<212> DNA
<213> Artificial Sequence
$$
<220>
<223> Description of Artificial Sequence: Synthetic DNA
<220>
5
CA 02385045 2002-03-14
WO 01/25438 PCT/US00/27781
<223> Clone ID 4BE10
<400> 13
tgtgatctgc ctcagaccca cagccttggt aacaggaggg ccttgatact cctggcacag 60
atgggacgaa tctctccttt ctcctgcctg aaggacagat atgatttcgg attcccccag 120
gaggagtttg atggcaacca gttccagaag gctcaagcca tctctgtcct ccatgagata 180
atgcagcaga ccttcaatct cttcagcaca aagaactcat ctgctgcttg ggatgagacc 240
ctcctagaaa aattttccac tgaactttac cagcaactga atgaactgga agcatgtgtg 300
atacaggggg ttggggtgga agagactccc ctgatgaatg aggactccat cttggctgtg 360
aggaaatact tccaaagaat cactctttat ctgacagaga agaagtatag.cccttgttcc 420
tgggaggttg tcagagcaga aatcatgaga tctttctctt tttcaacaaa cttgcaaaaa 480
agattaagga ggaaggaa 498
<210> 14
<211> 498
<212> DNA
<213> Artificial Sequence
<220>
<223> Description of Artificial Sequence: Synthetic DNA
<220>
<223> Clone ID 2DD9
<400> 14
tgtgatctgc ctcagaccca cagccttggt aacaggaggg ccttgatgct cctggcacaa 60
atgggaagaa tctccccttt ctcctgcctg aaggacagat atgatttcgg attcccccag 120
gaggagtttg atggcaacca gttccagaag gctcaagcca tctctgtcct ccatgagatg 180
atccagcaga ccttcaatct cttcagcaca aaggattcat ctgctgcttg ggaacagagc 240
ctcctagaaa aattttccac tggactctac cagcagctga atgacctgga agcctgcgtg 300
atacaggagg ttggggtgga agagaccccc ctgatgaatg aggactccat cctggctgtg 360
aagaaatact tccaaagaat cactctttat ctgacagaga agaagtatag cccttgttcc 420
tgggaggttg tcagagcaga aatcatgaga tctttctctt tttcaacaaa cttgcaaaaa 480
agattaagga ggaaggaa 498
<210> 15
<211> 498
<212> DNA
<213> Artificial Sequence
<220>
<223> Description of Artificial Sequence: Synthetic DNA
<220>
<223> Clone ID 3CA1
<400> 15
tgtgatctgc ctcagaccca cagccttggc aacaggaggg ccttgatact cctggcacaa 60
atgggaagaa tctctccttt ctcctgcctg aaggacagac atgactttgg attaccccag 120
gaggagtttg atggcaacca gttccagaag gctcaagcca tctctgtcct ccatgagatg 180
atccagcaga ccttcaatct cttcagcaca aagaactcat ctgctgcttg ggatgagacc 240
ctcctagaaa aattttccac tgaactttac cagcaactga ataacctgga agcatgtgtg 300
atacaggagg ttgggatgga agagactccc ctgatgaatg tggactccat cctggctgtg 360
aagaaatact tccaaagaat cactctttat ctgacagaga agaagtatag cccttgtgcc 420
tgggaggttg tcagagcaga aatcatgaga tctttctctt tttcaacaaa cttgcaaaaa 480
agattaagga ggaaggaa 498
<210> 16
<211> 498
6
CA 02385045 2002-03-14
WO 01/25438 PCT/US00/27781
<212> DNA
<213> Artificial Sequence
<220>
$ <223> Description of Artificial Sequence: Synthetic DNA
<220>
<223> Clone ID 2F8
<400> 16
tgtgatctgc ctcagaccca cagccttggt aacaggaggg ccttgatact cctggcacaa 60
atgggacgaa tctctccttt ctcctgcctg aaggacagat atgatttcgg attcccccag 120
gaggagtttg atggcaacca gttccagaag gctcaagcca tctctgtcct ccatgagatg 180
atgcagcaga ccttcaatct cttcagcaca aagaactcat ctgctgcttg ggatgagacc 240
ctcctagaaa aattttccac tgaactttac cagcaactga atgaactgga agcatgtgtg 300
atacaggagg ttggggtgga agagactccc ctgatgaatg aggactccat cctggctgtg 360
aagaaatact tccaaagaat cactctttat ctgacagaga agaagtatag cccttgttcc 420
tgggaggttg tcagagcaga aatcatgaga tctttctctt tttcaacaaa cttgcaaaaa 480
agattaagga ggaaggaa 498
<210> 17
<211> 498
<212> DNA
<213> Artificial Sequence
<220>
<223> Description of Artificial Sequence: Synthetic DNA
<220>
<223> Clone ID 6CG3
<400> 17
tgtgatctgc ctcagaccca cagccttggt aacaagaggg ccatgatgct cctggcacaa 60
atgggaagaa cctctccttt ctcctgtctg aaggacagac atgactttgg attcccccag 120
gaggagtttg atggcaacca gttccagagg gctcaagcca tctttgtcct ccatgagatg 180
atccagcaga ccttcaattt cttcagcaca aaggactcat ctgctgcttg ggaacagagc 240
ctcctagaaa aattttccac tgaacttaac cagcagctga atgacctgga agcctgcgtg 300
atacaggaag ttggggtgga agagactccc ctgatgaatg aggactccat cctggctgtg 360
aagaaatact tccaaagaat cactctttat ctgacagaga agaaatacag cccttgtgcc 420
tgggaggttg tcagagcaga aatcatgaga tctttctctt tttcaacaaa cttgcaaaaa 480
agattaagga ggaaggaa 498
<210> 18
<211> 498
<212> DNA
<213> Artificial Sequence
<220>
<223> Description of Artificial Sequence: Synthetic DNA
<2zo>
<223> Clone ID 3CG7
<400> 18
tgtgatctgc ctcagaccca cagccttggt aacagtaggg ccttgatgct cctggcacaa 60
atgggaagaa tctccccttt ctcctgcctg aaggacagac atgatttcgg attcccccag 120
gaggagtttg atggcaacca gttccagaag gctcaagcca tctctgcctt ccatgagatg 180
atccagcaga ccttcaatct cttcagcaca aaggattcat ctgctgcttg ggaacagaac 240
ctcctagaaa aattttccac tgaactttac cagcaactga ataacctgga agcatgtgtg 300
7
CA 02385045 2002-03-14
$
WO 01/25438 PCT/US00/27781
atacaggagg ttgggatgga agagactccc ctgatgaatg tggactccat cctggctgtg 360
aggaagtact tccaaagaat cactctttat ctaatagaga ggaaatacag cccttgtgcc 420
tgggaggttg tcagagcaga aatcatgaga tctttctctt tttcaacaaa cttgcaaaaa 480
agattaagga ggaaggaa 498
<210> 19
<211> 498
<212> DNA
<213> Artificial Sequence
<220>
<223> Description of Artificial Sequence: Synthetic DNA
<220>
1$ <223> Clone ID 1D3
<400> 19
tgtgatctgc ctcagaccca cagccttggt aacaggaggg ccttgatact cctggcacaa 60
atgggaagaa tctctcattt ctcctgcctg aaggacagac atgatttcgg attcccccag 120
2O gaggagtttg atggccacca gttccagaag actcaagcca tctctgtcct ccatgagatg 180
atccagcaga ccttcaatct cttcagcaca aaggactcat ctgctgcttg ggaacagagc 240
ctcctagaaa aattttccac tgaactttac cagcaactga atgacctgga agcatgtgtg 300
atacaggagg ttggggtgga agagactccc ctgatgaatg aggactccat cctggctgtg 360
aagaaatact tccaaagaat cactctttat ctgatggaga agaaatacag cccttgtgcc 420
2$ tgggaggttg tcagagcaga aatcatgaga tctttctctt tttcaacaaa cttgcaaaaa 480
agattaagga ggaaggaa 498
<210> 20
<211> 498
3~ <212> DNA
<213> Artificial Sequence
3$
<220>
<223> Description of Artificial Sequence: Synthetic DNA
<220>
<223> Clone ID 2G4
<400> 20
4O tgtgatctgc ctcagaccca cagccttggt aacaggaggg ccatgatgct cctggcacaa 60
atgagcagaa tctctccttc ctcc'tgtctg atggacagac atgactttga atttccccag 120
gaggaatttg atgataaaca gttccagaag gctccagcca tctctgtcct ccatgaggtg 180
attcagcaga ccttcaatct cttcagcaca gaggactcat ctgctgcttg ggaacagacc 240
ctcctagaaa aattttccac tgaactttac cagcaactga atgacctgga agcatgtgtg 300
4$ atgcaggagg agagggtggg agaaactccc ctgatgaatg cggactccat cttggctgtg 360
aggaaatact tccaaagaat cactctttat ctgacaaaga agaagtatag cccttgttcc 420
tgggaggttg tcagagcaga aatcatgaga tctttctctt tttcaacaaa cttgcaaaaa 480
agattaagga ggaaggaa 498
$~ <210> 21
<211> 498
<212> DNA
<213> Artificial Sequence
$$ <220>
<223> Description of Artificial Sequence: Synthetic DNA
<220>
<223> Clone ID lAl
8
CA 02385045 2002-03-14
WO 01/25438 PCT/US00/27781
<400> 21
tgtgatctgc ctcagaccca cagccttggt aacaggaggg ccttgatact cctggcacaa 60
atgggaagaa tctctcattt ctcctgcctg aaggacagat atgatttcgg attcccccag 120
$ gaggtgtttg atggcaacca gttccagaag gcccaagcca tctctgcctt ccatgagatg 180
atgcagcaga ccttcaatct cttcagcaca gaggactcat ctgctgcttg ggaacagagc 240
ctcctagaaa aattttccac tgaacttcac cagcaactga atgacctgga agcctgtgtg 300
atacaggagg ttggggtgga agagactccc ctgatgaatg aggactccat cctggctgtg 360
aggaaatact ttcaaagaat cactctttat ctaatggaga agaaatacag cccttgtgcc 420
tgggaggttg tcagagcaga aatcatgaga tctttctctt tttcaacaaa cttgcaaaaa 480
agattaagga ggaaggaa 498
<210> 22
<211> 498
1$ <212> DNA
<213> Artificial Sequence
<220>
<223> Description of Artificial Sequence: Synthetic DNA
<220>
<223> Clone ID 1D10
<400> 22
tgtgatctgc ctcagaccca cagccttggt aacaggaggg ccttgatact cctggcacaa 60
atgggaagaa tctctcattt ctcctgcctg aaggacagac atgatttcgg attcccccag 120
gaggagtttg atggccacca gttccagaag actcaagcca tctctgtcct ccatgagatg 180
atccagcaga ccttcaatct cttcagcaca aaggactcat ctgctgcttg ggaacagagc 240
ctcctagaaa aattttccac tgaactttac cagcaactga atgacctgga agcatgtgtg 300
atacaggagg ttggggtgga agagactccc ctgatgaatg aggactccat cctggctgtg 360
aagaaatact tccaaagaat cactctttat ctgatggaga agaaatacag cccttgtgcc 420
tgggaggttg tcagagcaga aatcatgaga tctttctctt tttcaacaaa cttgcaaaaa 480
agattaagga ggaaggaa 498
<210> 23
<211> 498
<212> DNA
<213> Artificial Sequence
<z2o>
<223> Description of Artificial Sequence: Synthetic DNA
<220>
<223> Clone ID 1F6
<400> 23
tgtgatctgc ctcagaccca cagccttggt aacaggagga ctttgatgat aatggcacaa 60
atgggaagaa tctctccttt ctcctgcctg aaggacagac atgactttgg atttccccag 120
gaggagtttg atggcaacca gttccagaag gctcaagcca tctctgtcct ccatgagatg 180
$0 atccagcaga ccttcaatct cttcagcaca aaggactcat ctgctacttg ggaacagagc 240
ctcctagaaa aattttccac tgaacttaac cagcagctga atgacctgga agcctgcgtg 300
atacaggagg ctggggtgga agagactccc ctgatgaatg tggactccat cctggctgtg 360
aagaaatact tccaaagaat cactctttat ctaacagaga agaaatacag cccttgtgcc 420
tgggaggttg tcagagcaga aatcatgaga tctttctctt tttcaacaaa cttgcaaaaa 480
agattaagga ggaaggaa 498
<210> 24
<211> 498
<212> DNA
9
CA 02385045 2002-03-14
WO 01/25438 PCT/US00/27781
<213> Artificial Sequence
<220>
<223> Description of Artificial Sequence: Synthetic DNA
<220>
<223> Clone ID 2A10
<400> 24
tgtgatctgc ctcagaccca cagccttggt aacaggaggg ccttgatact cctggcacaa 60
atgggaagaa tctctcattt ctcctgcctg aaggacagat atgatttcgg attcccccag 120
gaggtgtttg atggcaacca gttccagaag gctcaagcca tctctgcctt ccatgagatg 180
atccagcaga ccttcaatct cttcagcaca aaggactcat ctgctacttg ggaacagagc 240
ctcctagaaa aattttccac tgaactttac cagcaactga ataacctgga agcatgtgtg 300
1$ atacaggagg ttggggtgga agagactccc ctgatgaatg aggactccat cctggctgtg 360
aggaaatact ttcaaagaat cactctttat ctgatggaga agaaatacag cccttgtgcc 420
tgggaggttg tcagagcaga aatcatgaga tctttctctt tttcaacaaa cttgcaaaaa 480
agattaagga ggaaggaa 498
<210> 25
<211> 498
<212> DNA
<213> Artificial Sequence
2$ <220>
<223> Description of Artificial Sequence: Synthetic DNA
<220>
<223> Clone ID 2C3
<400> 25
tgtgatctgc ctcagaccca cagccttggt aacaggaggg ccttgatact cctggcacaa 60
atgggaagaa tctctccttt ctcctgcctg aaggacagac atgactttgg atttcctcag 120
gaggagtttg atggcaacca gtcccagaag gctcaagcca tctctgtcct ccatgagatg 180
3$ atccagcaga ccttcaatct cttcagcaca aaggactcat ctgatacttg ggatgcgacc 240
cttttagaaa aattttccac tgaacttaac cagcagctga atgacctgga agcctgcgtg 300
atacaggagg ttggggtgga agagaccccc ctgatgaatg tggactccat cctggctgtg 360
aagaaatact tccaaagaat cactctttat ctgacagaga agaaatacag cccttgtgcc 420
tgggaggttg tcagagcaga aatcatgaga tctttctctt tttcaacaaa cttgcaaaaa 480
agattaagga ggaaggaa 498
<210> 26
<211> 498
<212> DNA
4$ <213> Artificial Sequence
<220>
<223> Description of Artificial Sequence: Synthetic DNA
$0 <z2o>
<223> Clone ID 2D1
<400> 26
tgtgatctgc ctcagaccca cagccttggt aacaggaggg ccttgatact cctggcacaa 60
$$ atgggacgaa tctctccttt ctcctgcctg aaggacagac aagactttgg attcccccag 120
gaggagtttg atggcaaccg gttccagaag gctcaagcca tctctgtcct ccatgagatg 180
atccagcaga ccttcaatct cttcagcaca aagaactcat ctgctgcttg ggaacagagc 240
ctcctagaaa aattttccac tgaactctac cagcagctga atgacctgga agcctgcgtg 300
atacaggagg ttggggtgga agagaccccc ctgatgaatg aggactccat cctggctgtg 360
CA 02385045 2002-03-14
WO 01/25438 PCT/US00/27781
aagaaatact tccaaagaat cactctttat ctaatagaga ggaaatacag cccttgtgca 420
tgggaggttg tcagagcaga aatcatgaga tctttctctt tttcaacaaa cttgcaaaaa 480
agattaagga ggaaggaa 498
<210> 27
<211> 498
<212> DNA
<213> Artificial Sequence
<220>
<223> Description of Artificial Sequence: Synthetic DNA
<220>
<223> Clone ID 2D10
<400> 27
tgtgatctgc ctcagaccca cagccttggt aacaggaggg ccttgatact cctggcacaa 60
atgggaagag tctctccttt ctcctgcctg aaggacagac atgactttgg attcccccag 120
gaggagtttg atggcaacca gttccagaag gctcaagcca tctctgcctt ccatgagatg 180
2~ atccagcaga ccttcaatct cttcagcaca aaggactcat ctgctacttg ggaacagagc 240
ctcctagaaa aattttccac tgaactttac cagcaactga ataacctgga agcctgcgtg 300
atacaggagg ttggggtgga agagactccc ctgatgaatg tggactccat cctggctgtg 360
aagaaatac~t tccgaagaat cactctctat ctgacagaga agaaatacag cccttgtgcc 420
tgggaggttg tcagagcaga aatcatgaga tctttctctt tttcaacaaa cttgcaaaaa 480
agattaagga ggaaggaa 498
<210> 28
<211> 498
<212> DNA
<213> Artificial Sequence
<220>
<223> Description of Artificial Sequence: Synthetic DNA
<z2o>
<223> Clone ID 2D7
<400> 28
tgtgatctgc ctcagaccca cagccttggt aacaggcggg ccttgatact cctggcacaa 60
4O atgggaagaa tctctccttt ctcctgtctg aaggacagac atgacttcag atttccccag 120
gaggagtttg atggcaacca gttccagaag gctcaagcca tctctgtcct ccatgagatg 180
atccagcaga ccttcaatct cttcagcaca aaggactcat ctgctacttg ggaacagagc 240
ctcctagaaa aattttccac tgaactttac cagcaactga ataacctgga agcttgcgtg 300
atacaggagg ttggggtgga agagactccc ctgatgaatg tggactctat cctggctgtg 360
aagaaatact tccaaagaat cactctttat ctgacagaga ggaaatacag cccttgtgcc 420
tgggaggttg tcagagcaga aatcatgaga tctttctctt tttcaacaaa cttgcaaaaa 480
agattaagga ggaaggaa 498
<210> 29
~ <211> 498
<212> DNA
<213> Artificial Sequence
<220>
<223> Description of Artificial Sequence: Synthetic DNA
<220>
<223> Clone ID 2D9
11
CA 02385045 2002-03-14
WO 01/25438 PCT/US00/27781
<400> 29
tgtgatctgc ctcagaccca cagccttggt aacaggaggg ccttgatact cctggcacaa 60
atgggaagaa tctctccttt ctcctgcctg aaggacagac atgactttgg attcccccag 120
gaggagtttg atggcaacca gttccagaag gctcaagcca tctctgtcct ccatgagatg 180
atccagcaga ctttcaatct cttcagcaca aaggactcat ctgctacttg ggaacagagc 240
ctcctagaaa aattttccac tgaacttaac cagcagctga atgacctgga agcctgcgtg 300
atacaggagg ttggggtgga agagactccc ctggtgaatg tggactccat cctggctgtg 360
aagaaatact tccaaagaat cactctttat ctgacagaga agaaatacag cccttgtgcc 420
tgggaggttg tcagagcaga aatcatgaga tctttctctt tttcaacaaa cttgcaaaaa 480
1~ agattaagga ggaaggaa 498
<210> 30
<211> 498
<212> DNA
<213> Artificial Sequence
<220>
<223> Description of Artificial Sequence: Synthetic DNA
<220>
<223> Clone ID 2DA2
<400> 30
tgtgatctgc ctcagaccca cagccttggt aacaggaggc ccttgatact cctggcacaa 60
atgggaagaa tctctccttt ctcctgcctg aaggacagac aggacttcgg attcccccag 120
gaggagtttg atggcaacca gttccagaag gctcaagcca tctctgtcct ccatgagatg 180
atgcagcaga ccttcaatct cttcagcaca aagaactcat ctgctgcttg ggaacagagc 240
ctcctagaaa aattttccac tgaactccac cagcaactga atgaactgga agcatgtgtg 300
atacaggagg ttggggtgga agagactccc ctgatgaatg tggactccat cctggctgtg 360
3~ aagaaatact tccaaagaat cactctttat ctaatagaga ggaaatacag cccttgtgca 420
tgggaggttg tcagagcaga aatcatgaga tctttctctt tttcaacaaa cttgcaaaaa 480
agattaagga ggaaggaa 498
<210> 31
<211> 498
<212> DNA
<213> Artificial Sequence
<220>
<223> Description of Artificial Sequence: Synthetic DNA
<220>
<223> Clone ID 2DH9
4S <400> 31
tgtgatctgc ctcagaccca cagccctggt aacaggaggg ccttgatgct cctggcacaa 60
atgggacgaa tctctccttt ctcctgcctg aaggacagat atgatttcgg attcccccag 120
ggggagtttg atggcaacca gttccagaag gctcaagcca tctctgtcct ccatgagatg 180
atgcagcaga ccttcaatct cttcagcaca aaggattcat ctgctgcttg ggaacagagc 240
ctcctagaaa aattttccac tgaactctac cggcagctga atgacctgga agcctgtgtg 300
atacaggagg ttggggtgga agagaccccc ctgatgaatg tggactccat cctggctgtg 360
aggaagtact tccaaagaat cactctttat ctgacagaga agaagcatag cccttgttcc 420
tgggaggttg tcagagcaga aatcatgaga tctttctctt tttcaacaaa cttgcaaaaa 480
agattaagga ggaaggaa 498
<210> 32
<211> 498
<212> DNA
<213> Artificial Sequence
12
CA 02385045 2002-03-14
WO 01/25438 PCT/US00/27781
<220>
<223> Description of Artificial Sequence: Synthetic DNA
<220>
<223> Clone ID 2611
<400> 32
tgtgatctgc ctcagaccca cagccttggt aacaggaggg ccttgatact cctggcacaa 60
atgggaagaa tctctccttt ctcctgcctg aaggacagac atgactttgg acttccccag 120
gaggagtttg atggcaacca gttccagaag actcaagcca tctctgtcct ccatgagatg 180
atccagcaga ccttcaatct cttcagcaca aaggactcat ctgatacttg ggaacagagc 240
ctcctagaaa aattctacat tgaacttttc cagcagctga atgacctgga agcctgcgtg 300
atacaggagg ttggggtgga agagactccc ctgatgaatg tggactccat cctggctgtg 360
agaaaatact tccaaagaat cactctttat ctgacagagg agaaatacag cccttgtgcc 420
tgggaggttg tcagagcaga aatcatgaga tctttctctt tttcaacaaa cttgcaaaaa 480
agattaagga ggaaggaa 498
<210> 33
<211> 498
<212> DNA
<213> Artificial Sequence
<220>
<223> Description of Artificial Sequence: Synthetic DNA
<220>
<223> Clone ID 2612
<400> 33
tgtgatctgc ctcagaccca cagccttggt aacaggagga ctttgatgct catggcacaa 60
atgaggagaa tctctccttt cccccgcctg aaggacagat atgatttcgg attcccccag 120
gaggtgtttg atggcaacca gttccagaag gctcaagcta tcttcctttt ccatgagatg 180
atgcagcaga ccttcaatct cttcagcaca aagaactcat ctgctgcttg ggatgagacc 240
ctcctagaca aattctacac tgaactctac cagcagctga atgacttgga agcctgtgtg 300
atgcaggagg ggagggtggg agaaactccc ctgatgaatg cggactccat cttggctgtg 360
aagaaatact tccgaagaat cactctctat ctgacagaga agaaatacag cccttgtgcc 420
tgggaggctg tcagagcaga aatcatgaga tctttctctt tttcaacaaa cttgcaaaaa 480
agattaagga ggaaggaa 498
<210> 34
<211> 498
<212> DNA
<213> Artificial Sequence
<220>
<223> Description of Artificial Sequence: Synthetic DNA
<220>
SO <223> Clone ID 2H9
<400> 34
tgtgatctgc ctcagaccca cagccttggt aacaggaggg ccttgatact cctggcacaa 60
atgggaagaa tctctccttt ctcctgcctg aaggacagac atgactttgg attcccccag 120
gaggagtttg atggcaacca gttccagaag gctcaagcca tctctgtcct ccatgagatg 180
atccagcaga ccttcaatct cttcagcaca aaggactcat ctgctacttg ggaacagagc 240
ctcctagaaa aattttccac tgaacttaac cagcagctga atgacctaga agcctgtgtg 300
acacaggagg ttggggtgga agagactccc ctgatgaatg aggactctat cctggctgtg 360
aagaaatact tccaaagaat cactctttat ctgacagaga agaaatacag cccttgtgcc 420
13
CA 02385045 2002-03-14
WO 01/25438 PCT/US00/27781
tgggaggttg tcagagcaga aatcatgaga tctttctctt tttcaacaaa cttgcaaaaa 480
agattaagga ggaaggaa 498
<210> 35
<211> 498
<212> DNA
<213> Artificial Sequence
<220>
<223> Description of Artificial Sequence: Synthetic DNA
<220>
<223> Clone ID 6BC11
<400> 35
tgtgatctgc ctcagaccca cagccttggt aacaggaggg ccttgatact cctggcacaa 60
atgggaagaa tctctccttt ctcctgcctg aaggacagat atgatttcgg attcccccag 120
gaggagtttg atggcaacca gctccagaag gctcaagcca tctctgtcct ccatgagatg 180
atccagcaga ccttcaatct cttcagcaca aaggattcat ctgctgcttg ggaacagagc 240
ctcctagaaa aattttccac tgaacttaac cagcagctga atgacctgga agcctgcgtg 300
atacaggagg ttggagtgga agagactccc ctgatgaatg tggactccat cctggctgtg 360
aagaaatact tccaaagaat cactctttat ctgacagaga ggaaatacag cccttgtgcc 420
tgggaggttg tcagagcaga aatcatgaga tctttctctt tttcaacaaa cttgcaaaaa 480
agattaagga ggaaggaa 498
<210> 36
<211> 166
<212> PRT
<213> Artificial Sequence
<220>
<223> Description of Artificial Sequence: Synthetic amino acid
<220>
<223> Clone ID 2DH12
<400> 36
Cys Asp Leu Pro Gln Thr His Ser Leu Gly Asn Arg Arg Ala Leu Met
1 5 10 15
Leu Leu Ala Gln Met Gly Arg Ile Ser Pro Phe Ser Cys Leu Lys Asp
20 25 30
Arg Gln 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
55 60
50 Phe Asn Leu Phe Ser Thr Lys Asp Ser Ser Ala Ala Trp Glu Gln Thr
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 Lys Glu Thr Pro Leu Met
100 105 110
Asn Val Asp Ser Ile Leu Ala Val Arg Lys Tyr Phe Gln Arg Ile Thr
14
CA 02385045 2002-03-14
WO 01/25438 PCT/US00/27781
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 Phe Ser Phe Ser Thr Asn Leu Gln Lys
145 150 155 160
Arg Leu Arg Arg Lys Glu
165
<210> 37
<211> 166
<212> PRT
<213> Artificia l
Sequence
<220>
<223> Description Sequence: amino
of Artificial Synthetic acid
<220>
<223> CloneID 2CA3
<400> 37
Cys Asp ProGln ThrHisSer LeuGlyAsp ArgArgAla MetIle
Leu
1 5 10 15
Leu Leu GlnMet GlyArgIle SerProPhe SerCysLeu LysAsp
Ala
20 25 30
Arg Tyr PheGly PheProGln GluGluPhe AspGlyAsn GlnPhe
Asp
40 45
Gln Lys GlnAla IleSerVal LeuHisGlu MetIleGln GlnThr
Ala
35 50 55 60
Phe Asn PheSer ThrLysAsp SerSerAla AlaTrpGlu GlnSer
Leu
65 70 75 80
Leu Leu LysPhe SerThrGlu LeuTyrGln GlnLeuAsn GluLeu
Glu
85 90 95
Glu Ala ValIle GlnGluVal GlyValGly GluThrPro LeuMet
Cys
100 105 110
Asn Gly SerIle LeuAlaVal LysLysTyr PheGlnArg IleThr
Asp
115 120 125
Leu Tyr Leu Ile Glu Arg Lys Tyr Ser Pro Cys Ala Trp Glu Val Val
SO 130 135 140
Arg Ala Glu Ile Met Arg Ser Phe Ser Phe Ser Thr Asn Leu Gln Lys
145 150 155 160
5$ Arg Leu Arg Arg Lys Glu
165
<210> 38
CA 02385045 2002-03-14
WO 01/25438 PCT/US00/27781
<211> 166
<212> PRT
<213> Artificial Sequence
<220>
<223> Description of Artificial Sequence: Synthetic amino acid
<220>
<223> Clone ID 4AB9
<400> 38
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 AspPheGly PheProArg GluGluPhe AspGlyAsn GlnPhe
35 40 45
Gln Lys AlaGlnAla IleSerVal LeuHisGlu MetMetGln GlnThr
50 55 60
Phe Asn LeuPheSer ThrLysAsn SerSerAla AlaTrpAsp GluThr
65 70 75 80
Leu Leu GluLysPhe SerThrGlu LeuTyrGln GlnLeuAsn GluLeu
85 90 95
Glu Ala CysValIle GlnGluVal GlyValGlu GluThrPro LeuMet
100 105 110
Asn Glu AspSerIle LeuAlaVal LysLysTyr PheGlnArg IleThr
115 120 125
Leu Tyr LeuThrGlu LysLysTyr SerProCys SerTrpGlu ValVal
130 135 140
Arg Ala GluIleMet ArgSerPhe SerPheSer ThrAsnLeu GlnLys
145 150 155 160
Arg Leu ArgArgLys Glu
165
<210> 39
<211> 166
<212> PRT
<213> Artificial Sequence
<220>
<223> Description of Artificial Sequence: Synthetic amino acid
<220>
<223> Clone ID 2DA4
<400> 39
Cys Asp Leu Pro Gln Thr His Ser Leu Gly Asn Arg Arg Ala Leu Met
1 5 10 15
16
CA 02385045 2002-03-14
WO 01/25438 PCT/US00/27781
Leu Leu Gln MetGlyArgIle SerProPhe SerCysLeu LysAsp
Ala
20 25 30
$ Arg Gln Phe GlyPheProGln GluGluPhe AspSerAsn GlnPhe
Asp
35 40 45
Gln Lys Gln AlaIleSerVal LeuHisGlu MetMetGln GlnThr
Ala
50 55 60
Phe Asn Phe SerThrLysAsp SerSerAla AlaTrpAsp GluThr
Leu
65 70 75 80
Leu Leu Lys PheSerThrGlu LeuTyrGln GlnLeuAsn AspLeu
Glu
1$ 85 90 95
Glu Ala Val IleGlnGluVal GlyValGlu GluThrPro LeuMet -
Cys
100 105 110
Asn Val Ser IleLeuAlaVal ArgLysTyr PheGlnArg IleThr
Asp
115 120 125
Leu Tyr Ile GluArgLysTyr SerProCys AlaTrpGlu ValVal
Leu
130 135 140
2$
Arg Ala Ile MetArgSerPhe SerPheSer ThrAsnLeu GlnLys
Glu
145 150 155 160
Arg Leu Arg LysGlu
Arg
165
<210> 40
<211> 166
3$ <212> PRT
<213> Artificial Sequence
<220>
<223> Description Sequence: amino
of Artificial Synthetic acid
<220>
<223> CloneID DA11
3
<400> 40
4$ Cys Asp Pro GlnThrHisSer LeuGlyAsn ArgArgAla LeuVal
Leu
1 5 10 15
Leu Leu Ala Gln Met Gly Arg Ile Ser Pro Phe Ser Cys Leu Lys Asp
20 25 30
$0
Arg Tyr 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 Ala Trp'Asp Glu Thr
65 70 75 80
17
CA 02385045 2002-03-14
WO 01/25438 PCT/US00/27781
Leu Leu Glu Lys Phe Ser Thr Glu Leu Tyr Gln Gln Leu Asn Asp Leu
85 90 95
Glu Ala CysValIle GlnGluVal GlyValGlu GluThrPro LeuMet
100 105 110
Asn Glu AspSerIle LeuAlaVal LysLysTyr PheGlnArg IleThr
115 120 125
Leu Tyr LeuIleGlu ArgLysTyr SerProCys AlaTrpGlu ValVal
130 135 140
Arg Ala GluIleMet ArgSerPhe SerPheSer ThrAsnLeu GlnLys
145 150 155 160
Arg Leu ArgArgLys Glu
165
<210> 41
<211> 166
<212> PRT
<213> Artificial Sequence
2S <220>
<223> Descriptio n Artificial Sequence: amino
of Synthetic acid
<220>
<223> CloneID DB11
2
<400> 41
Cys Asp Pro GlnThrHisSer LeuGlyAsn ArgArg AlaLeuMet
Leu
1 5 10 15
Leu Leu Gln MetGlyArgIle SerProPhe SerCys LeuLysAsp
Ala
20 25 30
Arg Tyr Phe GlyPheProGln GluGluPhe AspGly AsnGlnPhe
Asp
35 40 45
Gln Lys Gln AlaIleSerVal LeuHisGlu MetIle GlnGlnThr
Ala
55 60
Phe Asn Phe SerThrLysAsp SerSerAla AlaTrp AspGluThr
Leu
45 65 70 75 80
Leu Leu Lys PheSerThrGlu LeuTyrGln GlnLeu AsnAspLeu
Glu
85 90 95
50 Glu Ala Val IleGlnGluVal GlyValGlu GluThr ProLeuMet
Cys
100 105 110
Asn Val Ser IleLeuAlaVal ArgLysTyr PheGln ArgIleThr
Asp
115 120 125
Leu Tyr Ile GluArgLysTyr SerProCys AlaTrp GluValVal
Leu
130 135 140
Arg Ala Ile MetArgSerPhe SerPheSer ThrAsn LeuGlnLys
Glu
18
CA 02385045 2002-03-14
WO 01/25438 PCT/US00/27781
145 150 155 160
Arg Leu Arg Arg Lys Glu
165
<210> 42
<211> 166
<212> PRT
<213> Artificial Sequence
<220>
<223> Description Sequence: amino
of Artificial Synthetic acid
1$ <220>
<223> CloneID CA5
2
<400> 42
Cys Asp Pro GlnThrHis SerLeuGly AsnArgArg AlaLeuIle
Leu
1 5 10 15
Leu Leu Gln MetGlyArg IleSerPro PheSerCys LeuLysAsp
Ala
20 25 30
2$ Arg Gln Phe GlyPhePro GlnGluGiu PheAspGly AsnArgPhe
Asp
35 40 45
Gln Lys Gln AlaIleSer ValLeuHis GluMetIle GlnGlnThr
Ala
50 55 60
Phe Asn Phe SerThrLys AsnSerSer AlaAlaTrp GluGlnSer
Leu
65 70 75 80
Leu Leu Lys PheSerThr GluLeuTyr GlnGlnLeu AsnAspLeu
Glu
3$ 85 90 95
Glu Ala Val IleGlnGlu ValGlyVal GluGluThr ProLeuMet
Cys
100 105 110
Asn Glu Ser IleLeuAla ValLysLys TyrPheGln ArgIleThr
Asp
115 120 ' 125
Leu Tyr Ile GluArgLys TyrSerPro CysAlaTrp GluValVal
Leu
130 135 140
4$
Arg Ala Ile MetArgSer PheSerPhe SerThrAsn LeuGlnLys
Glu
145 150 155 160
Arg Leu Arg LysGlu
Arg
$0 16S
<210> 43
<211> 166
$$ <212> PRT
<213> Artificial
Sequence
<220>
<223> Description f tificial Sequence: eticamino
o Ar Synth acid
19
CA 02385045 2002-03-14
WO 01/25438 PCT/US00/27781
<220>
<223> ID 2G6
Clone
<400>
43
Cys AspLeu ProGln ThrHisSer LeuGlyAsnArg ArgAla LeuIle
1 5 10 15
Leu LeuAla GlnMet GlyArgIle SerProPheSer CysLeu LysAsp
20 25 30
Arg HisAsp PheGly PheProGln GluGluPheAsp GlyAsn GlnPhe
35 40 45
Gln LysAla GlnAla IleSerVal LeuHisGluMet IleGln GlnThr
50 55 60
Phe AsnLeu PheSer ThrLysAsp SerSerAlaThr TrpGlu GlnSer
65 70 75 80
Leu LeuGlu LysPhe SerThrGlu LeuAsnGlnGln LeuAsn AspLeu
85 90 95
Glu AlaCys ValIle GlnGluVal GlyValGluGlu ThrPro LeuMet
100 105 110
Asn ValAsp ProIle LeuAlaVal LysLysTyrPhe GlnArg IleThr
115 120 125
Leu TyrLeu ThrGlu LysLysTyr SerProCysAla TrpGlu ValVal
130 135 140
Arg AlaGlu IleMet ArgSerPhe SerPheSerThr AsnLeu GlnLys
145 150 155 160
Arg LeuArg ArgLys Glu
165
<210> 44
<211> 166
<212> PRT
<213> Artificial Sequence
<220>
<223> Description of Artificial Sequence: Synthetic amino acid
<220>
<223> Clone ID 3AH7
<400> 44
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 Gly Phe Pro Gln Glu Glu Phe Asp Ser Asn Gln Phe
35 40 45
CA 02385045 2002-03-14
WO 01/25438 PCT/US00/27781
Gln Lys Ala Gln Ala Ile Ser Val Leu His Glu Met Ile Gln Gln Thr
50 55 60
Phe Asn Phe SerThrLys AspSerSer AlaAlaTrp GluGlnSer
Leu
65 70 75 80
Leu Leu Lys PheSerThr GluLeuHis GlnGlnLeu AsnGluLeu
Glu
85 90 95
Glu Ala Val ValGlnGlu ValGlyVal GluGluThr ProLeuMet
Cys
100 105 110
Asn Glu Ser IleLeuAla ValLysLys TyrLeuGln ArgIleThr
Asp
115 120 125
Leu Tyr Thr GluLysLys TyrSerPro CysAlaTrp GluValVal
Leu
130 135 140
Arg Ala Ile MetArgSer PheSerPhe SerThrAsn LeuGlnLys
Glu
145 150 155 160
Arg Leu Arg LysGlu
Arg
165
<210> 45
<211> 166
<212> PRT
<213> Artificial Sequence
<220>
<223> Descriptio n Artificial Sequence: amino
of Synthetic acid
<220>
<223> CloneID G5
2
<400> 45
Cys Asp Pro GlnThrHis SerLeuGly AsnArgArg AlaLeuMet
Leu
1 5 10 15
Leu Leu Ala Gln Met Gly Arg Ile Ser Pro Phe Ser Cys Leu Lys Asp
20 25 30
Arg GlnAspPhe GlyPheProGln GluGlu PheAspGly AsnGlnPhe
35 40 45
Gln LysAlaGln AlaIleSerVal LeuHis GluMetIle GlnGlnThr
50 55 60
Phe AsnLeuPhe SerThrLysAsp SerSer AlaAlaTrp GluGlnSer
65 70 75 80
Leu LeuGluLys PheSerThrGlu LeuTyr GlnGlnLeu AsnAspLeu
85 90 95
Glu AlaCysVal IleGlnGluVal GlyVal GluGluThr ProLeuMet
100 105 110
21
CA 02385045 2002-03-14
WO 01/25438 PCT/US00/27781
Asn Val 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 Phe Ser Phe Ser Thr Asn Leu Gln Lys
145 150 155 160
Arg Leu Arg Arg Lys Glu
165
<210> 46
<211> 166
<212> PRT
<213> Artificial Sequence
<220>
<223> Description of Artificial Sequence: Synthetic amino acid
<220>
<223> Clone ID 2BA8
<400> 46
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 TyrAsp PheGlyPhe ProGlnGlu GluPheAsp GlyAsn GlnPhe
35 40 45
Gln LysAla GlnAlaIle SerValLeu HisGluMet IleGln GlnThr
50 55 60
Phe AsnLeu PheSerThr LysAspSer SerAlaAla TrpGlu GlnSer
65 70 75 80
Leu LeuGlu LysPheSer ThrGluLeu TyrGlnGln LeuAsn AspLeu
85 90 95
Glu AlaCys ValIleGln GluValGly ValGluGlu ThrPro LeuMet
100 105 110
Asn ValAsp SerIleLeu AlaValArg LysTyrPhe GlnArg IleThr
115 120 125
Leu TyrLeu IleGluArg LysTyrSer ProCysAla TrpGlu ValVal
130 135 140
Arg AlaGlu IleMetArg SerPheSer PheSerThr AsnLeu GlnLys
145 150 155 160
Arg LeuArg ArgLysGlu
165
22
CA 02385045 2002-03-14
WO 01/25438 PCT/US00/27781
<210> 47
<211> 166
<212> PRT
<213> Artificial Sequence
$
<220>
<223> Description of Artificial Sequence: Synthetic amino acid
<220>
<223> CloneID 1F3
<400> 47
Cys Asp ProGlnThr HisSerLeu GlyAsn ArgArgAla LeuIle
Leu
1 5 10 15
1$
Leu Leu GlnMetGly ArgIleSer HisPhe SerCysLeu LysAsp
Gly
20 25 30
Arg His PheGlyPhe ProGlnGlu GluPhe AspGlyAsn GlnPhe
Asp
35 40 45
Gln Lys GlnAlaIle SerValLeu HisGlu MetIleGln GlnThr
Ala
50 55 60
2$ Phe Asn PheSerThr LysAspSer SerVal AlaTrpAsp GluArg
Leu
65 70 75 80
Leu Leu LysLeuTyr ThrGluLeu TyrGln GlnLeuAsn AspLeu
Asp
85 90 95
Glu Ala ValMetGln GluValTrp ValGly GlyThrPro LeuMet
Cys
100 105 110
Asn Glu SerIleLeu AlaValArg LysTyr PheGlnArg IleThr
Asp
3$ 115 120 125
Leu Tyr ThrGluLys LysTyrSer ProCys AlaTrpGlu ValVal
Leu
130 135 140
Arg Ala IleMetArg SerPheSer PheSer ThrAsnLeu GlnLys
Glu
145 150 155 160
Arg Leu ArgLysGlu
Arg
165
4$
<210> 48
<211> 166
<212> PRT
$0 <213> Artificia l
Sequence
<220>
<223> Description Sequence: amino
of Artificial Synthetic acid
$$ <220>
<223> CloneID 4BE10
<400> 48
Cys Asp ProGlnThr HisSerLeu GlyAsn ArgArgAla LeuIle
Leu
23
CA 02385045 2002-03-14
WO 01/25438 PCT/US00/27781
1 5 10 15
Leu LeuAlaGln MetGlyArg IleSerPro PheSerCys LeuLys Asp
20 25 30
$
Arg TyrAspPhe GlyPhePro GlnGluGlu PheAspGly AsnGln Phe
35 40 45
Gln LysAlaGln AlaIleSer ValLeuHis GluIleMet GlnGln Thr
50 55 60
Phe AsnLeuPhe SerThrLys AsnSerSer AlaAlaTrp AspGlu Thr
65 70 75 80
1$Leu LeuGluLys PheSerThr GluLeuTyr GlnGlnLeu AsnGlu Leu
85 90 95
Glu AlaCysVal IleGlnGly ValGlyVal GluGluThr ProLeu Met
100 105 110
Asn GluAspSer IleLeuAla ValArgLys TyrPheGln ArgIle Thr
115 120 125
Leu TyrLeuThr GluLysLys TyrSerPro CysSerTrp GluVal Val
2$ 130 135 140
Arg AlaGluIle MetArgSer PheSerPhe SerThrAsn LeuGln Lys
145 150 155 160
30Arg LeuArgArg LysGlu
165
<210> 49
3$ <211> 166
<212> PRT
<213> Artificial Sequence
<220>
40 <223> Description Artificial Sequence: amino
of Synthetic acid
<220>
<223> CloneID DD9
2
4$ <400> 49
Cys Asp ProGln ThrHisSerLeu GlyAsnArg ArgAlaLeu Met
Leu
1 5 10 15
Leu Leu GlnMet GlyArgIleSer ProPheSer CysLeuLys Asp
Ala
$0 20 25 30
Arg Tyr PheGly PheProGlnGlu GluPheAsp GlyAsnGln Phe
Asp
35 40 45
$$ Gln Lys GlnAla IleSerValLeu HisGluMet IleGlnGln Thr
Ala
50 55 60
Phe Asn PheSer ThrLysAspSer SerAlaAla TrpGluGln Ser
Leu
65 70 75 80
24
CA 02385045 2002-03-14
WO 01/25438 PCT/US00/27781
Leu Leu Glu Lys Phe Ser Thr Gly Leu Tyr Gln Gln Leu Asn Asp Leu
85 90 95
Glu AlaCys ValIle GlnGluVal GlyValGlu GluThrPro LeuMet
100 105 110
Asn GluAsp SerIle LeuAlaVal LysLysTyr PheGlnArg IleThr
115 120 125
Leu TyrLeu ThrGlu LysLysTyr SerProCys SerTrpGlu-ValVal
130 135 140
Arg AlaGlu IleMet ArgSerPhe SerPheSer ThrAsnLeu GlnLys
145 150 155 160
Arg LeuArg ArgLys Glu
165
<210> 50
<211> 166
<212> PRT
<213> Artificial Sequence
<220>
<223> Description Sequence: amino
of Artificial Synthetic acid
<220>
<223> CloneID CA1
3
<400> 50
Cys Asp Pro GlnThrHisSer LeuGlyAsn ArgArgAla LeuIle
Leu
1 5 10 15
Leu Leu Gln MetGlyArgIle SerProPhe SerCysLeu LysAsp
Ala
20 25 30
Arg His Phe GlyLeuProGln GluGluPhe AspGlyAsn GlnPhe
Asp
35 40 45
Gln Lys Gln AlaIleSerVal LeuHisGlu MetIleGln GlnThr
Ala
55 60
4$ Phe Asn Phe SerThrLysAsn SerSerAla AlaTrpAsp GluThr
Leu
65 70 75 80
Leu Leu Lys PheSerThrGlu LeuTyrGln GlnLeuAsn AsnLeu
Glu
85 90 95
50
Glu Ala Val IleGlnGluVal GlyMetGlu GluThrPro LeuMet
Cys
100 105 110
Asn Val Ser IleLeuAlaVal LysLysTyr PheGlnArg IleThr
Asp
115 120 125
Leu Tyr Thr GluLysLysTyr SerProCys AlaTrpGlu ValVal
Leu
130 135 140
CA 02385045 2002-03-14
WO 01/25438 PCT/US00/27781
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
<210> 51
<211> 166
<212> PRT
<213> Artificial Sequence
<220>
<223> Description of Artificial Sequence: Synthetic amino acid
1$
<220>
<223> Clone ID 2F8
<400> 51
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
2$
Arg TyrAsp PheGlyPhe ProGlnGlu GluPheAsp GlyAsnGln Phe
35 40 45
Gln LysAla GlnAlaIle SerValLeu HisGluMet MetGlnGln Thr
50 55 60
Phe AsnLeu PheSerThr LysAsnSer SerAlaAla TrpAspGlu Thr
65 70 75 80
3$ Leu LeuGlu LysPheSer ThrGluLeu TyrGlnGln LeuAsnGlu Leu
85 90 95
Glu AlaCys ValIleGln GluValGly ValGluGlu ThrProLeu Met
100 105 110
Asn GluAsp SerIleLeu AlaValLys LysTyrPhe GlnArgIle Thr
115 120 125
Leu Tyr Leu Thr Glu Lys Lys Tyr Ser Pro Cys Ser Trp Glu Val Val
4$ 130 135 140
Arg Ala Glu Ile Met Arg Ser Phe Ser Phe Ser Thr Asn Leu Gln Lys
145 150 155 160
$0 Arg Leu Arg Arg Lys Glu
165
<210> 52
$$ <211> 166
<212> PRT
<213> Artificial Sequence
<220>
26
CA 02385045 2002-03-14
WO 01/25438 PCT/US00/27781
<223> Description of Artificial Sequence: Synthetic amino acid
<220>
<223> Clone ID 6CG3
<400> 52
Cys Asp Leu Pro Gln Thr His Ser Leu Gly Asn Lys Arg Ala Met Met
1 5 10 15
Leu Leu Ala Gln Met Gly Arg Thr Ser Pro Phe Ser Cys Leu Lys Asp
25 30
Arg His Asp Phe Gly Phe Pro Gln Glu Glu Phe Asp Gly Asn Gln Phe
35 40 45
1$
Gln Arg Ala Gln Ala Ile Phe Val Leu His Glu Met Ile Gln Gln Thr
50 55 60
Phe Asn Phe Phe Ser Thr Lys Asp Ser Ser Ala Ala Trp Glu Gln Ser
20 65 70 75 80
Leu Leu Glu Lys Phe Ser Thr Glu Leu Asn Gln Gln Leu Asn Asp Leu
85 90 95
2$ Glu Ala Cys Val Ile Gln Glu Val Gly Val Glu Glu Thr Pro Leu Met
100 105 110
Asn Glu Asp Ser Ile Leu Ala Val Lys Lys Tyr Phe Gln Arg Ile Thr
115 120 125
Leu Tyr Leu Thr Glu Lys Lys Tyr Ser Pro Cys Ala Trp Glu Val Val
130 135 140
Arg Ala Glu Ile Met Arg Ser Phe Ser Phe Ser Thr Asn Leu Gln Lys
3$ 145 150 155 160
Arg Leu Arg Arg Lys Glu
165
<210> 53
<211> 166
<212> PRT
<213> Artificial Sequence
4$
<220>
<223> Description of Artificial Sequence: Synthetic amino acid
<220>
$0 <223> Clone ID 3CG7
<400> 53
Cys Asp Leu Pro Gln Thr His Ser Leu Gly Asn Ser Arg Ala Leu Met
1 5 10 15
$$
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
27
CA 02385045 2002-03-14
WO 01/25438 PCT/US00/27781
35 40 45
Gln Lys AlaGlnAla IleSerAla PheHisGlu MetIleGln GlnThr
50 55 60
Phe Asn LeuPheSer ThrLysAsp SerSerAla AlaTrpGlu GlnAsn
65 70 75 80
Leu Leu GluLysPhe SerThrGlu LeuTyrGln GlnLeuAsn AsnLeu
85 90 95
Glu Ala CysValIle GlnGluVal GlyMetGlu GluThrPro LeuMet
100 105 110
1$ Asn Val AspSerIle LeuAlaVal ArgLysTyr PheGlnArg IleThr
115 120 125
Leu Tyr LeuIleGlu ArgLysTyr SerProCys AlaTrpGlu ValVal
130 135 140
Arg Ala GluIleMet ArgSerPhe SerPheSer ThrAsnLeu GlnLys
145 150 155 160
Arg Leu ArgArgLys Glu
2$ 165
<210> 54
<211> 166
<212> PRT
<213> Artificia l
Sequence
<220>
<223> Description Artificial Sequence: amino
of Synthetic acid
3$
<220>
<223> CloneID 1D3
<400> 54
Cys Asp ProGln ThrHisSer LeuGlyAsnArg ArgAla LeuIle
Leu
1 5 10 15
Leu Leu GlnMet GlyArgIle SerHisPheSer CysLeu LysAsp
Ala
20 25 30
4$
Arg His PheGly PheProGln GluGluPheAsp GlyHis GlnPhe
Asp
35 40 45
Gln Lys GlnAla IleSerVal LeuHisGluMet IleGln GlnThr
Thr
$0 50 55 60
Phe Asn PheSer ThrLysAsp SerSerAlaAla TrpGlu GlnSer
Leu
65 70 75 80
$$ Leu Leu LysPhe SerThrGlu LeuTyrGlnGln LeuAsn AspLeu
Glu
85 90 95
Glu Ala ValIle GlnGluVal GlyValGluGlu ThrPro LeuMet
Cys
100 105 110
28
CA 02385045 2002-03-14
WO 01/25438 PCT/US00/27781
Asn Glu Asp Ser Ile Leu Ala Val Lys 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 Glu
165
1$ <210> 55
<211> 166
<212> PRT
<213> Artificial Sequence
<220>
<223> Description Artificial Sequence: amino
of Synthetic acid
<220>
<223> CloneID G4
2
2$
<400> 55
Cys Asp ProGln ThrHisSerLeu GlyAsnArg ArgAla MetMet
Leu
1 5 10 15
Leu Leu GlnMet SerArgIleSer ProSerSer CysLeu MetAsp
Ala
20 25 30
Arg His PheGlu PheProGlnGlu GluPheAsp AspLys GlnPhe
Asp
40 45
3$
Gln Lys ProAla IleSerValLeu HisGluVal IleGln GlnThr
Ala
50 55 60
Phe Asn PheSer ThrGluAspSer SerAlaAla TrpGlu GlnThr
Leu
65 70 75 80
Leu Leu LysPhe SerThrGluLeu TyrGlnGln LeuAsn AspLeu
Glu
85 90 95
4$ Glu Ala ValMet GlnGluGluArg ValGlyGlu ThrPro LeuMet
Cys
100 105 110
Asn Ala SerIle LeuAlaValArg LysTyrPhe GlnArg IleThr
Asp
115 120 125
$0
Leu Tyr ThrLys LysLysTyrSer ProCysSer TrpGlu ValVal
Leu
130 135 140
Arg Ala IleMet ArgSerPheSer PheSerThr AsnLeu GlnLys
Glu
55 145 150 155 160
Arg Leu ArgLys Glu
Arg
165
29
CA 02385045 2002-03-14
WO 01/25438 PCT/US00/27781
<210> 56
<211> 166
<212> PRT
$ <213> Artificial ce
Sequen
<220>
<223> Description f tificialSequence: eticamino
o Ar Synth acid
<220>
<223> CloneID 1A1
<400> 56
Cys Asp ProGlnThr HisSerLeu GlyAsn ArgArgAla LeuIle
Leu
1$ 1 5 10 15
Leu Leu GlnMetGly ArgIleSer HisPhe SerCysLeu LysAsp
Ala
20 25 30
Arg Tyr PheGlyPhe ProGlnGlu ValPhe AspGlyAsn GlnPhe
Asp
35 40 45
Gln Lys GlnAlaIle SerAlaPhe HisGlu MetMetGln GlnThr
Ala
50 55 60
2$
Phe Asn PheSerThr GluAspSer SerAla AlaTrpGlu GlnSer
Leu
65 70 75 80
Leu Leu LysPheSer ThrGluLeu HisGln GlnLeuAsn AspLeu
Glu
85 90 95
Glu Ala ValIleGln GluValGly ValGlu GluThrPro LeuMet
Cys
100 105 110
3$ Asn Glu SerIleLeu AlaValArg LysTyr PheGlnArg IleThr
Asp
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 Glu
4$ 165
<210> 57
<211> 166
$0 <212> PRT
<213> Artificial Sequence
<220>
<223> Description of Artificial Sequence: Synthetic amino acid
$$
<220>
<223> Clone ID 1D10
<400> 57
CA 02385045 2002-03-14
WO 01/25438 PCT/US00/27781
Cys Asp Leu Pro Gln Thr His Ser Leu Gly Asn Arg Arg Ala Leu Ile
1 5 10 15
Leu LeuAla GlnMetGly Ile SerProPhe SerCysLeu LysAsp
Arg
20 25 30
Arg HisAsp PheArgPhe ProGln GluGluPhe AspGlyAsn GlnLeu
35 40 45
Gln LysThr GlnAlaIle SerVal LeuHisGlu MetIleGln GlnThr
50 55 60
Phe AsnLeu PheSerThr LysAsp SerSerAla ThrTrpGlu GlnSer
65 70 75 80
Leu LeuGlu LysPheSer ThrGlu LeuAsnGln GlnLeuAsn AspLeu
85 90 95
Glu AlaCys ValIleGln GlyVal GlyValGlu GluThrPro ProMet
100 105 110
Asn ValAsp SerIleLeu AlaVal LysLysTyr PheGlnArg IleThr
115 120 125
2$ Leu TyrLeu ThrGluLys LysTyr SerProCys AlaTrpGlu ValVal
130 135 140
Arg AlaGlu IleMetArg SerPhe SerPheSer ThrAsnLeu GlnLys
145 150 155 160
Arg LeuArg ArgLysGlu
165
<210> ss
<211> 166
<212> PRT
<213> Artificial
Sequence
<220>
<223> Description Artificial Sequence: amino
of Synthetic acid
<220>
<223> CloneID 1F6
<400> 58
Cys Asp Pro ThrHisSer LeuGlyAsn ArgArgThr LeuMet
Leu Gln
1 5 10 15
Ile Met Gln GlyArgIle SerProPhe SerCysLeu LysAsp
Ala Met
20 25 30
Arg His Phe PheProGln GluGluPhe AspGlyAsn GlnPhe
Asp Gly
35 40 45
Gln Lys Gln IleSerVal LeuHisGlu MetIleGln GlnThr
Ala Ala
50 55 60
Phe Asn Phe ThrLysAsp SerSerAla ThrTrpGlu GlnSer
Leu Ser
31
CA 02385045 2002-03-14
WO 01/25438 PCT/US00/27781
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 CysValIle GlnGlu AlaGlyVal GluGluThr ProLeuMet
100 105 110
Asn Val AspSerIle LeuAla ValLysLys TyrPheGln ArgIleThr
115 120 125
Leu Tyr LeuThrGlu LysLys TyrSerPro CysAlaTrp GluValVal
130 135 140
1$ Arg Ala GluIleMet ArgSer PheSerPhe SerThrAsn LeuGlnLys
145 150 155 160
Arg Leu ArgArgLys Glu
165
<210> 59
<211> 166
<212> PRT
2$ <213> Artificial Sequence
<220>
<223> Description Artificial Sequence: amino
of Synthetic acid
<220>
<223> CloneID A10
2
<400> 59
Cys Asp Pro GlnThrHisSer LeuGlyAsn ArgArg AlaLeuIle
Leu
1 5 10 15
Leu Leu Gln MetGlyArgIle SerHisPhe SerCys LeuLysAsp
Ala
20 25 30
Arg Tyr Phe GlyPheProGln GluValPhe AspGly AsnGlnPhe
Asp
35 40 45
Gln Lys Gln AlaIleSerAla PheHisGlu MetIle GlnGlnThr
Ala
55 60
45
Phe Asn Phe SerThrLysAsp SerSerAla ThrTrp GluGlnSer
Leu
65 70 75 80
Leu Leu Lys PheSerThrGlu LeuTyrGln GlnLeu AsnAsnLeu
Glu
50 85 90 95
Glu Ala Val IleGlnGluVai GlyValGlu GluThr ProLeuMet
Cys
100 105 110
$$ Asn Glu Ser IleLeuAlaVal ArgLysTyr PheGln ArgIleThr
Asp
115 120 125
Leu Tyr Met GluLysLysTyr SerProCys AlaTrp GluValVal
Leu
130 135 140
32
CA 02385045 2002-03-14
WO 01/25438 PCT/US00/27781
Arg Ala Glu Ile Met Arg Ser Phe Ser Phe Ser Thr Asn Leu Gln Lys
145 150 155 160
$ Arg Leu Arg Arg Lys Glu
165
<210> 60
1O <211> 166
<212> PRT
<213> Artificial Sequence
<220>
1$ <223> Description of Artificial Sequence: Synthetic amino acid
<220>
<223> Clone ID 2C3
20 <400> 60
Cys Asp Leu Pro Gln Thr His Ser Leu Gly Asn Arg Arg Ala Leu Ile
1 5 10 15
Leu Leu Ala Gln Met Gly Arg Ile Ser Pro Phe Ser Cys Leu Lys Asp
2$ 20 25 30
Arg His AspPheGly PheProGln GluGluPhe AspGlyAsn GlnSer
35 40 45
30 Gln Lys AlaGlnAla IleSerVal LeuHisGlu MetIleGln GlnThr
50 55 60
Phe Asn LeuPheSer ThrLysAsp SerSerAsp ThrTrpAsp AlaThr
65 70 75 80
3$
Leu Leu GluLysPhe SerThrGlu LeuAsnGln GlnLeuAsn AspLeu
85 90 95
Glu Ala CysValIle GlnGluVal GlyValGlu GluThrPro LeuMet
4O 100 105 110
Asn Val AspSerIle LeuAlaVal LysLysTyr PheGlnArg IleThr
115 120 125
4$ Leu Tyr LeuThrGlu LysLysTyr SerProCys AlaTrpGlu ValVal
130 135 140
Arg Ala GluIleMet ArgSerPhe SerPheSer ThrAsnLeu GlnLys
145 150 155 160
$0
Arg Leu ArgArgLys Glu
165
$$ <210> 61
<211> 166
<212> PRT
<213> Artificial Sequence
33
CA 02385045 2002-03-14
WO 01/25438 PCT/US00/27781
<220>
<223> Descripti on Sequence: amino
of Synthetic acid
Artificial
<220>
<223> CloneID 2D1
<400> 61
Cys Asp ProGln ThrHisSer LeuGlyAsnArg ArgAla LeuIle
Leu
1 5 10 15
Leu Leu GlnMet ArgArgIle SerProPheSer CysLeu LysAsp
Ala
20 25 30
Arg His PheGly PheProGln GluGluPheAsp GlyAsn GlnPhe
Asp
1$ 35 40 45
Gln Lys GlnAla IleSerAla PheHisGluMet IleGln GlnThr
Ala
50 55 60
Phe Asn PheSer ThrLysAsp SerSerAlaAla TrpGlu GlnSer
Leu
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 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 Phe Ser Thr Asn Leu Gln Lys
145 150 155 160
Arg Leu Arg Arg Lys Glu
165
<210> 62
<211> 166
<212> PRT
4$ <213> Artificial Sequence
<220>
<223> Description of Artificial Sequence: Synthetic amino acid
<2zo>
<223> Clone ID 2D10
<400> 62
Cys Asp Leu Pro Gln Thr His Ser Leu Gly Asn Arg Arg Ala Leu Ile
5$ 1 5 10 15
Leu Leu Ala Gln Met Gly Arg Val Ser Pro Phe Ser Cys Leu Lys Asp
20 25 30
34
CA 02385045 2002-03-14
WO 01/25438 PCT/US00/27781
Arg His Phe GlyPheProGln GluGluPhe AspGlyAsn GlnPhe
Asp
35 40 45
Gln Lys Gln AlaIleSerAla PheHisGlu MetIleGln GlnThr
Ala
50 55 60
Phe Asn Phe SerThrLysAsp SerSerAla ThrTrpGlu GlnSer
Leu
65 70 75 80
Leu Leu Lys PheSerThrGlu LeuTyrGln GlnLeuAsn AsnLeu
Glu
85 90 95
Glu Ala Val IleGlnGluVal GlyValGlu GluThrPro LeuMet
Cys
100 105 110
1$
Asn Val Ser IleLeuAlaVal LysLysTyr PheArgArg IleThr
Asp
115 120 125
Leu Tyr Thr GluLysLysTyr SerProCys AlaTrpGlu ValVal
Leu
130 135 140
Arg Ala Ile MetArgSerPhe SerPheSer ThrAsnLeu GlnLys
Glu
145 150 155 160
2$ Arg Leu Arg LysGlu
Arg
165
<210> 63
<211> 166
<212> PRT
<213> Artificial Sequence
<220>
3$ <223> Descriptio n Artificial Sequence: amino
of Synthetic acid
<220>
<223> CloneID D7
2
<400> 63
Cys Asp Pro GlnThrHisSer LeuGlyAsn ArgArgAla LeuIle
Leu
1 5 10 15
Leu Leu Ala Gln Met Gly Arg Ile Ser Pro Phe Ser Cys Leu Lys Asp
20 25 . 30
Arg HisAsp PheArgPhe ProGlnGlu GluPheAsp GlyAsn GlnPhe
35 40 45
SO Gln LysAla GlnAlaIle SerValLeu HisGluMet IleGln GlnThr
55 60
Phe AsnLeu PheSerThr LysAspSer SerAlaThr TrpGlu GlnSer
65 70 75 80
Leu LeuGlu LysPheSer ThrGluLeu TyrGlnGln LeuAsn AsnLeu
85 90 95
Glu AlaCys ValIleGln GluValGly ValGluGlu ThrPro LeuMet
CA 02385045 2002-03-14
WO 01/25438 PCT/US00/27781
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 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 Lys
145 150 155 160
Arg Leu Arg Arg Lys Glu
165
1$
<210> 64
<211> 166
<212> PRT
<213> Artificia l
Sequence
<220>
_ <223> Descripti on Sequence: amino
of Synthetic acid
Artificial
<220>
2$ <223> CloneID 2D9
<400> 64
Cys Asp ProGlnThr HisSerLeu GlyAsnArg ArgAlaLeu Ile
Leu
1 5 10 15
Leu Leu GlnMetGly ArgIleSer ProPheSer CysLeuLys Asp
Ala
20 25 30
Arg His PheGlyPhe ProGlnGlu GluPheAsp GlyAsnGln Phe
Asp
3$ 35 40 45
Gln Lys GlnAlaIle SerValLeu HisGluMet IleGlnGln Thr
Ala
50 55 60
Phe Asn PheSerThr LysAspSer SerAlaThr TrpGluGln Ser
Leu
65 70 75 80
Leu Leu LysPheSer ThrGluLeu AsnGlnGln LeuAsnAsp Leu
Glu
85 90 95
4$
Glu Ala ValIleGln GluValGly ValGluGlu ThrProLeu Val
Cys
100 105 110
Asn Val SerIleLeu AlaValLys LysTyrPhe GlnArgIle Thr
Asp
$0 115 120 125
Leu Tyr ThrGluLys LysTyrSer ProCysAla TrpGluVal Val
Leu
130 135 140
$$ Arg Ala IleMetArg SerPheSer PheSerThr AsnLeuGln Lys
Glu
145 150 155 160
Arg Leu ArgLysGlu
Arg
165
36
CA 02385045 2002-03-14
WO 01/25438 PCT/US00/27781
<210> 65
<211> 166
<212> PRT
<213> Artificia l
Sequence
<220>
<223> Descripti on Sequence: amino
of Synthetic acid
Artificial
<220>
<223> CloneID 2DA2
<400> 65
Cys Asp ProGln ThrHisSerLeu GlyAsn ArgArgPro LeuIle
Leu
1 5 10 15
Leu Leu GlnMet GlyArgIleSer ProPhe SerCysLeu LysAsp
Ala
20 25 30
Arg Gln PheGly PheProGlnGlu GluPhe AspGlyAsn GlnPhe
Asp
35 40 45
Gln Lys GlnAla IleSerValLeu HisGlu MetMetGln GlnThr
Ala
50 55 60
Phe Asn PheSer ThrLysAsnSer SerAla AlaTrpGlu GlnSer
Leu
65 70 75 80
Leu Leu LysPhe SerThrGluLeu HisGln GlnLeuAsn GluLeu
Glu
85 90 95
Glu Ala ValIle GlnGluValGly ValGlu GluThrPro LeuMet
Cys
100 105 110
Asn Val SerIle LeuAlaValLys LysTyr PheGlnArg IleThr
Asp
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 Phe Ser Phe Ser Thr Asn Leu Gln Lys
145 150 155 160
Arg Leu Arg Arg Lys Glu
165
<210> 66
SO <211> 166
<212> PRT
<213> Artificial Sequence
<220>
<223> Description of Artificial Sequence: Synthetic amino acid
<220>
<223> Clone ID 2DH9
37
CA 02385045 2002-03-14
WO 01/25438 PCT/US00/27781
<400> 66
Cys Asp Leu Pro Gln Thr His Ser Pro Gly Asn Arg Arg Ala Leu Met
1 5 10 15
Leu Leu Ala Gln Met Gly Arg Ile Ser Pro Phe Ser Cys Leu Lys Asp
20 25 30
Arg Tyr Asp Phe Gly Phe Pro Gln Gly 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 Glu Gln Ser
65 70 75 80
Leu Leu Glu Lys Phe Ser Thr Glu Leu Tyr Arg Gln Leu Asn Asp Leu
85 90 95
Glu Ala Val IleGlnGluVal GlyVal GluGluThr ProLeuMet
Cys
100 105 110
Asn Val Ser IleLeuAlaVal ArgLys TyrPheGln ArgIleThr
Asp
115 120 125
Leu Tyr Thr GluLysLysHis SerPro CysSerTrp GluValVal
Leu
130 135 140
Arg Ala Ile MetArgSerPhe SerPhe SerThrAsn LeuGlnLys
Glu
145 150 155 160
Arg Leu Arg LysGlu
Arg
165
<210> 67
<211> 166
<212> PRT
<213> Artificial Sequence
<220>
<223> Description Artificial Sequence: amino
of Synthetic acid
<220>
<223> CloneID 611
2
<400> 67
Cys Asp Pro GlnThrHisSer LeuGly AsnArgArg AlaLeuIle
Leu
1 5 10 15
Leu Leu Gln MetGlyArgIle SerPro PheSerCys LeuLysAsp
Ala
20 25 30
Arg His Phe GlyLeuProGln GluGlu PheAspGly AsnGlnPhe
Asp
35 40 45
Gln Lys Gln AlaIleSerVal LeuHis GluMetIle GlnGlnThr
Thr
50 55 60
38
CA 02385045 2002-03-14
WO 01/25438 PCT/US00/27781
Phe Asn Leu Phe Ser Thr Lys Asp Ser Ser Asp Thr Trp Glu Gln Ser
65 70 75 80
Leu Leu Glu Lys Phe Tyr Ile Glu Leu Phe Gln Gln Leu Asn Asp Leu
85 90 95
Glu Ala Val IleGlnGlu ValGlyValGlu GluThr ProLeuMet
Cys
100 105 110
Asn Val Ser IleLeuAla ValArgLysTyr PheGln ArgIleThr
Asp
115 120 125
Leu Tyr Thr GluGluLys TyrSerProCys AlaTrp GluValVal
Leu
130 135 140
Arg Ala Ile MetArgSer PheSerPheSer ThrAsn LeuGlnLys
Glu
145 150 155 160
Arg Leu Arg LysGlu
Arg
165
<210> 68
<211> 166
<212> PRT
<213> Artificial Sequence
<220>
<223> Descriptio n Artificial Sequence: amino
of Synthetic acid
<220>
<223> CloneID 612
2
<400> 68
Cys Asp Pro GlnThrHis SerLeuGlyAsn ArgArg ThrLeuMet
Leu
1 5 10 15
Leu Met Ala Gln Met Arg Arg Ile Ser Pro Phe Pro Arg Leu Lys Asp
20 25 30
Arg Tyr AspPheGly PhePro GlnGluValPhe AspGly AsnGlnPhe
35 40 45
Gln Lys AlaGlnAla IlePhe LeuPheHisGlu MetMet GlnGlnThr
4S 50 55 60
Phe Asn LeuPheSer ThrLys AsnSerSerAla AlaTrp AspGluThr
65 70 75 80
Leu Leu AspLysPhe TyrThr GluLeuTyrGln GlnLeu AsnAspLeu
85 90 95
Glu Ala CysValMet GlnGlu GlyArgValGly GluThr ProLeuMet
100 105 110
Asn Ala AspSerIle LeuAla ValLysLysTyr PheArg ArgIleThr
115 120 125
Leu Tyr LeuThrGlu LysLys TyrSerProCys AlaTrp GluAlaVal
39
CA 02385045 2002-03-14
WO 01/25438 PCT/US00/27781
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
<210> 69
<211> 166
<212> PRT
<213> Artificia l
Sequence
15<220>
<223> Descripti on Sequence: amino
of Synthetic acid
Artificial
<220>
<223> CloneID 2H9
<400> 69
Cys Asp ProGln ThrHis SerLeuGly AsnArgArg AlaLeuIle
Leu
1 5 10 15
25Leu Leu GlnMet GlyArg IleSerPro PheSerCys LeuLysAsp
Ala
20 25 30
Arg His PheGly PhePro GlnGluGlu PheAspGly AsnGlnPhe
Asp
35 40 45
Gln Lys GlnAla IleSer ValLeuHis GluMetIle GlnGlnThr
Ala
50 55 60
Phe Asn PheSer ThrLys AspSerSer AlaThrTrp GluGlnSer
Leu
3$65 70 75 80
Leu Leu Glu Lys Phe Ser Thr Glu Leu Asn Gln Gln Leu Asn Asp Leu
85 90 95
40Glu AlaCysVal ThrGln GluValGly ValGluGlu ThrProLeu Met
100 105 110
Asn GluAspSer IleLeu AlaValLys LysTyrPhe GlnArgIle Thr
115 120 125
45
Leu TyrLeuThr GluLys LysTyrSer ProCysAla TrpGluVal Val
130 135 140
Arg AlaGluIle MetArg SerPheSer PheSerThr AsnLeuGln Lys
50145 150 155 160
Arg LeuArgArg LysGlu
165
<210> 70
<211> 166
<212> PRT
<213> Artificial Sequence
CA 02385045 2002-03-14
WO 01/25438 PCT/US00/27781
<220>
<223> on cialSequence: amino
Descripti of Synthetic acid
Artifi
<220>
<223> ID 6BC11
Clone
<400>
70
Cys Asp LeuProGln ThrHisSer LeuGlyAsn ArgArgAla LeuIle
1 5 10 15
Leu Leu AlaGlnMet GlyArgIle SerProPhe SerCysLeu LysAsp
20 25 30
IS Arg Tyr AspPheGly PheProGln GluGluPhe AspGlyAsn GlnLeu
35 40 45
Gln Lys AlaGlnAla IleSerVal LeuHisGlu MetIleGln GlnThr
50 55 60
Phe Asn LeuPheSer ThrLysAsp SerSerAla AlaTrpGlu GlnSer
65 70 75 80
Leu Leu GluLysPhe SerThrGlu LeuAsnGln GlnLeuAsn AspLeu
2$ 85 90 95
Glu Ala CysValIle GlnGluVal GlyValGlu GluThrPro LeuMet
100 105 110
Asn Val AspSerIle LeuAlaVal LysLysTyr PheGlnArg IleThr
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 Lys
145 150 155 160
Arg Leu Arg Arg Lys Glu
165
<210> 71
<211> 166
<212> PRT
<213> Artificial Sequence
<220>
<223> Description of Artificial Sequence: Synthetic amino acid
<220>
<223> Clone ID tl9bb
<220>
5$ <221> MOD_RES
<222> (11)
<223> N or D
<220>
41
CA 02385045 2002-03-14
WO 01/25438 PCT/US00/27781
<221> MOD_RES
<222> (12)
<223> R, S, or K
S <220>
<221>MOD_RES
<222>(15)
<223>L M
or
<220>
<221>MOD_RES
<222>(16)
<223>I, or
M V
1S <220>
<221>MOD_RES
<222>(19)
<223>A G
or
<220>
<221> MOD_RES
<222> (22)
<223> G R
or
<220>
<221> MOD_RES
<222> (24)
<223> I T
or
<220>
<221> MOD_RES
<222> (26)
<223> P H
or
<220>
<221> MOD_RES
<222> (34)
<223> H, or
Y Q
<220>
<221> MOD_RES
<222> (38)
<223> F L
or
<220>
<221> MOD_RES
<222> (40)
<223> Q R
or
<220>
<221> MOD_RES
<222> (45)
<223> G S
or
5$ <220>
<221> MOD_RES
<222> (46)
<223> N H
or
42
CA 02385045 2002-03-14
WO 01/25438 PCT/US00/27781
<220>
<221> MOD_RES
<222> (47)
<223> Q or R
<220>
<221> MOD_RES
<222> (50)
<223> K or R
<220>
<221> MOD_RES
<222> (51)
<223> A or T
1$
<220>
<221> MOD_RES
<222> (55)
<223> S or F
<220>
<221> MOD_RES
<222> (56)
<223> V or A
2$
<220>
<221> MOD_RES
<222> (57)
<223> L or F
<220>
<221> MOD_RES
<222> (60)
<223> M or I
3$
<220>
<221> MOD_RES
<222> (61)
<223> I or M
<220>
<221> MOD_RES
<222> (67)
<223> L or F
4$
<220>
<221> MOD_RES
<222> (72)
<223> D or N
$0
<220>
<221> MOD_RES
<222> (75)
<223> A or V
$$
<220>
<221> MOD_RES
<222> (76)
<223> A or T
43
CA 02385045 2002-03-14
WO 01/25438 PCT/US00/27781
<220>
<221> MOD_RES
<222> (78)
$ <223> E or D
<220>
<221> MOD_RES
<222> (79)
<223> Q or E
<220>
<221> MOD_RES
<222> (80)
<223> S, R, T,
or N
<220>
<221> MOD_RES
<222> (83)
<223> E or D
<220>
<221> MOD_RES
<222> (85)
<223> F or L
<220>
<221> MOD_RES
<222> (86)
<223> S or Y
<220>
<221> MOD_RES
<222> (88)
<223> E or G
<220>
<221> MOD_RES
<222> (90)
4O <223> Y, H, N
<220>
<221> MOD_RES
<222> (95)
4$ <223> D, E, or
N
<220>
<221> MOD_RES
<222> (101)
50 <223> I, M, or
V
<220>
<221> MOD_RES
<222> (103)
55 <223> E or G
<220>
<221> MOD_RES
<222> (105)
44
CA 02385045 2002-03-14
WO 01/25438 PCT/US00/27781
<223> G or 4J
<220>
<221> MOD_RES
$ <222> (106)
<223> V or M
<220>
<221> MOD_RES
<222> (107)
<223> E, G, or K
<220>
<221> MOD_RES
1$ <222> (108)
<223> E or G
<220>
<221> MOD_RES
<222> (114)
<223> V, E, or G
<220>
<221> MOD_RES
<222> (116)
<223> S or P
<220>
<221> MOD_RES
<222> (121)
<223> K or R
<220>
<221> MOD_RES
<222> (124)
<223> F or L
<220>
<221> MOD_RES
4O <222> (132)
<223> T, I, or M
<220>
<221> MOD_RES
<222> (134)
<223> K or R
<220>
<221> MOD_RES
$~ <222> (140)
<223> A or S
<400> 71
Cys
Asp
Leu
Pro
Gln
Thr
His
Ser
Leu
Gly
Xaa
Xaa
Arg
Ala
Xaa
Xaa
1 5 10 15
Leu
Leu
Xaa
Gln
Met
Xaa
Arg
Xaa
Ser
Xaa
Phe
Ser
Cys
Leu
Lys
Asp
20 25 30
CA 02385045 2002-03-14
WO 01/25438 PCT/US00/27781
Arg Xaa Asp Phe Gly Xaa Pro Xaa Glu Glu Phe Asp Xaa Xaa Xaa Phe
35 40 45
Gln Xaa Xaa Gln Ala Ile Xaa Xaa Xaa His Glu Xaa Xaa Gln Gln Thr
50 55 60
Phe Asn Xaa Phe Ser Thr Lys Xaa Ser Ser Xaa Xaa Trp Xaa Xaa Xaa
65 70 75 80
1~ Leu Leu Xaa Lys Xaa Xaa Thr Xaa Leu Xaa Gln Gln Leu Asn Xaa Leu
85 90 95
Glu Ala Cys Val Xaa Gln Xaa Val Xaa Xaa Xaa Xaa Thr Pro Leu Met
100 105 110
Asn Xaa Asp Xaa Ile Leu Ala Val Xaa Lys Tyr Xaa Gln Arg Ile Thr
115 120 125
Leu Tyr Leu Xaa Glu Xaa Lys Tyr Ser Pro Cys Xaa Trp Glu Val Val
130 135 140
Arg Ala Glu Ile Met Arg Ser Phe Ser Phe Ser Thr Asn Leu Gln Lys
145 150 155 160
2$ Arg Leu Arg Arg Lys Glu
165
<210> 72
30 <211> 498
<212> DNA
<213> Artificial Sequence
<220>
35 <223> Description of Artificial Sequence: Synthetic DNA
<220>
<223> Clone ID CH1.1
4~ <400> 72
tgtgatctgc ctcagaccca cagccttggt aacaggaggg ccttgatact cctggcacaa 60
atgggaagaa tctctccttt ctcctgtctg atggacagac atgactttgg atttccccag 120
gaggagtttg atgacaacca gttccagaag gctcaagcca tctctgtcct ccatgagatg 180
atccaacaga ccttcaatct cttcagcaca aaggactcat ctgctacttg ggatgagaca 240
45 cttctagaca aattctacac tgaactttac cagcagctga atgacctgga agcctgcgtg 300
atacaggagg ttggggtgga agagactccc ctgatgaatg aggactccat cttggctgtg 360
aagaaatact tccgaagaat cactctctat ctgacagaga agaaatacag cccttgtgcc 420
tgggaggttg tcagagcaga aatcatgaga tctttctctt tttcaacaaa cttgcaaaaa 480
agattaagga ggaaggaa 498
SO
<210> 73
<211> 498
<212> DNA
<213> Artificial Sequence
$$
<220>
<223> Description of Artificial Sequence: Synthetic DNA
<220>
46
CA 02385045 2002-03-14
WO 01/25438 PCT/US00/27781
<223> Clone ID CH1.2
<400> 73
tgtgatctgc ctcagaccca cagccttggt aacaggaggg ccttgatact cctggcacaa 60
$ atgggaagaa tctctccttt ctcctgcctg aaggacagac atgactttgg attcccccag 120
gaggagtttg atggcaacca gttccagaag gctcaaggca tctctgtcct ccatgagatg 180
atccagcaga ccttccatct cttcagcaca aaggactcat ctgctacttg ggaacagagc 240
ctcctagaaa aattttccac tgaacttaac cagcagctga atgacctgga agcctgcgtg 300
atacaggagg ttggggtgga agagactccc ctgatgaatg tggactccat cctggctgtg 360
aagaaatact tccgaagaat cactctttat ctgacagaga agaaatacag cccttgtgcc 420
tgggaggttg tcagagcaga aatcatgaga tctttctctt tttcaacaaa cttgcaaaaa 480
agattaagga ggaaggaa 498
<210> 74
1$ <211> 498
<212> DNA
<213> Artificial Sequence
<220>
<223> Description of Artificial Sequence: Synthetic DNA
<220>
<223> Clone ID CH1.3
<400> 74
tgtgatctgc ctcagaccca cagccttggt aacaggagga ctttgatgat aatggcacaa 60
atgggaagaa tctctccttt ctcctgcctg aaggacagac atgactttgg atttcctcag 120
gaggagtttg atggcaacca gttccagaag gctcaagcca tctctgtcct ccatgagatg 180
atccagcaga ccttcaatct cttcagcaca aaggactcat ctgctacttg ggatgagaca 240
cttctagaca aattctacac tgaactttac cagcagctga atgacctgga agcctgtatg 300
atgcaggagg ttggagtgga agacactcct ctgatgaatg tggactctat cctgactgtg 360
agaaaatact ttcgaagaat cactctttat ctgacagaga agaaatacag cccttgtgcc 420
tgggaggttg tcagagcaga aatcatgaga tctttctctt tttcaacaaa cttgcaaaaa 480
agattaagga ggaaggaa 498
<210> 75
<211> 498
<212> DNA
<213> Artificial Sequence
<220>
<223> Description of Artificial Sequence: Synthetic DNA
<220>
4S <223> Clone ID CH1.4
<400> 75
tgtgatctgc ctcagaccca cagcctgggt aataggaggg ccttgatact cctggcacaa 60
atgggaagaa tctctccttt ctcctgcctg aaggacagac atgactttgg attcccccag 120
$0 gaggagtttg gtggcaacca gttccagaag gctcaagcca tctctgtcct ccatgagatg 180
atccagcaga ccttcaatct cttcagcaca gaggactcat ctgctgcttg ggatgagacc 240
ctcctagaca aattctacat tgaacttttc cagcaactga atgacctgga agcctgtgtg 300
atgcaggagg agagggtggg agaaactccc ctgatgaatg cggactccat cttggctgtg 360
aagaaatact tccaaagaat cactctttat ctgacagaga agaaatacag cccttgtgcc 420
55 tgggaggttg tcagagcaga aatcatgaga tctttctctt tttcaacaaa cttgcaaaaa 480
agattaagga ggaaggaa 498
<210> 76
<211> 498
47
CA 02385045 2002-03-14
WO 01/25438 PCT/US00/27781
<212> DNA
<213> Artificial Sequence
<220>
<223> Description of Artificial Sequence: Synthetic DNA
<220>
<223> Clone ID CH2.1
<400> 76
tgtgatctgc ctcagaccca cagccttggt aacaggagga ctttgatgat aatggcacaa 60
atgggaagaa tctctccttt ctcctgcctg aaggacagac atgactttgg atttcctcag 120
gaggagtttg atggcaacca gttccagaag gctcaagcca tctctgtcct ccatgagatg 180
atccagcaga ccttcaatct cttcagcaca aaggactcat ctgctacttg ggatgagaca 240
cttctagaca aattctacac tgaactttac cagcagctga atgacctgga agcctgtatg 300
atacaggagg ttggggtgga agagactccc ctgatgaatg aggactccat cttggctgtg 360
aagaaatact tccgaagaat cactctctat ctgacagaga agaaatacag cccttgtgcc 420
tgggaggttg tcagagcaga aatcatgaga tctttctctt tttcaacaaa cttgcaaaaa 480
agattaagga ggaaggaa 498
<210> 77
<211> 498
<212> DNA
<213> Artificial Sequence
<220>
<223> Description of Artificial Sequence: Synthetic DNA
<220>
<223> Clone ID CH2.2
<400> 77
tgtgatctgc ctcagaccca cagccttggt aacaggaggg ccttgatact cctggcacaa 60
atgggaagaa tctctccttt ctcctgtctg atggacagac atgactttgg atttccccag 120
gaggagtttg atgacaacca gttccagaag gctcaagcca tctctgtcct ccatgagatg 180
atccaacaga ccttcaatct cttcagcaca aaggactcat ctgctacttg ggatgagaca 240
cttctagaca aattctacac tgaactttac cagcagctga atgacctgga agcctgtatg 300
atgcaggagg ttggagtgga agacactcct ctgatgaatg tggactctat cctgactgtg 360
aagaaatact tccgaagaat cactctttat ctgacagaga agaaatacag cccttgtgcc 420
tgggaggttg tcagagcaga aatcatgaga tctttctctt tttcaacaaa cttgcaaaaa 480
agattaagga ggaaggaa 498
<210> 78
<211> 498
4$ <212> DNA
<213> Artificial Sequence
0
<220>
<223> Description of Artificial Sequence: Synthetic DNA
<220>
<223> Clone ID CH2.3
<400> 78
$$ tgtgatctgc ctcagaccca cagccttggt aacaggagga ctttgatgat aatggcacaa 60
atgggaagaa tctctccttt ctcctgcctg aaggacagac atgactttgg atttcctcag 120
gaggagtttg atggcaacca gttccagaag gctcaagcca tctctgtcct ccatgagatg 180
atccagcaga ccttcaatct cttcagcaca aaggactcat ctgctacttg ggatgagaca 240
cttctagaca aattctacac tgaactttac cagcagctga atgacctgga agcctgtatg 300
48
CA 02385045 2002-03-14
WO 01/25438 PCT/US00/27781
atgcaggagg ttggagtgga agacactcct ctgatgaatg aggactccat cttggctgtg 360
aagaaatact tccgaagaat cactctctat ctgacagaga agaaatacag cccttgtgcc 420
tgggaggttg tcagagcaga aatcatgaga tctttctctt tctcaacaaa cttgcaaaaa 480
agattaagga ggaaggaa 498
<210> 79
<211> 166
<212> PRT
<213> Artificial Sequence
<220>
<223> Description of Artificial Sequence: Synthetic amino acid
<220>
<223> Clone ID CH1.1
<400> 79
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 Met Asp
20 25 30
Arg His Asp Phe Gly Phe Pro Gln Glu Glu Phe Asp Asp 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 Val Ile Gln Glu Val Gly Val Glu Glu Thr Pro Leu Met
100 105 110
Asn Glu 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 Phe Ser Phe Ser Thr Asn Leu Gln Lys
145 150 155 160
Arg Leu Arg Arg Lys Glu
165
<210> 80
<211> 166
<212> PRT
<213> Artificial Sequence
<220>
<223> Description of Artificial Sequence: Synthetic amino acid
49
CA 02385045 2002-03-14
WO 01/25438 PCT/US00/27781
<220>
<223> CloneID CH1.2
<400> 80
Cys Asp ProGln ThrHisSer LeuGlyAsn ArgArgAla LeuIle
Leu
1 5 10 15
Leu Leu GlnMet GlyArgIle SerProPhe SerCysLeu LysAsp
Ala
20 25 30
Arg His PheGly PheProGln GluGluPhe AspGlyAsn GlnPhe
Asp
35 40 45
Gln Lys GlnGly IleSerVal LeuHisGlu MetIleGln GlnThr
Ala
50 55 60
Phe His PheSer ThrLysAsp SerSerAla ThrTrpGlu GlnSer
Leu
65 70 75 80
Leu Leu LysPhe SerThrGlu LeuAsnGln GlnLeuAsn AspLeu
Glu
85 90 95
Glu Ala ValIle GlnGluVal GlyValGlu GluThrPro LeuMet
Cys
100 105 110
Asn Val SerIle LeuAlaVal LysLysTyr PheArgArg IleThr
Asp
115 120 125
Leu Tyr ThrGlu LysLysTyr SerProCys AlaTrpGlu ValVal
Leu
130 135 140
Arg Ala IleMet ArgSerPhe SerPheSer ThrAsnLeu GlnLys
Glu
145 150 155 160
Arg Leu ArgLys Glu
Arg
165
<210> 81
<211> 166
<212> PRT
<213> Artificial
Sequence
<220>
4$ <223> Description Artificial Sequence: amino
of Synthetic acid
<220>
<223> CloneID
CH1.3
<400> 81
Cys Asp ProGln ThrHisSer LeuGlyAsn ArgArgThr LeuMet
Leu
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
CA 02385045 2002-03-14
WO 01/25438 PCT/US00/27781
Gln Lys Ala Gln Ala Ile Ser Val Leu His Glu Met Ile Gln Gln Thr
50 55 60
Phe Asn LeuPheSer ThrLysAsp SerSerAla ThrTrpAsp GluThr
$ 65 70 75 80
Leu Leu AspLysPhe TyrThrGlu LeuTyrGln GlnLeuAsn AspLeu
85 90 95
Glu Ala CysMetMet GlnGluVal GlyValGlu AspThrPro LeuMet
100 105 110
Asn Val AspSerIle LeuThrVal ArgLysTyr PheArgArg IleThr
115 120 125
Leu Tyr LeuThrGlu LysLysTyr SerProCys AlaTrpGlu ValVal
130 135 140
Arg Ala GluIleMet ArgSerPhe SerPheSer ThrAsnLeu GlnLys
145 150 155 160
Arg Leu ArgArgLys Glu
165
<210> 82
<211> 166
<212> PRT
<213> Artificial Sequence
<220>
<223> Descriptio n Artificial Sequence: amino
of Synthetic acid
<220>
<223> CloneID H1.4
C
<400> 82
Cys Asp Pro GlnThrHisSer LeuGlyAsn ArgArg AlaLeuIle
Leu
1 5 10 15
Leu Leu Gln MetGly-ArgIle SerProPhe SerCys LeuLysAsp
Ala
20 25 30
Arg His Phe GlyPheProGln GluGluPhe GlyGly AsnGlnPhe
Asp
35 40 45
Gln Lys Gln AlaIleSerVal LeuHisGlu MetIle GlnGlnThr
Ala
55 60
50 Phe Asn Phe SerThrGluAsp SerSerAla AlaTrp AspGluThr
Leu
65 70 75 80
Leu Leu Lys PheTyrIleGlu LeuPheGln GlnLeu AsnAspLeu
Asp
85 90 95
Glu Ala Val MetGlnGluGlu ArgValGly GluThr ProLeuMet
Cys
100 105 110
Asn Ala Ser IleLeuAlaVal LysLysTyr PheGln ArgIleThr
Asp
51
CA 02385045 2002-03-14
WO 01/25438 PCT/US00/27781
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
<210> 83
<211> 166
1$ <212> PRT
<213> Artificial
Sequence
<220>
<223> Description Artificial Sequence: amino
of Synthetic acid
<220>
<223> CloneID
CH2.1
<400> 83
2$ Cys Asp ProGln ThrHisSer LeuGlyAsn ArgArgThr LeuMet
Leu
1 5 10 15
Ile Met GlnMet GlyArgIle SerProPhe SerCysLeu LysAsp
Ala
20 25 30
Arg His PheGly PheProGln GluGluPhe AspGlyAsn GlnPhe
Asp
40 45
Gln Lys GlnAla IleSerVal LeuHisGlu MetIleGln GlnThr
Ala
3$ 50 55 60
Phe Asn PheSer ThrLysAsp SerSerAla ThrTrpAsp GluThr
Leu
65 70 75 80
Leu Leu LysPhe TyrThrGlu LeuTyrGln GlnLeuAsn AspLeu
Asp
85 90 95
Glu Ala MetIle GlnGluVal GlyValGlu GluThrPro LeuMet
Cys
100 105 110
4$
Asn Glu SerIle LeuAlaVal LysLysTyr PheArgArg IleThr
Asp
115 120 125
Leu Tyr ThrGlu LysLysTyr SerProCys AlaTrpGlu ValVal
Leu
$0 130 135 140
Arg Ala IleMet ArgSerPhe SerPheSer ThrAsnLeu GlnLys
Glu
145 150 155 160
$$ Arg Leu ArgLys Glu
Arg
165
<210> 84
52
CA 02385045 2002-03-14
WO 01/25438 PCT/US00/27781
<211> 166
<212> PRT
<213> Artificia l
Sequence
$ <220>
<223> Descripti on Sequence: amino
of Synthetic acid
Artificial
<220>
<223> CloneID CH2.2
<400> 84
Cys Asp ProGln ThrHisSerLeu GlyAsnArg ArgAla LeuIle
Leu
1 5 10 15
1$ Leu Leu GlnMet GlyArgIleSer ProPheSer CysLeu MetAsp
Ala
20 25 30
Arg His PheGly PheProGlnGlu GluPheAsp AspAsn GlnPhe
Asp
35 40 45
Gln Lys GlnAla IleSerValLeu HisGluMet IleGln GlnThr
Ala
50 55 60
Phe Asn PheSer ThrLysAspSer SerAlaThr TrpAsp GluThr
Leu
2$ 65 70 75 80
Leu Leu LysPhe TyrThrGluLeu TyrGlnGln LeuAsn AspLeu
Asp
85 90 95
Glu Ala MetMet GlnGluValGly ValGluGlu ThrPro LeuMet
Cys
100 105 110
Asn Val SerIle LeuThrValLys LysTyrPhe ArgArg IleThr
Asp
115 120 125
3$
Leu Tyr ThrGlu LysLysTyrSer ProCysAla TrpGlu ValVal
Leu
130 135 140
Arg Ala IleMet ArgSerPheSer PheSerThr AsnLeu GlnLys
Glu
145 150 155 160
Arg Leu ArgLys Glu
Arg
165
4$
<210> 85
<211> 166
<212> PRT
<213> Artificial Sequence
$0
<220>
<223> Description of Artificial Sequence: Synthetic amino acid
<220>
$$ <223> Clone ID CH2.3
<400> 85
Cys Asp Leu Pro Gln Thr His Ser Leu Gly Asn Arg Arg Thr Leu Met
1 5 10 15
53
CA 02385045 2002-03-14
WO 01/25438 PCT/US00/27781
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
1$ 85 90 95
Glu AlaCys MetMet GlnGluValGly ValGluGlu ThrPro LeuMet
100 105 110
Asn GluAsp SerIle LeuAlaValLys LysTyrPhe ArgArg IleThr
115 120 125
Leu TyrLeu ThrGlu LysLysTyrSer ProCysAla TrpGlu ValVal
130 135 140
2$
Arg AlaGlu IleMet ArgSerPheSer PheSerThr AsnLeu GlnLys
145 150 155 ' 160
Arg LeuArg ArgLys Glu
165
<210> 86
<211> 15
3$ <212> DNA
<213> Artificial Sequence
<220>
<223> Description ArtificialSequence: Synthetic DNA
of
,
<400> 86 .
tgcgacttac cacaa 15
<210> 87
4$ <211> 26
<212> PRT
<213> Artificial
Sequence
<220>
$0 <223> Description ArtificialSequence: Synthetic amino acid
of
<400> 87
Trp Glu Val Val Ser Glu Met Arg Ser Phe 5er Tyr Ser
Arg Ile Thr
1 5 10 15
$$
Asn Leu Gln Arg Leu Arg Lys Asp
Arg Arg
20 25
54
CA 02385045 2002-03-14
WO 01/25438 PCT/US00/27781
<210> 88
<211> 26
<212> PRT
<213> Artificial Sequence
<220>
<223> Description of Artificial Sequence: Synthetic amino acid
<400> 88
1~ Trp Glu Leu Val Arg Ala Glu Ile Val Arg Ser Phe Ser Phe Ser Thr
1 5 10 15
Asn Leu Asn Lys Arg Leu Arg Lys Lys Glu
20 25
1$
ss
