U.S. patent application number 13/225004 was filed with the patent office on 2012-06-28 for interleukin-1 alpha antibodies and methods of use.
This patent application is currently assigned to XBIOTECH, INC.. Invention is credited to John Simard.
Application Number | 20120164665 13/225004 |
Document ID | / |
Family ID | 41380314 |
Filed Date | 2012-06-28 |
United States Patent
Application |
20120164665 |
Kind Code |
A1 |
Simard; John |
June 28, 2012 |
Interleukin-1 Alpha Antibodies and Methods of Use
Abstract
Fully human monoclonal Abs includes (i) an antigen-binding
variable region that exhibits very high binding affinity for
IL-1.alpha. and (ii) a constant region that is effective at both
activating the complement system though C1q binding and binding to
several different Fc receptors.
Inventors: |
Simard; John; (Austin,
TX) |
Assignee: |
XBIOTECH, INC.
Vancouver
BC
|
Family ID: |
41380314 |
Appl. No.: |
13/225004 |
Filed: |
September 2, 2011 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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12455458 |
Jun 1, 2009 |
8034337 |
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13225004 |
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61057586 |
May 30, 2008 |
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61121391 |
Dec 10, 2008 |
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61178350 |
May 14, 2009 |
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Current U.S.
Class: |
435/7.24 |
Current CPC
Class: |
A61P 37/00 20180101;
C07K 2317/76 20130101; C07K 16/245 20130101; A61P 29/00 20180101;
C12N 2330/50 20130101; A61P 9/10 20180101; A61K 2039/55527
20130101; C07K 2317/14 20130101; A61P 35/00 20180101; A61K 39/3955
20130101; C12N 5/0693 20130101; A61P 37/06 20180101; C07K 14/5412
20130101; C07K 2317/92 20130101; G01N 2333/545 20130101; C07K
2317/24 20130101; C12N 5/069 20130101; G01N 33/6863 20130101; G01N
33/6869 20130101; C07K 14/545 20130101; C07K 2317/21 20130101; C07K
16/24 20130101; A61P 43/00 20180101 |
Class at
Publication: |
435/7.24 |
International
Class: |
G01N 33/577 20060101
G01N033/577 |
Claims
1. A method of tracking inflammation in a human subject previously
subjected to the steps of: obtaining from the subject a first
sample of peripheral blood mononuclear cells at a first time;
contacting the first sample with a purified mAb that specifically
binds to human IL-1.alpha.; and determining the percent of cells in
the first sample that bind the mAb, the method comprising the steps
of: (a) obtaining from the subject a second sample of peripheral
blood mononuclear cells at a second time; (b) contacting the second
sample with the purified mAb that specifically binds to human
IL-1.alpha.; (c) determining the percent of cells in the second
sample that bind the monoclonal Ab; and (d) comparing the percent
of cells in the first sample that bind the mAb to the percent of
cells in the second sample that bind the monoclonal Ab.
2. The method of claim 1, wherein the purified mAb is a human IgG1
mAb comprising a heavy chain covalently joined to a light
chain.
3. The method of claim 2, wherein the heavy chain comprises the
amino acid sequence of SEQ ID NO: 9 and a light chain comprises the
amino acid sequence of SEQ ID NO:11.
Description
CROSS-REFERENCE TO RELATED APPLICATIONS
[0001] The present application is divisional application of U.S.
nonprovisional patent application Ser. No. 12/455,458, filed on
Jun. 1, 2009, which claims the priority of U.S. provisional patent
applications Ser. Nos. 61/057,586; 61/121,391; and 61/178,350 filed
on May 30, 2008; Dec. 10, 2008; and May 14, 2009, respectively.
STATEMENT AS TO FEDERALLY SPONSORED RESEARCH
[0002] Not applicable.
FIELD OF THE INVENTION
[0003] The invention relates generally to the fields of immunology,
inflammation, cancer, vascular disorders, and medicine. More
particularly, the invention relates to antibodies (Abs) which
specifically bind interleukin-1.alpha. (IL-1.alpha.) and methods of
using such Abs to treat, prevent, or detect a pathology associated
with aberrant IL-1.alpha. expression.
BACKGROUND
[0004] IL-1.alpha. is pro-inflammatory cytokine that plays a role
in a number of different activities including inflammation, immune
responses, tumor metastasis, and hematopoiesis. IgG autoantibodies
against IL-1.alpha. occur naturally in the general human population
and are thought to be beneficial in diseases such as
atherosclerosis.
SUMMARY
[0005] The invention is based on the development of fully human
monoclonal Abs (mAbs) that include (i) an antigen-binding variable
region that exhibits very high binding affinity for human
IL-1.alpha. and (ii) a constant region that is effective at both
activating the complement system though C1q binding and binding to
several different Fc receptors. The IL-1.alpha. specific mAbs
described herein was made by replacing the constant region of a
human IgG4 mAb having a variable region specific for human
IL-1.alpha. with the constant region of a human IgG1 mAb.
[0006] Accordingly, the invention features a purified human IgG1
mAb that specifically binds to human IL-1.alpha., the mAb including
a heavy chain covalently joined to a light chain. The heavy chain
can include the amino acid sequence of SEQ ID NO: 9 and the light
chain can include the amino acid sequence of SEQ ID NO:11.
[0007] Also within the invention is a set of isolated nucleic acids
including a first nucleic acid encoding the heavy chain of a human
IgG1 mAb that specifically binds to IL-1.alpha., and a second
nucleic acid encoding the light chain of the human IgG1 mAb that
specifically binds to human IL-1.alpha.. The first nucleic acid can
encode the amino acid sequence of SEQ ID NO: 9 and the second
nucleic acid can encode the amino acid sequence of SEQ ID NO:11.
The first nucleic acid can include the nucleotide sequence of SEQ
ID NO: 10 and the second nucleic acid can include the nucleotide
sequence of SEQ ID NO:12.
[0008] In another aspect, the invention features an expression
vector including a nucleic acid encoding the amino acid sequence of
SEQ ID NO: 9 or SEQ ID NO: 11.
[0009] Another feature of the invention is an isolated host cell
(e.g. a mammalian cell such as a CHO cell) including set of
isolated nucleic acids including a first nucleic acid encoding the
heavy chain of a human IgG1 mAb that specifically binds to
IL-1.alpha., and a second nucleic acid encoding the light chain of
the human IgG1 mAb that specifically binds to human IL-1.alpha..
The heavy chain can include the amino acid sequence of SEQ ID NO: 9
and a light chain can include the amino acid sequence of SEQ ID
NO:11.
[0010] The invention further features a method of killing a cell
expressing human IL-1.alpha.. This method can include the step of
contacting the cell with a purified human IgG1 mAb that
specifically binds to human IL-1.alpha..
[0011] A method of inhibiting migration of a human cell through a
basement membrane matrix is also within the invention. This method
can include the step of adding a purified mAb that specifically
binds to human IL-1.alpha. to a mixture including a basement
membrane matrix and the human cell.
[0012] Further within the invention is a method of inhibiting an
IL-1.alpha.-induced increase in ICAM-1 and/or E-selectin expression
on the surface of a human endothelial cell. This method can include
the step of adding a purified mAb that specifically binds to human
IL-1.alpha. to a mixture including the endothelial cell and
IL-1.alpha..
[0013] The invention additionally includes a method of tracking
inflammation in a human subject previously subjected to the steps
of: obtaining from the subject a first sample of peripheral blood
mononuclear cells at a first time; contacting the first sample with
a purified mAb that specifically binds to human IL-1.alpha.; and
determining the percent of cells in the first sample that bind the
monoclonal Ab. This method can include the steps of: (a) obtaining
from the subject a second sample of peripheral blood mononuclear
cells at a second time; (b) contacting the second sample with the
purified mAb that specifically binds to human IL-1.alpha.; (c)
determining the percent of cells in the second sample that bind the
monoclonal Ab; and (d) comparing the percent of cells in the first
sample that bind the mAb to the percent of cells in the second
sample that bind the monoclonal Ab.
[0014] In the foregoing methods, the purified mAb can be a human
IgG1 mAb including a heavy chain covalently joined to a light
chain, e.g., wherein the heavy chain includes the amino acid
sequence of SEQ ID NO: 9 and the light chain includes the amino
acid sequence of SEQ ID NO:11.
[0015] Another method within the invention features the steps of:
(a) enriching a biological sample obtained from a human subject
using a filter to separate molecules according to molecular weight
into a first fraction including intact IgG complexed with
IL-1.alpha. and second fraction including molecules less than 100
Kda; and (b) quantifying the amount of IL-1.alpha. in the first
fraction.
[0016] Yet another method within the invention features the steps
of: (a) enriching a sample of plasma obtained from a human subject
using a filter that separates molecules according to molecular
weight into a first fraction including intact IgG complexed with
IL-1.alpha. and second fraction including molecule less than 100
Kda; (b) adding the first fraction to a substrate including
immobilized anti-human IgG Abs under conditions that allow IgG in
the first fraction to specifically bind the anti-human IgG Abs
immobilized on the substrate; (c) washing the substrate to remove
material in the first fraction that does not specifically bind the
immobilized anti-human IgG Abs; (d) contacting the substrate washed
in step (c) with an Ab that specifically binds human IL-1.alpha.
under conditions that allows the Ab that specifically binds human
IL-1.alpha. to specifically bind any human IL-1.alpha. bound to the
substrate; (e) washing the substrate to remove any of the Ab that
specifically binds human IL-1.alpha. that is not bound to the
substrate; and (f) quantifying the amount of Ab that specifically
binds human IL-1.alpha. remaining bound to the substrate after step
(e).
[0017] Unless otherwise defined, all technical terms used herein
have the same meaning as commonly understood by one of ordinary
skill in the art to which this invention belongs. Commonly
understood definitions of biological terms can be found in Rieger
et al., Glossary of Genetics: Classical and Molecular, 5th edition,
Springer-Verlag: N.Y., 1991; and Lewin, Genes V, Oxford University
Press: New York, 1994.
[0018] The term "specifically binds", as used herein, when
referring to a polypeptide (including Abs) or receptor, refers to a
binding reaction which is determinative of the presence of the
protein or polypeptide or receptor in a heterogeneous population of
proteins and other biologics. Thus, under designated conditions
(e.g. immunoassay conditions in the case of an Ab), the specified
ligand or Ab binds to its particular "target" and does not bind in
a significant amount to other proteins present in the sample or to
other proteins to which the ligand or Ab may come in contact in an
organism. Generally, a first molecule that "specifically binds" a
second molecule has an equilibrium affinity constant greater than
about 10.sup.5 (e.g., 10.sup.6, 10.sup.7, 10.sup.8, 10.sup.9,
10.sup.10, 10.sup.11, and 10.sup.12 or more) liters/mole for that
second molecule.
[0019] When referring to a protein molecule such as an Ab,
"purified" means separated from components that naturally accompany
such molecules. Typically, an Ab or protein is purified when it is
at least about 10% (e.g., 9%, 10%, 20%, 30% 40%, 50%, 60%, 70%,
80%, 90%, 95%, 98%, 99%, 99.9%, and 100%), by weight, free from the
non-Ab proteins or other naturally-occurring organic molecules with
which it is naturally associated. Purity can be measured by any
appropriate method, e.g., column chromatography, polyacrylamide gel
electrophoresis, or HPLC analysis. A chemically-synthesized protein
or other recombinant protein produced in a cell type other than the
cell type in which it naturally occurs is "purified."
[0020] Although methods and materials similar or equivalent to
those described herein can be used in the practice or testing of
the present invention, suitable methods and materials are described
below. All publications mentioned herein are incorporated by
reference in their entirety. In the case of conflict, the present
specification, including definitions will control. In addition, the
particular embodiments discussed below are illustrative only and
not intended to be limiting.
DETAILED DESCRIPTION
[0021] The invention encompasses compositions and methods relating
to fully human mAbs that include (i) an antigen-binding variable
region that exhibits very high binding affinity for IL-1.alpha. and
(ii) a constant region that is effective at both activating the
complement system though C1q binding and binding to several
different Fc receptors. The below described preferred embodiments
illustrate adaptation of these compositions and methods.
Nonetheless, from the description of these embodiments, other
aspects of the invention can be made and/or practiced based on the
description provided below.
[0022] Methods involving conventional immunological and molecular
biological techniques are described herein. Immunological methods
(for example, assays for detection and localization of antigen-Ab
complexes, immunoprecipitation, immunoblotting, and the like) are
generally known in the art and described in methodology treatises
such as Current Protocols in Immunology, Coligan et al., ed., John
Wiley & Sons, New York. Techniques of molecular biology are
described in detail in treatises such as Molecular Cloning: A
Laboratory Manual, 2nd ed., vol. 1-3, Sambrook et al., ed., Cold
Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y., 2001; and
Current Protocols in Molecular Biology, Ausubel et al., ed., Greene
Publishing and Wiley-Interscience, New York. Ab methods are
described in Handbook of Therapeutic Abs, Dubel, S., ed.,
Wiley-VCH, 2007. Cell culture techniques are generally known in the
art and are described in detail in methodology treatises such as
Culture of Animal Cells: A Manual of Basic Technique, 4th edition,
by R Ian Freshney, Wiley-Liss, Hoboken, N.J., 2000; and General
Techniques of Cell Culture, by Maureen A Harrison and Ian F Rae,
Cambridge University Press, Cambridge, UK, 1994. Methods of protein
purification are discussed in Guide to Protein Purification:
Methods in Enzymology, Vol. 182, Deutscher M P, ed., Academic
Press, San Diego, Calif., 1990.
[0023] In one aspect, the invention features a fully human mAb that
includes (i) an antigen-binding variable region that exhibits very
high binding affinity for human IL-1.alpha. and (ii) a constant
region that is effective at both activating the complement system
though C1q binding and binding to several different Fc receptors.
The human Ab is preferably an IgG1. The Ka of the Ab is preferably
at least 1.times.10.sup.9 M.sup.-1 or greater (e.g., greater than
9.times.10.sup.10 M.sup.-1, 8.times.10.sup.10 M.sup.-1,
7.times.10.sup.10 M.sup.-1, 6.times.10.sup.10 M.sup.-1,
5.times.10.sup.10 M.sup.-1, 4.times.10.sup.10 M.sup.-1,
3.times.10.sup.10 M.sup.-1, 2.times.10.sup.10 M.sup.-1, or
1.times.10.sup.10 M.sup.-1).
[0024] Because B lymphocytes which express Ig specific for human
IL-1.alpha. occur naturally in human beings, a presently preferred
method for raising mAbs is to first isolate such a B lymphocyte
from a subject and then immortalize it so that it can be
continuously replicated in culture. Subjects lacking large numbers
of naturally occurring B lymphocytes which express Ig specific for
human IL-1.alpha. may be immunized with one or more human IL-la
antigens to increase the number of such B lymphocytes. Human mAbs
are prepared by immortalizing a human Ab secreting cell (e.g., a
human plasma cell). See, e.g., U.S. Pat. No. 4,634,664.
[0025] In an exemplary method, one or more (e.g., 5, 10, 25, 50,
100, 1000, or more) human subjects (e.g., subjects not previously
administered a human IL-1.alpha. vaccine) are screened for the
presence of such human IL-1.alpha.-specific Ab in their blood.
Those subjects that express the desired Ab can then be used as B
lymphocyte donors. In one possible method, peripheral blood is
obtained from a human donor that possesses B lymphocytes that
express human IL-1.alpha.-specific Ab. Such B lymphocytes are then
isolated from the blood sample, e.g., by cells sorting (e.g.,
fluorescence activated cell sorting, "FACS"; or magnetic bead cell
sorting) to select B lymphocytes expressing human
IL-1.alpha.-specific Ig. These cells can then be immortalized by
viral transformation (e.g., using EBV) or by fusion to another
immortalized cell such as a human myeloma according to known
techniques. The B lymphocytes within this population that express
Ig specific for human IL-1.alpha. can then be isolated by limiting
dilution methods (e.g., cells in wells of a microtiter plate that
are positive for Ig specific for human IL-1.alpha. are selected and
subcultured, and the process repeated until a desired clonal line
can be isolated). See, e.g., Goding, Monoclonal Abs: Principles and
Practice, pp. 59-103, Academic Press, 1986. Those clonal cell lines
that express Ig having at least nanomolar or picomolar binding
affinities for human IL-1.alpha. are preferred. MAbs secreted by
these clonal cell lines can be purified from the culture medium or
a bodily fluid (e.g., ascites) by conventional Ig purification
procedures such as salt cuts, size exclusion, ion exchange
separation, and affinity chromatography.
[0026] Although immortalized B lymphocytes might be used in in
vitro cultures to directly produce mAbs, in certain cases it might
be desirable to use heterologous expression systems to produce
mAbs. See, e.g., the methods described in U.S. patent application
Ser. No. 11/754,899. For example, the genes encoding an mAb
specific for human IL-1.alpha. might be cloned and introduced into
an expression vector (e.g., a plasmid-based expression vector) for
expression in a heterologous host cell (e.g., CHO cells, COS cells,
myeloma cells, and E. coli cells). Because Igs include heavy (H)
and light (L) chains in an H.sub.2L.sub.2 configuration, the genes
encoding each may be separately isolated and expressed in different
vectors.
[0027] Although generally less preferred, chimeric mAbs (e.g.,
"humanized" mAbs), which are antigen-binding molecules having
different portions derived from different animal species (e.g.,
variable region of a mouse Ig fused to the constant region of a
human Ig), might be used in the invention. Such chimeric Abs can be
prepared by methods known in the art. E. G., Morrison et al., Proc.
Nat'l. Acad. Sci. USA, 81:6851, 1984; Neuberger et al., Nature,
312:604, 1984; Takeda et al., Nature, 314:452, 1984. Similarly, Abs
can be humanized by methods known in the art. For example,
monoclonal Abs with a desired binding specificity can be
commercially humanized or as described in U.S. Pat. Nos. 5,693,762;
5,530,101; or 5,585,089.
[0028] The mAbs described herein might be affinity matured to
enhance or otherwise alter their binding specificity by known
methods such as VH and VL domain shuffling (Marks et al.
Bio/Technology 10:779-783, 1992), random mutagenesis of the
hypervariable regions (HVRs) and/or framework residues (Barbas et
al. Proc Nat. Acad. Sci. USA 91:3809-3813, 1994; Schier et al. Gene
169:147-155, 1995; Yelton et al. J. Immunol. 155:1994-2004, 1995;
Jackson et al., J. Immunol. 154(7):3310-9, 1995; and Hawkins et al,
J. Mol. Biol. 226:889-896, 1992. Amino acid sequence variants of an
Ab may be prepared by introducing appropriate changes into the
nucleotide sequence encoding the Ab. In addition, modifications to
nucleic acid sequences encoding mAbs might be altered (e.g.,
without changing the amino acid sequence of the mAb) for enhancing
production of the mAb in certain expression systems (e.g., intron
elimination and/or codon optimization for a given expression
system). The mAbs described herein can also be modified by
conjugation to another protein (e.g., another mAb) or non-protein
molecule. For example, a mAb might be conjugated to a water soluble
polymer such as polyethylene glycol or a carbon nanotube (See,
e.g., Kam et al., Proc. Natl. Acad. Sci. USA 102: 11600-11605,
2005). See, U.S. patent application Ser. No. 11/754,899.
[0029] Preferably, to ensure that high titers of human
IL-1.alpha.-specific mAb can be administered to a subject with
minimal adverse effects, the mAb compositions of the invention are
at least 0.5, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15,
20, 25, 30, 35, 40, 45, 50, 60, 70, 80, 90, 95, 96, 97, 98, 99,
99.9 or more percent by weight pure (excluding any excipients). The
mAb compositions of the invention might include only a single type
of mAb (i.e., one produced from a single clonal B lymphocyte line)
or might include a mixture of two or more (e.g., 2, 3, 4, 5, 6, 7,
8, 9, 10 or more) different types of mAbs. In addition to human
IL-la mAbs, the Ab compositions of the invention might also include
other mAbs that specifically bind antigens other than human
IL-1.alpha..
[0030] To modify or enhance their function, the human IL-1.alpha.
mAbs might be conjugated another molecule such as a cytotoxin or
detectable label. A human IL-1.alpha. specific mAb might be
conjugated with one or more cytotoxins to more effectively kill
cells expressing IL-1.alpha.. Cytotoxins for use in the invention
can be any cytotoxic agent (e.g., molecule that can kill a cell
after contacting the cell) that can be conjugated to a human
IL-1.alpha. specific mAb. Examples of cytotoxins include, without
limitation, radionuclides (e.g., .sup.35S, .sup.14C, .sup.32P,
.sup.125I, .sup.131I, .sup.90Y, .sup.89Zr, .sup.201Tl, .sup.186Re,
.sup.188Re, .sup.57Cu, .sup.213Bi, and .sup.211At), conjugated
radionuclides, and chemotherapeutic agents. Further examples of
cytotoxins include, but are not limited to, antimetabolites (e.g.,
5-fluorouricil (5-FU), methotrexate (MTX), fludarabine, etc.),
anti-microtubule agents (e.g., vincristine, vinblastine,
colchicine, taxanes (such as paclitaxel and docetaxel), etc.),
alkylating agents (e.g., cyclophasphamide, melphalan,
bischloroethylnitrosurea (BCNU), etc.), platinum agents (e.g.,
cisplatin (also termed cDDP), carboplatin, oxaliplatin, JM-216,
CI-973, etc.), anthracyclines (e.g., doxorubicin, daunorubicin,
etc.), antibiotic agents (e.g., mitomycin-C), topoisomerase
inhibitors (e.g., etoposide, tenoposide, and camptothecins), or
other cytotoxic agents such as ricin, diptheria toxin (DT),
Pseudomonas exotoxin (PE) A, PE40, abrin, saporin, pokeweed viral
protein, ethidium bromide, glucocorticoid, anthrax toxin and
others. See, e.g., U.S. Pat. No. 5,932,188.
[0031] The human IL-1.alpha. specific mAb can also be conjugated to
a detectable label. Useful detectable labels in the present
invention include biotin or streptavidin, magnetic beads,
fluorescent dyes (e.g., fluorescein isothiocyanate, texas red,
rhodamine, green fluorescent protein, and the like), radiolabels
(e.g., .sup.3H, .sup.125I, .sup.35S, .sup.14C, .sup.32P,
.sup.111In, .sup.97Ru, .sup.67Ga, .sup.68Ga, or .sup.72As),
radipaque substances such as metals for radioimaging, paramagnetic
agents for magnetic resonance imaging, enzymes (e.g., horseradish
peroxidase, alkaline phosphatase and others commonly used in an
ELISA), and colorimetric labels such as colloidal gold or colored
glass or plastic (e.g., polystyrene, polypropylene, latex, etc.)
beads. Means of detecting such labels are well known to those of
skill in the art. Thus, for example, radiolabels may be detected
using photographic film or scintillation counters. Fluorescent
markers may also be used and can be detected using a photodetector
to detect emitted illumination. Enzymatic labels are typically
detected by providing the enzyme with a substrate and detecting the
reaction product produced by the action of the enzyme on the
substrate, and colorimetric labels are detected by simply
visualizing the colored label.
[0032] The present invention also encompasses nucleic acid
molecules encoding fully human mAbs specific for human IL-1.alpha..
Although the same nucleic acid molecule might encode both the heavy
and light chains of a human IL-1.alpha.--specific mAb, two
different nucleic acid molecules, one encoding the heavy chain and
the other encoding the light chain might also be used. The amino
acid sequences of three IgG1 mAbs specific for human IL-1.alpha.
are presented herein. See SEQ ID NOs: 1, 3, 5, 7, 9, and 11.
Exemplary nucleic acid molecules encoding these amino acid
sequences are also described herein. See SEQ ID NOs: 2, 4, 6, 8,
10, and 12. Any other suitable nucleic acid that encodes the amino
acid sequences of the two described IgG1 mAbs or other mAbs within
the invention might also be used.
[0033] For production of mAbs, the nucleic acid molecules of the
invention might be incorporated into an expression vector in an
orientation wherein such nucleic acid molecules are operatively
linked to expression control sequences such as transcriptional and
translational control sequences. Examples of expression vectors
include vectors derived from plasmids and vectors derived from
viruses such as adenoviruses, adeno-associated viruses, and
retroviruses. The nucleic acid molecules encoding a light chain and
a heavy chain might be incorporated into a single vector or
different vectors. The vectors of the invention might also include
regulatory sequences such as promoters and/or enhancers (see, U.S.
Pat. No. 5,168,062, U.S. Pat. No. 4,510,245 and U.S. Pat. No.
4,968,615), selectable markers, or sequences encoding affinity tags
(for facilitating purification) or a detectable label.
[0034] For production of mAbs, the vectors of the invention can be
introduced into a suitable host cell, e.g., a prokaryotic cell such
as a bacteria or, preferably, a eukaryotic cell such as mammalian,
plant, or yeast host cell. Examples of methods for introducing
heterologous polynucleotides into host cells include use of viral
vectors, electroporation, encapsulation of the polynucleotide(s) in
liposomes, dextran-mediated transfection, calcium phosphate
precipitation, polybrene-mediated transfection, protoplast fusion,
Agrobacterium-mediated transformation, biolistic transformation,
and direct microinjection of the DNA into nuclei. Mammalian cell
lines are presently preferred for expression of mAbs from vectors.
Examples of mammalian host cells include Chinese hamster ovary
(CHO) cells (e.g., the DG44 CHO cell line), HeLa cells, baby
hamster kidney (BHK) cells, African green monkey kidney cells
(COS), human hepatocellular carcinoma cells (e.g., Hep G2), NS0
cells, SP2 cells, HEK-293T cells, 293 Freestyle cells, and NIH-3T3
cells. The mAbs of the invention might also be expressed in
transgenic animals or plants. See, e.g., U.S. Pat. Nos. 5,827,690;
5,756,687; 5,750,172; 5,741,957; 6,046,037; and 5,959,177.
[0035] The invention provides a method for detecting a human
IL-1.alpha.-expressing cell in a sample by contacting the cell with
a human IL-1.alpha.-specific mAb and detecting the mAb bound to the
cell. The invention also provides a method for killing a human
IL-1.alpha.-expressing cell by contacting the cell with a human
IL-1.alpha.-specific mAb. Such killing can be accomplished by
complement-mediated killing, Ab-dependent cell-mediated
cytotoxicity, or Ab-mediated delivery of a cytotoxin. The Abs
described herein have also been shown to be useful for other
methods.
[0036] For example, MABp1 has been to reduce IL-1.alpha. induced
ICAM1 and E-selectin expression on endothelial cells. MABp1 has
also been shown to be used in immunoassays for detecting and
quantifying IL-1.alpha. in a biological sample.
EXAMPLES
Example 1
Cloning of Anti-hIL-1.alpha. IgG1 and Kappa Chains
[0037] Variable region heavy chain (V-HC) and variable region light
chain (V-LC) sequences were gene synthesized using amino acid
sequence information provided in U.S. Pat. No. 5,959,085. V-HC was
PCR amplified introducing HindIII/ClaI sites upstream of the ATG
start codon and a NheI site at the 3' end. The human germline IgG1
constant region (including exons and introns) was PCR amplified
modifying the two 5' triplets encoding for the first two amino
acids Ala-Ser to an NheI site, and introducing a BamHI site at the
3' end. The human germline IgG1 constant region amino acid sequence
corresponded to Swiss-Prot entry P01857, except for a K171Q and a
V261L exchange. The V-HC and constant IgG1-HC sequence were ligated
using the NheI site and cloned into pcDNA3 using HindIII and BamHI
sites.
TABLE-US-00001 >hIL-1a-IgG1-HC (SEQ ID NO: 1)
MEFGLSWVFLVALLRGVQCQVQLVESGGGVVQPGRSLRLSCTASGFTFSMFG
VHWVRQAPGKGLEWVAAVSYDGSNKYYAESVKGRFTISRDNSKNILFLQMD
SLRLEDTAVYYCARGRPKVVIPAPLAHWGQGTLVTFSSASTKGPSVFPLAPSS
KSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSV
VTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGP
SVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAQTK
PREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQ
PREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIALEWESNGQPENNYKTTP
PVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK
>hIL-1a-IgG1-HC (SEQ ID NO: 2)
atggagttcgggctgagttgggtgttcctggtggctctgctgcggggcgtgcagtgccaggtgcagctggtgga-
gagtgg
gggtggcgtggtgcagcctggccggtctctgcgcctgtcttgcactgcctccggttttaccttttctatgtttg-
gtgtgcactgg
gtgcgccaggctcccggcaagggactggaatgggtggccgccgtgagttacgacgggtccaacaaatattacgc-
tgaga
gcgtgaaaggcagattcaccatcagcagagataattccaagaatattctgttcctgcagatggacagtctgaga-
ctggagg
acactgctgtgtactactgcgctcgtggacgccctaaggtggtcatccccgcccccatggcacattggggccag-
ggaactc
tggtgaccttttctagcgctagcaccaagggcccatcggtcttccccctggcaccctcctccaagagcacctct-
gggggca
cagcggccctgggctgcctggtcaaggactacttccccgaaccggtgacggtgtcgtggaactcaggcgccctg-
accag
cggcgtccacaccttcccggctgtcctacagtcctcaggactctactccctcagcagcgtagtgaccgtgccct-
ccagcag
cttgggcacccagacctacatctgcaacgtgaatcacaagcccagcaacaccaaggtggacaagaaagttgagc-
ccaaa
tattgtgacaaaactcacacatgcccaccgtgcccagcacctgaactcctggggggaccgtcagtattcctctt-
ccccccaa
aacccaaggacaccctcatgatctcccggacccctgaggtcacatgcgtggtggtggacgtgagccacgaagac-
cctga
ggtcaagttcaactggtacgtggacggcgtggaggtgcataatgcccagacaaagccgcgggaggagcagtaca-
acag
cacgtaccgtgtggtcagcgtcctcaccgtcctgcaccaggactggctgaatggcaaggagtacaagtgcaagg-
tctcca
acaaagccctcccagcccccatcgagaaaaccatctccaaagccaaagggcagccccgagaaccacaggtgtac-
accc
tgcccccatcccgggatgagctgaccaagaaccaggtcagcctgacctgcctggtcaaaggcttctatcccagc-
gacatc
gccctggagtgggagagcaatgggcagccggagaacaactacaagaccacgcctcccgtgctggactccgacgg-
ctcc
ttcttcctctacagcaagctcaccgtggacaagagcaggtggcagcaggggaacgtcttctcatgctccgtgat-
gcatgag gctctgcacaaccactacacgcagaagagcctctccttaagtccgggaaaataa
[0038] The V-LC was PCR amplified introducing HindIII/ClaI sites
upstream of the ATG start codon and a BsiWI site at the 3' end. The
human constant Kappa-LC sequence was PCR amplified introducing a 5'
BsiWI site encoding an additional Arg and the first amino acids
Thr, and a BamHI site at the 3' end. The human constant Kappa-LC
amino acid sequence corresponded to Swiss-Prot entry P01834. V-HC
and constant Kappa-LC sequences were ligated using the BsiWI site
and cloned into pcDNA3 using HindIII and BamHI sites.
TABLE-US-00002 >hIL-1a-K-LC [SEQ ID NO: 3]
MDMRVPAQLLGLLLLWFPGSRCDIQMTQSPSSVSASVGDRVTITCRASQGISS
WLAWYQQKPGKAPKLLIYEASNLETGVPSRFSGSGSGSDFTLTISSLQPEDFA
TYYCQQTSSFLLSFGGGTKVEHRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNF
YPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHK
VYACEVTHQGLSSPVTKSFNRGEC >hIL-1a-K-LC [SEQ ID NO: 4]
atggacatgcgcgtgcccgcccagctgctggggctgctgctgctgtggttccctggatctaggtgcgacattca-
gatgacc
cagtcccccagctcagtgtcagcctccgtgggcgacagagtgacaatcacctgccgcgcctctcagggaatctc-
tagttgg
ctggcctggtaccagcagaagcctggaaaggcccccaagctgctgatctatgaagcctccaacctggagaccgg-
cgtgc
cctctcgcttcagcggctcaggctcaggcagtgattttactctgaccatcagctccctgcagccagaggatttc-
gctacttact
actgccagcagacctcttccttcctgctgtccttcgggggaggcacaaaggtggagcaccgtacggtggctgca-
ccatctg
tattcatcttcccgccatctgatgagcagttgaaatctggaactgcctctgttgtgtgcctgctgaataacttc-
tatcccagaga
ggccaaagtacagtggaaggtggataacgccctccaatcgggtaactcccaggagagtgtcacagagcaggaca-
gcaa
ggacagcacctacagcctcagcagcaccctgacgctgagcaaagcagactacgagaaacacaaagtctacgcct-
gcga
agtcacccatcagggcctgagttcaccggtgacaaagagcttcaacaggggagagtgttag
Example 2
Generation of NATHMAB-hIL-1a IgG1 and Kappa Chain
[0039] The complete sequence encoding the NATHMAB-h1L-1a/lgG1 heavy
chain was gene synthesized. The V-HC sequence corresponded to the
amino acid sequence described in U.S. Pat. No. 5,959,085. The human
constant IgG1-HC sequence corresponded to Swiss-Prot entry P01857.
The nucleotide sequence was codon optimized for expression in CHO
cells. A Kozac sequence (gccacc) was added upstream of the start
ATG.
TABLE-US-00003 >NATHMAB-hIL-1A-IGG1-HC [SEQ ID NO: 5]
MEFGLSWVFLVALLRGVQCQVQLVESGGGVVQPGRSLRLSCTASGFTFSMFG
VHWVRQAPGKGLEWVAAVSYDGSNKYYAESVKGRFTISRDNSKNILFLQMD
SLRLEDTAVYYCARGRPKVVIPAPLAHWGQGTLVTFSSASTKGPSVFPLAPSS
KSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSV
VTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGP
SVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTK
PREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQ
PREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTP
PVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK
>NATHMAB-hIL-1A-IGG1.sup.-HC [SEQ ID NO: 6]
gccaccatggagtttggtatgtcctgggtgttcttggtggctctgctgaggggggtgcagtgccaggtccagct-
ggtggagt
ctggtgggggagtggtgcagcctgggagatctctgcggctgtcttgcactgcctctggtttcactttctctatg-
tttggtgtgca
ttgggtcaggcaagcaccaggcaaaggactcgagtgggtcgcagctgtgagctatgacgggtctaacaaatatt-
acgctg
agtctgtcaagggtaggtttaccatcagccgggataattccaaaaatatcctgttcctgcaaatggactctctg-
aggctggaa
gatactgcagtctactattgtgcaagggggaggccaaaggtggtgatccccgctcccctcgctcactggggaca-
gggaac
cctggtgactttcagctctgctagcaccaagggccctagcgtgttcccattggctccttcctccaaatctactt-
ctggaggcac
cgccgccctgggatgtctcgtgaaagattattttcctgagcccgtcaccgtgagctggaacagcggcgccctga-
ctagcgg
cgtgcacacctttcccgcagtgctgcaatctagcgggctgtactccctgagctctgtcgtgaccgtgccctcca-
gcagcctc
ggaactcagacctacatctgcaatgtcaatcataaaccctctaataccaaagtcgataagaaggtcgaacctaa-
atcttgcga
taaaacccatacctgccccccttgcccagcacccgaactgctgggcggtccctctgtgtttctgttccccccca-
aacccaaa
gataccctgatgatctctaggacccccgaggtcacttgtgtcgtggtggatgtgtcccacgaagatccagaagt-
caaattca
actggtatgtggacggggtcgaagtgcacaacgcaaagaccaagcctagggaggaacagtataatagcacatat-
agggt
ggtcagcgtcctgaccgtcctgcatcaggactggctgaatggcaaagaatataagtgtaaagtgtccaacaagg-
ccctgcc
agccccaatcgaaaagacaatctctaaagccaaggggcaaccccgggaacctcaggtctatacactgccaccct-
ctcgg
gatgaactgaccaagaatcaggtgagcctgacatgtcttgtgaagggtttttatccctccgacattgccgtgga-
gtgggaga
gcaatggacaaccagaaaataactacaaaaccacaccccctgtgctggactccgatggttccttcttcctctac-
tctaagctg
acagtggataagtctaggtggcagcaggggaatgtgttctcctgctctgtgatgcacgaggcactgcacaatca-
ttatacac aaaagtctctgtctctgtctccaggaaagtaa
[0040] The complete sequence encoding the NATHMAB-hIL-1a/Kappa
light chain was gene synthesized. The V-LC sequence corresponded to
the amino acid sequence described in U.S. Pat. No. 5,959,085. The
human constant Kappa-LC sequence corresponded to Swiss-Prot entry
P01834. The nucleotide sequence was codon optimized for expression
in CHO cells. A Kozac sequence (gccacc) was added upstream of
ATG.
TABLE-US-00004 >NATHMAB-hIL-1A-K-LC [SEQ ID NO: 7]
MDMRVPAQLLGLLLLWFPGSRCDIQMTQSPSSVSASVGDRVTITCRASQGISS
WLAWYQQKPGKAPKLLIYEASNLETGVPSRFSGSGSGSDFTLTISSLQPEDFA
TYYCQQTSSFLLSFGGGTKVEHTVAAPSVFIFPPSDEQLKSGTASVVCLLNNF
YPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHK
VYACEVTHQGLSSPVTKSFNRGEC NATHMAB-hIL-1A-K-LC [SEQ ID NO: 8]
gccaccatggacatgcgcgttcctgcccagctcctcggactgctgctgctttggttcccaggctcccggtgtga-
tattcag
atgacacagtctccctcctccgtatctgcatccgtgggcgacagggtcacaatcacttgtagggccagccaggg-
gatctc
tagttggctcgcatggtaccaacaaaagccaggtaaggctccgaaactgctcatttacgaagctagtaacctcg-
aaacag
gcgtgccaagccggtttagcggctccggttccggttctgacttcaccctcactatttcctccctgcaacctgag-
gattttgcc
acatattactgtcagcaaacttcttcttttctgactcctttggtgggggaactaaggtggagcacacagtggcc-
gccccca
gcgtctttatcttccccccaagcgatgaacagctgaagtcagggaccgccagcgtggtctgcctgctcaataat-
ttttaccc
tcgcgaggctaaggtccaatggaaagtggataacgccctccagagcggtaactctcaggagtctgtcacagagc-
aaga
cagcaaggatagcacctattccctctccagcaccctgacactgtctaaggccgactacgagaaacacaaagtgt-
acgctt
gtgaggtgactcaccagggactgagtagccctgtgacaaaatctttcaataggggagaatgctga
Example 3
Expression of NATHMAB-IL1-a (IgG1/k Subtype)
[0041] NATHMAB-IL-1.alpha. was expressed and purified using a
transient transfection method. Cell culture supernatant or protein
G affinity purified Ab was subjected to further analysis as
described below. Human embryonic kidney (HEK) 293T cells were
cultured in DMEM containing 10% FCS, and transiently transfected
using jetPEI reagent (Polyplus) according to manufacturer's
protocol. Cells were seeded on 10 cm dishes (3.times.10.sup.6 cells
per 10 cm dish) 24 h prior to transfection to reach approximately
50% density at the time point of transfection. 5 .mu.g per dish of
pcDNA3-anti-hIL-1.alpha.-IgG1-HC and a 2-fold molar excess of
pcDNA3-anti-hIL-1.alpha.-Kappa were used for transfection. After
recovery, medium was changed to DMEM containing 2% FCS (10 ml per
dish) and Ab was collected for 5 to 6 days. The supernatant was
collected, filtered, pH adjusted to 7.5-8, and stored at 4.degree.
C. until further use.
[0042] Part of the supernatant (250 ml) was incubated with protein
G sepharose (GE Healthcare) for 3 h at 4.degree. C. on a rotation
wheel. Then, the protein G sepharose was loaded onto a gravity flow
column and washed with PBS. Ab was eluted in 1 ml fractions using
100 mM glycine/150 mM NaCl into 100 .mu.l Tris (pH 8), followed by
dialysis with PBS containing 10% glycerol. The total protein
concentration of each fraction was measured using the BCA Protein
Detection Kit (Pierce). Correct size of heavy and light chains, and
of the assembled native Ab was confirmed by SDS-PAGE.
[0043] Supernatant containing NATHMAB-hIL-1.alpha. purified Ab and
Triton X-100 cell lysates of producer HEK 293T cells were tested
for antigen binding in a radioimmunoassay (RIA) using
.sup.125I-hIL-1.alpha.. Binding was assayed by absorption to
protein G. All samples bound .sup.125I-hIL-1.alpha. with highest
activity in the eluate. Binding of purified NATHMAB-hIL-1.alpha. in
a concentration of 0.012% (half-max activity in RIA) to
.sup.125I-hIL-1.alpha. was used for measuring the affinity
coefficient. The Ka of NATHMAB-hIL-1.alpha. under these conditions
was 3.03.times.10.sup.10 M.sup.-1. Back calculation revealed an
estimated concentration of approximately 30 .mu.g/ml active
anti-hIL-1.alpha.-IgG in the purified eluate.
[0044] Neutralizing activity of NATHMAB-hIL1.alpha. was tested in a
bioassay using the murine EL4-6.1 subline which produces high
levels of IL-2 when treated with murine or human IL-1.alpha.
(Zubler et al., J. Immunol. 134:3662-3668, 1985). The indicated
concentrations of NATHMAB-hIL-1.alpha. (eluate) were incubated for
30 min at 37.degree. C. with various concentrations of recombinant
hIL-la (eBioscience) in a final volume of 100 .mu.l/well in a
96-well culture plate (flat bottomed). Each point was carried out
in triplicate and in culture medium (DMEM, 5% FCS). To each well
were added 100 .mu.l of a suspension of EL4-6.1 cells
(5.times.10.sup.5 cells/ml) in culture medium containing 0.2
.mu.g/ml ionomycin. After incubation for 24 h at 37.degree. C. in a
5% CO.sub.2 incubator, cell free supernatants were harvested and
assayed for IL-2 concentrations using a commercially available
ELISA (R&D Systems). The results showed that
NATHMAB-IL-1.alpha. effectively neutralized hIL-1.alpha.-induced
IL-2 secretion by EL-4 cells.
[0045] To test for neutralization of membrane-bound hIL-1.alpha.,
the same EL-4 cell-based assay as described above was used with
following modifications. Different concentrations of
NATHMAB-hIL-1.alpha. (eluate) were incubated with various numbers
of human activated monocytes. For monocyte preparation, PBMC were
isolated from buffy coat using Ficoll-Paque centrifugation.
Monocytes were allowed to adhere for 1.5 h at 37.degree. C. in RPMI
on plastic dishes. Non-adherent lymphocytes were washed away to
yield a nearly pure monocyte culture. Monocytes were cultured in
RPMI containing Gln, Pyr, and 10% FCS for 24 h with LPS (1
.mu.g/ml) at 37.degree. C. in a 5% CO.sub.2 incubator. Cells were
detached with PBS/2 mM EDTA, carefully scraped from plates, and
transferred into Falcon tubes. Cells were washed twice with PBS,
resuspended in PBS/1% PFA and fixed for 10 min at 20.degree. C.
Cells were washed with glycine buffer (150 mM glycine, 75 mM NaCl,
pH 7.4), then with culture medium and counted. The results showed
that NATHMAB-hIL-la effectively neutralized IL-2 secretion by EL-4
cells induced by membrane-bound hIL-1.alpha.. In an experiment
similar to that described above, NATHMAB-hIL-1.alpha. was tested
for neutralization of murine IL-1.alpha.. Indicated amounts of
NATHMAB-hIL-1.alpha. supernatant were incubated with recombinant
human (h) or murine (m) IL-1.alpha. (eBioscience). The supernatant
containing the Ab neutralized human, but not murine,
IL-1.alpha..
Example 4
Ab-Mediated Killing of Cancer Cells
[0046] Human peripheral blood mononuclear cells (PBMC) isolated
from the buffy coat by standard Ficoll Paque preparation were
incubated in either RPMI-1640 CM or RPMI-1640-CM containing rhIL-2
(30 ng/ml, ebioscience) at 37.degree. C. and 5% CO2 overnight and
used as effector cells (E). THP1 cells were used as the targets
(T). The assay was carried out in 96-well plates with each point in
triplicate. After 1.times.10.sup.4 targets that were incubated with
different concentration of MABp1 for 15 mins, effector cells were
added in an ET ratio of 25:1 and 50:1 to 1.times.10.sup.4 targets
and incubated for another 4 hours. 75 ul of assay volume were
transferred to a new 96-well plate and cytotoxicity was assayed
using the LDH cytotoxicity detection kit (Roche) according to
manufacturer's protocol. % specific lysis=(mean experimental
release-mean spontaneous release without antibody).times.100/(mean
maximal release from targets-mean spontaneous release from targets)
A. untreated PBMC were used as effector cells. B. rhIL-2-treated
PBMC were used as effector cells. In both cases, increasing
concentrations (1.25 to 20 ug/ml) of MABp1 resulted in increased
target cell killing (up to about 90%) at both ET ratios.
Example 5
Human Anti-IL1.alpha. Specific mAb Sequences
[0047] The complete sequence encoding for another human
anti-hIL-1algG.sub.1/Kappa light chain specific for human
IL1.alpha. (MABp1) was synthesized and expressed as described
above. In the nucleic acids encoding the heavy and light chains, a
Kozac sequence (gccacc) was added upstream of the start ATG.
TABLE-US-00005 Heavy Chain [SEQ ID NO: 9]
MEFGLSWVFLVALLRGVQCQVQLVESGGGVVQPGRSLRLSCTASGFTFSMFG
VHWVRQAPGKGLEWVAAVSYDGSNKYYAESVKGRFTISRDNSKNILFLQMD
SLRLEDTAVYYCARGRPKVVIPAPLAHWGQGTLVTFSSASTKGPSVFPLAPSS
KSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSV
VTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKSCDKTHTCPPCPAPELLGGP
SVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTK
PREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQ
PREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTP
PVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK [SEQ ID NO:
10]
gccaccatggagtttggtctgtcctgggtgttcttggtggctctgctgaggggggtgcagtgccaggtccagct-
ggtggagt
ctggtgggggagtggtgcagcctgggagatctctgcggctgtcttgcactgcctctggtttcactttctctatg-
tttggtgtgca
ttgggtcaggcaagcaccaggcaaaggactcgagtgggtcgcagctgtgagctatgacgggtctaacaaatatt-
acgctg
agtctgtcaagggtaggtttaccatcagccgggataattccaaaaatatcctgttcctgcaaatggactctctg-
aggctggaa
gatactgcagtctactattgtgcaagggggaggccaaaggtggtgatccccgctcccctcgctcactggggaca-
gggaac
cctggtgactttcagctctgctagcaccaagggccctagcgtgttcccattggctccttcctccaaatctactt-
ctggaggcac
cgccgccctgggatgtctcgtgaaagattattttcctgagcccgtcaccgtgagctggaacagcggcgccctga-
ctagcgg
cgtgcacacctttcccgcagtgctgcaatctagcgggctgtactccctgagctctgtcgtgaccgtgccctcca-
gcagcctc
ggaactcagacctacatctgcaatgtcaatcataaaccctctaataccaaagtcgataagagggtcgaacctaa-
atcttgcg
ataaaacccatacctgccccccttgcccagcacccgaactgctgggcggtccctctgtgtttctgttccccccc-
aaacccaa
agataccctgatgatctctaggacccccgaggtcacttgtgtcgtggtggatgtgtcccacgaagatccagaag-
tcaaattc
aactggtatgtggacggggtcgaagtgcacaacgcaaagaccaagcctagggaggaacagtataatagcacata-
taggg
tggtcagcgtcctgaccgtcctgcatcaggactggctgaatggcaaagaatataagtgtaaagtgtccaacaag-
gccctgc
cagccccaatcgaaaagacaatctctaaagccaaggggcaaccccgggaacctcaggtctatacactgccaccc-
tctcgg
gaggaaatgaccaagaatcaggtgagcctgacatgtcttgtgaagggtttttatccctccgacattgccgtgga-
gtgggaga
gcaatggacaaccagaaaataactacaaaaccacaccccctgtgctggactccgatggttccttcttcctctac-
tctaagctg
acagtggataagtctaggtggcagcaggggaatgtgttctcctgctctgtgatgcacgaggcactgcacaatca-
ttatacac aaaagtctctgtctctgtctccaggaaagtaa Light Chain [SEQ ID NO:
11] MDMRVPAQLLGLLLLWFPGSRCDIQMTQSPSSVSASVGDRVTITCRASQGISS
WLAWYQQKPGKAPKLLIYEASNLETGVPSRFSGSGSGSDFTLTISSLQPEDFA
TYYCQQTSSFLLSFGGGTKVEHKRTVAAPSVFIFPPSDEQLKSGTASVVCLLN
NFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKH
KVYACEVTHQGLSSPVTKSFNRGEC [SEQ ID NO: 12]
gccaccatggacatgcgcgttcctgcccagctcctcggactgctgctgctttggttcccaggctcccggtgtga-
tattcagat
gacacagtctccctcctccgtatctgcatccgtgggcgacagggtcacaatcacttgtagggccagccagggga-
tctctag
ttggctcgcatggtaccaacaaaagccaggtaaggctccgaaactgctcatttacgaagctagtaacctcgaaa-
caggcgt
gccaagccggtttagcggctccggttccggttctgacttcaccctcactatttcctccctgcaacctgaggatt-
ttgccacatat
tactgtcagcaaacttcttcttttctgctctcctttggtggaggaactaaggtggagcacaagcggacagttgc-
tgctcctagc
gtctttatcttccctccaagcgatgaacagctgaagtcagggaccgccagcgtggtctgcctgctcaataattt-
ttaccctcgc
gaggctaaggtccaatggaaagtggataacgccctccagagcggtaactctcaggagtctgtcacagagcaaga-
cagca
aggatagcacctattccctctccagcaccctgacactgtctaaggccgactacgagaaacacaaagtgtacgct-
tgtgaggt
gactcaccagggactgagtagccctgtgacaaaatctttcaataggggagaatgctga
Example 6
MABp1 Binding Affinity
[0048] The binding affinity of purified MABp1 was determined using
surface plasmon resonanace (SPR) on a BIAcore 2000 instrument (GE
Health Sciences). A mouse monoclonal anti-human IgG (Fc) Ab was
covalently immobilized on the flow cells of a CM5 sensor chip using
a human Ab capture kit and amine coupling kit (GE Health Sciences).
Immobilization levels of 8000-14000 RU would typically be achieved.
After immobilization of the mouse anti-human IgG (Fc) capture Ab,
three start-up cycles with HBS-EP running buffer (GE Health
Sciences) and two start-up cycles with MABp1 were run to stabilize
the CM5 surface and to remove any non-covalently bound Ab. For
analysis, MABp1 Ab was diluted into HBS-EP running buffer to a
final concentration of 1 .mu.g/mL and immobilized to 700 RU on one
flow cell of the CM5 sensor chip. Carrier-free human IL-1A cytokine
(eBioscience, #34-8019) was serially diluted in HBS-EP running
buffer over a test range from 100 nM to 0.05 nM. Flow rate was 30
.mu.l/min. Dissociation data for each cytokine dilution was
recorded for 15 minutes. The CM5 surface was regenerated after each
cycle using a single injection of 3 M MgCl.sub.2 for 25 seconds at
a flow rate of 30 .mu.l/min. BiaEvaluation software and a Langmuir
binding model was used to fit the data. The K.sub.D for MABp1 was
determined to be less than 2.0.times.10.sup.-10 M.
Example 7
MABp1 Inhibits Tumor Cell Invasion of a Basement Membrane
Matrix
[0049] Matrigel (BD), a basement membrane matrix, was thawed at
4.degree. C. overnight and the dilute (5 mg/ml to 1 mg/ml) in serum
free cold cell culture media. 100 ul of the diluted matrigel was
placed into the upper chambers of a 24-well transwell (Costar) and
the transwell was incubated at 370C for at least 4 to 5 h for
gelling. Tumor cells (MDA-MB-231 and THP-1) were harvested from
tissue culture flasks by Trypsin/EDTA, washed with culture media,
and resuspended in medium containing 1% FBS at a density of
1.times.10.sup.6 cells/ml. The gelled matrigel was gently washed
with warmed serum free-culture media, and 100 ul of the cell
suspension was added in each well. The lower chamber of the
transwell was filled with 600 ul of culture media, and the plates
was incubated at 37.degree. C. for 12 to 24 h. The cells that did
not invade the matrigel were gently scraped off the top of each
transwell with a cotton swab. The transwells were then removed from
the 24-well plates and stained with crystal violet after fixing the
invaded cells with 70% ethanol or methanol. The invaded cells were
counted under a light microscope. The percent of cells invading the
matrigel was significantly inhibited in the presence of MABp1.
Example 8
MABp1 Blocks Increase in ICAM1 Expression in Endothelial Cells
[0050] Human umbilical vein endothelial cells (HUVEC) (BD
Biosciences) were seeded to 24-well plates at 5.times.10.sup.5 per
well in 1 mL of M-200 medium supplemented with low-serum growth
supplement (Invitrogen). Cells were allowed to settle for 3-4
hours. Medium was aspirated and a fresh 1 mL of M-200 was added per
well. MABp1 was added directly to cells @ 4.26 .mu.g/mL,
co-incubated for 15 minutes at room temperature, and then
recombinant human IL-1.alpha. (rhIL1A, eBioscience) was added to a
final concentration of 40 pg/mL. Positive control wells received
the addition of IL-1.alpha. only. HUVEC cells in the absence of
IL-1a or the absence of MABp1 served as negative controls. After
17-20 hours incubation at 37.degree. C., 5% CO.sub.2, cells were
lifted from the plates by a non-enzymatic treatment for 20 minutes
using CellStripper reagent (Cellgro Mediatech) and then immediately
assayed for CD54 (ICAM-1) expression using standard flow cytometry
protocols. Staining buffer comprised Dulbecco's PBS supplemented
with 2% heat-inactivated fetal bovine serum. PE-conjugated mouse
anti-human CD54 (ICAM-1) mAb (eBioscience, clone HA58) or a
PE-conjugated mouse IgG1k isotype control (eBiocience, #12-4714)
were used per manufacturer's instructions to stain HUVEC cells in a
100 microliter staining volume for 20 minutes in the dark at room
temperature. Two washes in staining buffer were subsequently
performed and then samples were acquired on a FACSCalibur flow
cytometer (BD Biosciences). Among several independent experiments
(n=5) the upregulation of ICAM-1 adhesion molecules induced by
rhIL1A on the surface of HUVEC cells was neutralized by MABp1 to
baseline levels exhibited by the unstimulated HUVEC cells.
Example 9
MABp1 Blocks Increase in E-selectin Expression in Endothelial
Cells
[0051] Similar to its effects on ICAM-1 induction, MABp1-mediated
neutralization of induction of CD62E (E-selectin) on HUVEC cells
was also observed. This effect was most pronounced when HUVEC cells
were stimulated not by soluble rhIL-1a but by membranous IL-1a
anchored by glycosyl-phosphatidylinositol to the surface of DG44
CHO cells (GPI-IL1A cells). In this experiment, confluent cultures
of HUVEC cells in 6-well plates were co-cultured overnight with
5.times.10.sup.6 GPI-IL1A DG44 cells in M-200 medium, either alone,
in the presence of 10 .mu.g/mL MABp1, or in the presence of 10
.mu.g/mL D5 isotype control Ab. After 17-20 hours, HUVEC monolayers
were washed extensively with Dulbecco's PBS and then lifted by
non-enzymatic treatment for 20 minutes with CellStripper reagent
(Cellgro Mediatech) and then immediately assayed for CD62E
(E-selectin) expression using standard flow-cytometry protocols.
Staining buffer comprised Dulbecco's PBS supplemented with 2%
heat-inactivated fetal bovine serum. PE-conjugated mouse anti-human
CD62E mAb (eBioscience, clone P2H3) or a PE-conjugated mouse IgG1k
isotype control (eBiocience, clone P3) were used per manufacturer's
instructions to stain HUVEC cells in a 100 microliter staining
volume for 20 minutes in the dark at room temperature. Two washes
in staining buffer were subsequently performed and then samples
were acquired on a FACSCalibur flow cytometer (BD Biosciences).
Upregulated E-selectin expression on the surface of HUVEC cells
induced by membranous GPI-IL-1a was neutralized by MABp1 to
baseline levels exhibited by unstimulated HUVEC cells.
Example 10
MRC-5 Bioassay for MABp1 Potency (Neutralization of rhIL1A)
[0052] The MRC-5 cell line, derived from fetal human lung
fibroblasts, was obtained from the ATCC collection (CCL-171). The
IL-1 neutralizing potency of MABp1 was assayed by measuring IL-1A
induced release of IL-6 from MRC-5 cells. MRC-5 cells were seeded
at 5.times.10.sup.3 per well to a 96-well plate in 100 microliters
of DMEM complete medium. Cells were cultured overnight at 37
.degree. C. in a humidified 5% CO.sub.2 incubator. Confluent MRC-5
cells were subsequently cultured another 24 hours with 20 pg/mL of
recombinant human IL-1A (rhIL1A, eBioscience) either alone or in
the presence of increasing concentrations of MABp1. Negative
control cells were not stimulated with rhIL1A. After the 24 hours,
supernatants were collected and assayed for IL-6 release using and
IL-6 ELISA kit from eBioscience. The IC.sub.50, or concentration of
MABp1 required to inhibit 50% of the maximal IL-6 release, was in
the range of 0.001-0.01 .mu.g/mL.
Example 11
MABp1 Identifies IL-1a+Cells
[0053] One hundred microliters of sodium heparin anti-coagulated
whole blood was aliquoted to polystyrene FACS tubes. Samples were
incubated at room temperature for 15 minutes with 1 mg of human IgG
(protein-A purified) plus 2 ml of heat-inactivated fetal bovine
serum to block Fc receptors. Primary Abs were then added to the
sample: Either 1 mg of Alexa-488 labeled MABp1, 1 mg of
FITC-labeled monoclonal anti-membrane human IL1A Ab (FAB200F,
R&D Systems), or 1 mg of a murine isotype control (IC002F,
R&D Systems). Primary Abs were incubated with sample for 30
minutes at room temperature in the dark. Sample erythrocytes were
then lysed (BD Biosciences PharmLyse solution) at room temperature
for 15 minutes, centrifuged at 300.times.g for 5 minutes, and
aspirated. Sample pellets were washed three times with 1 mL Hank's
balanced salt solution (HBSS) containing 2% heat-inactivated fetal
bovine serum. Sample was resuspended in 0.3 mL HBSS+2% FBS and data
was acquired on a FACSCalibur flow cytometer and analyzed using
CellQuest software. Flow cytometric analysis of human PBMC using
MABp1 showed that only 0.2% of PBMC were positive for
IL-1.alpha..
Example 12
MABp1 for Detecting and Tracking Infections and Inflammation
[0054] Flow cytometric analysis (as in Example 11) of human PBMC
using MABp1 showed a 3.6-fold increase in the percent of PBMC
positive for IL-1.alpha..sup.+ in a subject with a sub-clinical
infection compared to a normal control. Similarly, in a subject
with an inflamed wisdom tooth, an increase in the percent of PBMC
positive for IL-1.alpha..sup.+. A substantial decrease in the
number of IL-1.alpha..sup.+ PBMC was observed from 14 to 45 days
after removal of the wisdom tooth.
Example 13
Immunoassay for Detecting and/or Quantifying IL-1.alpha.
[0055] In general, very low levels of IL-1.alpha. are present in
the plasma of human subjects. Because these levels are often beyond
the detection threshold of conventional immunoassays, an ELISA with
improved sensitivity was developed. In this ELISA, exogenous
anti-IL-1.alpha. Ab (e.g., MABp1) can be added to a biological
sample being tested (e.g., human plasma) under conditions that
allow the Ab to bind IL-1.alpha. in the sample. Because, it was
observed that almost all IL-1.alpha. in human plasma samples exists
already bound to endogenous anti-IL-1.alpha. Ab, the latter step
can often be omitted. The sample with IL-1a-Ab complexes is then
applied to a filter (Amicon centrifugal device) with a molecular
weight cutoff of about 100 kDa to separate the IL-1.alpha.-Ab
complexes from molecules in the sample less than the molecular
weight cutoff. In one experiment, this resulted in a 50-fold
concentration. The processed sample (and dilutions thereof) was
then added to wells of a microtiter plate coated with an anti-human
IgG capture Ab (2 ug/ml mouse anti-human IgG, Fc-specific, Southern
Biotech product code #9042-01). After allowing time to bind the
IL-1.alpha.-Ab complexes in the sample, the wells were washed to
remove non-binding material. A labeled anti-human IL-1.alpha.
secondary Ab was then added to the wells (0.2 ug/ml
biotin-conjugated monoclonal mouse anti-human IL-1A Ab, clone CRM6,
eBioscience catalog #13-7017). After allowing time to bind the
IL-1.alpha. in the wells, the plate was washed and the amount of
labeled anti-human IL-1.alpha. in each well was quantified as an
indication of the concentration of IL-1.alpha. in the sample being
tested.
Other Embodiments
[0056] It is to be understood that while the invention has been
described in conjunction with the detailed description thereof, the
foregoing description is intended to illustrate and not limit the
scope of the invention, which is defined by the scope of the
appended claims. Other aspects, advantages, and modifications are
within the scope of the following claims.
Sequence CWU 1
1
121471PRThomo sapiens 1Met Glu Phe Gly Leu Ser Trp Val Phe Leu Val
Ala Leu Leu Arg Gly1 5 10 15Val Gln Cys Gln Val Gln Leu Val Glu Ser
Gly Gly Gly Val Val Gln 20 25 30Pro Gly Arg Ser Leu Arg Leu Ser Cys
Thr Ala Ser Gly Phe Thr Phe 35 40 45Ser Met Phe Gly Val His Trp Val
Arg Gln Ala Pro Gly Lys Gly Leu 50 55 60Glu Trp Val Ala Ala Val Ser
Tyr Asp Gly Ser Asn Lys Tyr Tyr Ala65 70 75 80Glu Ser Val Lys Gly
Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn 85 90 95Ile Leu Phe Leu
Gln Met Asp Ser Leu Arg Leu Glu Asp Thr Ala Val 100 105 110Tyr Tyr
Cys Ala Arg Gly Arg Pro Lys Val Val Ile Pro Ala Pro Leu 115 120
125Ala His Trp Gly Gln Gly Thr Leu Val Thr Phe Ser Ser Ala Ser Thr
130 135 140Lys Gly Pro Ser Val Phe Pro Leu Ala Pro Ser Ser Lys Ser
Thr Ser145 150 155 160Gly Gly Thr Ala Ala Leu Gly Cys Leu Val Lys
Asp Tyr Phe Pro Glu 165 170 175Pro Val Thr Val Ser Trp Asn Ser Gly
Ala Leu Thr Ser Gly Val His 180 185 190Thr Phe Pro Ala Val Leu Gln
Ser Ser Gly Leu Tyr Ser Leu Ser Ser 195 200 205Val Val Thr Val Pro
Ser Ser Ser Leu Gly Thr Gln Thr Tyr Ile Cys 210 215 220Asn Val Asn
His Lys Pro Ser Asn Thr Lys Val Asp Lys Lys Val Glu225 230 235
240Pro Lys Ser Cys Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro
245 250 255Glu Leu Leu Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys
Pro Lys 260 265 270Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr
Cys Val Val Val 275 280 285Asp Val Ser His Glu Asp Pro Glu Val Lys
Phe Asn Trp Tyr Val Asp 290 295 300Gly Val Glu Val His Asn Ala Gln
Thr Lys Pro Arg Glu Glu Gln Tyr305 310 315 320Asn Ser Thr Tyr Arg
Val Val Ser Val Leu Thr Val Leu His Gln Asp 325 330 335Trp Leu Asn
Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu 340 345 350Pro
Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg 355 360
365Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg Asp Glu Leu Thr Lys
370 375 380Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro
Ser Asp385 390 395 400Ile Ala Leu Glu Trp Glu Ser Asn Gly Gln Pro
Glu Asn Asn Tyr Lys 405 410 415Thr Thr Pro Pro Val Leu Asp Ser Asp
Gly Ser Phe Phe Leu Tyr Ser 420 425 430Lys Leu Thr Val Asp Lys Ser
Arg Trp Gln Gln Gly Asn Val Phe Ser 435 440 445Cys Ser Val Met His
Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser 450 455 460Leu Ser Leu
Ser Pro Gly Lys465 47021416DNAHomo sapiens 2atggagttcg ggctgagttg
ggtgttcctg gtggctctgc tgcggggcgt gcagtgccag 60gtgcagctgg tggagagtgg
gggtggcgtg gtgcagcctg gccggtctct gcgcctgtct 120tgcactgcct
ccggttttac cttttctatg tttggtgtgc actgggtgcg ccaggctccc
180ggcaagggac tggaatgggt ggccgccgtg agttacgacg ggtccaacaa
atattacgct 240gagagcgtga aaggcagatt caccatcagc agagataatt
ccaagaatat tctgttcctg 300cagatggaca gtctgagact ggaggacact
gctgtgtact actgcgctcg tggacgccct 360aaggtggtca tccccgcccc
cctggcacat tggggccagg gaactctggt gaccttttct 420agcgctagca
ccaagggccc atcggtcttc cccctggcac cctcctccaa gagcacctct
480gggggcacag cggccctggg ctgcctggtc aaggactact tccccgaacc
ggtgacggtg 540tcgtggaact caggcgccct gaccagcggc gtccacacct
tcccggctgt cctacagtcc 600tcaggactct actccctcag cagcgtagtg
accgtgccct ccagcagctt gggcacccag 660acctacatct gcaacgtgaa
tcacaagccc agcaacacca aggtggacaa gaaagttgag 720cccaaatctt
gtgacaaaac tcacacatgc ccaccgtgcc cagcacctga actcctgggg
780ggaccgtcag tcttcctctt ccccccaaaa cccaaggaca ccctcatgat
ctcccggacc 840cctgaggtca catgcgtggt ggtggacgtg agccacgaag
accctgaggt caagttcaac 900tggtacgtgg acggcgtgga ggtgcataat
gcccagacaa agccgcggga ggagcagtac 960aacagcacgt accgtgtggt
cagcgtcctc accgtcctgc accaggactg gctgaatggc 1020aaggagtaca
agtgcaaggt ctccaacaaa gccctcccag cccccatcga gaaaaccatc
1080tccaaagcca aagggcagcc ccgagaacca caggtgtaca ccctgccccc
atcccgggat 1140gagctgacca agaaccaggt cagcctgacc tgcctggtca
aaggcttcta tcccagcgac 1200atcgccctgg agtgggagag caatgggcag
ccggagaaca actacaagac cacgcctccc 1260gtgctggact ccgacggctc
cttcttcctc tacagcaagc tcaccgtgga caagagcagg 1320tggcagcagg
ggaacgtctt ctcatgctcc gtgatgcatg aggctctgca caaccactac
1380acgcagaaga gcctctcctt aagtccggga aaataa 14163235PRTHomo sapiens
3Met Asp Met Arg Val Pro Ala Gln Leu Leu Gly Leu Leu Leu Leu Trp1 5
10 15Phe Pro Gly Ser Arg Cys Asp Ile Gln Met Thr Gln Ser Pro Ser
Ser 20 25 30Val Ser Ala Ser Val Gly Asp Arg Val Thr Ile Thr Cys Arg
Ala Ser 35 40 45Gln Gly Ile Ser Ser Trp Leu Ala Trp Tyr Gln Gln Lys
Pro Gly Lys 50 55 60Ala Pro Lys Leu Leu Ile Tyr Glu Ala Ser Asn Leu
Glu Thr Gly Val65 70 75 80Pro Ser Arg Phe Ser Gly Ser Gly Ser Gly
Ser Asp Phe Thr Leu Thr 85 90 95Ile Ser Ser Leu Gln Pro Glu Asp Phe
Ala Thr Tyr Tyr Cys Gln Gln 100 105 110Thr Ser Ser Phe Leu Leu Ser
Phe Gly Gly Gly Thr Lys Val Glu His 115 120 125Arg Thr Val Ala Ala
Pro Ser Val Phe Ile Phe Pro Pro Ser Asp Glu 130 135 140Gln Leu Lys
Ser Gly Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe145 150 155
160Tyr Pro Arg Glu Ala Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln
165 170 175Ser Gly Asn Ser Gln Glu Ser Val Thr Glu Gln Asp Ser Lys
Asp Ser 180 185 190Thr Tyr Ser Leu Ser Ser Thr Leu Thr Leu Ser Lys
Ala Asp Tyr Glu 195 200 205Lys His Lys Val Tyr Ala Cys Glu Val Thr
His Gln Gly Leu Ser Ser 210 215 220Pro Val Thr Lys Ser Phe Asn Arg
Gly Glu Cys225 230 2354708DNAHomo sapiens 4atggacatgc gcgtgcccgc
ccagctgctg gggctgctgc tgctgtggtt ccctggatct 60aggtgcgaca ttcagatgac
ccagtccccc agctcagtgt cagcctccgt gggcgacaga 120gtgacaatca
cctgccgcgc ctctcaggga atctctagtt ggctggcctg gtaccagcag
180aagcctggaa aggcccccaa gctgctgatc tatgaagcct ccaacctgga
gaccggcgtg 240ccctctcgct tcagcggctc aggctcaggc agtgatttta
ctctgaccat cagctccctg 300cagccagagg atttcgctac ttactactgc
cagcagacct cttccttcct gctgtccttc 360gggggaggca caaaggtgga
gcaccgtacg gtggctgcac catctgtctt catcttcccg 420ccatctgatg
agcagttgaa atctggaact gcctctgttg tgtgcctgct gaataacttc
480tatcccagag aggccaaagt acagtggaag gtggataacg ccctccaatc
gggtaactcc 540caggagagtg tcacagagca ggacagcaag gacagcacct
acagcctcag cagcaccctg 600acgctgagca aagcagacta cgagaaacac
aaagtctacg cctgcgaagt cacccatcag 660ggcctgagtt caccggtgac
aaagagcttc aacaggggag agtgttag 7085471PRTHomo sapiens 5Met Glu Phe
Gly Leu Ser Trp Val Phe Leu Val Ala Leu Leu Arg Gly1 5 10 15Val Gln
Cys Gln Val Gln Leu Val Glu Ser Gly Gly Gly Val Val Gln 20 25 30Pro
Gly Arg Ser Leu Arg Leu Ser Cys Thr Ala Ser Gly Phe Thr Phe 35 40
45Ser Met Phe Gly Val His Trp Val Arg Gln Ala Pro Gly Lys Gly Leu
50 55 60Glu Trp Val Ala Ala Val Ser Tyr Asp Gly Ser Asn Lys Tyr Tyr
Ala65 70 75 80Glu Ser Val Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn
Ser Lys Asn 85 90 95Ile Leu Phe Leu Gln Met Asp Ser Leu Arg Leu Glu
Asp Thr Ala Val 100 105 110Tyr Tyr Cys Ala Arg Gly Arg Pro Lys Val
Val Ile Pro Ala Pro Leu 115 120 125Ala His Trp Gly Gln Gly Thr Leu
Val Thr Phe Ser Ser Ala Ser Thr 130 135 140Lys Gly Pro Ser Val Phe
Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser145 150 155 160Gly Gly Thr
Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu 165 170 175Pro
Val Thr Val Ser Trp Asn Ser Gly Ala Leu Thr Ser Gly Val His 180 185
190Thr Phe Pro Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser
195 200 205Val Val Thr Val Pro Ser Ser Ser Leu Gly Thr Gln Thr Tyr
Ile Cys 210 215 220Asn Val Asn His Lys Pro Ser Asn Thr Lys Val Asp
Lys Lys Val Glu225 230 235 240Pro Lys Ser Cys Asp Lys Thr His Thr
Cys Pro Pro Cys Pro Ala Pro 245 250 255Glu Leu Leu Gly Gly Pro Ser
Val Phe Leu Phe Pro Pro Lys Pro Lys 260 265 270Asp Thr Leu Met Ile
Ser Arg Thr Pro Glu Val Thr Cys Val Val Val 275 280 285Asp Val Ser
His Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp 290 295 300Gly
Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr305 310
315 320Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu His Gln
Asp 325 330 335Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn
Lys Ala Leu 340 345 350Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala
Lys Gly Gln Pro Arg 355 360 365Glu Pro Gln Val Tyr Thr Leu Pro Pro
Ser Arg Asp Glu Leu Thr Lys 370 375 380Asn Gln Val Ser Leu Thr Cys
Leu Val Lys Gly Phe Tyr Pro Ser Asp385 390 395 400Ile Ala Val Glu
Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys 405 410 415Thr Thr
Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser 420 425
430Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser
435 440 445Cys Ser Val Met His Glu Ala Leu His Asn His Tyr Thr Gln
Lys Ser 450 455 460Leu Ser Leu Ser Pro Gly Lys465 47061422DNAHomo
sapiens 6gccaccatgg agtttggtct gtcctgggtg ttcttggtgg ctctgctgag
gggggtgcag 60tgccaggtcc agctggtgga gtctggtggg ggagtggtgc agcctgggag
atctctgcgg 120ctgtcttgca ctgcctctgg tttcactttc tctatgtttg
gtgtgcattg ggtcaggcaa 180gcaccaggca aaggactcga gtgggtcgca
gctgtgagct atgacgggtc taacaaatat 240tacgctgagt ctgtcaaggg
taggtttacc atcagccggg ataattccaa aaatatcctg 300ttcctgcaaa
tggactctct gaggctggaa gatactgcag tctactattg tgcaaggggg
360aggccaaagg tggtgatccc cgctcccctc gctcactggg gacagggaac
cctggtgact 420ttcagctctg ctagcaccaa gggccctagc gtgttcccat
tggctccttc ctccaaatct 480acttctggag gcaccgccgc cctgggatgt
ctcgtgaaag attattttcc tgagcccgtc 540accgtgagct ggaacagcgg
cgccctgact agcggcgtgc acacctttcc cgcagtgctg 600caatctagcg
ggctgtactc cctgagctct gtcgtgaccg tgccctccag cagcctcgga
660actcagacct acatctgcaa tgtcaatcat aaaccctcta ataccaaagt
cgataagaag 720gtcgaaccta aatcttgcga taaaacccat acctgccccc
cttgcccagc acccgaactg 780ctgggcggtc cctctgtgtt tctgttcccc
cccaaaccca aagataccct gatgatctct 840aggacccccg aggtcacttg
tgtcgtggtg gatgtgtccc acgaagatcc agaagtcaaa 900ttcaactggt
atgtggacgg ggtcgaagtg cacaacgcaa agaccaagcc tagggaggaa
960cagtataata gcacatatag ggtggtcagc gtcctgaccg tcctgcatca
ggactggctg 1020aatggcaaag aatataagtg taaagtgtcc aacaaggccc
tgccagcccc aatcgaaaag 1080acaatctcta aagccaaggg gcaaccccgg
gaacctcagg tctatacact gccaccctct 1140cgggatgaac tgaccaagaa
tcaggtgagc ctgacatgtc ttgtgaaggg tttttatccc 1200tccgacattg
ccgtggagtg ggagagcaat ggacaaccag aaaataacta caaaaccaca
1260ccccctgtgc tggactccga tggttccttc ttcctctact ctaagctgac
agtggataag 1320tctaggtggc agcaggggaa tgtgttctcc tgctctgtga
tgcacgaggc actgcacaat 1380cattatacac aaaagtctct gtctctgtct
ccaggaaagt aa 14227234PRTHomo sapiens 7Met Asp Met Arg Val Pro Ala
Gln Leu Leu Gly Leu Leu Leu Leu Trp1 5 10 15Phe Pro Gly Ser Arg Cys
Asp Ile Gln Met Thr Gln Ser Pro Ser Ser 20 25 30Val Ser Ala Ser Val
Gly Asp Arg Val Thr Ile Thr Cys Arg Ala Ser 35 40 45Gln Gly Ile Ser
Ser Trp Leu Ala Trp Tyr Gln Gln Lys Pro Gly Lys 50 55 60Ala Pro Lys
Leu Leu Ile Tyr Glu Ala Ser Asn Leu Glu Thr Gly Val65 70 75 80Pro
Ser Arg Phe Ser Gly Ser Gly Ser Gly Ser Asp Phe Thr Leu Thr 85 90
95Ile Ser Ser Leu Gln Pro Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln
100 105 110Thr Ser Ser Phe Leu Leu Ser Phe Gly Gly Gly Thr Lys Val
Glu His 115 120 125Thr Val Ala Ala Pro Ser Val Phe Ile Phe Pro Pro
Ser Asp Glu Gln 130 135 140Leu Lys Ser Gly Thr Ala Ser Val Val Cys
Leu Leu Asn Asn Phe Tyr145 150 155 160Pro Arg Glu Ala Lys Val Gln
Trp Lys Val Asp Asn Ala Leu Gln Ser 165 170 175Gly Asn Ser Gln Glu
Ser Val Thr Glu Gln Asp Ser Lys Asp Ser Thr 180 185 190Tyr Ser Leu
Ser Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu Lys 195 200 205His
Lys Val Tyr Ala Cys Glu Val Thr His Gln Gly Leu Ser Ser Pro 210 215
220Val Thr Lys Ser Phe Asn Arg Gly Glu Cys225 2308711DNAHomo
sapiens 8gccaccatgg acatgcgcgt tcctgcccag ctcctcggac tgctgctgct
ttggttccca 60ggctcccggt gtgatattca gatgacacag tctccctcct ccgtatctgc
atccgtgggc 120gacagggtca caatcacttg tagggccagc caggggatct
ctagttggct cgcatggtac 180caacaaaagc caggtaaggc tccgaaactg
ctcatttacg aagctagtaa cctcgaaaca 240ggcgtgccaa gccggtttag
cggctccggt tccggttctg acttcaccct cactatttcc 300tccctgcaac
ctgaggattt tgccacatat tactgtcagc aaacttcttc ttttctgctc
360tcctttggtg ggggaactaa ggtggagcac acagtggccg cccccagcgt
ctttatcttc 420cccccaagcg atgaacagct gaagtcaggg accgccagcg
tggtctgcct gctcaataat 480ttttaccctc gcgaggctaa ggtccaatgg
aaagtggata acgccctcca gagcggtaac 540tctcaggagt ctgtcacaga
gcaagacagc aaggatagca cctattccct ctccagcacc 600ctgacactgt
ctaaggccga ctacgagaaa cacaaagtgt acgcttgtga ggtgactcac
660cagggactga gtagccctgt gacaaaatct ttcaataggg gagaatgctg a
7119471PRTHomo sapiens 9Met Glu Phe Gly Leu Ser Trp Val Phe Leu Val
Ala Leu Leu Arg Gly1 5 10 15Val Gln Cys Gln Val Gln Leu Val Glu Ser
Gly Gly Gly Val Val Gln 20 25 30Pro Gly Arg Ser Leu Arg Leu Ser Cys
Thr Ala Ser Gly Phe Thr Phe 35 40 45Ser Met Phe Gly Val His Trp Val
Arg Gln Ala Pro Gly Lys Gly Leu 50 55 60Glu Trp Val Ala Ala Val Ser
Tyr Asp Gly Ser Asn Lys Tyr Tyr Ala65 70 75 80Glu Ser Val Lys Gly
Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn 85 90 95Ile Leu Phe Leu
Gln Met Asp Ser Leu Arg Leu Glu Asp Thr Ala Val 100 105 110Tyr Tyr
Cys Ala Arg Gly Arg Pro Lys Val Val Ile Pro Ala Pro Leu 115 120
125Ala His Trp Gly Gln Gly Thr Leu Val Thr Phe Ser Ser Ala Ser Thr
130 135 140Lys Gly Pro Ser Val Phe Pro Leu Ala Pro Ser Ser Lys Ser
Thr Ser145 150 155 160Gly Gly Thr Ala Ala Leu Gly Cys Leu Val Lys
Asp Tyr Phe Pro Glu 165 170 175Pro Val Thr Val Ser Trp Asn Ser Gly
Ala Leu Thr Ser Gly Val His 180 185 190Thr Phe Pro Ala Val Leu Gln
Ser Ser Gly Leu Tyr Ser Leu Ser Ser 195 200 205Val Val Thr Val Pro
Ser Ser Ser Leu Gly Thr Gln Thr Tyr Ile Cys 210 215 220Asn Val Asn
His Lys Pro Ser Asn Thr Lys Val Asp Lys Arg Val Glu225 230 235
240Pro Lys Ser Cys Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro
245 250 255Glu Leu Leu Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys
Pro Lys 260 265 270Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr
Cys Val Val Val 275 280 285Asp Val Ser His Glu Asp Pro Glu Val Lys
Phe Asn Trp Tyr Val Asp 290 295 300Gly Val Glu Val His Asn Ala Lys
Thr Lys Pro Arg Glu Glu Gln Tyr305 310 315 320Asn Ser Thr Tyr Arg
Val Val Ser Val Leu Thr Val Leu His Gln Asp 325 330 335Trp Leu Asn
Gly
Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu 340 345 350Pro Ala
Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg 355 360
365Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg Glu Glu Met Thr Lys
370 375 380Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro
Ser Asp385 390 395 400Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro
Glu Asn Asn Tyr Lys 405 410 415Thr Thr Pro Pro Val Leu Asp Ser Asp
Gly Ser Phe Phe Leu Tyr Ser 420 425 430Lys Leu Thr Val Asp Lys Ser
Arg Trp Gln Gln Gly Asn Val Phe Ser 435 440 445Cys Ser Val Met His
Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser 450 455 460Leu Ser Leu
Ser Pro Gly Lys465 470101422DNAHomo sapiens 10gccaccatgg agtttggtct
gtcctgggtg ttcttggtgg ctctgctgag gggggtgcag 60tgccaggtcc agctggtgga
gtctggtggg ggagtggtgc agcctgggag atctctgcgg 120ctgtcttgca
ctgcctctgg tttcactttc tctatgtttg gtgtgcattg ggtcaggcaa
180gcaccaggca aaggactcga gtgggtcgca gctgtgagct atgacgggtc
taacaaatat 240tacgctgagt ctgtcaaggg taggtttacc atcagccggg
ataattccaa aaatatcctg 300ttcctgcaaa tggactctct gaggctggaa
gatactgcag tctactattg tgcaaggggg 360aggccaaagg tggtgatccc
cgctcccctc gctcactggg gacagggaac cctggtgact 420ttcagctctg
ctagcaccaa gggccctagc gtgttcccat tggctccttc ctccaaatct
480acttctggag gcaccgccgc cctgggatgt ctcgtgaaag attattttcc
tgagcccgtc 540accgtgagct ggaacagcgg cgccctgact agcggcgtgc
acacctttcc cgcagtgctg 600caatctagcg ggctgtactc cctgagctct
gtcgtgaccg tgccctccag cagcctcgga 660actcagacct acatctgcaa
tgtcaatcat aaaccctcta ataccaaagt cgataagagg 720gtcgaaccta
aatcttgcga taaaacccat acctgccccc cttgcccagc acccgaactg
780ctgggcggtc cctctgtgtt tctgttcccc cccaaaccca aagataccct
gatgatctct 840aggacccccg aggtcacttg tgtcgtggtg gatgtgtccc
acgaagatcc agaagtcaaa 900ttcaactggt atgtggacgg ggtcgaagtg
cacaacgcaa agaccaagcc tagggaggaa 960cagtataata gcacatatag
ggtggtcagc gtcctgaccg tcctgcatca ggactggctg 1020aatggcaaag
aatataagtg taaagtgtcc aacaaggccc tgccagcccc aatcgaaaag
1080acaatctcta aagccaaggg gcaaccccgg gaacctcagg tctatacact
gccaccctct 1140cgggaggaaa tgaccaagaa tcaggtgagc ctgacatgtc
ttgtgaaggg tttttatccc 1200tccgacattg ccgtggagtg ggagagcaat
ggacaaccag aaaataacta caaaaccaca 1260ccccctgtgc tggactccga
tggttccttc ttcctctact ctaagctgac agtggataag 1320tctaggtggc
agcaggggaa tgtgttctcc tgctctgtga tgcacgaggc actgcacaat
1380cattatacac aaaagtctct gtctctgtct ccaggaaagt aa 142211236PRTHomo
sapiens 11Met Asp Met Arg Val Pro Ala Gln Leu Leu Gly Leu Leu Leu
Leu Trp1 5 10 15Phe Pro Gly Ser Arg Cys Asp Ile Gln Met Thr Gln Ser
Pro Ser Ser 20 25 30Val Ser Ala Ser Val Gly Asp Arg Val Thr Ile Thr
Cys Arg Ala Ser 35 40 45Gln Gly Ile Ser Ser Trp Leu Ala Trp Tyr Gln
Gln Lys Pro Gly Lys 50 55 60Ala Pro Lys Leu Leu Ile Tyr Glu Ala Ser
Asn Leu Glu Thr Gly Val65 70 75 80Pro Ser Arg Phe Ser Gly Ser Gly
Ser Gly Ser Asp Phe Thr Leu Thr 85 90 95Ile Ser Ser Leu Gln Pro Glu
Asp Phe Ala Thr Tyr Tyr Cys Gln Gln 100 105 110Thr Ser Ser Phe Leu
Leu Ser Phe Gly Gly Gly Thr Lys Val Glu His 115 120 125Lys Arg Thr
Val Ala Ala Pro Ser Val Phe Ile Phe Pro Pro Ser Asp 130 135 140Glu
Gln Leu Lys Ser Gly Thr Ala Ser Val Val Cys Leu Leu Asn Asn145 150
155 160Phe Tyr Pro Arg Glu Ala Lys Val Gln Trp Lys Val Asp Asn Ala
Leu 165 170 175Gln Ser Gly Asn Ser Gln Glu Ser Val Thr Glu Gln Asp
Ser Lys Asp 180 185 190Ser Thr Tyr Ser Leu Ser Ser Thr Leu Thr Leu
Ser Lys Ala Asp Tyr 195 200 205Glu Lys His Lys Val Tyr Ala Cys Glu
Val Thr His Gln Gly Leu Ser 210 215 220Ser Pro Val Thr Lys Ser Phe
Asn Arg Gly Glu Cys225 230 23512717DNAHomo sapiens 12gccaccatgg
acatgcgcgt tcctgcccag ctcctcggac tgctgctgct ttggttccca 60ggctcccggt
gtgatattca gatgacacag tctccctcct ccgtatctgc atccgtgggc
120gacagggtca caatcacttg tagggccagc caggggatct ctagttggct
cgcatggtac 180caacaaaagc caggtaaggc tccgaaactg ctcatttacg
aagctagtaa cctcgaaaca 240ggcgtgccaa gccggtttag cggctccggt
tccggttctg acttcaccct cactatttcc 300tccctgcaac ctgaggattt
tgccacatat tactgtcagc aaacttcttc ttttctgctc 360tcctttggtg
gaggaactaa ggtggagcac aagcggacag ttgctgctcc tagcgtcttt
420atcttccctc caagcgatga acagctgaag tcagggaccg ccagcgtggt
ctgcctgctc 480aataattttt accctcgcga ggctaaggtc caatggaaag
tggataacgc cctccagagc 540ggtaactctc aggagtctgt cacagagcaa
gacagcaagg atagcaccta ttccctctcc 600agcaccctga cactgtctaa
ggccgactac gagaaacaca aagtgtacgc ttgtgaggtg 660actcaccagg
gactgagtag ccctgtgaca aaatctttca ataggggaga atgctga 717
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