U.S. patent application number 10/813646 was filed with the patent office on 2006-01-26 for monoclonal antibodies to type i interferon receptor.
This patent application is currently assigned to Genentech, Inc.. Invention is credited to Anan Chuntharapai, Kyung Jin Kim, Richard B. Love, Ji Lu.
Application Number | 20060020118 10/813646 |
Document ID | / |
Family ID | 31996448 |
Filed Date | 2006-01-26 |
United States Patent
Application |
20060020118 |
Kind Code |
A1 |
Chuntharapai; Anan ; et
al. |
January 26, 2006 |
Monoclonal antibodies to type I interferon receptor
Abstract
Anti-IFNAR1 monoclonal antibodies with neutralizing activities
against the anti-viral cytopathic effects of various type I
interferons are provided.
Inventors: |
Chuntharapai; Anan; (Colma,
CA) ; Kim; Kyung Jin; (Los Altos, CA) ; Love;
Richard B.; (San Francisco, CA) ; Lu; Ji;
(Fremont, CA) |
Correspondence
Address: |
MERCHANT & GOULD PC
P.O. BOX 2903
MINNEAPOLIS
MN
55402-0903
US
|
Assignee: |
Genentech, Inc.
South San Francisco
CA
|
Family ID: |
31996448 |
Appl. No.: |
10/813646 |
Filed: |
March 29, 2004 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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09056461 |
Apr 7, 1998 |
6713609 |
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10813646 |
Mar 29, 2004 |
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08888140 |
Jul 3, 1997 |
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09056461 |
Apr 7, 1998 |
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60058212 |
Jul 16, 1996 |
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Current U.S.
Class: |
530/388.22 |
Current CPC
Class: |
G01N 33/6866 20130101;
C07K 2317/92 20130101; C07K 2317/76 20130101; G01N 2333/56
20130101; C07K 16/2866 20130101; A61P 43/00 20180101; C07K 2319/30
20130101 |
Class at
Publication: |
530/388.22 |
International
Class: |
C07K 16/28 20060101
C07K016/28 |
Claims
1. An anti-IFNAR1 monoclonal antibody that inhibits the anti-viral
activity of a first type I interferon and does not inhibit the
anti-viral activity of a second type I interferon selected from the
group consisting of IFN-.alpha.A, IFN-.alpha.B,
IFN-.alpha..sub.II1, and IFN-.beta..
2. The monoclonal antibody of claim 1 wherein the second type I
interferon is IFN-.beta..
3. The monoclonal antibody of claim 2 wherein the first type I
interferon is selected from the group consisting of IFN-.alpha.A,
IFN-.alpha.B, and IFN-.alpha.G.
4. The monoclonal antibody of claim 3 that inhibits the anti-viral
activity of IFN-.alpha.A, IFN-.alpha.B, and IFN-.alpha.G.
5. The monoclonal antibody of claim 4 that is designated 4A7,
having ATCC Deposit No. HB 12132.
6. The monoclonal antibody of claim 4, wherein the antibody
recognizes a conformational epitope on IFNAR1.
7. The monoclonal antibody of claim 6, wherein the antibody does
not bind to a peptide consisting of the amino acid sequence of
domain 1 (amino acids 1-200) of IFNAR1 and does not bind to a
peptide consisting of the amino acid sequence of domain 2 (amino
acids 204-404) of IFNAR1.
8. The monoclonal antibody of claim 4 that binds to one or more
amino acids in situ in the sequence of amino acids 244-249 of
IFNAR1, and binds to one or more amino acids in situ in the
sequence of amino acids 291-298 of IFNAR1.
9. The monoclonal antibody of claim 8 that binds to amino acids
249, 291 and 296 of IFNAR1 in situ.
10. The monoclonal antibody of claim 9 that is designated 2E1,
having ATCC Deposit No. HB 12133.
Description
[0001] This is a continuation-in-part of co-pending non-provisional
application U.S. Ser. No. 08/888,140 filed Jul. 3, 1997, which
claims priority under 35 U.S.C. .sctn. 119(e) to provisional
application U.S. Ser. No. 60/058,212 filed Jul. 16, 1996, now
abandoned, which non-provisional application is incorporated herein
by reference, and to which non-provisional application priority is
claimed under 35 U.S.C. .sctn.120.
FIELD OF THE INVENTION
[0002] This invention relates to the field of anti-type I
interferon receptor antibodies, and more particularly to anti-type
1 interferon receptor antibodies that neutralize the anti-viral
cytopathic effects of various type I interferons.
BACKGROUND OF THE INVENTION
[0003] The type 1 interferons (IFNs) are cytokines that have
pleiotropic effects on a wide variety of cell types. IFNs are best
known for their anti-viral activity, but they also have
anti-bacterial, anti-protozoal, immunomodulatory, and cell-growth
regulatory functions. The type 1 interferons include
interferon-.alpha. (IFN-.alpha.) and interferon-.beta.
(IFN-.beta.). Human IFN-.alpha. (hIFN-.alpha.) is a heterogeneous
family with at least 23 polypeptides while there is only one
IFN-.beta. polypeptide (J. Interferon Res., 13: 443-444 (1993)).
The hIFN-.alpha. subtypes show more than 70% amino acid sequence
homology, and there is approximately 25% amino acid identity with
hIFN-.beta.. The hIFNs-.alpha. and hIFN-.beta. share a common
receptor.
[0004] Three components of the hIFN-.alpha. receptor complex have
recently been cloned. The cDNA for the first hIFN-.alpha. receptor
(hIFNAR1) encodes a 63 kD receptor protein (reported in Uze et al.,
Cell, 60: 225-234 (1990)). This receptor undergoes extensive
glycosylation, which causes it to migrate in gel electrophoresis as
a much larger 135 kD protein. The second interferon receptor,
hIFNAR2 (hIFN-.alpha..beta.R long), is a 115 kD protein which
mediates a functional signaling complex when associated with
hIFNAR1 (reported in Domanski et al., J. Biol. Chem., 270:
21606-21611 (1995)). The third hIFN-.alpha. receptor, an
IFN-.alpha./.beta. receptor (hIFN-.alpha..beta.R short), is a 55 kD
protein that can bind to type 1 hIFNs but cannot form a functional
complex when associated with hIFNAR1 (reported in Novick et al.,
Cell; 77: 391400 (1994)). This IFN-.alpha./.beta. receptor appears
to be an alternatively spliced variant of hIFNAR2.
[0005] The unprocessed hIFNAR1 expression product is composed of
557 amino acids including an extracellular domain (ECD) of 409
residues, a transmembrane domain of 21 residues, and an
intracellular domain of 100 residues as shown in FIG. 5 on page 229
of Uze et al., supra. The ECD of IFNAR1 is composed of two domains,
domain 1 and domain 2, which are separated by a three-proline
motif. There is 19% sequence identity and 50% sequence homology
between domains 1 and 2 (Uze et al., supra). Each domain (D200) is
composed of approximately 200 residues and can be further
subdivided into two homologous subdomains (SD100) of approximately
100 amino acids.
[0006] Cytokine receptors have been categorized into two classes
based on the distribution of cysteine residues. The class 1
cytokine receptor family includes receptors for human growth
hormone (hGHR), erythropoietin, IL-3 and IL4, while the class 2
cytokine receptor family includes the IFN.gamma. receptor, tissue
factor, CRF2-4 and IL-10 receptors. Sequence analysis of the
hIFN.alpha. receptors shows that they are related to the class 2
cytokine receptor family.
[0007] Through the use of IFNAR1 gene knockout mice, IFNAR1 has
been shown to be essential for the response to all type 1 IFNs
(Muller et al., Science, 264: 1918-1921 (1994); Cleary et al., J.
Biol. Chem., 269: 18747-18749 (1994)) and for the mediation of
species-specific IFN signal transduction (Constantinescu et al.,
Proc. Natl. Acad. Sci USA, 91: 9602-9606 (1994)).
[0008] Benoit et al., J. Immunol., 150: 707-716 (1993) reported an
anti-IFNAR1 mAb, 64G12, that was found to inhibit the binding of
IFN-.alpha.2 (IFN-.alpha.A) and IFN-.alpha.B (IFN-.alpha.8) to
Daudi cells and to inhibit the antiviral activity of IFN-.alpha.2,
IFN-.beta. and IFN-.omega. (IFN-.alpha..sub.II1) on Daudi cells.
Benoit et al. also reported that 64G12 recognizes an epitope
present in domain 1 of IFNAR1. Eid and Tovey, J. Interferon
Cytokine Res., 15: 205-211 (1995) reported that 64G12 cannot
immunoprecipitate cross-linked IFN-.alpha.2-receptor complexes from
Daudi cells.
SUMMARY OF THE INVENTION
[0009] In one aspect, the invention provides an anti-IFNAR1
monoclonal antibody that inhibits the anti-viral activity of a
first type I interferon and does not inhibit the anti-viral
activity of a second type I interferon.
[0010] In another aspect, the invention provides an anti-IFNAR1
monoclonal antibody that inhibits anti-viral activity of a first
type I interferon and does not inhibit the anti-viral activity of
IFN-.alpha.A.
[0011] In still another aspect, the invention provides an
anti-IFNAR1 monoclonal antibody that inhibits anti-viral activity
of a first type I interferon and does not inhibit the anti-viral
activity of IFN-.alpha.B.
[0012] In yet another aspect, the invention provides an anti-IFNAR1
monoclonal antibody that inhibits anti-viral activity of a first
type I interferon and does not inhibit the anti-viral activity of
IFN-.alpha..sub.II1.
[0013] In a further aspect, the invention provides an anti-IFNAR1
monoclonal antibody that inhibits anti-viral activity of a first
type I interferon and does not inhibit the anti-viral activity of
IFN-.beta..
[0014] In an additional aspect, the invention provides an
anti-IFNAR1 monoclonal antibody that inhibits the anti-viral
activity of a first type I interferon and does not inhibit the
anti-viral activity of a second type I interferon selected from the
group consisting of IFN-.alpha.A, IFN-.alpha.B,
IFN-.alpha..sub.II1, and IFN-.beta..
[0015] The invention also encompasses an anti-IFNAR1 monoclonal
antibody that binds to one or more amino acids in situ in the
sequence of amino acids 244-249 of IFNAR1, and binds to one or more
amino acids in situ in the sequence of amino acids 291-298 of IFNAR
1.
BRIEF DESCRIPTION OF THE DRAWINGS
[0016] FIG. 1 is a graph depicting mAb 2E1 binding to U266 human
myeloma cell line as determined by FACS analysis. U266 cells were
incubated with hybridoma culture supernatant and then contacted
with FITC-goat anti-mouse IgG.
[0017] FIGS. 2A-E are graphs depicting epitope mapping for mAbs
2E1, 2E8, 2H6, 4A7 and 5A8, respectively, as determined by
competitive binding ELISA. IFNAR1 (ECD)-IgG captured by goat
anti-human IgG was incubated with predetermined concentrations of
biotinylated (Bio)-mAb in the presence of 500-1,000 fold excess of
unlabeled mAbs. The level of Bio-mAb bound was detected by the
addition of horse radish peroxidase (HRP)-streptavidin.
[0018] FIG. 3 is a collection of autoradiographs depicting the
effect of mAbs 2E1, 2E8, 2H6, 4A7 and 5A8 on ISGF3 formation in
Hela cells induced by IFN-.alpha.8 (IFN-.alpha.D) in an
electrophoretic mobility shift assay (EMSA).
[0019] FIG. 4 is a graph depicting a hydropathy profile and the
location of certain alanine-substituted mutants of hIFNAR1.
[0020] FIG. 5 is a graph depicting mAb binding to IFNAR1 ECD-IgG
(closed columns), IFNAR1 domain 1-IgG (shaded columns), IFNAR1
domain 2-IgG (diagonally hatched columns), and to a control with no
antigen (open columns) as determined by ELISA. Microtiter wells
coated with goat anti-human IgG were incubated with culture
supernatants containing 2 mg/ml of each immunoadhesin followed by
the addition of 10 mg/ml of mAbs. The mAb bound to the
immunoadhesin was detected by HRP-goat anti-mouse IgG.
[0021] FIG. 6 is a model of hIFNAR1 displaying its protein sequence
on the structural backbone of tissue factor. Subdomain SD100A of
domain 1 and subdomain SD100A' of domain 2 are shown in dark gray.
Subdomain SD100B of domain 1 and SD100B' of domain 2 are shown in
light gray. Regions involved in the binding of anti-IFNAR1 mAbs are
shown in orange. Amino acid residues involved in the binding of
anti-IFNAR1 mAbs are shown in red.
[0022] FIGS. 7A-7F (hereinafter collectively referred to as FIG. 7)
depict the DNA sequence (SEQ ID NO. 21) and amino acid sequence
(SEQ ID NO. 22) of the IFNAR1 ECD-IgG coding insert in pRK5
hIFNAR1-IgG clone 53.65. The DNA sequence encoding the leader
peptide amino acid sequence (corresponding to amino acids 1-29 in
FIG. 5 on page 229 of Uze et al., Cell, 60: 225-234 (1990)) of
IFNAR1 is shown as bases 38-124 of SEQ ID NO. 21 in FIG. 7. The
leader peptide amino acid sequence is omitted from FIG. 7 in order
to present the mature IFNAR1 ECD sequence as amino acids 1-404 of
the IFNAR1 ECD-IgG fusion protein sequence (SEQ ID NO. 22). Unless
otherwise indicated, the amino acid numbering scheme for IFNAR1 ECD
shown in FIG. 7 is used throughout the application.
METHODS OF CARRYING OUT THE INVENTION
A. Definitions
[0023] As used herein, the terms "type I interferon" and "human
type I interferon" are defined as all species of native human
interferon which fall within the human interferon-.alpha.,
interferon-.omega. and interferon-.beta. classes and which bind to
a common cellular receptor. Natural human interferon-.alpha.
comprises 23 or more closely related proteins encoded by distinct
genes with a high degree of structural homology (Weissmann and
Weber, Prog. Nucl. Acid. Res. Mol. Biol., 33: 251 (1986); J.
Interferon Res., 13: 443-44 (1993)). The human IFN-.alpha. locus
comprises two subfamilies. The first subfamily consists of at least
14 functional, non-allelic genes, including genes encoding
IFN-.alpha.A (IFN-.alpha.2), IFN-.alpha.B (IFN-.alpha.8),
IFN-.alpha.C (IFN-.alpha.10), IFN-.alpha.D (IFN-.alpha.1),
IFN-.alpha.E (IFN-.alpha.22), IFN-.alpha.F (IFN-.alpha.21),
IFN-.alpha.G (IFN-.alpha.5), and IFN-.alpha.H (IFN-.alpha.14), and
pseudogenes having at least 80% homology. The second subfamily,
.alpha..sub.II or .omega., contains at least 5 pseudogenes and 1
functional gene (denoted herein as "IFN-.alpha..sub.II1" or
"IFN-.omega.") which exhibits 70% homology with the IFN-.alpha.
genes (Weissmann and Weber (1986)). The human IFN-.beta. is encoded
by a single copy gene.
[0024] As used herein, the terms "first human interferon-.alpha.
(hIFN-.alpha.) receptor", "hIFNAR1", "IFNAR1", and "Uze chain" are
defined as the 557 amino acid receptor protein cloned by Uze et
al., Cell, 60: 225-234 (1990), including an extracellular domain of
409 residues, a transmembrane domain of 21 residues, and an
intracellular domain of 100 residues, as shown in FIG. 5 on page
229 of Uze et al. Also encompassed by the foregoing terms are
fragments of IFNAR1 that contain the extracellular domain (ECD) (or
fragments of the ECD) of IFNAR1.
[0025] As used herein, the term "anti-IFNAR1 antibody" is defined
as an antibody that is capable of binding to IFNAR1.
[0026] "Polymerase chain reaction" or "PCR" refers to a procedure
or technique in which minute amounts of a specific piece of nucleic
acid, RNA and/or DNA, are amplified as described in U.S. Pat. No.
4,683,195 issued 28 Jul. 1987. Generally, sequence information from
the ends of the region of interest or beyond needs to be available,
such that oligonucleotide primers can be designed; these primers
will be identical or similar in sequence to opposite strands of the
template to be amplified. The 5' terminal nucleotides of the two
primers can coincide with the ends of the amplified material. PCR
can be used to amplify specific RNA sequences, specific DNA
sequences from total genomic DNA, and cDNA transcribed from total
cellular RNA, bacteriophage or plasmid sequences, etc. See
generally Mullis et al., Cold Spring Harbor Symp. Quant. Biol.
51:263 (1987); Erlich, ed., PCR Technology (Stockton Press, NY,
1989). As used herein, PCR is considered to be one, but not the
only, example of a nucleic acid polymerase reaction method for
amplifying a nucleic acid test sample comprising the use of a known
nucleic acid as a primer and a nucleic acid polymerase to amplify
or generate a specific piece of nucleic acid.
[0027] "Antibodies" (Abs) and "immunoglobulins" (Igs) are
glycoproteins having the same structural characteristics. While
antibodies exhibit binding specificity to a specific antigen,
immunoglobulins include both antibodies and other antibody-like
molecules that lack antigen specificity. Polypeptides of the latter
kind are, for example, produced at low levels by the lymph system
and at increased levels by myelomas.
[0028] "Native antibodies and immunoglobulins" are usually
heterotetrameric glycoproteins of about 150,000 daltons, composed
of two identical light (L) chains and two identical heavy (H)
chains. Each light chain is linked to a heavy chain by one covalent
disulfide bond, while the number of disulfide-linkages varies
between the heavy chains of different immunoglobulin isotypes. Each
heavy and light chain also has regularly spaced intrachain
disulfide bridges. Each heavy chain has at one end a variable
domain (VH) followed by a number of constant domains. Each light
chain has a variable domain at one end (VL) and a constant domain
at its other end; the constant domain of the light chain is aligned
with the first constant domain of the heavy chain, and the light
chain variable domain is aligned with the variable domain of the
heavy chain. Particular amino acid residues are believed to form an
interface between the light- and heavy-chain variable domains
(Clothia et al., J. Mol. Biol. 186:651 (1985); Novotny and Haber,
Proc. Natl. Acad. Sci. U.S.A. 82:4592 (1985)).
[0029] The term "variable" refers to the fact that certain portions
of the variable domains differ extensively in sequence among
antibodies and are used in the binding and specificity of each
particular antibody for its particular antigen. However, the
variability is not evenly distributed throughout the variable
domains of antibodies. It is concentrated in three segments called
complementarity-determining regions (CDRs) or hypervariable regions
both in the light-chain and the heavy-chain variable domains. The
more highly conserved portions of variable domains are called the
framework (FR). The variable domains of native heavy and light
chains each comprise four FR regions, largely adopting a
.beta.-sheet configuration, connected by three CDRs, which form
loops connecting, and in some cases forming part of, the
.beta.-sheet structure. The CDRs in each chain are held together in
close proximity by the FR regions and, with the CDRs from the other
chain, contribute to the formation of the antigen-binding site of
antibodies (see Kabat et al., Sequences of Proteins of
immunological Interest, Fifth Edition, National Institute of
Health, Bethesda, Md. (1991)). The constant domains are not
involved directly in binding an antibody to an antigen, but exhibit
various effector functions, such as participation of the antibody
in antibody-dependent cellular toxicity.
[0030] Papain digestion of antibodies produces two identical
antigen-binding fragments, called "Fab" fragments, each with a
single antigen-binding site, and a residual "Fc" fragment, whose
name reflects its ability to crystallize readily. Pepsin treatment
yields an F(ab').sub.2 fragment that has two antigen-combining
sites and is still capable of cross-linking antigen.
[0031] "Fv" is the minimum antibody fragment that contains a
complete antigen-recognition and -binding site. In a two-chain Fv
species, this region consists of a dimer of one heavy- and one
light-chain variable domain in tight, non-covalent association. In
a single-chain Fv species, one heavy- and one light-chain variable
domain can be covalently linked by a flexible peptide linker such
that the light and heavy chains can associate in a "dimeric"
structure analogous to that in a two-chain Fv species. It is in
this configuration that the three CDRs of each variable domain
interact to define an antigen-binding site on the surface of the
VH-VL dimer. Collectively, the six CDRs confer antigen-binding
specificity to the antibody. However, even a single variable domain
(or half of an Fv comprising only three CDRs specific for an
antigen) has the ability to recognize and bind antigen, although at
a lower affinity than the entire binding site.
[0032] The Fab fragment also contains the constant domain of the
light chain and the first constant domain (CH1) of the heavy chain.
Fab' fragments differ from Fab fragments by the addition of a few
residues at the carboxy terminus of the heavy chain CH1 domain
including one or more cysteines from the antibody hinge region.
Fab'-SH is the designation herein for Fab' in which the cysteine
residue(s) of the constant domains bear a free thiol group.
F(ab').sub.2 antibody fragments originally were produced as pairs
of Fab' fragments which have hinge cysteines between them. Other
chemical couplings of antibody fragments are also known.
[0033] The "light chains" of antibodies (immunoglobulins) from any
vertebrate species can be assigned to one of two clearly distinct
types, called kappa (.kappa.) and lambda (.lamda.), based on the
amino acid sequences of their constant domains.
[0034] Depending on the amino acid sequence of the constant domain
of their heavy chains, immunoglobulins can be assigned to different
classes. There are five major classes of immunoglobulins: IgA, IgD,
IgE, IgG, and IgM, and several of these can be further divided into
subclasses (isotypes), e.g., IgG.sub.1, IgG.sub.2, IgG.sub.3,
IgG.sub.4, IgA.sub.1, and IgA.sub.2. The heavy-chain constant
domains that correspond to the different classes of immunoglobulins
are called .alpha., .delta., .epsilon., .gamma., and .mu.,
respectively. The subunit structures and three-dimensional
configurations of different classes of immunoglobulins are well
known.
[0035] The term "antibody" specifically covers monoclonal
antibodies, including antibody fragment clones.
[0036] "Antibody fragments" comprise a portion of an intact
antibody, generally the antigen binding or variable region of the
intact antibody. Examples of antibody fragments include Fab, Fab',
F(ab').sub.2, and Fv fragments; diabodies; single-chain antibody
molecules, including single-chain Fv (scFv) molecules; and
multispecific antibodies formed from antibody fragments.
[0037] The term "monoclonal antibody" as used herein refers to an
antibody (or antibody fragment) obtained from a population of
substantially homogeneous antibodies, i.e., the individual
antibodies comprising the population are identical except for
possible naturally occurring mutations that may be present in minor
amounts. Monoclonal antibodies are highly specific, being directed
against a single antigenic site. Furthermore, in contrast to
conventional (polyclonal) antibody preparations that typically
include different antibodies directed against different
determinants (epitopes), each monoclonal antibody is directed
against a single determinant on the antigen. In addition to their
specificity, the monoclonal antibodies are advantageous in that
they are synthesized by the hybridoma culture, uncontaminated by
other immunoglobulins. The modifier "monoclonal" indicates the
character of the antibody as being obtained from a substantially
homogeneous population of antibodies, and is not to be construed as
requiring production of the antibody by any particular method. For
example, the monoclonal antibodies to be used in accordance with
the present invention may be made by the hybridoma method first
described by Kohler et al., Nature, 256:495 (1975), or may be made
by recombinant DNA methods (see, e.g., U.S. Pat. No. 4,816,567).
The "monoclonal antibodies" also include clones of
antigen-recognition and binding-site containing antibody fragments
(Fv clones) isolated from phage antibody libraries using the
techniques described in Clackson et al., Nature, 352:624-628 (1991)
and Marks et al., J. Mol. Biol., 222:581-597 (1991), for
example.
[0038] The monoclonal antibodies herein specifically include
"chimeric" antibodies (immunoglobulins) in which a portion of the
heavy and/or light chain is identical with or homologous to
corresponding sequences in antibodies derived from a particular
species or belonging to a particular antibody class or subclass,
while the remainder of the chain(s) is identical with or homologous
to corresponding sequences in antibodies derived from another
species or belonging to another antibody class or subclass, as well
as fragments of such antibodies, so long as they exhibit the
desired biological activity (U.S. Pat. No. 4,816,567 to Cabilly et
al.; Morrison et al., Proc. Natl. Acad. Sci. USA, 81:6851-6855
(1984)).
[0039] "Humanized" forms of non-human (e.g., murine) antibodies are
chimeric immunoglobulins, immunoglobulin chains or fragments
thereof (such as Fv, Fab, Fab', F(ab').sub.2 or other
antigen-binding subsequences of antibodies) which contain minimal
sequence derived from non-human immunoglobulin. For the most part,
humanized antibodies are human immunoglobulins (recipient antibody)
in which residues from a complementarity-determining region (CDR)
of the recipient are replaced by residues from a CDR of a non-human
species (donor antibody) such as mouse, rat or rabbit having the
desired specificity, affinity, and capacity. In some instances, Fv
framework region (FR) residues of the human immunoglobulin are
replaced by corresponding non-human residues. Furthermore,
humanized antibodies may comprise residues that are found neither
in the recipient antibody nor in the imported CDR or framework
sequences. These modifications are made to further refine and
optimize antibody performance. In general, the humanized antibody
will comprise substantially all of at least one, and typically two,
variable domains, in which all or substantially all of the CDR
regions correspond to those of a non-human immunoglobulin and all
or substantially all of the FR regions are those of a human
immunoglobulin sequence. The humanized antibody optimally also will
comprise at least a portion of an immunoglobulin constant region
(Fc), typically that of a human immunoglobulin. For further
details, see Jones et al., Nature, 321:522-525 (1986); Reichmann et
al., Nature, 332:323-329 (1988); and Presta, Curr. Op. Struct.
Biol., 2:593-596 (1992). The humanized antibody includes a
Primatized.TM.antibody wherein the antigen-binding region of the
antibody is derived from an antibody produced by immunizing macaque
monkeys with the antigen of interest.
[0040] "Single-chain Fv" or "scFv" antibody fragments comprise the
VH and VL domains of antibody, wherein these domains are present in
a single polypeptide chain. Generally, the scFv polypeptide further
comprises a polypeptide linker between the VH and VL domains which
enables the scFv to form the desired structure for antigen binding.
For a review of scFv see Pluckthun, in The Pharmacology of
Monoclonal Antibodies, vol. 113, Rosenburg and Moore eds.,
Springer-Verlag, New York, pp. 269-315 (1994).
[0041] The term "diabodies" refers to small antibody fragments with
two antigen-binding sites, which fragments comprise a heavy-chain
variable domain (VH) connected to a light-chain variable domain
(VL) in the same polypeptide chain (VH-VL). By using a linker that
is too short to allow pairing between the two domains on the same
chain, the domains are forced to pair with the complementary
domains of another chain and create two antigen-binding sites.
Diabodies are described more fully in, for example, EP 404,097; WO
93/11161; and Hollinger et al., Proc. Natl. Acad. Sci. USA,
90:6444-6448 (1993).
[0042] An "isolated" antibody is one that has been identified and
separated and/or recovered from a component of its natural
environment. Contaminant components of its natural environment are
materials that would interfere with diagnostic or therapeutic uses
for the antibody, and may include enzymes, hormones, and other
proteinaceous or nonproteinaceous solutes. In preferred
embodiments, the antibody will be purified (1) to greater than 95%
by weight of antibody as determined by the Lowry method, and most
preferably more than 99% by weight, (2) to a degree sufficient to
obtain at least 15 residues of N-terminal or internal amino acid
sequence by use of a spinning cup sequenator, or (3) to homogeneity
by SDS-PAGE under reducing or nonreducing conditions using
Coomassie blue or, preferably, silver stain. Isolated antibody
includes the antibody in situ within recombinant cells since at
least one component of the antibody's natural environment will not
be present. Ordinarily, however, isolated antibody will be prepared
by at least one purification step.
[0043] "Treatment" refers to both therapeutic treatment and
prophylactic or preventative measures. Those in need of treatment
include those already with the disorder as well as those in which
the disorder is to be prevented.
[0044] "Mammal" for purposes of treatment refers to any animal
classified as a mammal, including humans, domestic and farm
animals, and zoo, sports, or pet animals, such as dogs, horses,
cats, cows, etc. Preferably, the mammal is human.
[0045] As used herein, the terms "each member of the group
consisting of" and "each of" are synonymous.
[0046] As used herein, the terms "any member of the group
consisting of" and "any of" are synonymous.
B. General Methods
[0047] In general, the invention provides anti-IFNAR1 antibodies
that are useful for treatment of immune-mediated disorders in which
a partial or total blockade of type I interferon activity is
desired. In one embodiment, the anti-IFNAR1 antibodies of the
invention are used to treat autoimmune disorders, such as type I
and type II diabetes, systemic lupus erythematosis (SLE), and
rheumatoid arthritis. In another embodiment, the anti-IFNAR1
antibodies provided herein are used to treat graft rejection or
graft versus host disease. The unique properties of the anti-IFNAR1
antibodies of the invention make them particularly useful for
effecting target levels of immunosuppression in a patient. For
patients requiring acute intervention, the anti-IFNAR1 antibodies
provided herein which cause broad spectrum ablation of type I
interferon activity can be used to effect the largest possible
compromise of an undesired immune response. For patients requiring
maintenance immunosuppression, the anti-IFNAR1 antibodies provided
herein which block the activity of one or more (but not all)
species of type I interferon can be used to effect partial
compromise of the patient's immune system in order to reduce the
risk of undesirable immune responses while leaving some components
of the patient's type I interferon-mediated immunity intact in
order to avoid infection.
[0048] In another aspect, the anti-IFNAR1 antibodies of the
invention find utility as reagents for detection and isolation of
IFNAR1, such as detection of IFNAR1 expression in various cell
types and tissues, including the determination of IFNAR1 receptor
density and distribution in cell populations, and cell sorting
based on IFNAR1 expression. In yet another aspect, the present
anti-IFNAR1 antibodies are useful for the development of IFNAR1
antagonists with type I interferon inhibition activity patterns
similar to those of the subject antibodies. The anti-IFNAR1
antibodies of the invention can be used in competition binding
assays with IFNAR1 to screen for small molecule antagonists of
IFNAR1 that will exhibit similar pharmacological effects in
blocking the activities of type I interferons to IFNAR1.
[0049] I. Methods of Making Synthetic Anti-IFNAR1 Fv Clones
[0050] The anti-IFNAR1 antibodies of the invention can be made by
using combinatorial libraries to screen for synthetic antibody
clones with the desired activity or activities. In principle,
synthetic antibody clones are selected by screening phage libraries
containing phage that display various fragments of antibody
variable region (Fv) fused to phage coat protein. Such phage
libraries are panned by affinity chromatography against the desired
ligand. Clones expressing Fv fragments capable of binding to the
desired ligand are adsorbed to the ligand and thus separated from
the non-binding clones in the library. The binding clones are then
eluted from the ligand, and can be further enriched by additional
cycles of ligand adsorption/elution. Any of the anti-IFNAR1
antibodies of the invention can be obtained by designing a suitable
ligand screening procedure to select for the phage clone of
interest followed by construction of a full length anti-IFNAR1
antibody clone using the Fv sequences from the phage clone of
interest and suitable constant region (Fc) sequences described in
Kabat et al., Sequences of Proteins of Immunological Interest,
Fifth Edition, NIH Publication 91-3242, Bethesda Md. (1991), vols.
1-3.
[0051] 1. Construction of Phage Libraries
[0052] The antigen-binding domain of an antibody is formed from two
variable (V) regions of about 110 amino acids, one each from the
light (VL) and heavy (VH) chains, that both present three
hypervariable loops or complementarity-determining regions (CDRs).
Variable domains can be displayed functionally on phage, either as
single-chain Fv (scFv) fragments, in which VH and VL are covalently
linked through a short, flexible peptide, or as Fab fragments, in
which they are each fused to a constant domain and interact
non-covalently, as described in Winter et al., Ann. Rev. Immunol.,
12: 433455 (1994). As used herein, scFv encoding phage clones and
Fab encoding phage clones are collectively referred to as "Fv phage
clones" or "Fv clones".
[0053] The naive repertoire of an animal (the repertoire before
antigen challenge) provides it with antibodies that can bind with
moderate affinity (K.sub.a of about 10.sup.6 to 10.sup.7 M.sup.-1)
to essentially any non-self molecule. The sequence diversity of
antibody binding sites is not encoded directly in the germline but
is assembled in a combinatorial manner from V gene segments.
Inhuman heavy chains, the first two hypervariable loops (H1 and H2)
are drawn from less than 50 VH gene segments, which are combined
with D segments and JH segments to create the third hypervariable
loop (H3). In human light chains, the first two hypervariable loops
(L1 and L2) and much of the third (L3) are drawn from less than
approximately 30 V.lamda. and less than approximately 30 V.kappa.
segments to complete the third hypervariable loop (L3).
[0054] Each combinatorial rearrangement of V-gene segments in stem
cells gives rise to a B cell that expresses a single VH-VL
combination. Immunizations triggers any B cell making a VH-VL
combination that binds the immunogen to proliferate (clonal
expansion) and to secrete the corresponding antibody. These naive
antibodies are then matured to high affinity (Ka.gtoreq.10.sup.9
M.sup.-1) by a process of mutagenesis and selection known as
affinity maturation. It is after this point that cells are normally
removed to prepare hybridomas and generate high-affinity monoclonal
antibodies.
[0055] At three stages of this process, repertoires of VH and VL
genes can be separately cloned by polymerase chain reaction (PCR)
and recombined randomly in phage libraries, which can then be
searched for antigen-binding clones as described in Winter et al.,
Ann. Rev. Immunol., 12: 433-455 (1994). Libraries from immunized
sources provide high-affinity antibodies to the immunogen without
the requirement of constructing hybridomas. Alternatively, the
naive repertoire can be cloned to provide a single source of human
antibodies to a wide range of non-self and also self antigens
without any immunization as described by Griffiths et al., EMBO J,
12: 725-734 (1993). Finally, naive libraries can also be made
synthetically by cloning the unrearranged V-gene segments from stem
cells, and using PCR primers containing random sequence to encode
the highly variable CDR3 regions and to accomplish rearrangement in
vitro as described by Hoogenboom and Winter, J. Mol. Biol., 227:
381-388 (1992).
[0056] Phage display mimics the B cell. Filamentous phage is used
to display antibody fragments by fusion to the minor coat protein
pIII. The antibody fragments can be displayed as single chain Fv
fragments, in which VH and VL domains are connected on the same
polypeptide chain by a flexible polypeptide spacer, e.g. as
described by Marks et al., J. Mol. Biol., 222: 581-597 (1991), or
as Fab fragments, in which one chain is fused to pIII and the other
is secreted into the bacterial host cell periplasm where assembly
of a Fab-coat protein structure which becomes displayed on the
phage surface by displacing some of the wild type coat proteins,
e.g. as described in Hoogenboom et al., Nucl. Acids Res., 19:
41334137 (1991). When antibody fragments are fused to the
N-terminus of pIII, the phage is infective. However, if the
N-terminal domain of pIII is excised and fusions made to the second
domain, the phage is not infective, and wild type pII must be
provided by helper phage.
[0057] The pIII fusion and other proteins of the phage can be
encoded entirely within the same phage replicon, or on different
replicons. When two replicons are used, the pIII fusion is encoded
on a phagemid, a plasmid containing a phage origin of replication.
Phagemids can be packaged into phage particles by "rescue" with a
helper phage such as M13K07 that provides all the phage proteins,
including pIII, but due to a defective origin is itself poorly
packaged in competitions with the phagemids as described in Vieira
and Messing, Meth. Enzymol., 153: 3-11 (1987). In a preferred
method, the phage display system is designed such that the
recombinant phage can be grown in host cells under conditions
permitting no more than a minor amount of phage particles to
display more than one copy of the Fv-coat protein fusion on the
surface of the particle as described in Bass et al., Proteins, 8:
309-314 (1990) and in WO 92/09690 (PCT/US91/09133 published Jun.
11, 1992).
[0058] In general, nucleic acids encoding antibody gene fragments
are obtained from immune cells harvested from humans or animals. If
a library biased in favor of anti-IFNAR1 clones is desired, the
subject is immunized with IFNAR1 to generate an antibody response,
and spleen cells and/or circulating B cells other peripheral blood
lymphocytes (PBLs) are recovered for library construction. In a
preferred embodiment, a human antibody gene fragment library biased
in favor of anti-human IFNAR1 clones is obtained by generating an
anti-human IFNAR1 antibody response in transgenic mice carrying a
functional human immunoglobulin gene array (and lacking a
functional endogenous antibody production system) such that IFNAR1
immunization gives rise to B cells producing human antibodies
against IFNAR1. The generation of human antibody-producing
transgenic mice is described in Section B(III)(b) below.
[0059] Additional enrichment for anti-IFNAR1 reactive cell
populations can be obtained by using a suitable screening procedure
to isolate B cells expressing IFNAR1-specific membrane bound
antibody, e.g., by cell separation with IFNAR1 affinity
chromatography or adsorption of cells to fluorochrome-labelled
IFNAR1 followed by flow-activated cell sorting (FACS).
[0060] Alternatively, the use of spleen cells and/or B cells or
other PBLs from an unimmunized donor provides a better
representation of the possible antibody repertoire, and also
permits the construction of an antibody library using any animal
(human or non-human) species in which IFNAR1 is not antigenic. For
libraries incorporating in vitro antibody gene construction, stem
cells are harvested from the subject to provide nucleic acids
encoding unrearranged antibody gene segments. The immune cells of
interest can be obtained from a variety of animal species, such as
human, mouse, rat, lagomorpha, luprine, canine, feline, porcine,
bovine, equine, and avian species, etc.
[0061] Nucleic acid encoding antibody variable gene segments
(including VH and VL segments) are recovered from the cells of
interest and amplified. In the case of rearranged VH and VL gene
libraries, the desired DNA can be obtained by isolating genomic DNA
or mRNA from lymphocytes followed by polymerase chain reaction
(PCR) with primers matching the 5' and 3' ends of rearranged VH and
VL genes as described in Orlandi et al., Proc. Natl. Acad. Sci.
(USA), 86: 3833-3837 (1989), thereby making diverse V gene
repertoires for expression. The V genes can be amplified from cDNA
and genomic DNA, with back primers at the 5' end of the exon
encoding the mature V-domain and forward primers based within the
J-segment as described in Orlandi et al. (1989) and in Ward et al.,
Nature, 341: 544-546 (1989). However, for amplifying from cDNA,
back primers can also be based in the leader exon as described in
Jones et al., Biotechnol, 9: 88-89 (1991), and forward primers
within the constant region as described in Sastry et al., Proc.
Natl. Acad. Sci. (USA), 86: 5728-5732 (1989). To maximize
complementarity, degeneracy can be incorporated in the primers as
described in Orlandi et al. (1989) or Sastry et al. (1989).
Preferably, the library diversity is maximized by using PCR primers
targeted to each V-gene family in order to amplify all available VH
and VL arrangements present in the immune cell nucleic acid sample,
e.g. as described in the method of Marks et al., J. Mol. Biol.,
222: 581-597 (1991) or as described in the method of Orum et al.,
Nucleic Acids Res., 21: 4491-4498 (1993). For cloning of the
amplified DNA into expression vectors, rare restriction sites can
be introduced within the PCR primer as a tag at one end as
described in Orlandi et al. (1989), or by further PCR amplification
with a tagged primer as described in Clackson et al., Nature, 352:
624-628 (1991).
[0062] Repertoires of synthetically rearranged V genes can be
derived in vitro from V gene segments. Most of the human VH-gene
segments have been cloned and sequenced (reported in Tomlinson et
al., J. Mol. Biol., 227: 776-798 (1992)), and mapped (reported in
Matsuda et al., Nature Genet., 3: 88-94 (1993); these cloned
segments (including all the major conformations of the H1 and H2
loop) can be used to generate diverse VH gene repertoires with PCR
primers encoding H3 loops of diverse sequence and length as
described in Hoogenboom and Winter, J. Mol. Biol., 227: 381-388
(1992). VH repertoires can also be made with all the sequence
diversity focussed in a long H3 loop of a single length as
described in Barbas et al., Proc. Natl. Acad. Sci. USA, 89:
4457-4461 (1992). Human V.kappa. and V.lamda. segments have been
cloned and sequenced (reported in Williams and Winter, Eur. J.
Immunol., 23: 1456-1461 (1993)) and can be used to make synthetic
light chain repertoires. Synthetic V gene repertoires, based on a
range of VH and VL folds, and L3 and H3 lengths, will encode
antibodies of considerable structural diversity. Following
amplification of V-gene encoding DNAs, germline V-gene segments can
be rearranged in vitro according to the methods of Hoogenboom and
Winter, J. Mol. Biol., 227: 381-388 (1992).
[0063] Repertoires of antibody fragments can be constructed by
combining VH and VL gene repertoires together in several ways. Each
repertoire can be created in different vectors, and the vectors
recombined in vitro, e.g., as described in Hogrefe et al., Gene,
128: 119-126 (1993), or in vivo by combinatorial infection, e.g.,
the loxp system described in Waterhouse et al., Nucl. Acids Res.,
21: 2265-2266 (1993). The in vivo recombination approach exploits
the two-chain nature of Fab fragments to overcome the limit on
library size imposed by E. coli transformation efficiency. Naive VH
and VL repertoires are cloned separately, one into a phagemid and
the other into a phage vector. The two libraries are then combined
by phage infection of phagemid-containing bacteria so that each
cell contains a different combination and the library size is
limited only by the number of cells present (about 10.sup.12
clones). Both vectors contain in vivo recombination signals so that
the VH and VL genes are recombined onto a single replicon and are
co-packaged into phage virions. These huge libraries provide large
numbers of diverse antibodies of good affinity (K.sub.a of about
10.sup.8).
[0064] Alternatively, the repertoires may be cloned sequentially
into the same vector, e.g. as described in Barbas et al., Proc.
Natl. Acad. Sci. USA, 88: 7978-7982 (1991), or assembled together
by PCR and then cloned, e.g. as described in Clackson et al.,
Nature, 352: 624-628 (1991). PCR assembly can also be used to join
VH and VL DNAs with DNA encoding a flexible peptide spacer to form
single chain Fv (scFv) repertoires. In yet another technique, "in
cell PCR assembly" is used to combine VH and VL genes within
lymphocytes by PCR and then clone repertoires of linked genes as
described in Embleton et al., Nucl. Acids Res., 20: 3831-3837
(1992).
[0065] The antibodies produced by naive libraries (either natural
or synthetic) can be of moderate affinity (K.sub.a of about
10.sup.6 to 10.sup.7 M.sup.-1), but affinity maturation can also be
mimicked in vitro by constructing and reselecting from secondary
libraries as described in Winter et al. (1994), supra. For example,
mutation can be introduced at random in vitro by using error-prone
polymerase (reported in Leung et al., Technique, 1: 11-15 (1989))
in the method of Hawkins et al., J. Mol. Biol., 2-26: 889-896
(1992) or in the method of Gram et al., Proc. Natl. Acad. Sci USA,
89: 3576-3580 (1992). Additionally, affinity maturation can be
performed by randomly mutating one or more CDRs, e.g. using PCR
with primers carrying random sequence spanning the CDR of interest,
in selected individual Fv clones and screening for higher affinity
clones. Another effective approach is to recombine the VH or VL
domains selected by phage display with repertoires of naturally
occurring V domain variants obtained from unimmunized donors and
screen for higher affinity in several rounds of chain reshuffling
as described in Marks et al., Biotechnol., 10: 779-783 (1992). This
technique allows the production of antibodies and antibody
fragments with affinities in the 109 M range.
[0066] 2. Panning Phage Display Libraries for Anti-IFNAR1
Clones
[0067] a: Synthesis of IFNAR1 and IFNAR1 Ligands
[0068] Nucleic acid sequence encoding the IFNAR1s used herein can
be designed using the amino acid sequence of the desired region of
IFNAR1, e.g. the extracellular domain spanning amino acids 28 to
434 of FIG. 2 of WO 93/20187 (PCT/EP93/00770 published Oct. 14,
1993). Alternatively, the cDNA sequence of FIG. 2 of WO 93/20187
can be used. In addition, nucleic acid encoding an immunoglobulin G
(IgG)-IFNAR1 extracellular domain fusion protein can be obtained
from the amino acid or cDNA sequence shown in FIG. 8 below.
Likewise, nucleic acid sequence encoding the human type I
interferons used herein can be designed using published amino acid
and nucleic acid sequences, e.g. see the J. Interferon Res., 13:
443444 (1993) compilation of references containing genomic and cDNA
sequences for various type I interferons, and the references cited
therein. For the IFN-.alpha.A, IFN-.alpha.B, IFN-.alpha.C,
IFN-.alpha.D, IFN-.alpha.E, IFN-.alpha.F, IFN-.alpha.G, and
IFN-.alpha.H amino acid sequences or cDNA sequences, see FIGS. 3
and 4 on pages 23-24 of Goeddel et al., Nature, 290: 20-26 (1981).
For cDNA encoding the amino acid sequence of IFN-.alpha..sub.II1
(IFN-.omega.), see Capon et al., Mol. Cell. Biol., 5: 768-779
(1985) and Hauptmann and Swetly, Nucleic Acids Res., 13: 47394749
(1985). For cDNA encoding the amino acid sequence of IFN-.beta.,
see Taniguchi et al., Proc. Jpn. Acad Ser. B. 55: 464-469 (1979);
Taniguchi et al., Gene, 10: 11-15 (1980); and U.S. Pat. No.
5,460,811 to Goeddel and Crea. DNAs encoding the IFNAR1 s or type I
interferons of interest can be prepared by a variety of methods
known in the art. These methods include, but are not limited to,
chemical synthesis by any of the methods described in Engels et
al., Agnew. Chem. Int. Ed. Engl., 28: 716-734 (1989), such as the
triester, phosphite, phosphoramidite and H-phosphonate methods. In
one embodiment, codons preferred by the expression host cell are
used in the design of the IFNAR1 or type I interferon-encoding DNA.
Alternatively, DNA encoding the IFNAR1 or type I interferon can be
isolated from a genomic or cDNA library.
[0069] For production of the mutant IFNAR Is used herein, DNA
sequence encoding wild type IFNAR1 can be altered to encode the
desired IFNAR1 mutant by using recombinant DNA techniques, such as
site specific mutagenesis (Kunkel et al., Methods Enzymol.
204:125-139 (1991); Carter, P., et al., Nucl. Acids. Res. 13:4331
(1986); Zoller, M. J. et al., Nucl. Acids Res. 10:6487 (1982)),
cassette mutagenesis (Wells, J. A., et al., Gene 34:315 (1985)),
restriction selection mutagenesis (Wells, J. A., et al., Philos.
Trans, R. Soc. London SerA 317: 415 (1986)), and the like.
[0070] Following construction of the DNA molecule encoding the
IFNAR1 or type I interferon of interest, the DNA molecule is
operably linked to an expression control sequence in an expression
vector, such as a plasmid, wherein the control sequence is
recognized by a host cell transformed with the vector. In general,
plasmid vectors contain replication and control sequences that are
derived from species compatible with the host cell. The vector
ordinarily carries a replication site, as well as sequences which
encode proteins that are capable of providing phenotypic selection
in transformed cells.
[0071] For expression in prokaryotic hosts, suitable vectors
include pBR322 (ATCC No. 37,017), phGH107 (ATCC No. 40,011),
pBO475, pS0132, pRIT5, any vector in the pRIT20 or pRIT30 series
(Nilsson and Abrahmsen, Meth. Enzymol., 185: 144-161 (1990)),
pRIT2T, pKK233-2, pDR540 and pPL-lambda. Prokaryotic host cells
containing the expression vectors suitable for use herein include
E. coli K12 strain 294 (ATCC NO. 31446), E coli strain JM101
(Messing et al., Nucl. Acid Res., 9: 309 (1981)), E. coli strain B,
E. coli strain .chi.1776 (ATCC No. 31537), E. coli c600 (Appleyard,
Genetics, 39: 440 (1954)), E. coli W3110 (F-, gamma-, prototrophic,
ATCC No. 27325), E. coli strain 27C7 (W3110, tonA, phoA E15,
(argF-lac) 169, ptr3, degP41, ompT, kan') U.S. Pat. No. 5,288,931,
ATCC No. 55,244), Bacillus subtilis, Salmonella typhimurium,
Serratia marcesans, and Pseudomonas species.
[0072] In addition to prokaryotes, eukaryotic organisms, such as
yeasts, or cells derived from multicellular organisms can be used
as host cells. For expression in yeast host cells, such as common
baker's yeast or Saccharomyces cerevisiae, suitable vectors include
episomally replicating vectors based on the 2-micron plasmid,
integration vectors, and yeast artificial chromosome (YAC) vectors.
For expression in insect host cells, such as Sf9 cells, suitable
vectors include baculoviral vectors. For expression in plant host
cells, particularly dicotyledonous plant hosts, such as tobacco,
suitable expression vectors include vectors derived from the Ti
plasmid of Agrobacterium tumefaciens.
[0073] However, interest has been greatest in vertebrate host
cells. Examples of useful mammalian host cells include monkey
kidney CVI line transformed by SV40 (COS-7, ATCC CRL 1651); human
embryonic kidney line (293 or 293 cells subcloned for growth in
suspension culture, Graham et al., J. Gen Virol., 36: 59 (1977));
baby hamster kidney cells (BHK, ATCC CCL 10); Chinese hamster ovary
cells/-DHFR (CHO, Urlaub and Chasin, Proc. Natl. Acad. Sci. USA,
77: 4216 (1980)); mouse sertoli cells (TM4, Mather, Biol. Reprod.,
23: 243-251 (1980)); monkey kidney cells (CVI ATCC CCL 70); African
green monkey kidney cells (VERO-76, ATCC CRL-1587); human cervical
carcinoma cells (HELA, ATCC CCL 2); canine kidney cells (MDCK, ATCC
CCL 34); buffalo rat liver cells (BRL 3A, ATCC CRL 1442); human
lung cells (WI 38, ATCC CCL 75); human liver cells (Hep G2, HB
8065); mouse mammary tumor (MMT 060562, ATCC CCL51); TR1 cells
(Mather et al., Annals N.Y. Acad. Sci., 383: 44-68 (1982)); MRC 5
cells; FS4 cells; and a human hepatoma cell line (Hep G2). For
expression in mammalian host cells, useful vectors include vectors
derived from SV40, vectors derived from cytomegalovirus such as the
pRK vectors, including pRK5 and pRK7 (Suva et al., Science, 237:
893-896 (1987), EP 307,247 (Mar. 15, 1989), EP 278,776 (Aug. 17,
1988)) vectors derived from vaccinia viruses or other pox viruses,
and retroviral vectors such as vectors derived from Moloney's
murine leukemia virus (MoMLV).
[0074] Optionally, the DNA encoding the IFNAR1 or type I interferon
of interest is operably linked to a secretory leader sequence
resulting in secretion of the expression product by the host cell
into the culture medium. Examples of secretory leader sequences
include stil, ecotin, lamB, herpes GD, lpp, alkaline phosphatase,
invertase, and alpha factor. Also suitable for use herein is the 36
amino acid leader sequence of protein A (Abrahmsen et al., EMBO J.,
4: 3901 (1985)).
[0075] Host cells are transfected and preferably transformed with
the above-described expression or cloning vectors of this invention
and cultured in conventional nutrient media modified as appropriate
for inducing promoters, selecting transformants, or amplifying the
genes encoding the desired sequences.
[0076] Transfection refers to the taking up of an expression vector
by a host cell whether or not any coding sequences are in fact
expressed. Numerous methods of transfection are known to the
ordinarily skilled artisan, for example, CaPO.sub.4 precipitation
and electroporation. Successful transfection is generally
recognized when any indication of the operation of this vector
occurs within the host cell.
[0077] Transformation means introducing DNA into an organism so
that the DNA is replicable, either as an extrachromosomal element
or by chromosomal integrant. Depending on the host cell used,
transformation is done using standard techniques appropriate to
such cells. The calcium treatment employing calcium chloride, as
described in section 1.82 of Sambrook et al., Molecular Cloning
(2nd ed.), Cold Spring Harbor Laboratory, NY (1989), is generally
used for prokaryotes or other cells that contain substantial
cell-wall barriers. Infection with Agrobacterium tumefaciens is
used for transformation of certain plant cells, as described by
Shaw et al., Gene, 23: 315 (1983) and WO 89/05859 published 29 Jun.
1989. For mammalian cells without such cell walls, the calcium
phosphate precipitation method described in sections 16.30-16.37 of
Sambrook et al, supra, is preferred. General aspects of mammalian
cell host system transformations have been described by Axel in
U.S. Pat. No. 4,399,216 issued 16 Aug. 1983. Transformations into
yeast are typically carried out according to the method of Van
Solingen et al., J. Bact., 130: 946 (1977) and Hsiao et al., Proc.
Natl. Acad. Sci. (USA), 76: 3829 (1979). However, other methods for
introducing DNA into cells such as by nuclear injection,
electroporation, or by protoplast fusion may also be used.
[0078] Prokaryotic host cells used to produce the IFNAR1 or type I
interferon of interest can be cultured as described generally in
Sambrook et al., supra.
[0079] The mammalian host cells used to produce the IFNAR1 or type
I interferon of interest can be cultured in a variety of media.
Commercially available media such as Ham's F10 (Sigma), Minimal
Essential Medium ((MEM), Sigma), RPMI-1640 (Sigma), and Dulbecco's
Modified Eagle's Medium ((DMEM), Sigma) are suitable for culturing
the host cells. In addition, any of the media described in Ham and
Wallace, Meth. Enz., 58: 44 (1979), Barnes and Sato, Anal.
Biochem., 102: 255 (1980), U.S. Pat. No. 4,767,704; 4,657,866;
4,927,762; or 4,560,655; WO 90/03430; WO 87/00195; U.S. Pat. Re.
30,985; or U.S. Pat. No. 5,122,469, the disclosures of all of which
are incorporated herein by reference, may be used as culture media
for the host cells. Any of these media may be supplemented as
necessary with hormones and/or other growth factors (such as
insulin, transferrin, or epidermal growth factor), salts (such as
sodium chloride, calcium, magnesium, and phosphate), buffers (such
as HEPES), nucleosides (such as adenosine and thymidine),
antibiotics (such as Gentamycin.TM. drug), trace elements (defined
as inorganic compounds usually present at final concentrations in
the micromolar range), and glucose or an equivalent energy source.
Any other necessary supplements may also be included at appropriate
concentrations that would be known to those skilled in the art. The
culture conditions, such as temperature, pH, and the like, are
those previously used with the host cell selected for expression,
and will be apparent to the ordinarily skilled artisan.
[0080] The host cells referred to in this disclosure encompass
cells in in vitro culture as well as cells that are within a host
animal.
[0081] In an intracellular expression system or periplasmic space
secretion system, the recombinantly expressed IFNAR1 or type I
interferon protein can be recovered from the culture cells by
disrupting the host cell membrane/cell wall (e.g. by osmotic shock
or solubilizing the host cell membrane in detergent).
Alternatively, in an extracellular secretion system, the
recombinant protein can be recovered from the culture medium. As a
first step, the culture medium or lysate is centrifuged to remove
any particulate cell debris. The membrane and soluble protein
fractions are then separated. Usually, the IFNAR1 or type I
interferon is purified from the soluble protein fraction. If the
IFNAR1 is expressed as a membrane bound species, the membrane bound
peptide can be recovered from the membrane fraction by
solubilization with detergents. The crude peptide extract can then
be further purified by suitable procedures such as fractionation on
immunoaffinity or ion-exchange columns; ethanol precipitation;
reverse phase HPLC; chromatography on silica or on a cation
exchange resin such as DEAE; chromatofocusing; SDS-PAGE; ammonium
sulfate precipitation; gel filtration using, for example, Sephadex
G-75; hydrophobic affinity resins and ligand affinity using IFNAR1
(for type I interferon purification) or type I interferons or
anti-IFNAR1 antibodies (for IFNAR1 purification) immobilized on a
matrix.
[0082] Many of the human type I interferons used herein can be
obtained from commercial sources, e.g. human IFN-.beta. is
available from Sigma (St. Louis, Mo.).
[0083] b. Immobilization of IFNAR1
[0084] The purified IFNAR1 can be attached to a suitable matrix
such as agarose beads, acrylamide beads, glass beads, cellulose,
various acrylic copolymers, hydroxyl methacrylate gels, polyacrylic
and polymethacrylic copolymers, nylon; neutral and ionic carriers,
and the like, for use in the affinity chromatographic separation of
phage display clones. Attachment of the IFNAR1 protein to the
matrix can be accomplished by the methods described in Methods in
Enzymology, vol. 44 (1976). A commonly employed technique for
attaching protein ligands to polysaccharide matrices, e.g. agarose,
dextran or cellulose, involves activation of the carrier with
cyanogen halides and subsequent coupling of the peptide ligand's
primary aliphatic or aromatic amines to the activated matrix.
[0085] Alternatively, IFNAR1 can be used to coat the wells of
adsorption plates, expressed on host cells affixed to adsorption
plates or used in cell sorting, or conjugated to biotin for capture
with streptavidin-coated beads, or used in any other art-known
method for panning phage display libraries.
[0086] c. Panning Procedures
[0087] The phage library samples are contacted with immobilized
IFNAR1 under conditions suitable for binding of at least a portion
of the phage particles with the adsorbent. Normally, the
conditions, including pH, ionic strength, temperature and the like
are selected to mimic physiological conditions. The phages bound to
the solid phase are washed and then eluted by acid, e.g. as
described in Barbas et al., Proc. Natl. Acad. Sci USA, 88:
7978-7982 (1991), or by alkali, e.g. as described in Marks et al.,
J. Mol. Biol., 222: 581-597 (1991), or by IFNAR1 antigen or type I
interferon ligand competition, e.g. in a procedure similar to the
antigen competition method of Clackson et al., Nature, 352: 624-628
(1991). Phages can be enriched 20-1,000-fold in a single round of
selection. Moreover, the enriched phages can be grown in bacterial
culture and subjected to further rounds of selection.
[0088] The efficiency of selection depends on many factors,
including the kinetics of dissociation during washing, and whether
multiple antibody fragments on a single phage can simultaneously
engage with antigen. Antibodies with fast dissociation kinetics
(and weak binding affinities) can be retained by use of short
washes, multivalent phage display and high coating density of
antigen in solid phase. The high density not only stabilizes the
phage through multivalent interactions, but also favors rebinding
of phage that has dissociated. The selection of antibodies with
slow dissociation kinetics (and good binding affinities) can be
promoted by use of long washes and monovalent phage display as
described in Bass et al., Proteins, 8: 309-314 (1990) and in WO
92/09690, and a low coating density of antigen as described in
Marks et al., Biotechnol., 10: 779-783 (1992).
[0089] It is possible to select between phage antibodies of
different affinities, even with affinities that differ slightly,
for IFNAR1. However, random mutation of a selected antibody (e.g.
as performed in some of the affinity maturation techniques
described above) is likely to give rise to many mutants, most
binding to antigen, and a few with higher affinity. With limiting
IFNAR1, rare high affinity phage could be competed out. To retain
all the higher affinity mutants, phages can be incubated with
excess biotinylated IFNAR1, but with the biotinylated IFNAR1 at a
concentration of lower molarity than the target molar affinity
constant for IFNAR1. The high affinity-binding phages can then be
captured by streptavidin-coated paramagnetic beads. Such
"equilibrium capture" allows the antibodies to be selected
according to their affinities of binding, with sensitivity that
permits isolation of mutant clones with as little as two-fold
higher affinity from a great excess of phages with lower affinity.
Conditions used in washing phages bound to a solid phase can also
be manipulated to discriminate on the basis of dissociation
kinetics.
[0090] 3. Activity Selection of Anti-IFNAR1 Clones
[0091] In one embodiment, the invention provides anti-IFNAR1
antibodies which bind to specific determinant(s) on IFNAR1 and/or
which do not bind other specific determinant(s) on IFNAR1. Fv
clones corresponding to such anti-IFNAR1 antibodies can be
conveniently selected by adsorbing library clones to immobilized
IFNAR1 mutants containing Ala substitutions at the specific
determinants of interest. If clones which do not bind the selected
IFNAR1 determinant(s) are desired, then the clones which adsorb to
the IFNAR1 mutant are recovered, e.g. by eluting the adsorbed
clones with wild type IFNAR1. The separation occurs because of the
difference in the affinities of the desired and undesired clones
for the IFNAR1 mutant. Since the IFNAR1 determinant(s) bound by the
desired clones do not include the amino acid(s) at the
Ala-substituted position(s) in the IFNAR1 mutant, the desired
clones will bind to the immobilized, mutant IFNAR1 whereas the
undesired clones will not. Accordingly, the adsorption of library
clones to immobilized, mutant IFNAR1 will yield a population of
clones bound to solid phase that is enriched for the property of
not being able to bind to the selected IFNAR1 determinant(s). The
desired clones will exhibit similar or approximately the same
binding activities with the corresponding Ala-substituted IFNAR1
mutant and wild type IFNAR1.
[0092] If clones which bind to the selected IFNAR1 determinant(s)
are desired, then library clones which fail to adsorb to
immobilized, mutant IFNAR1 are recovered (i.e. collected from the
column flow-through fractions), the recovered clones are adsorbed
to immobilized, wild type IFNAR1, and then the adsorbed clones are
recovered, e.g. by elution with excess wild type IFNAR1. The first
adsorption step removes clones that bind to IFNAR1 but do not bind
to the selected determinant(s), and the second adsorption step
removes clones that do not bind to IFNAR1 at all, leaving a
population of clones enriched for binding to the selected IFNAR1
determinant(s). The desired clone will exhibit binding activity
with wild type IFNAR1 that is greater than the clone's binding
activity with the corresponding Ala-substituted IFNAR1 mutant (i.e.
a binding level with wild type IFNAR1 that is above the background
binding level with mutant IFNAR1). Optionally, the desired clone
will exhibit binding activity with the corresponding
Ala-substituted IFNAR1 mutant that is less than about 50%, or less
than about 30%, or less than about 20%, or less than about 10%, or
less than about 7%, or less than about 6%, or less than about 5%,
or less than about 4%, or less than about 3%, or less than about
2%, or less than about 1%, or about 0% of the clone's binding
activity with wild type IFNAR1.
[0093] Optionally, clones that bind or do not bind to selected
IFNAR1 determinants can be further enriched by repeating the
selection procedures described herein one or more times.
[0094] Also provided herein are anti-IFNAR1 antibodies and Fv
clones which bind to one or more amino acids in situ in the
sequence of amino acids 103-111 of IFNAR1 and which do not bind to
one or more amino acids in situ in the sequence of amino acids
244-249 of IFNAR1. These Fv clones can be selected by (1) isolating
anti-IFNAR1 clones from a phage library as described in Section
B(I)(2) above, and optionally amplifying the isolated population of
phage clones by growing up the population in a suitable bacterial
host; (2) adsorbing the anti-IFNAR1 phage clones to immobilized
mutant IFNAR1 containing Ala substitutions at amino acid positions
244-249 in order to separate desired clones from clones that
require wild type amino acids at positions 244-249 for binding to
IFNAR1; (3) eluting the adsorbed clones with an excess of IFNAR1;
(4) contacting the eluted clones with immobilized, mutant IFNAR1
containing Ala substitutions at amino acid positions 103-111 in
order to adsorb undesired clones which bind to determinants on
IFNAR1 that do not overlap with amino acid positions 103-111; and
(5) recovering the clones which fail to adsorb to the immobilized,
mutant IFNAR1 from the flow-through fractions in step (4).
[0095] Additionally provided herein are anti-IFNAR1 antibodies and
Fv clones which bind to one or more amino acids in situ in the
sequence of amino acids 103-111 of IFNAR1 and which do not bind to
amino acid 249 of IFNAR1 in situ. These Fv clones can be selected
by (1) isolating anti-IFNAR1 clones from a phage library as
described in Section B(I)(2) above, and optionally amplifying the
isolated population of phage clones by growing up the population in
a suitable bacterial host; (2) adsorbing anti-IFNAR1 phage clones
to immobilized mutant IFNAR1 containing an Ala substitution at
amino acid position 249 in order to separate desired clones from
clones that require the wild type amino acid at position 249 for
binding to IFNAR1; (3) eluting the adsorbed clones with an excess
of IFNAR1; (4) contacting the eluted clones with immobilized,
mutant IFNAR1 containing Ala substitutions at amino acid positions
103-111 in order to adsorb undesired clones which bind to
determinants on IFNAR1 that do not overlap with amino acid
positions 103-111; and (5) recovering the clones which fail to
adsorb to the immobilized, mutant IFNAR1 from the flow-through
fractions in step (4).
[0096] Also encompassed herein are anti-IFNAR1 antibodies and Fv
clones which bind to one or more amino acids in situ in the
sequence of amino acids 103-111 of IFNAR1, and which bind to amino
acids 291 and 296 of IFNAR1 in situ, and which do not bind to amino
acid 249 of IFNAR1 in situ. These Fv clones can be selected by (1)
isolating anti-IFNAR1 clones from a phage library as described in
Section B(I)(2) above, and optionally amplifying the isolated
population of phage clones by growing up the population in a
suitable bacterial host; (2) adsorbing anti-IFNAR1 phage clones to
immobilized mutant IFNAR1 containing an Ala substitution at amino
acid position 249 in order to separate desired clones from clones
that require the wild type amino acid at position 249 for binding
to IFNAR1; (3) eluting the adsorbed clones with excess IFNAR1; (4)
contacting the eluted clones with immobilized, mutant IFNAR1
containing Ala substitutions at amino acid positions 103-111 in
order to adsorb undesired clones which bind to determinants on
IFNAR1 that do not overlap with amino acid positions 103-111; (5)
recovering the clones that fail to adsorb to immobilized, mutant
IFNAR1 from the flow-through fractions in step (4); (6) contacting
the recovered clones with immobilized, mutant IFNAR1 containing Ala
substitutions at amino acids 291 and 296 in order to adsorb
undesired clones which bind to determinants on IFNAR1 that do not
overlap with amino acid positions 291 and 296; and (7) recovering
the clones which fail to adsorb to immobilized, mutant IFNAR1 from
the flow-through fractions in step (6).
[0097] Also provided herein are anti-IFNAR1 antibodies and Fv
clones that bind to one or more amino acids in situ in the sequence
of amino acids 244-249 of IFNAR1. These Fv clones can be selected
by (1) isolating anti-IFNAR1 clones from a phage library as
described in Section B(I)(2) above, and optionally amplifying the
isolated population of phage clones by growing up the population in
a suitable bacterial host; (2) adsorbing the anti-IFNAR1 clones to
immobilized IFNAR1; (3) subjecting the adsorbed clones to elution
with a mutant IFNAR1 containing Ala substitutions at amino acid
positions 244-249 in order to elute the undesired clones which bind
determinants on IFNAR1 that do not overlap with amino acids at
positions 244-249 on IFNAR1; and (4) recovering the remaining
adsorbed clones by elution with excess IFNAR1.
[0098] Additionally provided herein are anti-IFNAR1 antibodies and
Fv clones that bind to one or more amino acids in situ in the
sequence of amino acids 291-298 of IFNAR1. These Fv clones can be
selected by (1) isolating anti-IFNAR1 clones from a phage library
as described in Section B(I)(2) above, and optionally amplifying
the isolated population of phage clones by growing up the
population in a suitable bacterial host; (1) adsorbing the
anti-IFNAR1 clones to immobilized IFNAR1; (3) subjecting the
adsorbed clones to elution with a mutant IFNAR1 containing Ala
substitutions at amino acid positions 291-298 in order to elute
undesired clones which bind determinants on IFNAR1 that do not
overlap with amino acid positions 291-298 on IFNAR1; and (4)
recovering the remaining adsorbed clones by elution with excess
IFNAR1.
[0099] The invention also provides anti-IFNAR1 antibodies and Fv
clones which bind to one or more amino acids in situ in the
sequence of amino acids 244-249 of IFNAR1 and bind to one or more
amino acids in situ in the sequence of amino acids 291-298 of
IFNAR1. Fv clones corresponding to such anti-IFNAR1 antibodies can
be selected by (1) isolating anti-IFNAR1 clones from a phage
library as described in Section B(I)(2) above, and optionally
amplifying the isolated population of phage clones by growing up
the population in a suitable bacterial host; (2) adsorbing the
resulting clones to immobilized IFNAR1; (3) subjecting the adsorbed
anti-IFNAR1 clones to elution with a cocktail of excess mutant
IFNAR1 containing Ala substitutions at amino acids positions
244-249 and excess mutant IFNAR1 containing Ala substitutions at
amino acid positions 291-298, or subjecting the adsorbed clones to
consecutive elutions with each of the IFNAR1 mutants, in order to
elute undesired clones which bind to determinants on IFNAR1 that do
not overlap with both amino acid positions 244-249 and amino acid
positions 291-298 on IFNAR1; and (4) recovering the remaining
adsorbed clones by elution with excess IFNAR1.
[0100] In another embodiment, the invention provides anti-IFNAR1
antibodies and Fv clones that bind to amino acid 249 of IFNAR1. Fv
clones corresponding to such anti-IFNAR1 antibodies can be selected
by (1) isolating anti-IFNAR1 clones from a phage library as
described in Section B(I)(2) above, and optionally amplifying the
isolated population of phage clones by growing up the anti-IFNAR1
population in a suitable bacterial host; (2) adsorbing the
resulting anti-IFNAR1 clones to immobilized IFNAR1; (3) subjecting
the adsorbed clones to elution with mutant IFNAR1 containing an Ala
substitution at amino acid position 249 of IFNAR1 in order to elute
undesired clones which bind determinants on IFNAR1 that do not
overlap with amino acid position 249 on IFNAR1; and (4) recovering
the remaining adsorbed clones by elution with excess IFNAR1.
[0101] In another embodiment, the invention provides anti-IFNAR1
antibodies and Fv clones that bind to amino acid 291 of IFNAR1. Fv
clones corresponding to such anti-IFNAR1 antibodies can be selected
by (1) isolating anti-IFNAR1 clones from a phage library as
described in Section B(I)(2) above, and optionally amplifying the
isolated population of phage clones by growing up the anti-IFNAR1
population in a suitable bacterial host; (2) adsorbing the
resulting anti-IFNAR1 clones to immobilized IFNAR1; (3) subjecting
the adsorbed clones to elution with mutant IFNAR1 containing an Ala
substitution at amino acid position 291 of IFNAR1 in order to elute
undesired clones which bind determinants on IFNAR1 that do not
overlap with amino acid position 291 on IFNAR1; and (4) recovering
the remaining adsorbed clones by elution with excess IFNAR1.
[0102] In another embodiment, the invention provides anti-IFNAR1
antibodies and Fv clones that bind to amino acid 296 of IFNAR1. Fv
clones corresponding to such anti-IFNAR1 antibodies can be selected
by (1) isolating anti-IFNAR1 clones from a phage library as
described in Section B(I)(2) above, and optionally amplifying the
isolated population of phage clones by growing up the anti-IFNAR1
population in a suitable bacterial host; (2) adsorbing the
resulting anti-IFNAR1 clones to immobilized IFNAR1; (3) subjecting
the adsorbed clones to elution with mutant IFNAR1 containing an Ala
substitution at amino acid position 296 of IFNAR1 in order to elute
undesired clones which bind determinants on IFNAR1 that do not
overlap with amino acid position 296 on IFNAR1; and (4) recovering
the remaining adsorbed clones by elution with excess IFNAR1.
[0103] The invention further provides anti-IFNAR1 antibodies and Fv
clones that bind to amino acids 249, 291 and 296 of IFNAR1 in situ.
Fv clones corresponding to such anti-IFNAR1 antibodies can be
selected by (1) isolating anti-IFNAR1 clones from a phage library
as described in Section B(I)(2) above, and optionally amplifying
the isolated population of phage clones by growing up the
population in a suitable bacterial host; (2) adsorbing the
resulting anti-IFNAR1 clones to immobilized IFNAR1; (3) subjecting
the adsorbed clones to elution with a cocktail of excess mutant
IFNAR1 containing an Ala substitution at amino acid position 249,
excess mutant IFNAR1 containing an Ala substitution at amino acid
position 291, and excess mutant IFNAR1 containing an Ala
substitution at amino acid position 296, or subjecting the adsorbed
clones to consecutive elutions with each of the IFNAR1 mutants, in
order to elute undesired clones which bind to determinants on
IFNAR1 which do not overlap with amino acids 249, 291 or 296 of
IFNAR1 in situ; and (4) recovering the remaining adsorbed clones by
elution with excess IFNAR1.
[0104] In another embodiment, the invention provides any of the
anti-IFNAR1 antibodies described above that additionally binds to a
conformational epitope on IFNAR1. Fv clones corresponding to such
anti-IFNAR1 antibodies can be selected according to the procedures
described above modified to include the additional step of
screening clones for binding to denatured IFNAR1, e.g., by layering
clone suspensions on plates coated with denatured IFNAR1, and
collecting non-binding clones from the plate washes. It will be
appreciated that the denatured IFNAR1-coated plate adsorption step
can be performed before or after the other selection procedures for
the Fv clone of interest, or can be performed at any point in such
selection procedures that is immediately preceded by the elution of
the clones of interest from a particular adsorbent.
[0105] Also provided herein are anti-IFNAR1 Fv clones that bind to
the amino acid sequence of amino acids 103-111 of IFNAR1 in situ,
do not bind to the amino acid sequence of amino acids 244-249 of
IFNAR1 in situ, and bind to a conformational epitope of IFNAR1.
[0106] Additionally provided herein are anti-IFNAR1 Fv clones that
bind to the amino acid sequence of amino acids 103-111 of IFNAR1 in
situ, do not bind to amino acid 249 of IFNAR1 in situ, and bind to
a conformational epitope of IFNAR1.
[0107] Further encompassed herein are anti-IFNAR1 Fv clones that
bind to the amino acid sequence of amino acids 103-111 of IFNAR1 in
situ, bind to amino acids 291 and 296 of IFNAR1 in situ, do not
bind to amino acid 249 of IFNAR1 in situ, and bind to a
conformational epitope of IFNAR1.
[0108] Also included herein are any of the anti-IFN antibodies
described above that additionally bind to a conformational epitope
formed by domain 1 and domain 2 of IFNAR1. Fv clones corresponding
to such anti-IFNAR1 antibodies can be selected according to the
procedures described above modified to include selection steps that
exclude clones that bind to a peptide consisting of the amino acid
sequence of domain 1 (amino acids 1-200 of IFNAR1) or bind to a
peptide consisting of the amino acid sequence of domain 2 (amino
acids 204-404). In one embodiment, the clones of interest are
selected by layering a clone suspension on plates coated with
domain 1 peptide, recovering the non-binding clones from the plate
washes, layering a suspension of the recovered clones on plates
coated with domain 2 peptide, and recovering the non-binding
clones. In another embodiment, the clones of interest are selected
by adsorbing clones to immobilized IFNAR1, subjecting the adsorbed
clones to elution with a cocktail of excess domain 1 peptide and
excess domain 2 peptide (or alternatively subjecting the adsorbed
clones to serial elutions with the individual peptides), discarding
the eluted clones, and recovering the clones that remain bound to
adsorbent. The domain 1 peptide and domain 2 peptide binding
selection step can be performed before or after the other selection
procedures for the Fv clone of interest, or can be performed at any
point in such selection procedures immediately preceding which the
clones of interest are either (1) eluted from a particular
adsorbent (e.g. if peptide-coated plates are used for selection) or
(2) adsorbed to immobilized IFNAR1 (e.g. if elution with a peptide
cocktail is used for selection).
[0109] Also provided herein are anti-IFNAR1 Fv clones that bind to
the amino acid sequence of amino acids 103-111 of IFNAR1 in situ,
do not bind to the amino acid sequence of amino acids 244-249 of
IFNAR1 in situ, do not bind to a peptide consisting of the amino
acid sequence of domain 1 (amino acids 1-200 of IFNAR1), and do not
bind to a peptide consisting of the amino acid sequence of domain 2
(amino acids 204-404 of IFNAR1).
[0110] Additionally provided herein are anti-IFNAR1 Fv clones that
bind to the amino acid sequence of amino acids 103-111 of IFNAR1 in
situ, do not bind to amino acid 249 of IFNAR1 in situ, do not bind
to a peptide consisting of the amino acid sequence of domain 1
(amino acids 1-200 of IFNAR1), and do not bind to a peptide
consisting of the amino acid sequence of domain 2 (amino acids
204-404 of IFNAR1).
[0111] Further encompassed herein are anti-IFNAR1 Fv clones that
bind to the amino acid sequence of amino acids 103-111 of IFNAR1 in
situ, bind to amino acids 291 and 296 of IFNAR1 in situ, do not
bind to amino acid 249 of IFNAR1 in situ, do not bind to a peptide
consisting of the amino acid sequence of domain 1 (amino acids
1-200 of IFNAR1), and do not bind to a peptide consisting of the
amino acid sequence of domain 2 (amino acids 204-404 of
IFNAR1).
[0112] In yet another embodiment, the invention provides
anti-IFNAR1 Fv clones that bind to one or more of amino acids
244-249 of IFNAR1 in situ, bind to one or more of amino acids
291-298 of IFNAR1 in situ, and bind to a conformational epitope of
IFNAR1.
[0113] In still another embodiment, the invention provides
anti-IFNAR1 Fv clones that bind to amino acids 249, 291 and 296 of
IFNAR1 in situ, and bind to a conformational epitope of IFNAR1.
[0114] Further provided herein are anti-IFNAR1 Fv clones that bind
to one or more of amino acids 244-249 of IFNAR1 in situ, bind to
one or more of amino acids 291-298 of IFNAR1 in situ, do not bind
to a peptide consisting of the amino acid sequence of domain 1
(amino acids 1-200) of IFNAR1, and do not bind to a peptide
consisting of the amino acid sequence of domain 2 (amino acids
204-404) of IFNAR1.
[0115] Additionally provided herein are anti-IFNAR1 Fv clones that
bind to amino acids 249, 291 and 296 of IFNAR1 in situ, do not bind
to a peptide consisting of the amino acid sequence of domain 1
(amino acids 1-200) of IFNAR1, and do not bind to a peptide
consisting of the amino acid sequence of domain 2 (amino acids
204-404) of IFNAR1.
[0116] II. Methods of Making Anti-IFNAR1 Hybridomas
[0117] The anti-IFNAR1 antibodies of the invention are preferably
monoclonal. Also encompassed within the scope of the invention are
Fab, Fab', Fab'-SH and F(ab').sub.2 fragments of the anti-IFNAR1
antibodies provided herein. These antibody fragments can be created
by traditional means, such as enzymatic digestion, or may be
generated by recombinant techniques. Such antibody fragments may be
chimeric or humanized. These fragments are useful for the
diagnostic and therapeutic purposes set forth below.
[0118] Monoclonal antibodies are obtained from a population of
substantially homogeneous antibodies, i.e., the individual
antibodies comprising the population are identical except for
possible naturally occurring mutations that may be present in minor
amounts. Thus, the modifier "monoclonal" indicates the character of
the antibody as not being a mixture of discrete antibodies.
[0119] The anti-IFNAR1 monoclonal antibodies of the invention can
be made using the hybridoma method first described by Kohler et
al., Nature, 256:495 (1975), or may be made by recombinant DNA
methods (U.S. Pat. No. 4,816,567).
[0120] In the hybridoma method, a mouse or other appropriate host
animal, such as a hamster, is immunized to elicit lymphocytes that
produce or are capable of producing antibodies that will
specifically bind to the protein used for immunization. Antibodies
to IFNAR1 generally are raised in animals by multiple subcutaneous
(sc) or intraperitoneal (ip) injections of IFNAR1 and an adjuvant.
In one embodiment, animals are immunized with a derivative of
IFNAR1 that contains the extracellular domain (ECD) of IFNAR1 fused
to the Fc portion of an immunoglobulin heavy chain. In a preferred
embodiment, animals are immunized with an IFNAR1-IgG1 fusion
protein as described in the Example below. Animals ordinarily are
immunized against immunogenic conjugates or derivatives of IFNAR1
with monophosphoryl lipid A (MPL)/trehalose dicrynomycolate (TDM)
(Ribi Immunochem. Research, Inc., Hamilton, Mont.) and the solution
is injected intradermally at multiple sites. Two weeks later the
animals are boosted. 7 to 14 days later animals are bled and the
serum is assayed for anti-IFNAR1 titer. Animals are boosted until
titer plateaus.
[0121] Alternatively, lymphocytes may be immunized in vitro.
Lymphocytes then are fused with myeloma cells using a suitable
fusing agent, such as polyethylene glycol, to form a hybridoma cell
(Goding, Monoclonal Antibodies: Principles and Practice, pp. 59-103
(Academic Press, 1986)).
[0122] The hybridoma cells thus prepared are seeded and grown in a
suitable culture medium that preferably contains one or more
substances that inhibit the growth or survival of the unfused,
parental myeloma cells. For example, if the parental myeloma cells
lack the enzyme hypoxanthine guanine phosphoribosyl transferase
(HGPRT or HPRT), the culture medium for the hybridomas typically
will include hypoxanthine, aminopterin, and thymidine (HAT medium),
which substances prevent the growth of HGPRT-deficient cells.
[0123] Preferred myeloma cells are those that fuse efficiently,
support stable high-level production of antibody by the selected
antibody-producing cells, and are sensitive to a medium such as HAT
medium. Among these, preferred myeloma cell lines are murine
myeloma lines, such as those derived from MOPC-21 and MPC-11 mouse
tumors available from the Salk Institute Cell Distribution Center,
San Diego, Calif. USA, and SP-2 or X63-Ag8-653 cells available from
the American Type Culture Collection, Rockville, Md. USA. Human
myeloma and mouse-human heteromyeloma cell lines also have been
described for the production of human monoclonal antibodies
(Kozbor, J. Immunol., 133:3001 (1984); Brodeur et al., Monoclonal
Antibody Production Techniques and Applications, pp. 51-63 (Marcel
Dekker, Inc., New York, 1987)).
[0124] Culture medium in which hybridoma cells are growing is
assayed for production of monoclonal antibodies directed against
IFNAR1. Preferably, the binding specificity of monoclonal
antibodies produced by hybridoma cells is determined by
immunoprecipitation or by an in vitro binding assay, such as
radioimmunoassay (RIA) or enzyme-linked immunoadsorbent assay
(ELISA).
[0125] The binding affinity of the monoclonal antibody can, for
example, be determined by the Scatchard analysis of Munson et al.,
Anal. Biochem., 107:220 (1980).
[0126] After hybridoma cells are identified that produce antibodies
of the desired specificity, affinity, and/or activity, the clones
may be subcloned by limiting dilution procedures and grown by
standard methods (Goding, Monoclonal Antibodies:Principles and
Practice, pp. 59-103 (Academic Press, 1986)). Suitable culture
media for this purpose include, for example, D-MEM or RPMI-1640
medium. In addition, the hybridoma cells may be grown in vivo as
ascites tumors in an animal.
[0127] The monoclonal antibodies secreted by the subclones are
suitably separated from the culture medium, ascites fluid, or serum
by conventional immunoglobulin purification procedures such as, for
example, protein A-Sepharose, hydroxylapatite chromatography, gel
electrophoresis, dialysis, or affinity chromatography.
[0128] Anti-IFNAR1 antibodies of the invention possessing the
unique properties described in Section I above can be obtained by
screening anti-IFNAR1 hybridoma clones for the desired properties
by any convenient method. For example, if an anti-IFNAR1 monoclonal
antibody that binds or does not bind to a particular IFNAR1
determinant(s) is desired, the candidate antibody can be screened
for the presence or absence of differential affinity to wild type
IFNAR1 and to mutant IFNAR1 that contains Ala substitution(s) at
the determinant(s) of interest as described above. In one aspect,
the candidate antibody can be tested for binding to wild type
IFNAR1 and mutant IFNAR1 in an immunoprecipitation or
immunoadsorption assay. For example, a capture ELISA can be used
wherein plates are coated with a given density of wild type IFNAR1
or an equal density of mutant IFNAR1, the coated plates are
contacted with equal concentrations of the candidate antibody, and
the bound antibody is detected enzymatically, e.g. by contacting
the bound antibody with HRP-conjugated anti-Ig antibody or
biotinylated anti-Ig antibody, developing the bound anti-Ig
antibody with streptavidin-HRP and/or hydrogen peroxide, and
detecting the HRP color reaction by spectrophotometry at 490 nm
with an ELISA plate reader. The candidate antibody that binds to
the particular IFNAR1 determinant(s) of interest will exhibit
binding activity with wild type IFNAR1 that is greater than the
candidate antibody's binding activity with the corresponding
Ala-substituted IFNAR1 mutant (i.e. a binding level with wild type
IFNAR1 that is above the background binding level with mutant
IFNAR1). Optionally, the candidate antibody that binds to the
particular IFNAR1 determinant(s) of interest will exhibit binding
activity with the corresponding Ala-substituted IFNAR1 mutant that
is less than about 50%, or less than about 30%, or less than about
20%, or less than about 10%, or less than about 7%, or less than
about 6%, or less than about 5%, or less than about 4%, or less
than about 3%, or less than about 2%, or less than about 1%, or
about 0% of the antibody's binding activity with wild type IFNAR1,
e.g. as determined by dividing the HRP color reaction optical
density observed for capture ELISA with IFNAR1 mutant adsorbent by
the HRP color reaction optical density observed for capture ELISA
with wild type IFNAR1 adsorbent. The candidate antibody that does
not bind to the particular IFNAR1 determinant(s) of interest will
exhibit similar or approximately the same binding activities with
the corresponding Ala-substituted IFNAR1 mutant and wild type
IFNAR1.
[0129] An anti-IFNAR1 monoclonal antibody that (1) binds to a
conformational epitope on IFNAR1 or (2) does not bind to a peptide
consisting of the amino acid sequence of domain 1 or domain 2 of
IFNAR1 as provided herein can be detected by screening for failure
to bind to completely denatured IFNAR1, or failure to bind to
domain 1 peptide or domain 2 peptide, as desired, in an immunoblot
system, e.g. using the candidate antibody to probe a Western blot
of denaturing gel electrophoresed IFNAR1 or domain 1 or domain 2
peptides. Alternatively, the candidate antibody's inability to bind
to completely denatured IFNAR1, domain 1 peptide or domain 2
peptide can be determined by immunoprecipitation or
immunoadsorption techniques, e.g. a capture ELISA wherein plates
are coated with the denatured IFNAR1, domain 1 peptide or domain 2
peptide, the coated plates are contacted with a solution of the
candidate antibody, and the bound antibody is detected
enzymatically, e.g. contacting the bound antibody with
HRP-conjugated anti-Ig antibody and developing the HRP color
reaction.
[0130] In another embodiment, the invention provides anti-IFNAR1
monoclonal antibodies that inhibit the anti-viral activity of a
first type I interferon and do not inhibit the anti-viral activity
of a second type I interferon. The anti-IFNAR1 antibodies of the
invention can be obtained by screening candidate anti-IFNAR1
antibodies in any convenient type I interferon viral infectivity
inhibition assay. Such assays are well known in the art, and
include, for example, type I interferon-induced inhibition of
encephatomyocarditis virus (EMC) infectivity in A549 cells as
described in Current Protocols in Immunology, Coligan, J. E.,
Kruisbeek, A. M., Margulies, D. H., Shevach, E. M., and Strober,
W., eds, Greene Publishing Associates and Wiley-Interscience,
(1992), vol. 1, unit 6.9.1. In another example, the assay uses type
I interferon-induced inhibition of vesicular stomatitis virus (VSV)
infectivity in Daudi cells as described by Dron and Tovey, J. Gen.
Virol., 64: 2641-2647 (1983). Generally, cells are seeded in
attached cell culture plates, grown for 1 day, and then incubated
for an additional day in the presence of various concentrations of
a selected type I interferon and in the presence or absence of an
excess of the candidate IFNAR1 antibody or a control antibody.
Cells are challenged with virus, incubated for an additional day,
and then viral activity is quantitated by detection of remaining
viable cells (e.g. by cell staining) or by lysing cells, collecting
culture supernatants and titering the virus concentrations present
in the supernatants. The candidate antibody that inhibits the
anti-viral activity of a selected type I interferon will inhibit
more anti-viral activity than the baseline level of anti-viral
activity inhibition measured in the presence of an equivalent
concentration of control antibody. Optionally, the candidate
antibody that inhibits the anti-viral activity of a selected type I
interferon will inhibit at least about 50%, or at least about 70%,
or at least about 80%, or at least about 90%, or at least about
95%, or at least about 99%, or about 100% of the activity of the
type I interferon in the anti-viral activity assay as compared to
baseline activity measured in the presence of an equivalent
concentration of control antibody. The candidate antibody that does
not inhibit the anti-viral activity of a selected type I interferon
will exhibit similar or approximately the same level of anti-viral
activity inhibition as control antibody.
[0131] In another embodiment, each type I interferon species used
in the viral infectivity assay is titrated to a concentration that
provides the same level of inhibition of viral activity as that
induced by a preselected number of units of an IFN-.alpha.
standard. This concentration serves to provide the normalized units
of the subject type I interferon species. In order to assess the
ability of an anti-IFNAR1 antibody to inhibit the anti-viral
activity of various type I interferons, the effective concentration
(EC50) of anti-IFNAR1 antibody for inhibiting 50% of a particular
type I interferon's anti-viral activity (at the concentration
titrated to provide the normalized units of activity) is determined
for each type I interferon to be tested. In another embodiment,
each type I interferon to be tested is normalized to at least at or
about 1 unit/ml, or at or about 1 unit/ml to at or about 1,000
units/ml, or at or about 1 unit/ml to at or about 100 units/ml, of
human IFN-.alpha.2. In yet another embodiment, each type I
interferon to be tested is normalized to 10 units/ml of the NIH
reference standard for recombinant human IFN-.alpha.2
(IFN-.alpha.A).
[0132] In still another embodiment, the candidate anti-IFNAR1
antibody that does not inhibit the anti-viral activity of a
selected type I interferon will exhibit no effect at a
concentration of up to at or about 1 .mu.g/ml, or up to ator about
10 .mu.g/ml, or up to at or about 20 .mu.g/ml, or up to at or about
30 .mu.g/ml, or up to at or about 50 .mu.g/ml, or up to at or about
75 .mu.g/ml, or up to at or about 100 .mu.g/ml, against the
anti-viral activity of the selected type I interferon in the A549
cell EMC viral infectivity assay described in Current Protocols in
Immunology, Coligan, J. E., Kruisbeek, A. M., Margulies, D. H.,
Shevach, E. M., and Strober, W., eds, Greene Publishing Associates
and Wiley-Interscience, (1992), vol. 2, unit 6.9.1, wherein the
selected type I interferon is normalized to 10 units/ml of NIH
reference standard for recombinant human IFN-.alpha.2
(IFN-.alpha.A).
[0133] In another embodiment, the candidate anti-IFNAR1 antibody
that inhibits the anti-viral activity of a first type I interferon
and does not inhibit the anti-viral activity of a second type I
interferon will (1) exhibit an EC50 of up to at or about 1
.mu.g/ml, or up to at or about 3 .mu.g/ml, or up to at or about 6
.mu.g/ml, or up to at or about 10 .mu.g/ml, or up to at or about 20
.mu.g/ml, or up to at or about 30 .mu.g/ml, or up to at or about 40
.mu.g/ml, or up to at or about 50 .mu.g/ml, or up to at or about 75
.mu.g/ml, or up to at or about 100 .mu.g/ml, against the anti-viral
activity of the first type I interferon in an A549 cell EMC viral
infectivity assay, such as the A549 cell EMC viral infectivity
assay described in Current Protocols in Immunology, supra, and (2)
exhibit no effect at a concentration of up to at or about 30
.mu.g/ml, or up to at or about 40 .mu.g/ml, or up to at or about 50
.mu.g/ml, or up to at or about 75 .mu.g/ml, or up to at or about
100 .mu.g/ml, against the anti-viral activity of the second type I
interferon in the A549 cell EMC viral infectivity assay, wherein in
the A549 cell EMC viral infectivity assay the first and second type
I interferons are normalized to 10 units/ml of NIH reference
standard for recombinant IFN-.alpha.2 (IFN-.alpha.A).
[0134] In yet another embodiment, the candidate anti-IFNAR1
antibody that inhibits the anti-viral activity of a first type I
interferon and does not inhibit the anti-viral activity of a second
type I interferon will (1) exhibit an EC50 of up to at or about 20
.mu.g/ml against the anti-viral activity of the first type I
interferon in an A549 cell EMC viral infectivity assay, such as the
A549 cell EMC viral infectivity assay described in Current
Protocols in Immunology, supra, and (2) exhibit no effect at a
concentration of 30 .mu.g/ml against the anti-viral activity of the
second type I interferon in the A549 cell EMC viral infectivity
assay, wherein in the A549 cell EMC viral infectivity assay the
first and second type I interferons are normalized to 10 units/ml
of NIH reference standard for recombinant IFN-.alpha.2
(IFN-.alpha.A).
[0135] In yet another embodiment, the candidate anti-IFNAR1
antibody that inhibits the anti-viral activity of a first type I
interferon and does not inhibit the anti-viral activity of a second
type I interferon will (1) exhibit an EC50 of up to at or about 10
.mu.g/ml against the anti-viral activity of the first type I
interferon in an A549 cell EMC viral infectivity assay, such as the
A549 cell EMC viral infectivity assay described in Current
Protocols in Immunology, supra, and (2) exhibit no effect at a
concentration of 30 .mu.g/ml against the anti-viral activity of the
second type I interferon in the A549 cell EMC viral infectivity
assay, wherein in the A549 cell EMC viral infectivity assay the
first and second type I interferons are normalized to 10 units/ml
of NIH reference standard for recombinant IFN-.alpha.2
(IFN-.alpha.A).
[0136] In yet another embodiment, the candidate anti-IFNAR1
antibody that inhibits the anti-viral activity of a first type I
interferon and does not inhibit the anti-viral activity of a second
type I interferon will (1) exhibit an EC50 of up to at or about 6
.mu.g/ml against the anti-viral activity of the first type I
interferon in an A549 cell EMC viral infectivity assay, such as the
A549 cell EMC viral infectivity assay described in Current
Protocols in Immunology, supra, and (2) exhibit no effect at a
concentration of 30 .mu.g/ml against the anti-viral activity of the
second type I interferon in the A549 cell EMC viral infectivity
assay, wherein in the A549 cell EMC viral infectivity assay the
first and second type I interferons are normalized to 10 units/ml
of NIH reference standard for recombinant IFN-.alpha.2
(IFN-.alpha.A).
[0137] In yet another embodiment, the candidate anti-IFNAR1
antibody that inhibits the anti-viral activity of a first type I
interferon and does not inhibit the anti-viral activity of a second
type I interferon will (1) exhibit an EC50 of up to at or about 3
.mu.g/ml against the anti-viral activity of the first type I
interferon in an A549 cell EMC viral infectivity assay, such as the
A549 cell EMC viral infectivity assay described in Current
Protocols in Immunology, supra, and (2) exhibit no effect at a
concentration of 30 .mu.g/ml against the anti-viral activity of the
second type I interferon in the A549 cell EMC viral infectivity
assay, wherein in the A549 cell EMC viral infectivity assay the
first and second type I interferons are normalized to 10 units/ml
of NIH reference standard for recombinant IFN-.alpha.2
(IFN-.alpha.A).
[0138] In yet another embodiment, the candidate anti-IFNAR1
antibody that inhibits the anti-viral activity of a first type I
interferon and does not inhibit the anti-viral activity of a second
type I interferon will (1) exhibit an EC50 of up to at or about 1
.mu.g/ml against the anti-viral activity of the first type I
interferon in an A549 cell EMC viral infectivity assay, such as the
A549 cell EMC viral infectivity assay described in Current
Protocols in Immunology, supra, and (2) exhibit no effect at a
concentration of 30 .mu.g/ml against the anti-viral activity of the
second type I interferon in the A549 cell EMC viral infectivity
assay, wherein in the A549 cell EMC viral infectivity assay the
first and second type I interferons are normalized to 10 units/ml
of NIH reference standard for recombinant IFN-.alpha.2
(IFN-.alpha.A).
[0139] In another aspect, the invention provides an anti-IFNAR1
antibody that inhibits the anti-viral activity of a first type I
interferon selected from the group consisting of IFN-.alpha.A,
IFN-.alpha.B, and IFN-.alpha.G and does not inhibit the anti-viral
activity of a second type I interferon.
[0140] Also provided herein is an anti-IFNAR1 antibody that
inhibits the anti-viral activity of a first type I interferon and
does not inhibit the anti-viral activity of a second type I
interferon selected from the group consisting of IFN-.alpha.A,
IFN-.alpha.B, IFN-.alpha..sub.II1, and IFN-.beta..
[0141] In yet another embodiment, the anti-IFNAR1 inhibits the
anti-viral activity of a first type 1 interferon and does not
inhibit the anti-viral activity of a second type I interferon
selected from the group consisting of IFN-.alpha.D, IFN-.alpha.F,
and IFN-.beta..
[0142] Additionally provided herein is an anti-IFNAR1 antibody that
inhibits the anti-viral activity of a first type I interferon
selected from the group consisting of IFN-.alpha.A, IFN-.alpha.B,
and IFN-.alpha.G and does not inhibit the anti-viral activity of a
second type I interferon selected from the group consisting of
IFN-.alpha.D, IFN-.alpha.F, and IFN-.beta..
[0143] Further provided herein is anti-IFNAR1 antibody that
inhibits the anti-viral activity of a first type I interferon
selected from the group consisting of IFN-.alpha.A, IFN-.alpha.D,
IFN-.alpha.F, and IFN-.beta..
[0144] Also encompassed herein is an anti-IFNAR1 antibody that
inhibits the anti-viral activity of a first type I interferon and
does not inhibit the anti-viral activity of a second type I
interferon selected from the group consisting of IFN-.alpha.B and
IFN-.alpha.G.
[0145] Further encompassed herein is an anti-IFNAR1 antibody that
inhibits the anti-viral activity of a first type I interferon
selected from the group consisting of IFN-.alpha.A, IFN-.alpha.D,
IFN-.alpha.F, and IFN-.beta. and does not inhibit the anti-viral
activity of a second type I interferon selected from the group
consisting of IFN-.alpha.B and IFN-.alpha.G.
[0146] The invention further provides an anti-IFNAR1 antibody that
inhibits the anti-viral activity of more than one selected type I
interferon and does not inhibit the anti-viral activity of another
selected type I interferon; [0147] In one embodiment, the invention
provides an anti-IFNAR1 antibody that inhibits the anti-viral
activity of IFN-.alpha.A, IFN-.alpha.B, and IFN-.alpha.G and does
not inhibit the anti-viral activity of another type I interferon.
In another embodiment, the invention provides an anti-IFNAR1
antibody that inhibits the anti-viral activity of IFN-.alpha.A,
IFN-.alpha.B and IFN-.alpha.G and does not inhibit the anti-viral
activity of another type I interferon selected from the group
consisting of IFN-.alpha.D, IFN-.alpha.F, and IFN-.beta..
[0148] In another embodiment, the invention provides an anti-IFNAR1
antibody that inhibits the anti-viral activity of IFN-.alpha.A,
IFN-.alpha.B, IFN-.alpha.D and IFN-.alpha.G and does not inhibit
the anti-viral activity of IFN-.beta..
[0149] In another embodiment, the invention provides an anti-IFNAR1
antibody that inhibits the anti-viral activity of IFN-.alpha.A,
IFN-.alpha.B, IFN-.alpha.D and IFN-.alpha.G and does not inhibit
the anti-viral activity of IFN-.beta., wherein (1) the antibody
exhibits an EC50 of up to at or about 1 .mu.g/ml, or up to at or
about 3 .mu.g/ml, or up to at or about 6 .mu.g/ml, or up to at or
about 10 .mu.g/ml, or up to at or about 20 .mu.g/ml, or up to at or
about 30 .mu.g/ml, or up to at or about 40 .mu.g/ml, or up to at or
about 50 .mu.g/ml, or up to at or about 75 .mu.g/ml, or up to at or
about 100 .mu.g/ml, against the anti-viral activities of
IFN-.alpha.A, IFN-.alpha.B, IFN-.alpha.D and IFN-.alpha.G in an
A549 cell EMC viral infectivity assay, such as the A549 cell EMC
viral infectivity assay described in Current Protocols in
Immunology, supra, and (2) the antibody exhibits no effect at a
concentration of up to at or about 30 .mu.g/ml, or up to at or
about 40 .mu.g/ml, or up to at or about 50 .mu.g/ml, or up to at or
about 75 .mu.g/ml, or up to at or about 100 .mu.g/ml, against the
anti-viral activity of the IFN-.beta. in the A549 cell EMC viral
infectivity assay, and wherein in the A549 cell EMC viral
infectivity assay each type I interferon is normalized to 10
units/ml of NIH reference standard for recombinant IFN-.alpha.2
(IFN-.alpha.A).
[0150] In another embodiment, the invention provides an anti-IFNAR1
antibody that inhibits the anti-viral activity of IFN-.alpha.A,
IFN-.alpha.B, IFN-.alpha.D and IFN-.alpha.G and does not inhibit
the anti-viral activity of IFN-.beta., wherein (1) the antibody
exhibits an EC50 of up to at or about 10 .mu.g/ml against the
anti-viral activity of IFN-.alpha.D in an A549 cell EMC viral
infectivity assay, such as the A549 cell EMC viral infectivity
assay described in Current Protocols in Immunology, supra, (2) the
antibody exhibits an EC50 of up to at or about 10 .mu.g/ml against
the anti-viral activity of IFN-.alpha.A in the A549 cell EMC viral
infectivity assay, (3) the antibody exhibits an EC50 of up to at or
about 6 .mu.g/ml against the anti-viral activity of IFN-.alpha.G in
the A549 cell EMC viral infectivity assay, (4) the antibody
exhibits an EC50 of up to at or about 3 .mu.g/ml against the
anti-viral activity of IFN-.alpha.B in the A549 cell EMC viral
infectivity assay, and (5) the antibody exhibits no effect at a
concentration of up to at or about 30 .mu.g/ml against the
anti-viral activity of the IFN-.beta. in the A549 cell EMC viral
infectivity assay, and wherein in the A549 cell EMC viral
infectivity assay each type I interferon is normalized to 10
units/ml of NIH reference standard for recombinant IFN-.alpha.2
(IFN-.alpha.A).
[0151] In another embodiment, the invention provides an anti-IFNAR1
antibody that inhibits the anti-viral activity of IFN-.alpha.A,
IFN-.alpha.B, IFN-.alpha.D and IFN-.alpha.G and does not inhibit
the anti-viral activity of IFN-.beta., wherein (1) the antibody
exhibits an EC50 of up to at or about 3 .mu.g/ml against the
anti-viral activity of IFN-.alpha.D in an A549 cell EMC viral
infectivity assay, such as the A549 cell EMC viral infectivity
assay described in Current Protocols in Immunology, supra, (2) the
antibody exhibits an EC50 of up to at or about 1 .mu.g/ml against
the anti-viral activity of IFN-.alpha.A in the A549 cell EMC viral
infectivity assay, (3) the antibody exhibits an EC50 of up to at or
about 1 .mu.g/ml against the anti-viral activity of IFN-.alpha.G in
the A549 cell EMC viral infectivity assay, (4) the antibody
exhibits an EC50 of up to at or about 1 .mu.g/ml against the
anti-viral activity of IFN-.alpha.B in the A549 cell EMC viral
infectivity assay, and (5) the antibody exhibits no effect at a
concentration of up to at or about 30 .mu.g/ml against the
anti-viral activity of the IFN-.beta. in the A549 cell EMC viral
infectivity assay, and wherein in the A549 cell EMC viral
infectivity assay each type I interferon is normalized to 10
units/ml of NIH reference standard for recombinant IFN-.alpha.2
(IFN-.alpha.A).
[0152] In yet another embodiment, the invention provides an
anti-IFNAR1 antibody that inhibits the anti-viral activity of
IFN-.alpha.A, IFN-.alpha.D, IFN-.alpha.F, and IFN-.beta. and does
not inhibit the anti-viral activity of another type I interferon.
In still another embodiment, the invention provides an anti-IFNAR1
antibody that inhibits the anti-viral activity of IFN-.alpha.A,
IFN-.alpha.D, IFN-.alpha.F, and IFN-.beta. and does not inhibit the
anti-viral activity of another type I interferon selected from the
group consisting of IFN-.alpha.B and IFN-.alpha.G.
[0153] In a further embodiment, the invention provides an
anti-IFNAR1 antibody that inhibits the anti-viral activity of
IFN-.alpha.D and IFN-.beta. and does not inhibit the anti-viral
activity of another type I interferon. In an additional embodiment,
the invention provides an anti-IFNAR1 antibody that inhibits the
anti-viral activity of IFN-.alpha.D and IFN-.beta. and does not
inhibit the anti-viral activity of another type I interferon
selected from the group consisting of IFN-.alpha.A, IFN-.alpha.B,
IFN-.alpha.F and IFN-.alpha.G.
[0154] The invention additionally provides an anti-IFNAR1 antibody
that inhibits the anti-viral activity of a first type I interferon
and that does not inhibit the anti-viral activity of more than one
other type I interferon.
[0155] In one embodiment, the invention provides an anti-IFNAR1
that inhibits the anti-viral activity of a first type I interferon
and does not inhibit the anti-viral activity of any type I
interferon in the group consisting of IFN-.alpha.D, IFN-.alpha.F,
and IFN-.beta.. In another embodiment, the invention provides an
anti-IFNAR1 antibody that inhibits the anti-viral activity of a
first type I interferon and does not inhibit the anti-viral
activity of any type I interferon in the group consisting of
IFN-.alpha.B and IFN-.alpha.G. In yet another embodiment, the
invention provides an anti-IFNAR1 antibody that inhibits the
anti-viral activity of a first type I interferon and does not
inhibit the anti-viral activity of any type I interferon in the
group consisting of IFN-.alpha.A, IFN-.alpha.B, IFN-.alpha.F, and
IFN-.alpha.G.
[0156] Further provided herein is an anti-IFNAR1 antibody that
inhibits the anti-viral activity of at least two species of type I
interferon and that does not inhibit the anti-viral activity of at
least two more species of type I interferon.
[0157] In another embodiment, the invention provides an anti-IFNAR1
antibody that inhibits the anti-viral activity of IFN-.alpha.A,
IFN-.alpha.B, and IFN-.alpha.G does not inhibit the anti-viral
activity of any type I interferon in the group consisting of
IFN-.alpha.D, IFN-.alpha.F, and IFN-.beta..
[0158] In yet another embodiment, the invention provides an
anti-IFNAR1 antibody that inhibits the anti-viral activity of
IFN-.alpha.A, IFN-.alpha.D, IFN-.alpha.F, and IFN-.beta. and does
not inhibit the anti-viral activity of any type I interferon in the
group consisting of IFN-.alpha.B and IFN-.alpha.G.
[0159] In still another embodiment, the invention provides an
anti-IFNAR1 antibody that inhibits the anti-viral activity of
IFN-.alpha.D and IFN-.beta. and does not inhibit the anti-viral
activity of any type I interferon in the group consisting of
IFN-.alpha.A, IFN-.alpha.B, IFN-.alpha.F, and IFN-.alpha.G.
[0160] In other embodiments, the invention provides anti-IFNAR1
antibodies which possess combinations of the type I interferon
anti-viral inhibiting and/or non-inhibiting properties and the
IFNAR1 determinant binding and/or non-binding properties described
herein. Anti-IFNAR1 antibodies corresponding to these embodiments
can be obtained by using combinations of the type I anti-viral
activity inhibitions assays described above for selection of
antibodies with unique type I interferon inhibiting/non-inhibiting
properties and immunoprecipitation or immunoadsorption screening
procedures for selection of antibodies with unique IFNAR1
determinant binding/non-binding properties.
[0161] For example, the invention provides an anti-IFNAR1 antibody
that inhibits the anti-viral activity of a first type I interferon
and does not inhibit the anti-viral activity of a second type I
interferon, binds to one or more amino acids in situ in the
sequence of amino acids 103-111 of IFNAR1, and does not bind to one
or more amino acids in situ in the sequence of amino acids 244-249
of IFNAR1.
[0162] In a preferred embodiment, the invention provides an
anti-IFNAR1 antibody that inhibits the anti-viral activity of a
first type I interferon selected from the group consisting of
IFN-.alpha.A, IFN-.alpha.B, and IFN-.alpha.G and does not inhibit
the anti-viral activity of a second type I interferon, binds to one
or more amino acids in situ in the sequence of amino acids 103-111
of IFNAR1, and does not bind to one or more amino acids in situ in
the sequence of amino acids 244-249 of IFNAR1.
[0163] In another preferred embodiment, the invention provides an
anti-IFNAR1 antibody that inhibits the anti-viral activity of a
first type I interferon and does not inhibit the anti-viral
activity of a second type I interferon selected from the group
consisting of IFN-.alpha.D, IFN-.alpha.F, and IFN-.beta., binds to
one or more amino acids in situ in the sequence of amino acids
103-111 of IFNAR1, and does not bind to one or more amino acids in
situ in the sequence of amino acids 244-249 of IFNAR1.
[0164] In yet another preferred embodiment, the invention provides
an anti-IFNAR1 antibody that inhibits the anti-viral activity of
more than one selected type I interferon, does not inhibit the
anti-viral activity of another selected type I interferon to
IFNAR1, binds to one or more amino acids in situ in the sequence of
amino acids 103-111 of IFNAR1, and does not bind to one or more
amino acids in situ in the sequence of amino acids 244-249 of
IFNAR1.
[0165] In one preferred embodiment, the invention provides an
anti-IFNAR1 antibody that inhibits the anti-viral activity of
IFN-.alpha.A, IFN-.alpha.B, and IFN-.alpha.G, does not inhibit the
anti-viral activity of another selected type I interferon to
IFNAR1, binds to one or more amino acids in situ in the sequence of
amino acids 103-111 of IFNAR1, and does not bind to one or more
amino acids in situ in the sequence of amino acids 244-249 of
IFNAR1. In another preferred embodiment, the invention provides an
anti-IFNAR1 antibody that inhibits the anti-viral activity of
IFN-.alpha.A, IFN-.alpha.B and IFN-.alpha.G, does not inhibit the
anti-viral activity of another type I interferon selected from the
group consisting of IFN-.alpha.D, IFN-.alpha.F, and IFN-.beta.,
binds to one or more amino acids in situ in the sequence of amino
acids 103-111 of IFNAR1, and does not bind to one or more amino
acids in situ in the sequence of amino acids 244-249 of IFNAR1.
[0166] In yet another preferred embodiment, the invention provides
an anti-IFNAR1 antibody that inhibits the anti-viral activity of
IFN-.alpha.D and IFN-.beta., does not inhibit the anti-viral
activity of another selected type I interferon to IFNAR1, binds to
one or more amino acids in situ in the sequence of amino acids
103-111 of IFNAR1, and does not bind to one or more amino acids in
situ in the sequence of amino acids 244-249 of IFNAR1. In still
another preferred embodiment, the invention provides an anti-IFNAR1
antibody that inhibits the anti-viral activity of IFN-.alpha.D and
IFN-.beta., does not inhibit the anti-viral activity of another
type I interferon selected from the group consisting of
IFN-.alpha.A, IFN-.alpha.B, IFN-.alpha.F and IFN-.alpha.G, binds to
one or more amino acids in situ in the sequence of amino acids
103-111 of IFNAR1, and does not bind to one or more amino acids in
situ in the sequence of amino acids 244-249 of IFNAR1.
[0167] Additionally preferred is an anti-IFNAR1 antibody that
inhibits the anti-viral activity of a selected type I interferon to
IFNAR1, does not inhibit the anti-viral activity of more than one
other type I interferon to IFNAR1, binds to one or more amino acids
in situ in the sequence of amino acids 103-111 of IFNAR1, and does
not bind to one or more amino acids in situ in the sequence of
amino acids 244-249 of IFNAR1.
[0168] In another preferred embodiment, the invention provides an
anti-IFNAR1 antibody that inhibits the anti-viral activity of a
selected type I interferon, does not inhibit the anti-viral
activity of any type I interferon in the group consisting of
IFN-.alpha.D, IFN-.alpha.F, and IFN-.beta., binds to one or more
amino acids in situ in the sequence of amino acids 103-111 of
IFNAR1, and does not bind to one or more amino acids in situ in the
sequence of amino acids 244-249 of IFNAR1. In yet another preferred
embodiment, the invention provides an anti-IFNAR1 antibody that
inhibits the anti-viral activity of a selected type I interferon,
does not inhibit the anti-viral activity of any type I interferon
in the group consisting of IFN-.alpha.A, IFN-.alpha.B,
IFN-.alpha.F, and IFN-.alpha.G, binds to one or more amino acids in
situ in the sequence of amino acids 103-111 of IFNAR1, and does not
bind to one or more amino acids in situ in the sequence of amino
acids 244-249 of IFNAR1.
[0169] Further preferred embodiments include an anti-IFNAR1
antibody that inhibits the anti-viral activity of at least two
species of type I interferon, does not inhibit the anti-viral
activity of at least two more species of type I interferon, binds
to one or more amino acids in situ in the sequence of amino acids
103-111 of IFNAR1, and does not bind to one or more amino acids in
situ in the sequence of amino acids 244-249 of IFNAR1.
[0170] In another preferred embodiment, the invention provides an
anti-IFNAR1 antibody that inhibits the anti-viral activity of
IFN-.alpha.A, IFN-.alpha.B and IFN-.alpha.G, does not inhibit the
anti-viral activity of any type I interferon in the group
consisting of IFN-.alpha.D, IFN-.alpha.F and IFN-.beta., binds to
one or more amino acids in situ in the sequence of amino acids
103-111 of IFNAR1, and does not bind to one or more amino acids in
situ in the sequence of amino acids 244-249 of IFNAR I.
[0171] In yet another preferred embodiment, the invention provides
an anti-IFNAR1 antibody that inhibits the anti-viral activity of
IFN-.alpha.D and IFN-.beta., does not inhibit the anti-viral
activity of any type I interferon in the group consisting of
IFN-.alpha.A, IFN-.alpha.B, IFN-.alpha.F, and IFN-.alpha.G, binds
to one or more amino acids in situ in the sequence of amino acids
103-111 of IFNAR1, and does not bind to one or more amino acids in
situ in the sequence of amino acids 244-249 of IFNAR1.
[0172] In a further preferred embodiment, the invention provides
anti-IFNAR1 antibodies that inhibit the anti-viral activity of a
first type I interferon, do not inhibit the anti-viral activity of
a second type I interferon and IFNAR1, bind to one or more amino
acids in situ in the sequence of amino acids 103-111 of IFNAR1, and
do not bind to amino acid 249 of IFNAR1.
[0173] In a preferred embodiment, the invention provides an
anti-IFNAR1 antibody that inhibits the anti-viral activity of a
first type I interferon selected from the group consisting of
IFN-.alpha.A, IFN-.alpha.B, and IFN-.alpha.G, do not inhibit the
anti-viral activity of a second type I interferon and IFNAR1, bind
to one or more amino acids in situ in the sequence of amino acids
103-111 of IFNAR1, and do not bind to amino acid 249 of IFNAR1.
[0174] In another preferred embodiment, the invention provides an
anti-IFNAR1 antibody that inhibits the anti-viral activity of a
first type I interferon, does not inhibit the anti-viral activity
of a second type I interferon selected from the group consisting of
IFN-.alpha.D, IFN-.alpha.F, and IFN-.beta., bind to one or more
amino acids in situ in the sequence of amino acids 103-111 of
IFNAR1, and do not bind to amino acid 249 of IFNAR1.
[0175] In yet another preferred embodiment, the invention provides
an anti-IFNAR1 antibody that inhibits the anti-viral activity of
more than one selected type I interferon, does not inhibit the
anti-viral activity of another selected type I interferon, binds to
one or more amino acids in situ in the sequence of amino acids
103-111 of IFNAR1, and does not bind to amino acid 249 of IFNAR1 in
situ.
[0176] In one preferred embodiment, the invention provides an
anti-IFNAR1 antibody that inhibits the anti-viral activity of
IFN-.alpha.A, IFN-.alpha.B, and IFN-.alpha.G, does not inhibit the
anti-viral activity of another selected type I interferon, binds to
one or more amino acids in situ in the sequence of amino acids
103-111 of IFNAR 1, and does not bind to amino acid 249 of IFNAR1
in situ. In another preferred embodiment, the invention provides an
anti-IFNAR1 antibody that inhibits the anti-viral activity of
IFN-.alpha.A, IFN-.alpha.B and IFN-.alpha.G, does not inhibit the
anti-viral activity of another type I interferon selected from the
group consisting of IFN-.alpha.D, IFN-.alpha.F, and IFN-.beta.,
binds to one or more amino acids in situ in the sequence of amino
acids 103-111 of IFNAR1, and does not bind to amino acid 249 of
IFNAR1 in situ.
[0177] In yet another preferred embodiment, the invention provides
an anti-IFNAR1 antibody that inhibits the anti-viral activity of
IFN-.alpha.D and IFN-.beta., does not inhibit the anti-viral
activity of another selected type I interferon, binds to one or
more amino acids in situ in the sequence of amino acids 103-111 of
IFNAR1, and does not bind to amino acid 249 of IFNAR1 in situ. In
still another preferred embodiment, the invention provides an
anti-IFNAR1 antibody that inhibits the anti-viral activity of
IFN-.alpha.D and IFN-.beta., does not inhibit the anti-viral
activity of another type I interferon selected from the group
consisting of IFN-.alpha.A, IFN-.alpha.B, IFN-.alpha.F and
IFN-.alpha.G, binds to one or more amino acids in situ in the
sequence of amino acids 103-111 of IFNAR1, and does not bind to
amino acid 249 of IFNAR I in situ.
[0178] Additionally preferred is an anti-IFNAR1 antibody that
inhibits the anti-viral activity of a first type I interferon, does
not inhibit the anti-viral activity of more than one other type I
interferon to IFNAR1, binds to one or more amino acids in situ in
the sequence of amino acids 103-111 of IFNAR1, and does not bind to
amino acid 249 of IFNAR1 in situ. In another preferred embodiment,
the invention provides an anti-IFNAR1 Fv antibody that inhibits the
anti-viral activity of a first type I interferon, does not inhibit
the anti-viral activity of any type I interferon in the group
consisting of IFN-.alpha.D, IFN-.alpha.F, and IFN-.beta., binds to
one or more amino acids in situ in the sequence of amino acids
103-111 of IFNAR1, and does not bind to amino acid 249 of IFNAR1 in
situ.
[0179] Further preferred embodiments include anti-IFNAR1 antibodies
that inhibit the anti-viral activity of at least two species of
type I interferon, do not inhibit the anti-viral activity of at
least two more species of type I interferon, bind to one or more
amino acids in situ in the sequence of amino acids 103-111 of
IFNAR1, and do not bind to amino acid 249 of IFNAR1 in situ. In yet
another preferred embodiment, the invention provides an anti-IFNAR1
antibody that inhibits the anti-viral activity of IFN-.alpha.A,
IFN-.alpha.B, and IFN-.alpha.G, does not inhibit the anti-viral
activity of any type I interferon in the group consisting of
IFN-.alpha.D, IFN-.alpha.F, and IFN-.beta., bind to one or more
amino acids in situ in the sequence of amino acids 103-111 of
IFNAR1, and do not bind to amino acid 249 of IFNAR1 in situ. In a
further embodiment, the invention provides an anti-IFNAR1 antibody
that inhibits the antiviral activity of IFN-.alpha.D and
IFN-.beta., does not inhibit the anti-viral activity of any type I
interferon in the group consisting of IFN-.alpha.A, IFN-.alpha.B,
IFN-.alpha.F, and IFN-.alpha.G, binds to one or more amino acids in
situ in the sequence of amino acids 103-111 of IFNAR1, and does not
bind to amino acid 249 of IFNAR1 in situ.
[0180] In another preferred embodiment, the invention provides
anti-IFNAR1 antibodies that inhibit the anti-viral activity of a
first type I interferon and IFNAR1, do not inhibit the anti-viral
activity of a second type I interferon and IFNAR1, bind to one or
more amino acids in situ in the sequence of amino acids 103-111 of
IFNAR1, bind to amino acids 291 and 296 of IFNAR1, and do not bind
to amino acid 249 of IFNAR1.
[0181] In one preferred embodiment, the anti-IFNAR1 antibody
inhibits the anti-viral activity of a first type I interferon
selected from the group consisting of IFN-.alpha.A, IFN-.alpha.B,
and IFN-.alpha.G, does not inhibit the anti-viral activity of a
second type I interferon, binds to one or more amino acids in situ
in the sequence of amino acids 103-111 of IFNAR1, binds to amino
acids 291 and 296 of IFNAR1, and does not bind to amino acid 249 of
IFNAR1.
[0182] In another preferred embodiment, the anti-IFNAR1 antibody
inhibits the anti-viral activity of a first type I interferon, does
not inhibit the anti-viral activity of a second type I interferon
selected from the group consisting of IFN-.alpha.D, IFN-.alpha.F,
and IFN-.beta., binds to one or more amino acids in situ in the
sequence of amino acids 103-111 of IFNAR1, binds to amino acids 291
and 296 of IFNAR1, and does not bind to amino acid 249 of
IFNAR1.
[0183] In yet another preferred embodiment, the invention provides
anti-IFNAR1 antibodies that inhibit the anti-viral activity of more
than one selected type I interferon to IFNAR1, do not inhibit the
anti-viral activity of another selected type I interferon to
IFNAR1, bind to one or more amino acids in situ in the sequence of
amino acids 103-111 of IFNAR1, bind to amino acids 291 and 296 of
IFNAR1 in situ, and do not bind to amino acid 249 of IFNAR1 in
situ. In one preferred embodiment, the invention provides an
anti-IFNAR1 antibody that inhibits the anti-viral activity of
IFN-.alpha.A, IFN-.alpha.B, and IFN-.alpha.G, do not inhibit the
anti-viral activity of another selected type. I interferon to
IFNAR1, bind to one or more amino acids in situ in the sequence of
amino acids 103-111 of IFNAR1, bind to amino acids 291 and 296 of
IFNAR1 in situ, and do not bind to amino acid 249 of IFNAR1 in
situ. In still another preferred embodiment, the invention provides
an anti-IFNAR1 antibody that inhibits the anti-viral activity of
IFN-.alpha.A, IFN-.alpha.B and IFN-.alpha.G, does not inhibit the
anti-viral activity of another type I interferon selected from the
group consisting of IFN-.alpha.D, IFN-.alpha.F, and IFN-.beta.,
binds to one or more amino acids in situ in the sequence of amino
acids 103-111 of IFNAR1, binds to amino acids 291 and 296 of IFNAR1
in situ, and do not bind to amino acid 249 of IFNAR1 in situ.
[0184] Additionally preferred are anti-IFNAR1 antibodies that
inhibit the anti-viral activity of a selected type I interferon, do
not inhibit the anti-viral activity of more than one other type I
interferon, bind to one or more amino acids in situ in the sequence
of amino acids 103-111 of IFNAR1, bind to amino acids 291 and 296
of IFNAR1 in situ, and do not bind to amino acid 249 of IFNAR1 in
situ. Also preferred are anti-IFNAR1 antibodies that inhibit the
anti-viral activity of a selected type I interferon, do not inhibit
the anti-viral activity of any type I interferon in the group
consisting of IFN-.alpha.D, IFN-.alpha.F, and IFN-.beta., bind to
one or more amino acids in situ in the sequence of amino acids
103-111 of IFNAR1, bind to amino acids 291 and 296 of IFNAR1 in
situ, and do not bind to amino acid 249 of IFNAR1 in situ.
[0185] Further preferred embodiments include anti-IFNAR1 antibodies
that inhibit the anti-viral activity of at least two species of
type I interferon, do not inhibit the anti-viral activity of at
least two more species of type I interferon, bind to one or more
amino acids in situ in the sequence of amino acids 103-111 of
IFNAR1, bind to amino acids 291 and 296 in situ, and do not bind to
amino acid 249 of IFNAR1 in situ. In yet another preferred
embodiment, the invention provides an anti-IFNAR1 antibody that
inhibits the anti-viral activity of IFN-.alpha.A, IFN-.alpha.B, and
IFN-.alpha.G, does not inhibit the anti-viral activity of any type
I interferon in the group consisting of IFN-.alpha.D, IFN-.alpha.F,
and IFN-.beta., bind to one or more amino acids in situ in the
sequence of amino acids 103-111 of IFNAR1, bind to amino acids 291
and 296 in situ, and do not bind to amino acid 249 of IFNAR1 in
situ.
[0186] The invention additionally provides anti-IFNAR1 antibodies
which inhibit the anti-viral activity of IFN-.alpha.A,
IFN-.alpha.B, and IFN-.alpha.G, which do not block the anti-viral
activity of IFN-.beta., and which bind to one or more amino acids
in situ in the sequence of amino acids 244-249 of IFNAR1 and bind
to one or more amino acids in situ in the sequence of amino acids
291-298 of IFNAR1. Thus, the invention includes an anti-IFNAR1
antibody (1) which possesses any pattern of
IFN-.beta.-non-inhibiting and IFN-.alpha.A-, IFN-.alpha.B-, and
IFN-.alpha.G-inhibiting activity described above (2) which binds to
one or more amino acids in situ in the sequence of amino acids
244-249 of IFNAR1 and (3) which binds to one or more amino acids in
situ in the sequence of amino acids 291-298 of IFNAR1.
[0187] The invention also provides anti-IFNAR1 antibodies which
inhibit the anti-viral activity of IFN-.alpha.A, IFN-.alpha.B,
IFN-.alpha.D, and IFN-.alpha.G, which do not block the anti-viral
activity of IFN-.beta., and which bind to one or more amino acids
in situ in the sequence of amino acids 244-249 of IFNAR1 and bind
to one or more amino acids in situ in the sequence of amino acids
291-298 of IFNAR1. Thus, the invention includes an anti-IFNAR1
antibody (1) which possesses any pattern of
IFN-.beta.-non-inhibiting and IFN-.alpha.A-, IFN-.alpha.B-,
IFN-.alpha.D-, and IFN-.alpha.G-inhibiting activity described above
(2) which binds to one or more amino acids in situ in the sequence
of amino acids 244-249 of IFNAR1 and (3) which binds to one or more
amino acids in situ in the sequence of amino acids 291-298 of
IFNAR1.
[0188] The invention also encompasses anti-IFNAR1 antibodies which
inhibit the anti-viral activity of IFN-.alpha.A, IFN-.alpha.B, and
IFN-.alpha.G, which do not inhibit the anti-viral activity of
IFN-.beta., and which bind to amino acids 249, 291 and 296 of
IFNAR1 in situ. Thus, the invention includes an anti-IFNAR1
antibody (1) which possesses any pattern of
IFN-.beta.-non-inhibiting and IFN-.alpha.A-, IFN-.alpha.B-, and
IFN-.alpha.G-inhibiting activity described above and (2) which
binds to amino acids 249, 291 and 296 of IFNAR1 in situ.
[0189] The invention further provides anti-IFNAR1 antibodies which
inhibit the anti-viral activity of IFN-.alpha.A, IFN-.alpha.B,
IFN-.alpha.D, and IFN-.alpha.G, which do not inhibit the anti-viral
activity of IFN-.beta., and which bind to amino acids 249, 291 and
296 of IFNAR1 in situ. Thus, the invention includes an anti-IFNAR1
antibody (1) which possesses any pattern of
IFN-.beta.-non-inhibiting and IFN-.alpha.A-, IFN-.alpha.B-,
IFN-.alpha.D-, and IFN-.alpha.G-inhibiting activity described above
and (2) which binds to amino acids 249, 291 and 296 of IFNAR1 in
situ.
[0190] In another embodiment, the invention provides any of the
anti-IFNAR1 antibodies described above that additionally binds to a
conformational epitope on IFNAR1. Such anti-IFNAR1 antibodies can
be obtained by adding the above-described denatured IFNAR1
immunoblotting or immunoadsorption assay to the series of
procedures used to screen for the other desired antibody properties
described above. It will be appreciated that the denatured IFNAR1
immunoblotting or immunoadsorption assay can be performed before,
after, or at any convenient point during the other selection
procedures for the anti-IFNAR1 antibody of interest.
[0191] In a further embodiment, the invention provides anti-IFNAR1
antibodies that inhibit the anti-viral activity of a first type I
interferon, do not inhibit the anti-viral activity of a second type
I interferon, and bind to a conformational epitope of IFNAR1.
[0192] In yet another embodiment, the invention provides
anti-IFNAR1 antibodies that inhibit the anti-viral activity of a
first type I interferon selected from the group consisting of
IFN-.alpha.A, IFN-.alpha.B, and IFN-.alpha.G, do not inhibit the
anti-viral activity of a second type I interferon, and bind to a
conformational epitope of IFNAR1.
[0193] In still another embodiment, the invention provides
anti-IFNAR1 antibodies that inhibit the anti-viral activity of a
first type I interferon, do not inhibit the anti-viral activity of
a second type I interferon selected from the group consisting of
IFN-.alpha.D, IFN-.alpha.F, and IFN-.beta., and bind to a
conformational epitope of IFNAR1.
[0194] In a further embodiment, the invention provides anti-IFNAR1
antibodies that inhibit the anti-viral activity of a first type I
interferon selected from the group consisting of IFN-.alpha.A,
IFN-.alpha.B, and IFN-.alpha.G, do not inhibit the anti-viral
activity of a second type I interferon selected from the group
consisting of IFN-.alpha.D, IFN-.alpha.F, and IFN-.beta., and bind
to a conformational epitope of IFNAR1.
[0195] Also provided herein are anti-IFNAR1 antibodies that inhibit
the anti-viral activity of each type I interferon in the group
consisting of IFN-.alpha.A, IFN-.alpha.B, and IFN-.alpha.G, do not
inhibit the anti-viral activity of a type I interferon selected
from the group consisting of IFN-.alpha.D, IFN-.alpha.F, and
IFN-.beta., and bind to a conformational epitope of IFNAR1.
[0196] Further provided herein are anti-IFNAR1 antibodies that
inhibit the anti-viral activity of each type I interferon in the
group consisting of IFN-.alpha.A, IFN-.alpha.B, and IFN-.alpha.G,
do not inhibit the anti-viral activity of any type I interferon in
the group consisting of IFN-.alpha.D, IFN-.alpha.F, and IFN-.beta.,
and bind to a conformational epitope of IFNAR1.
[0197] Also included herein are any of the anti-IFNAR1 antibodies
described above that additionally binds to a conformational epitope
formed by domain 1 and domain 2 of IFNAR-1. Such anti-IFNAR1
antibodies can be obtained by adding the above-described
immunoprecipitation or immunoadsorption assays for determining
domain 1 peptide or domain 2 peptide binding, e.g. ELISA capture
assays, to the series of procedures used to screen for the other
desired antibody properties described above. It will be appreciated
that the domain 1 peptide and/or domain 2 peptide
immunoprecipitation or immunoadsorption screen can be performed
before, after, or at any convenient point during the other
selection procedures for the anti-IFNAR1 antibody of interest.
[0198] In a further embodiment, the invention provides anti-IFNAR1
antibodies that inhibit the anti-viral activity of a first type I
interferon, do not inhibit the anti-viral activity of a second type
I interferon, do not bind to a peptide consisting of the amino acid
sequence of domain 1 (amino acids 1-200 of IFNAR1), and do not bind
to a peptide consisting of the amino acid sequence of domain 2
(amino acids 204-404 of IFNAR1).
[0199] In yet another embodiment, the invention provides
anti-IFNAR1 antibodies that inhibit the anti-viral activity of a
first type I interferon selected from the group consisting of
IFN-.alpha.A, IFN-.alpha.B, and IFN-.alpha.G, do not inhibit the
anti-viral activity of a second type I interferon, do not bind to a
peptide consisting of the amino acid sequence of domain 1 (amino
acids 1-200 of IFNAR1), and do not bind to a peptide consisting of
the amino acid sequence of domain 2 (amino acids 204-404 of
IFNAR1).
[0200] In still another embodiment, the invention provides
anti-IFNAR1 antibodies that inhibit the anti-viral activity of a
first type I interferon, do not inhibit the anti-viral activity of
a second type I interferon selected from the group consisting of
IFN-.alpha.D, IFN-.alpha.F, and IFN-.beta., do not bind to a
peptide consisting of the amino acid sequence of domain 1 (amino
acids 1-200 of IFNAR1), and do not bind to a peptide consisting of
the amino acid sequence of domain 2 (amino acids 204-404 of
IFNAR1).
[0201] In a further embodiment, the invention provides anti-IFNAR1
antibodies that inhibit the anti-viral activity of a first type I
interferon selected from the group consisting of IFN-.alpha.A,
IFN-.alpha.B, and IFN-.alpha.G, do not inhibit the anti-viral
activity of a second type I interferon selected from the group
consisting of IFN-.alpha.D, IFN-.alpha.F, and IFN-.beta., do not
bind to a peptide consisting of the amino acid sequence of domain 1
(amino acids 1-200 of IFNAR1), and do not bind to a peptide
consisting of the amino acid sequence of domain 2 (amino acids
204-404 of IFNAR1).
[0202] Also provided herein are anti-IFNAR1 antibodies that inhibit
the anti-viral activity of each type I interferon in the group
consisting of IFN-.alpha.A, IFN-.alpha.B, and IFN-.alpha.G, do not
inhibit the anti-viral activity of a type I interferon selected
from the group consisting of IFN-.alpha.D, IFN-.alpha.F, and
IFN-.beta., do not bind to a peptide consisting of the amino acid
sequence of domain 1 (amino acids 1-200 of IFNAR1), and do not bind
to a peptide consisting of the amino acid sequence of domain 2
(amino acids 204-404 of IFNAR1).
[0203] Further provided herein are anti-IFNAR1 antibodies that
inhibit the anti-viral activity of each type I interferon in the
group consisting of IFN-.alpha.A, IFN-.alpha.B, and IFN-.alpha.G,
do not inhibit the anti-viral activity of any type I interferon in
the group consisting of IFN-.alpha.D, IFN-.alpha.F, and IFN-.beta.,
do not bind to a peptide consisting of the amino acid sequence of
domain 1 (amino acids 1-200 of IFNAR1), and do not bind to a
peptide consisting of the amino acid sequence of domain 2 (amino
acids 204-404 of IFNAR1).
[0204] In another embodiment, the invention provides anti-IFNAR1
antibodies that inhibit the anti-viral activity of IFN-.alpha.A,
IFN-.alpha.B, IFN-.alpha.D, and IFN-.alpha.G, do not inhibit the
anti-viral activity of IFN-.beta., and bind to a conformational
epitope of IFNAR1. Thus, the invention includes an anti-IFNAR1
antibody (1) which possesses any pattern of
IFN-.beta.-non-inhibiting and IFN-.alpha.A-, IFN-.alpha.B-,
IFN-.alpha.D-, and IFN-.alpha.G-inhibiting activity described above
and (2) which binds to a conformational epitope of IFNAR1.
[0205] In yet another embodiment, the invention provides
anti-IFNAR1 antibodies that inhibit the anti-viral activity of
IFN-.alpha.A, IFN-.alpha.B, IFN-.alpha.D, and IFN-.alpha.G, do not
inhibit the anti-viral activity of IFN-.beta., bind to one or more
of amino acids 244-249 of IFNAR1 in situ, bind to one or more of
amino acids 291-298 of IFNAR1 in situ, and bind to a conformational
epitope of IFNAR1. Thus, the invention includes an anti-IFNAR1
antibody (1) which possesses any pattern of
IFN-.beta.-non-inhibiting and IFN-.alpha.A-, IFN-.alpha.B-,
IFN-.alpha.D-, and IFN-.alpha.G-inhibiting activity described above
(2) which binds to one or more of amino acids 244-249 of IFNAR1 in
situ (3) which binds to one or more of amino acids 291-298 of
IFNAR1 in situ and (4) which binds to a conformational epitope of
IFNAR1.
[0206] In still another embodiment, the invention provides
anti-IFNAR1 antibodies that inhibit the anti-viral activity of
IFN-.alpha.A, IFN-.alpha.B, IFN-.alpha.D, and IFN-.alpha.G, do not
inhibit the anti-viral activity of IFN-.beta., bind to amino acids
249, 291 and 296 of IFNAR1 in situ, and bind to a conformational
epitope of IFNAR1. Thus, the invention includes an anti-IFNAR1
antibody (1) which possesses any pattern of
IFN-.beta.-non-inhibiting and IFN-.alpha.A-, IFN-.alpha.B-,
IFN-.alpha.D-, and IFN-.alpha.G-inhibiting activity described above
(2) which binds to amino acids 249, 291 and 296 of IFNAR1 in situ
and (3) which binds to a conformational epitope of IFNAR1.
[0207] Also provided herein are anti-IFNAR1 antibodies that inhibit
the anti-viral activity of IFN-.alpha.A, IFN-.alpha.B,
IFN-.alpha.D, and IFN-.alpha.G, do not inhibit the anti-viral
activity of IFN-.beta., do not bind to a peptide consisting of the
amino acid sequence of domain 1 (amino acids 1-200) of IFNAR1, and
do not bind to a peptide consisting of the amino acid sequence of
domain 2 (amino acids 204-404) of IFNAR1. Thus, the invention
includes an anti-IFNAR1 antibody (1) which possesses any pattern of
IFN-.beta.-non-inhibiting and IFN-.alpha.A-, IFN-.alpha.B-,
IFN-.alpha.D-, and IFN-.alpha.G-inhibiting activity described above
(2) which does not bind to a peptide consisting of the amino acid
sequence of domain 1 (amino acids 1-200) of IFNAR1 and (3) which
does not bind to a peptide consisting of the amino acid sequence of
domain 2 (amino acids 204-404) of IFNAR1.
[0208] Further provided herein are anti-IFNAR1 antibodies that
inhibit the anti-viral activity of IFN-.alpha.A, IFN-.alpha.B,
IFN-.alpha.D, and IFN-.alpha.G, do not inhibit the anti-viral
activity of IFN-.beta., bind to one or more of amino acids 244-249
of IFNAR1 in situ, bind to one or more of amino acids 291-298 of
IFNAR1 in situ, do not bind to a peptide consisting of the amino
acid sequence of domain 1 (amino acids 1-200) of IFNAR1, and do not
bind to a peptide consisting of the amino acid sequence of domain 2
(amino acids 204-404) of IFNAR1. Thus, the invention includes an
anti-IFNAR1 antibody (1) which possesses any pattern of
IFN-.beta.-non-inhibiting and IFN-.alpha.A-, IFN-.alpha.B-,
IFN-.alpha.D-, and IFN-.alpha.G-inhibiting activity described above
(2) which binds to one or more of amino acids 244-249 of IFNAR1 in
situ (3) which binds to one or more of amino acids 291-298 of
IFNAR1 in situ (4) which does not bind to a peptide consisting of
the amino acid sequence of domain 1 (amino acids 1-200) of IFNAR1
and (5) which does not bind to a peptide consisting of the amino
acid sequence of domain 2 (amino acids 204-404) of IFNAR1.
[0209] Additionally provided herein are anti-IFNAR1 antibodies that
inhibit the anti-viral activity of IFN-.alpha.A, IFN-.alpha.B,
IFN-.alpha.D, and IFN-.alpha.G, do not inhibit the anti-viral
activity of IFN-.beta., bind to amino acids 249, 291 and 298 in
IFNAR1 in situ, do not bind to a peptide consisting of the amino
acid sequence of domain 1 (amino acids 1-200) of IFNAR1, and do not
bind to a peptide consisting of the amino acid sequence of domain 2
(amino acids 204-404) of IFNAR1. Thus, the invention includes an
anti-IFNAR1 antibody (1) which possesses any pattern of
IFN-.beta.-non-inhibiting and IFN-.alpha.A-, IFN-.alpha.B-,
IFN-.alpha.D-, and IFN-.alpha.G-inhibiting activity described above
(2) which binds to amino acids 249, 291 and 296 of IFNAR1 in situ
(3) which does not bind to a peptide consisting of the amino acid
sequence of domain 1 (amino acids 1-200) of IFNAR1 and (4) which
does not bind to a peptide consisting of the amino acid sequence of
domain 2 (amino acids 204-404) of IFNAR1.
[0210] In another embodiment, the invention provides the
anti-IFNAR1 monoclonal antibody produced by hybridoma cell line 5A8
(ATCC Deposit No. HB 12129).
[0211] In yet another embodiment, the invention provides the
anti-IFNAR1 monoclonal antibody produced by hybridoma cell line 2E8
(ATCC Deposit No. HB 12130).
[0212] In still another embodiment, the invention provides the
anti-IFNAR1 monoclonal antibody produced by hybridoma cell line 2H6
(ATCC Deposit No. HB 12131).
[0213] In a further embodiment, the invention provides the
anti-IFNAR1 monoclonal antibody produced by hybridoma cell line 4A7
(ATCC Deposit No. HB 12132).
[0214] In an additional embodiment, the invention provides the
anti-IFNAR1 monoclonal antibody produced by hybridoma cell line 2E1
(ATCC Deposit No. HB 12133).
[0215] In still another embodiment, the invention provides
anti-IFNAR1 monoclonal antibodies that compete with 5A8 antibody,
2E8 antibody, 2H6 antibody, 4A7 antibody, or 2E1 antibody for
binding to IFNAR1. Such competitor antibodies include antibodies
that recognize an IFNAR1 epitope that is the same as or overlaps
with the IFNAR1 epitope recognized by an antibody selected from the
group consisting of the 5A8, 2E8, 2H6, 4A7 and 2E1 antibodies. Such
competitor antibodies can be obtained by screening anti-IFNAR1
hybridoma supernatants for binding to immobilized IFNAR1 in
competition with labeled 5A8 antibody, 2E8 antibody, 2H6 antibody,
4A7 antibody or 2E1 antibody. A hybridoma supernatant containing
competitor antibody will reduce the amount of bound, labeled
antibody detected in the subject competition binding mixture as
compared to the amount of bound, labeled antibody detected in a
control binding mixture containing irrelevant (or no) antibody. Any
of the competition binding assays described in Section IV below is
suitable for use in the foregoing procedure.
[0216] III. Methods of Constructing Recombinant Anti-IFNAR1
Antibodies
[0217] DNA encoding the hybridoma-derived monoclonal antibodies or
phage display Fv clones of the invention is readily isolated and
sequenced using conventional procedures (e.g. by using
oligonucleotide primers designed to specifically amplify the heavy
and light chain coding regions of interest from hybridoma or phage
DNA template). Once isolated, the DNA can be placed into expression
vectors, which are then transfected into host cells such as E. coli
cells, simian COS cells, Chinese hamster ovary (CHO) cells, or
myeloma cells that do not otherwise produce immunoglobulin protein,
to obtain the synthesis of the desired monoclonal antibodies in the
recombinant host cells. Review articles on recombinant expression
in bacteria of antibody-encoding DNA include Skerra et al., Curr.
Opinion in Immunol., 5: 256 (1993) and Pluckthun, Immunol. Revs,
130; 151 (1992).
[0218] DNA encoding the Fv clones of the invention can be combined
with known DNA sequences encoding heavy chain and/or light chain
constant regions (e.g. the appropriate DNA sequences can be
obtained from Kabat et al., supra) to form clones encoding full or
partial length heavy and/or light chains. It will be appreciated
that constant regions of any isotype can be used for this purpose,
including IgG, IgM, IgA, IgD, and IgE constant regions, and that
such constant regions can be obtained from any human or animal
species. A Fv clone derived from the variable domain DNA of one
animal (such as human) species and then fused to constant region
DNA of another animal species to form coding sequence(s) for
"hybrid", full length heavy chain and/or light chain is included in
the definition of "chimeric" and "hybrid" antibody as used herein.
In a preferred embodiment, a Fv clone derived from human variable
DNA is fused to human constant region DNA to form coding
sequence(s) for all human, full or partial length heavy and/or
light chains.
[0219] DNA encoding anti-IFNAR1 antibody derived from a hybridoma
of the invention can also be modified, for example, by substituting
the coding sequence for human heavy- and light-chain constant
domains in place of homologous murine sequences derived from the
hybridoma clone (e.g. as in the method of Morrison et al., Proc.
Natl. Acad. Sci. USA, 81: 6851-6855 (1984)). DNA encoding a
hybridoma or Fv clone-derived antibody or fragment can be further
modified by covalently joining to the immunoglobulin coding
sequence all or part of the coding sequence for a
non-immunoglobulin polypeptide. In this manner, "chimeric" or
"hybrid" antibodies are prepared that have the binding specificity
of the Fv clone or hybridoma clone-derived antibodies of the
invention.
[0220] Typically, such non-immunoglobulin polypeptides are
substituted for the constant domains of an antibody of the
invention, or they are substituted for the variable domains of one
antigen-combining site of an antibody of the invention to create a
chimeric bivalent antibody comprising one antigen-combining site
having specificity for IFNAR1 and another antigen-combining site
having specificity for a different antigen.
[0221] Chimeric or hybrid antibodies also can be prepared in vitro
using known methods in synthetic protein chemistry, including those
involving crosslinking agents. For example, immunotoxins can be
constructed using a disulfide-exchange reaction or by forming a
thioether bond. Examples of suitable reagents for this purpose
include iminothiolate and methyl-4-mercaptobutyrimidate.
[0222] a. Humanized Antibodies
[0223] Methods for humanizing non-human antibodies are well known
in the art. Generally, a humanized antibody has one or more amino
acid residues introduced into it from a source that is non-human.
These non-human amino acid residues are often referred to as
"import" residues, which are typically taken from an "import"
variable domain. It will be appreciated that variable domain
sequences obtained from any non-human animal phage display
library-derived Fv clone or from any non-human animal
hybridoma-derived antibody clone provided as described herein can
serve as the "import" variable domain used in the construction of
the humanized antibodies of the invention. Humanization can be
essentially performed following the method of Winter and co-workers
(Jones et al., Nature, 321: 522 (1986); Riechmann et al., Nature,
332: 323 (1988); Verhoeyen et al., Science, 239: 1534 (1988)), by
substituting non-human animal, e.g. rodent, CDRs or CDR sequences
for the corresponding sequences of a human antibody. Accordingly,
such "humanized" antibodies are chimeric antibodies (Cabilly et
al., supra), wherein substantially less than an intact human
variable domain has been substituted by the corresponding sequence
from a non-human species. In practice, humanized antibodies are
typically human antibodies in which some CDR residues and possibly
some FR residues are substituted by residues from analogous sites
in non-human animal, e.g. rodent, antibodies.
[0224] The choice of human variable domains, both light and heavy,
to be used in making the humanized antibodies is very important to
reduce antigenicity. According to the so-called "best-fit" method,
the sequence of the variable domain of a non-human animal, e.g.
rodent, antibody is screened against the entire library of known
human variable-domain sequences. The human sequence that is closest
to that of the non-human animal is then accepted as the human
framework (FR) for the humanized antibody (Sims et al., J.
Immunol., 151: 2296 (1993); Chothia and Lesk, J. Mol. Biol., 196:
901 (1987)). Another method uses a particular framework derived
from the consensus sequence of all human antibodies of a particular
subgroup light or heavy chains. The same framework can be used for
several different humanized antibodies (Carter et al., Proc. Natl.
Acad. Sci USA, 89: 4285 (1992); Presta et al., J. Immunol., 151:
2623 (1993)).
[0225] It is also important that antibodies be humanized with
retention of high affinity for the antigen and other favorable
biological properties. To achieve this goal, according to a
preferred method, humanized antibodies are prepared by a process of
analysis of the parental sequences and various conceptual humanized
products using three-dimensional models of the parental and
humanized sequences. Three-dimensional immunoglobulin models are
commonly available and are familiar to those skilled in the art.
Computer programs are available which illustrate and display
probable three-dimensional Informational structures of selected
candidate immunoglobulin sequences. Inspection of these displays
permits analysis of the likely role of the residues in the
functioning of the candidate immunoglobulin sequence, i.e., the
analysis of residues that influence the ability of the candidate
immunoglobulin to bind to its antigen. In this way, FR residues can
be selected and combined from the consensus and import sequences so
that the desired antibody characteristic, such as increased
affinity for the target antigen(s), is achieved. In general, the
CDR residues are directly and most substantially involved in
influencing antigen binding.
[0226] b. Human Antibodies
[0227] Human anti-IFNAR1 antibodies of the invention can be
constructed by combining Fv clone variable domain sequence(s)
selected from human-derived phage display libraries with known
human constant domain sequences(s) as described above.
Alternatively, human monoclonal anti-IFNAR1 antibodies of the
invention can be made by the hybridoma method. Human myeloma and
mouse-human heteromyeloma cell lines for the production of human
monoclonal antibodies have been described, for example, by Kozbor
J. Immunol., 133: 3001 (1984); Brodeur et al., Monoclonal Antibody
Production Techniques and Applications, pp. 51-63 (Marcel Dekker,
Inc., New York, 1987); and Boerner et al., J. Immunol., 147: 86
(1991).
[0228] It is now possible to produce transgenic animals (e.g. mice)
that are capable, upon immunization, of producing a full repertoire
of human antibodies in the absence of endogenous immunoglobulin
production. For example, it has been described that the homozygous
deletion of the antibody heavy-chain joining region (JH) gene in
chimeric and germ-line mutant mice results in complete inhibition
of endogenous antibody production. Transfer of the human germ-line
immunoglobulin gene array in such germ-line mutant mice will result
in the production of human antibodies upon antigen challenge. See,
e.g., Jakobovits et al., Proc. Natl. Acad. Sci USA, 90: 2551
(1993); Jakobovits et al., Nature, 362: 255 (1993); Bruggermann et
al., Year in Immunol., 7:33 (1993).
[0229] Gene shuffling can also be used to derive human antibodies
from non-human, e.g. rodent, antibodies, where the human antibody
has similar affinities and specificities to the starting non-human
antibody. According to this method, which is also called "epitope
imprinting", either the heavy or light chain variable region of a
non-human antibody fragment obtained by phage display techniques as
described above is replaced with a repertoire of human V domain
genes, creating a population of non-human chain/human chain scFv or
Fab chimeras. Selection with antigen results in isolation of a
non-human chain/human chain chimeric scFv or Fab wherein the human
chain restores the antigen binding site destroyed upon removal of
the corresponding non-human chain in the primary phage display
clone, i.e. the epitope governs (imprints) the choice of the human
chain partner. When the process is repeated in order to replace the
remaining non-human chain, a human antibody is obtained (see PCT WO
93/06213 published Apr. 1, 1993). Unlike traditional humanization
of non-human antibodies by CDR grafting, this technique provides
completely human antibodies, which have no FR or CDR residues of
non-human origin.
[0230] c. Bispecific Antibodies
[0231] Bispecific antibodies are monoclonal, preferably human or
humanized, antibodies that have binding specificities for at least
two different antigens. In the present case, one of the binding
specificities is for IFNAR1 and the other is for any other antigen.
Exemplary bispecific antibodies may bind to two different epitopes
of the IFNAR1 protein. Bispecific antibodies may also be used to
localize cytotoxic agents to cells that express IFNAR1. These
antibodies possess an IFNAR1-binding arm and an arm which binds the
cytotoxic agent (e.g. saporin, anti-interferon-.alpha., vinca
alkaloid, ricin A chain, methotrexate or radioactive isotope
hapten). Bispecific antibodies can be prepared as full length
antibodies or antibody fragments (e.g. F(ab).sub.2 bispecific
antibodies).
[0232] Methods for making bispecific antibodies are known in the
art. Traditionally, the recombinant production of bispecific
antibodies is based on the co-expression of two immunoglobulin
heavy chain-light chain pairs, where the two heavy chains have
different specificities (Milstein and Cuello, Nature, 305: 537
(1983)). Because of the random assortment of immunoglobulin heavy
and light chains, these hybridomas (quadromas) produce a potential
mixture of 10 different antibody molecules, of which only one has
the correct bispecific structure. The purification of the correct
molecule, which is usually done by affinity chromatography steps,
is rather cumbersome, and the product yields are low. Similar
procedures are disclosed in WO 93/08829 published May 13, 1993, and
in Traunecker et al., EMBO J., 10: 3655 (1991).
[0233] According to a different and more preferred approach,
antibody variable domains with the desired binding specificities
(antibody-antigen combining sites) are fused to immunoglobulin
constant domain sequences. The fusion preferably is with an
immunoglobulin heavy chain constant domain, comprising at least
part of the hinge, CH2, and CH3 regions. It is preferred to have
the first heavy-chain constant region (CH1), containing the site
necessary for light chain binding, present in at least one of the
fusions. DNAs encoding the immunoglobulin heavy chain fusions and,
if desired, the immunoglobulin light chain, are inserted into
separate expression vectors, and are co-transfected into a suitable
host organism. This provides for great flexibility in adjusting the
mutual proportions of the three polypeptide fragments in
embodiments when unequal ratios of the three polypeptide chains
used in the construction provide the optimum yields. It is,
however, possible to insert the coding sequences for two or all
three polypeptide chains in one expression vector when the
expression of at least two polypeptide chains in equal ratios
results in high yields or when the ratios are of no particular
significance.
[0234] In a preferred embodiment of this approach, the bispecific
antibodies are composed of a hybrid immunoglobulin heavy chain with
a first binding specificity in one arm, and a hybrid immunoglobulin
heavy chain-light chain pair (providing a second binding
specificity) in the other arm. It was found that this asymmetric
structure facilitates the separation of the desired bispecific
compound from unwanted immunoglobulin chain combinations, as the
presence of an immunoglobulin light chain in only one half of the
bispecific molecule provides for a facile way of separation. This
approach is disclosed in WO 94/04690. For further details of
generating bispecific antibodies see, for example, Suresh et al.,
Methods in Enzymology, 121:210 (1986).
[0235] According to another approach, the interface between a pair
of antibody molecules can be engineered to maximize the percentage
of heterodimers that are recovered from recombinant cell culture.
The preferred interface comprises at least a part of the C.sub.H3
domain of an antibody constant domain. In this method, one or more
small amino acid side chains from the interface of the first
antibody molecule are replaced with larger side chains (e.g.
tyrosine or tryptophan). Compensatory "cavities" of identical or
similar size to the large side chain(s) are created on the
interface of the second antibody molecule by replacing large amino
acid side chains with smaller ones (e.g. alanine or threonine).
This provides a mechanism for increasing the yield of the
heterodimer over other unwanted end-products such as
homodimers.
[0236] Bispecific antibodies include cross-linked or
"heteroconjugate" antibodies. For example, one of the antibodies in
the heteroconjugate can be coupled to avidin, the other to biotin.
Such antibodies have, for example, been proposed to target immune
system cells to unwanted cells (U.S. Pat. No. 4,676,980), and for
treatment of HIV infection (WO 91/00360, WO 92/00373, and EP
03089). Heteroconjugate antibodies may be made using any convenient
cross-linking methods. Suitable cross-linking agents are well known
in the art, and are disclosed in U.S. Pat. No. 4,676,980, along
with a number of cross-linking techniques.
[0237] Techniques for generating bispecific antibodies from
antibody fragments have also been described in the literature. For
example, bispecific antibodies can be prepared using chemical
linkage. Brennan et al., Science, 229: 81 (1985) describe a
procedure wherein intact antibodies are proteolytically cleaved to
generate F(ab').sub.2 fragments. These fragments are reduced in the
presence of the dithiol complexing agent sodium arsenite to
stabilize vicinal dithiols and prevent intermolecular disulfide
formation. The Fab' fragments generated are then converted to
thionitrobenzoate (TNB) derivatives. One of the Fab'-TNB
derivatives is then-reconverted to the Fab'-thiol by reduction with
mercaptoethylamine and is mixed with an equimolar amount of the
other Fab'-TNB derivative to form the bispecific antibody. The
bispecific antibodies produced can be used as agents for the
selective immobilization of enzymes.
[0238] Recent progress has facilitated the direct recovery of
Fab'-SH fragments from E. coli, which can be chemically coupled to
form bispecific antibodies. Shalaby et al., J. Exp. Med., 175:
217-225 (1992) describe the production of a fully humanized
bispecific antibody F(ab').sub.2 molecule. Each Fab' fragment was
separately secreted from E. coli and subjected to directed chemical
coupling in vitro to form the bispecific antibody. The bispecific
antibody thus formed was able to bind to cells overexpressing the
HER2 receptor and normal human T cells, as well as trigger the
lytic activity of human cytotoxic lymphocytes against human breast
tumor targets.
[0239] Various techniques for making and isolating bispecific
antibody fragments directly from recombinant cell culture have also
been described. For example, bispecific antibodies have been
produced using leucine zippers. Kostelny et al., J. Immunol.,
148(5):1547-1553 (1992). The leucine zipper peptides from the Fos
and Jun proteins were linked to the Fab' portions of two different
antibodies by gene fusion. The antibody homodimers were reduced at
the hinge region to form monomers and then re-oxidized to form the
antibody heterodimers. This method can also be utilized for the
production of antibody homodimers. The "diabody" technology
described by Hollinger et al., Proc. Natl. Acad. Sci. USA,
90:6444-6448 (1993) has provided an alternative mechanism for
making bispecific antibody fragments. The fragments comprise a
heavy-chain variable domain (VH) connected to a light-chain
variable domain (VL) by a linker that is too short to allow pairing
between the two domains on the same chain. Accordingly, the VH and
VL domains of one fragment are forced to pair with the
complementary VL and VH domains of another fragment, thereby
forming two antigen-binding sites. Another strategy for making
bispecific antibody fragments by the use of single-chain Fv (sFv)
dimers has also been reported. See Gruber et al., J. Immunol.,
152:5368 (1994).
[0240] Antibodies with more than two valencies are contemplated For
example, trispecific antibodies can be prepared. Tutt et al. J.
Immunol. 147: 60 (1991).
[0241] IV. Diagnostic Uses of Anti-IFNAR1 Antibodies
[0242] The anti-IFNAR1 antibodies of the invention are unique
research reagents which provide anti-type I interferon activity
templates for use in chemical library screening, wherein the
practitioner can use a signal transduction assay as an initial,
high volume screen for agents that exhibit an anti-type I
interferon activity pattern that is similar to the anti-type I
interferon activity pattern of an anti-IFNAR1 antibody of the
invention. In this way, candidate agents likely to exhibit a
desired type I interferon activity inhibition profile can be
obtained with ease, avoiding prohibitively expensive and
logistically impossible numbers of type I interferon induced viral
inhibition assays or cell proliferation inhibition assays on large
chemical libraries.
[0243] In one embodiment, the anti-IFNAR1 antibodies of the
invention are used to screen chemical libraries in a Kinase
Receptor Activation (KIRA) Assay as described in WO 95/14930
(published 1 Jun. 1995). The KIRA assay is suitable for use herein
because ligand binding to the type I interferon receptor complex in
situ in on the surface of host cells expressing the receptor
induces a rapid increase in the phosphorylation of tyrosine
residues in the intracellular domains of both IFNAR1 and IFNAR2
components of the receptor as taught in Platanias and Colamonici,
J. Biol. Chem., 269: 17761-17764 (1994). The level of tyrosine
phosphorylation can be used as a measure of signal transduction.
The effect of an anti-IFNAR1 antibody of the invention on the
levels of tyrosine phosphorylation induced by various type I
interferons in the KIRA assay can be used as a bench mark activity
pattern for comparison to the activity patterns generated by the
library compounds in the assay.
[0244] The KIRA assay suitable for use herein employs a host cell
that expresses the type I interferon receptor (both IFNAR1 and
IFNAR2 components of the receptor) and the particular series of
type I interferons which define the inhibitor profile of interest.
Cells which naturally express the human type I interferon receptor,
such as the human Daudi cells and U-266 human myeloma cells
described in Colamonici and Domanski, J. Biol. Chem., 268:
10895-10899 (1993), can be used. In addition, cells which are
transfected with the IFNAR1 and IFNAR2 components and contain
intracellular signaling proteins necessary for type I interferon
signal transduction, such as mouse L-929 cells as described in
Domanski et al., J. Biol. Chem., 270: 21606-21611 (1995), can be
used. In the KIRA assay, the candidate antagonist is incubated with
each type I interferon ligand to be tested, and each incubation
mixture is contacted with the type I interferon receptor-expressing
host cells. The treated cells are lysed, and IFNAR1 protein in the
cell lysate is immobilized by capture with solid phase anti-IFNAR1
antibody. Signal transduction is assayed by measuring the amount of
tyrosine phosphorylation that exists in the intracellular domain
(ICD) of captured IFNAR1 and the amount of tyrosine phosphorylation
that exists in the intracellular domain of any co-captured IFNAR2.
Alternatively, cell lysis and immunoprecipitation can be performed
under denaturing conditions in order to avoid co-capture of IFNAR2
and permit measurement of IFNAR1 tyrosine phosphorylation alone,
e.g. in a manner similar to the procedure described in Platanias et
al., J. Biol. Chem., 271: 23630-23633 (1996). The level of tyrosine
phosphorylation can be accurately measured with labeled
anti-phosphotyrosine antibody that identifies phosphorylated
tyrosine residues.
[0245] In another embodiment, a host cell coexpressing IFNAR2 and a
chimeric construct containing IFNAR1 fused at its carboxy terminus
to an affinity handle polypeptide is used in the KIRA assay. The
chimeric IFNAR1 construct permits capture of the construct from
cell lysate by use of a solid phase capture agent (in place of an
anti-IFNAR1 antibody) specific for the affinity handle polypeptide.
In a preferred embodiment, the affinity handle polypeptide is
Herpes simplex virus glycoprotein D (gD) and the capture agent is
an anti-gD monoclonal antibody as described in Examples 2 and 3 of
WO 95/14930.
[0246] In this system, the anti-IFNAR1 antibody of the invention
that possesses the type I interferon inhibition activity profile of
interest is used as a standard for analysis of the tyrosine
phosphorylation patterns generated by the members of the chemical
library that is screened. The IFNAR1 ICD tyrosine phosphorylation
pattern generated by the anti-IFNAR1 antibody standard is compared
to the tyrosine phosphorylation patterns produced in the library
screen, and patterns found to match that of the anti-IFNAR1
antibody standard identify candidate agents that are likely to have
a type I interferon activity inhibition profile similar to that of
the anti-IFNAR1 antibody standard. Accordingly, the anti-IFNAR1
antibody of the invention provides a useful means to quickly and
efficiently screen large chemical libraries for compounds likely to
exhibit the particular type I interferon activity inhibition
profile of the antibody.
[0247] The anti-IFNAR1 antibodies of the invention are useful in
diagnostic assays for IFNAR1 expression in specific cells or
tissues wherein the antibodies are labeled as described below
and/or are immobilized on an insoluble matrix. Anti-IFNAR1
antibodies also are useful for the affinity purification of IFNAR1
from recombinant cell culture or natural sources.
[0248] Anti-IFNAR1 antibodies can be used for the detection of
IFNAR1 in any one of a number of well known diagnostic assay
methods. For example, a biological sample may be assayed for IFNAR1
by obtaining the sample from a desired source, admixing the sample
with anti-IFNAR1 antibody to allow the antibody to form
antibody/IFNAR1 complex with any IFNAR1 present in the mixture, and
detecting any antibody/IFNAR1 complex present in the mixture. The
biological sample may be prepared for assay by methods known in the
art which are suitable for the particular sample. The methods of
admixing the sample with antibodies and the methods of detecting
antibody/IFNAR1 complex are chosen according to the type of assay
used. Such assays include competitive and sandwich assays, and
steric inhibition assays. Competitive and sandwich methods employ a
phase-separation step as an integral part of the method while
steric inhibition assays are conducted in a single reaction
mixture.
[0249] Analytical methods for IFNAR1 all use one or more of the
following reagents: labeled IFNAR1 analogue, immobilized IFNAR1
analogue, labeled anti-IFNAR1 antibody, immobilized anti-IFNAR1
antibody and steric conjugates. The labeled reagents also are known
as "tracers."
[0250] The label used is any detectable functionality that does not
interfere with the binding of IFNAR1 and anti-IFNAR1 antibody.
Numerous labels are known for use in immunoassay, examples
including moieties that may be detected directly, such as
fluorochrome, chemiluminescent, and radioactive labels, as well as
moieties, such as enzymes, that must be reacted or derivatized to
be detected. Examples of such labels include the radioisotopes
.sup.32P, .sup.14C, .sup.125I, .sup.3H, and .sup.131I, fluorophores
such as rare earth chelates or fluorescein and its derivatives,
rhodamine and its derivatives, dansyl, umbelliferone,
luceriferases, e.g., firefly luciferase and bacterial luciferase
(U.S. Pat. No. 4,737,456), luciferin, 2,3-dihydrophthalazinediones,
horseradish peroxidase (HRP), alkaline phosphatase,
.beta.-galactosidase, glucoamylase, lysozyme, saccharide oxidases,
e.g., glucose oxidase, galactose oxidase, and glucose-6-phosphate
dehydrogenase, heterocyclic oxidases such as uricase and xanthine
oxidase, coupled with an enzyme that employs hydrogen peroxide to
oxidize a dye precursor such as HRP, lactoperoxidase, or
microperoxidase, biotin/avidin, spin labels, bacteriophage labels,
stable free radicals, and the like.
[0251] Conventional methods are available to bind these labels
covalently to proteins or polypeptides. For instance, coupling
agents such as dialdehydes, carbodiimides, dimaleimides,
bis-imidates, bis-diazotized benzidine, and the like may be used to
tag the antibodies with the above-described fluorescent,
chemiluminescent, and enzyme labels. See, for example, U.S. Pat.
No. 3,940,475 (fluorimetry) and U.S. Pat. No. 3,645,090 (enzymes);
Hunter et al., Nature, 144: 945 (1962); David et al., Biochemistry,
13: 1014-1021 (1974); Pain et al., J. Immunol. Methods, 40: 219-230
(1981); and Nygren, J. Histochem. and Cytochem., 30: 407-412
(1982). Preferred labels herein are enzymes such as horseradish
peroxidase and alkaline phosphatase.
[0252] The conjugation of such label, including the enzymes, to the
antibody is a standard manipulative procedure for one of ordinary
skill in immunoassay techniques. See, for example, O'Sullivan et
al., "Methods for the Preparation of Enzyme-antibody Conjugates for
Use in Enzyme Immunoassay," in Methods in Enzymology, ed. J. J.
Langone and H. Van Vunakis, Vol. 73 (Academic Press, New York,
N.Y., 1981), pp. 147-166.
[0253] Immobilization of reagents is required for certain assay
methods. Immobilization entails separating the anti-IFNAR1 antibody
from any IFNAR1 that remains free in solution. This conventionally
is accomplished by either insolubilizing the anti-IFNAR1 antibody
or IFNAR1 analogue before the assay procedure, as by adsorption to
a water-insoluble matrix or surface (Bennich et al., U.S. Pat. No.
3,720,760), by covalent coupling (for example, using glutaraldehyde
cross-linking), or by insolubilizing the anti-IFNAR1 antibody or
IFNAR1 analogue afterward, e.g., by immunoprecipitation.
[0254] Other assay methods, known as competitive or sandwich
assays, are well established and widely used in the commercial
diagnostics industry.
[0255] Competitive assays rely on the ability of a tracer IFNAR1
analogue to compete with the test sample IFNAR1 for a limited
number of anti-IFNAR1 antibody antigen-binding sites. The
anti-IFNAR1 antibody generally is insolubilized before or after the
competition and then the tracer and IFNAR1 bound to the anti-IFNAR1
antibody are separated from the unbound tracer and IFNAR1. This
separation is accomplished by decanting (where the binding partner
was preinsolubilized) or by centrifuging (where the binding partner
was precipitated after the competitive reaction). The amount of
test sample IFNAR1 is inversely proportional to the amount of bound
tracer as measured by the amount of marker substance. Dose-response
curves with known amounts of IFNAR1 are prepared and compared with
the test results to quantitatively determine the amount of IFNAR1
present in the test sample. These assays are called ELISA systems
when enzymes are used as the detectable markers.
[0256] Another species of competitive assay, called a "homogeneous"
assay, does not require a phase separation. Here, a conjugate of an
enzyme with the IFNAR1 is prepared and used such that when
anti-IFNAR1 antibody binds to the IFNAR1 the presence of the
anti-IFNAR1 antibody modifies the enzyme activity. In this case,
the IFNAR1 or its immunologically active fragments are conjugated
with a bifunctional organic bridge to an enzyme such as peroxidase.
Conjugates are selected for use with anti-IFNAR1 antibody so that
binding of the anti-IFNAR1 antibody inhibits or potentiates the
enzyme activity of the label. This method per se is widely
practiced under the name of EMIT.
[0257] Steric conjugates are used in steric hindrance methods for
homogeneous assay. These conjugates are synthesized by covalently
linking a low-molecular-weight hapten to a small IFNAR1 fragment so
that antibody to hapten is substantially unable to bind the
conjugate at the same time as anti-IFNAR1 antibody. Under this
assay procedure the IFNAR1 present in the test sample will bind
anti-IFNAR1 antibody, thereby allowing anti-hapten to bind the
conjugate, resulting in a change in the character of the conjugate
hapten, e.g., a change in fluorescence when the hapten is a
fluorophore.
[0258] Sandwich assays particularly are useful for the
determination of IFNAR1 or anti-IFNAR1 antibodies. In sequential
sandwich assays an immobilized anti-IFNAR1 antibody is used to
adsorb test sample IFNAR1, the test sample is removed as by
washing, the bound IFNAR1 is used to adsorb a second, labeled
anti-IFNAR1 antibody and bound material is then separated from
residual tracer. The amount of bound tracer is directly
proportional to test sample IFNAR1. In "simultaneous" sandwich
assays the test sample is not separated before adding the labeled
anti-IFNAR1. A sequential sandwich assay using an anti-IFNAR1
monoclonal antibody as one antibody and a polyclonal anti-IFNAR1
antibody as the other is useful in testing samples for IFNAR1.
[0259] The foregoing are merely exemplary diagnostic assays for
IFNAR1. Other methods now or hereafter developed that use
anti-IFNAR1 antibody for the determination of IFNAR1 are included
within the scope hereof, including the bioassays described
above.
[0260] V. Therapeutic Compositions and Administration of
Anti-IFNAR1 Antibodies
[0261] Therapeutic formulations of the anti-IFNAR1 antibodies of
the invention are prepared for storage by mixing antibody having
the desired degree of purity with optional physiologically
acceptable carriers, excipients, or stabilizers (Remington: The
Science and Practice of Pharmacy, 19th Edition, Alfonso, R., ed,
Mack Publishing Co. (Easton, Pa.: 1995)), in the form of
lyophilized cake or aqueous solutions. Acceptable carriers,
excipients or stabilizers are nontoxic to recipients at the dosages
and concentrations employed, and include buffers such as phosphate,
citrate, and other organic acids; antioxidants including ascorbic
acid; low molecular weight (less than about 10 residues)
polypeptides; proteins, such as serum albumin, gelatin, or
immunoglobulins; hydrophilic polymers such as polyvinylpyrrolidone;
amino acids such as glycine, glutamine, asparagine, arginine or
lysine; monosaccharides, disaccharides, and other carbohydrates
including glucose, mannose, or dextrins; chelating agents such as
EDTA; sugar alcohols such as mannitol or sorbitol; salt-forming
counterions such as sodium; and/or nonionic surfactants such as
Tween, Pluronics or polyethylene glycol (PEG).
[0262] The anti-IFNAR1 antibody to be used for in vivo
administration-must be sterile. This is readily accomplished by
filtration through sterile filtration membranes, prior to or
following lyophilization and reconstitution. The anti-IFNAR1
antibody ordinarily will be stored in lyophilized form or in
solution.
[0263] Therapeutic anti-IFNAR1 antibody compositions generally are
placed into a container having a sterile access port, for example,
an intravenous solution bag or vial having a stopper pierceable by
a hypodermic injection needle.
[0264] The route of anti-IFNAR1 antibody administration is in
accord with known methods, e.g. injection or infusion by
intravenous, intraperitoneal, intracerebral, subcutaneous,
intramuscular, intraocular, intraarterial, intracerebrospinal, or
intralesional routes, or by sustained release systems as noted
below. Preferably the antibody is given systemically.
[0265] Suitable examples of sustained-release preparations include
semipermeable polymer matrices in the form of shaped articles, e.g.
films, or microcapsules. Sustained release matrices include
polyesters, hydrogels, polylactides (U.S. Pat. No. 3,773,919, EP
58,481), copolymers of L-glutamic acid and gamma ethyl-L-glutamate
(Sidman et al, Biopolymers, 22: 547-556 (1983)), poly
(2-hydroxyethyl-methacrylate). (Langer et al., J. Biomed. Mater.
Res., 15: 167-277 (1981) and Langer, Chem. Tech., 12: 98-105
(1982)), ethylene vinyl acetate (Langer et al., supra) or
poly-D-(-)-3-hydroxybutyric acid (EP 133,988). Sustained-release
anti-IFNAR1 antibody compositions also include liposomally
entrapped antibody. Liposomes containing antibody are prepared by
methods known per se: DE 3,218,121; Epstein et al., Proc. Natl.
Acad. Sci. USA, 82: 3688-3692 (1985); Hwang et al., Proc. Natl.
Acad. Sci USA, 77: 40304034 (1980); EP 52,322; EP 36,676; EP
88,046; EP 143,949; EP 142,641; Japanese patent application
83-118008; U.S. Pat. Nos. 4,485,045 and 4,544,545; and EP 102,324.
Ordinarily the liposomes are of the small (about 200-800 Angstroms)
unilamelar type in which the lipid content is greater than about 30
mol. % cholesterol, the selected proportion being adjusted for the
optimal antibody therapy.
[0266] Anti-IFNAR1 antibody can also be administered by inhalation.
Commercially available nebulizers for liquid formulations,
including jet nebulizers and ultrasonic nebulizers are useful for
administration. Liquid formulations can be directly nebulized and
lyophilized powder can be nebulized after reconstitution.
Alternatively, anti-IFNAR1 antibody can be aerosolized using a
fluorocarbon formulation and a metered dose inhaler, or inhaled as
a lyophilized and milled powder.
[0267] An "effective amount" of anti-IFNAR1 antibody to be employed
therapeutically will depend, for example, upon the therapeutic
objectives, the route of administration, the type of anti-IFNAR1
antibody employed, and the condition of the patient. Accordingly,
it will be necessary for the therapist to titer the dosage and
modify the route of administration as required to obtain the
optimal therapeutic effect. Typically, the clinician will
administer the anti-IFNAR1 antibody until a dosage is reached that
achieves the desired effect. The progress of this therapy is easily
monitored by conventional assays.
[0268] The patients to be treated with the anti-IFNAR1 antibody of
the invention include preclinical patients or those with recent
onset of immune-mediated disorders, and particularly autoimmune
disorders. Patients are candidates for therapy in accord with this
invention until such point as no healthy tissue remains to be
protected from immune-mediated destruction. For example, a patient
suffering from insulin-dependent diabetes mellitus (IDDM) can
benefit from therapy with an anti-IFNAR1 antibody of the invention
until the patient's pancreatic islet cells are no longer viable. It
is desirable to administer an anti-IFNAR1 antibody as early as
possible in the development of the immune-mediated or autoimmune
disorder, and to continue treatment for as long as is necessary for
the protection of healthy tissue from destruction by the patient's
immune system. For example, the IDDM patient is treated until
insulin monitoring demonstrates adequate islet response and other
indicia of islet necrosis diminish (e.g. reduction in anti-islet
antibody titers), after which the patient can be withdrawn from
anti-IFNAR1 antibody treatment for a trial period during which
insulin response and the level of anti-islet antibodies are
monitored for relapse.
[0269] In the treatment and prevention of an immune-mediated or
autoimmune disorder by an anti-IFNAR1 antibody, the antibody
composition will be formulated, dosed, and administered in a
fashion consistent with good medical practice. Factors for
consideration in this context include the particular disorder being
treated, the particular mammal being treated, the clinical
condition of the individual patient, the cause of the disorder, the
site of delivery of the antibody, the particular type of antibody,
the method of administration, the scheduling of administration, and
other factors known to medical practitioners. The "therapeutically
effective amount" of antibody to be administered will be governed
by such considerations, and is the minimum amount necessary to
prevent, ameliorate, or treat the disorder, including treating
chronic autoimmune conditions and immunosuppression maintenance in
transplant recipients. Such amount is preferably below the amount
that is toxic to the host or renders the host significantly more
susceptible to infections.
[0270] As a general proposition, the initial pharmaceutically
effective amount of the antibody administered parenterally will be
in the range of about 0.1 to 50 mg/kg of patient body weight per
day, with the typical initial range of antibody used being 0.3 to
20 mg/kg/day, more preferably 0.3 to 15 mg/kg/day. The desired
dosage can be delivered by a single bolus administration, by
multiple bolus administrations, or by continuous infusion
administration of antibody, depending on the pattern of
pharmacokinetic decay that the practitioner wishes to achieve.
[0271] As noted above, however, these suggested amounts of antibody
are subject to a great deal of therapeutic discretion. The key
factor in selecting an appropriate dose and scheduling is the
result obtained, as indicated above.
[0272] The antibody need not be, but is optionally formulated with
one or more agents currently used to prevent or treat the
immune-mediated or autoimmune disorder in question. For example, in
rheumatoid arthritis, the antibody may be given in conjunction with
a glucocorticosteroid. The effective amount of such other agents
depends on the amount of anti-IFNAR1 antibody present in the
formulation, the type of disorder or treatment, and other factors
discussed above. These are generally used in the same dosages and
with administration routes as used hereinbefore or about from 1 to
99% of the heretofore employed dosages.
[0273] Further details of the invention can be found in the
following example, which further defines the scope of the
invention. All references cited throughout the specification, and
the references cited therein, are hereby expressly incorporated by
reference in their entirety.
EXAMPLE
Materials and Methods
Preparation of Soluble IFNAR1-IgG.
[0274] A cDNA encoding the human immunoglobulin fusion proteins
(immunoadhesins) based on the ECD of the hIFNAR1 (pRK5 hIFNAR1-IgG
clone 53.65) was generated using methods similar to those described
by Haak-Frendscho et. al., Immunology 79: 594-599 (1993) for the
construction of a murine IFN--Y receptor immunoadhesin. Briefly,
the plasmid pRKCD4.sub.2Fc, was constructed as described in Example
4 of WO 89/02922 (PCT/US88/03414 published Apr. 6, 1989). The cDNA
coding sequence for the 404 amino acid ECD of mature hIFNAR1 shown
in FIG. 7 was obtained from the published sequence (Uze et al.,
Cell, 60: 225-234 (1990)). The CD4 coding sequence in the
pRKCD4.sub.2Fc, was replaced with the hIFNAR1 ECD encoding cDNA to
form pRK5hIFNAR1-IgG clone 53.65. The nucleic acid sequence (SEQ ID
NO. 21) and amino acid sequence (SEQ ID NO. 22) for the hIFNAR1
ECD-IgG encoding insert of clone 53.65 are shown in FIG. 7.
hIFNAR1-IgG was expressed in human embryonic kidney 293 cells by
transient transfection using a calcium phosphate precipitation
technique. The immunoadhesin was purified from serum-free cell
culture supernatants in a single step by affinity chromatography on
a protein A-sepharose column as described in Haak-Frendscho et al.
(1993), supra. Bound hIFNAR1-IgG was eluted with 0.1 M citrate
buffer, pH 3.0, containing 20% (w/v) glycerol. The hIFNAR1-IgG
purified was >95% pure, as judged by SDS-PAGE.
Production of hIFN-.alpha. Subtypes.
[0275] Standard cloning procedures described in Maniatis et al.,
Molecular Cloning: A Laboratory Manual, Cold Spring Harbor
Laboratory Press, Cold Spring Harbor, N.Y. (1989) were used to
construct plasmids that direct the translocation of the various
species of hIFN-.alpha. into the periplasmic space of E. coli. PCR
reactions were performed on cDNA clones of the various subspecies
of hIFN-.alpha. disclosed in Goeddel et al., Nature 290: 20-26
(1981) with NsiI and StyI restriction sites added to the primers.
These PCR products were then subcloned into the corresponding sites
of the expression vector pB0720 described in Cunningham et al.,
Science 243:1330-1336 (1989). The resulting plasmids placed
production of the hIFN-.alpha. subtypes under control of the E.
coli phoA promoter and the heat-stable enterotoxin II signal
peptide as described in Chang et al., Gene 55: 189-196 (1987). The
correct DNA sequence of each gene was confirmed using the United
States Biochemical Sequenase Kit version 2.0. Each plasmid was
transformed into the E. coli strain 27C7 (ATCC # 55244) and grown
in 10 liter fermentors as described in Carter et al.,
Bio/Technology 10: 163-167 (1992). Human hIFNs were purified from
E. coli paste containing each IFN-.alpha. by affinity
chromatography. Bacterial cells were lysed, and the lysate was
centrifuged at 10,000.times.g to remove debris. The supernatant was
applied to an immunoaffinity column containing a mouse
anti-hIFN-.alpha.B antibody (LI-1) that was obtained as described
in Staehelin et al., Proc. Natl. Acad. Sci. 78:1848-1852 (1981).
LI-1 was immobilized on controlled pore glass by a modification of
the method of Roy et al., Journal of Chromatography, 303: 225-228
(1984). The bound interferon was eluted from the column with 0.1 M
citrate, pH 3.0, containing 20% (w/v) glycerol. The purified IFN
was analyzed by SDS-PAGE and immunoblotting, and was assayed for
bioactivity by the hIFN-induced anti-viral assay as described
herein. hIFN.beta. was obtained from Sigma (St. Louis, Mo.) and
IFN-.alpha.1/2 was obtained as described in Rehberg et al., J.
Biol. Chem., 257: 11497-11502 (1992) or Horisberger and Marco,
Pharmac. Ther., 66: 507-534 (1995).
Generation of mAbs to hIFNAR1.
[0276] Balb/c mice were immunized into each hind foot pad 11 times
at two week intervals, with 2.5 .mu.g of hIFNAR1-IgG fusion protein
resuspended in MPL-TDM (Ribi Immunochem. Research Inc., Hamilton,
Mont.). Three days after the final boost, popliteal lymph node
cells were fused with murine myeloma cells, P3X63AgU.1 (ATCC
CRL1597), using 35% polyethylene glycol. Hybridomas were selected
in HAT medium. Ten days after the fusion, hybridoma culture
supernatants were first screened for mAbs binding to the
hIFNAR1-IgG fusion protein in a capture ELISA. The selected culture
supernatants were then tested by flow cytometric analysis for their
ability to recognize the hIFNAR1 on U266 cells as described in
Chuntharapai et al., J. Immunol., 152:1783-1789 (1994). The
blocking mAbs were selected for their ability to inhibit the
anti-viral cytopathic effect of IFN as described below.
[0277] The affinities of these mAbs were determined in a
competitive binding radioimmunoprecipitation assay according to the
method of Kim et al., J. Immunol. Method, 156: 9-17 (1992).
Briefly, .sup.125-hIFNAR1-IgG (specific activity 11.6 .mu.Ci/.mu.g)
was prepared using a lactoperoxidase labeling method. mAbs were
allowed to bind to .sup.125I-hIFNAR1-IgG in the presence of various
concentrations of unlabeled hIFNAR1-IgG for 1 hour at room
temperature (RT). These mixtures were then incubated with goat
anti-mouse IgG for 1 hour at RT in the presence of 5% human serum.
The immune complexes were then precipitated by the addition of cold
6% polyethylene glycol (MW 8,000) followed by centrifugation at
200.times.g for 20 minutes at 4.degree. C. Supernatants were
removed and the radioactivity remaining in the pellet was
determined using a gamma counter. The affinity of each mAb was
determined according to the method of Munson et al., Anal. Biochem.
107: 220-239 (1980).
Capture ELISA.
[0278] Microtiter plates (Maxisorb; Nunc, Kamstrup, Denmark) were
coated with 50 .mu.l/well of 2 .mu.g/ml of goat antibodies specific
to the Fc portion of human IgG (Goat anti-hIgG-Fc, Cappel), in PBS,
overnight at 4.degree. C. and blocked with 2% BSA for 1 hour at
room temperature. After washing the plates, 50 .mu.l/well of 2
.mu.g/ml of IFNAR1-IgG (or IFNAR1-IgG mutant) was added, and plates
were incubated for 1 hour. After washing the plates, the remaining
anti-Fc binding sites were blocked with PBS containing 3% human
serum and 10 .mu.g/ml of CD4-IgG for 1 hour. After washing, plates
were then incubated with 50 .mu.l/well of 2 .mu.g/ml of anti-IFNAR1
mAbs (or hybridoma culture supernatants) for 1 hour. After washing,
plates were then incubated with 50 .mu.l/well of HRP-Goat
anti-mouse IgG. The bound enzyme was detected by the addition of
the substrate and the plates were read at 490 nM with an ELISA
plate reader. Between each step, plates were washed in wash buffer
(PBS containing 0.05% Tween 20).
[0279] During the IFNAR1-IgG mutant analysis, the concentrations of
immunoadhesin molecules in 293 transfected culture supernatants
were determined using CD4-IgG as a standard and were adjusted to be
equal to the lowest concentration of immunoadhesin molecules. The
degree of mAb binding to these mutants were then compared to the
wild type of the same concentration.
Western Blot.
[0280] Reduced hIFNAR1 was prepared by treating the hIFNAR1-IgG
fusion protein with 5 mM of 2-mercaptoethanol at 95.degree. C. for
5 minutes. The ability of the mAbs to bind to the native and
reduced hIFNAR1-IgG was determined by immunoblotting using 12%
SDS-PAGE as described in Kim et al., J. Immunol. Method 156: 9-17
(1992).
Epitope Mapping Using a Competitive Binding ELISA.
[0281] To determine whether the mAbs recognized the same or
different epitopes, a competitive binding ELISA was performed as
described in Kim et al., (1992), supra, using biotinylated mAbs
(Bio-mAb). mAbs were biotinylated using N-hydroxyl succinimide as
described in Antibodies (A Laboratory Manual), Harlow, E. and Lane,
D., eds, Cold Spring Harbor (1988), p. 341. Microtiter wells were
coated with 50 .mu.l of Goat anti-hIgG-Fc and kept overnight at
4.degree. C., blocked with assay buffer for 1 hour, and incubated
with 251 .mu.l/well of IFNAR1-IgG (1 .mu.g/ml) for 1 hour at room
temperature. After washing microtiter wells, a mixture of a
predetermined optimal concentration of Bio-mAb and a thousand-fold
excess of unlabeled mAb was added into each well. Following 1 hour
incubation at room temperature, plates were washed and the amount
of Bio-mAb was detected by the addition of HRP-streptavidin. After
washing the microtiter wells, the bound enzyme was detected by the
addition of the substrate, and the plates were read at 490 nm with
an ELISA plate reader.
Electrophoretic Mobility Shift Assay (EMSA)
[0282] Briefly, .alpha.-IFNs (25 ng/ml) plus various concentrations
(5-500 .mu.g/ml) of anti-hIFNAR1 mAbs were incubated with
5.times.10.sup.5 Hela cells in 200 .mu.l of DMEM for 30 minutes at
37.degree. C. Cells were washed in PBS and resuspended in 125 .mu.l
of buffer A (10 mM HEPES, pH 7.9, 10 mM KCL, 0.1 mM ETDA, 1 mM DTT,
1 mM phenylmethylsulfonyl fluoride, 10 .mu.g/ml leupeptin, 10
.mu.g/ml aprotinin) as described in Kurabayashi et al., Mol. Cell
Biol., 15: 6386 (1995). After a 15 minute incubation on ice, cells
were lysed by the addition of 0.025% NP40. The nuclear pellet was
obtained by centrifugation and was resuspended in 50 .mu.l of
buffer B (20 mM HEPES, pH 7.9, 400 mM NaCl, 0.1 mM EDTA, 1 mM DTT,
1 mM phenylmethylsulfonyl fluoride, 10 .mu.g/ml leupeptin, 10
.mu.g/ml aprotinin) and incubated on ice for 30 minutes. The
nuclear fraction was clarified by centrifugation and the
supernatant stored at -70.degree. C. until use. Double-stranded
probes were prepared from single-stranded oligonucleotides (ISG15
top: 5'-GATCGGGAAAGGGAAACGAAACTGAAGCC-3' (SEQ ID NO:23)), ISG15
bottom: 5'-GATCGGCTTCAGTTTCGGTTTCCCTTTCCC-3' (SEQ ID NO:24))
utilizing a DNA polymerase I Klenow fill-in reaction with
.sup.32P-DATP (3,000 Ci/mM, Amersham). Labeled oligonucleotides
were purified from unincorporated radioactive nucleotides using
BIO-Spin 30 columns (Bio-Rad). Binding reactions, containing 5
.mu.l of nuclear extract, 25,000 cpm of labeled probe and 2 .mu.g
of non-specific competitor poly (dI-dC)-poly (dI-dC) in 15 .mu.l of
binding buffer (10 mM Tris-HCL, pH 7.5, 50 mM NaCl, 1 mM EDTA, 1 mM
DTT, 1 mM phenylmethylsulfonyl fluoride and 15% glycerol) were
incubated at room temperature for 30 minutes. DNA-protein complexes
were resolved in 6% non-denaturing polyacrylamide gels (Novex) and
analyzed by autoradiography. The specificity of the assay was
determined by the addition of 350 ng of unlabeled ISG15 probe in
separate reaction mixtures. Formation of an ISGF3 specific complex
was confirmed by a super shift assay with anti-STATI antibody.
Assay for hIFN.alpha. Induced Anti-Viral Activity.
[0283] The assay was done as described in Current Protocols in
Immunol., Coligan, J. E., Kruisbeek, A. M., Margulies, D. H.,
Shevach, E. M., and Strober, W., eds, Greene Publishing Associates
and Wiley-Interscience (1992), Vol. 1, Unit 6.9.1, using the human
lung carcinoma cell line A549 challenged with encephalomyocarditis
virus (EMC). Briefly, A549 cells seeded at 2.times.10.sup.5
cells/100 .mu.l were grown in DMEM containing 2 mM glutamine,
antibiotics, and 5% FCS for 24 hours. Serial dilutions of mAbs in
50 .mu.l DMEM were incubated with various units of type 1 IFNs in
50 .mu.l DMEM for 1 hour at 37.degree. C. These mixtures were then
incubated with A549 cells (5.times.10.sup.5 cells/100 .mu.l of DMEM
containing 4% FCS) for another 24 hours. Culture supernatants were
removed and cells were challenged with 2.times.10.sup.5 pfu of EMC
virus in 100 .mu.l for an additional 24 hours. At the end of the
incubation, cell viability was determined by visual microscopic
examination. The neutralizing antibody titer (EC50) was defined as
the concentration of antibody that neutralizes 50% of the
anti-viral cytopathic effect by 10 unit/ml of type 1 IFNs. The
units of type 1 IFNs used in this study were determined using NIH
reference recombinant human IFN-.alpha.2 (IFN-.alpha.A) as a
standard. The specific activities of the various type 1 IFNs
utilized were IFN-.alpha.2/1 (2.times.10.sup.7 IU/mg), IFN-.alpha.1
(3.times.10.sup.7 IU/mg), IFN-.alpha.2 (2.times.10.sup.7 IU/mg),
IFN-.alpha.5 (8.times.10.sup.7 IU/mg), IFN-.alpha.8
(19.times.10.sup.7 IU/mg) and IFN-.beta. (1.5.times.10.sup.5
IU/mg).
Generation of Domain 1-IgG, Domain 2-IgG and Various Mutants to the
hIFNAR1.
[0284] The cDNAs encoding domain 1 (1-200 residues) and domain 2
(204-404 residues) of IFNAR1 were separately constructed and
expressed as immunoadhesins.
[0285] Single alanine substitution mutants were generated according
to the method of Kunkel et al., Methods Enzymol. 154: 367-414
(1987), and Hebert et al, J. Biol. Chem., 268: 18549-18553 (1993).
The plasmid DNA was isolated using an RPM Kit (BIO 101 Inc., La
Jolla, Calif.) and was sequenced by the Sanger method using an ABI
373A DNA sequencer to verify the mutation. Mutant receptor-IgGs
were expressed transiently in human 293 cells as described above.
Transfected 293 cells were grown overnight in F-12:DMEM (50:50)
containing 10% FCS, 2 mM glutamine, 100 .mu.g/ml of penicillin, 100
.mu.g/ml of streptomycin, 10 .mu.g/ml of glycine, 15 .mu.g/ml of
hypoxanthine, and 5 .mu.g/ml of thymidine, and then were placed in
serum-free media. Three days later, culture supernatants were
collected and used in a capture ELISA. For the hIFNAR1-IgG mutant
analysis, the concentrations of immunoadhesin molecules in 293
transfected culture supernatants were determined using CD4-IgG as a
standard and were adjusted to be equal to the lowest concentration
of immunoadhesin molecules. The degree of mAb binding to these
mutants was then compared to the wild type of the same
concentration.
Results
mAb Binding to Different Sites on hIFNAR1.
[0286] Five anti-hIFNAR1 mAbs (2E1, 2E8, 2H6, 4A7, and 5A8)
producing hybridomas (generated as described above) that exhibited
different binding epitopes and blocking activities described below
were selected for further characterization; All of these mAbs are
of the IgG2a isotype and recognized the IFNAR1 expressed on U266
human myeloma cells as determined by FACS analysis (Table I below).
Western blot analysis determined that only mAbs 2H6, 4A7, and 5A8
bind to the reduced IFNAR1 as shown in Table I below. This
indicated that mAbs 2E1 and 2E8 recognize conformational epitopes
while mAbs 2H6, 4A7, and 5A8 recognize linear epitopes. The
dissociation constants of these mAbs for IFNAR1-IgG were determined
to be in the range of 52-3,120 .mu.M as shown in Table I below, as
determined by competitive radioimmunoprecipitation followed by
Scatchard analysis. TABLE-US-00001 TABLE I General Characteristics
of mAbs to hIFNAR1 mAbs FACS.sup.a Immunoblot.sup.b Kd.sup.-1
(pM).sup.c epitope.sup.d Blocking act..sup.e 2E1 ++ - 66 A1
.alpha.2/1, .alpha.1, .alpha.2, .alpha.5, .alpha.8 2E8 ++ - 97 A2
None 2H6 ++ + 3120 B None 4A7 ++ + 52 C .alpha.2/1, .alpha.1,
.alpha.2, .alpha.5, .alpha.8 5A8 ++ + 174 D .alpha.8.sup.f
.sup.aFACS staining was done using the human myeloma cell line
U266. .sup.bThe immunoblot was performed using reduced hIFNAR1.
.sup.cThe affinities of these mAbs for soluble hIFNAR1-IgG were
determined by Scatchard analysis. .sup.dThe epitopes recognized by
these mAbs as determined by competitive binding ELISA were named
arbitrarily. .sup.eSummary of results from anti-viral assay and
ISGF3 EMSA. .sup.fThe blocking activity was observed only in the
EMSA.
[0287] To determine whether each mAb recognizes the same or
different epitopes, competitive binding ELISAs were performed to
detect the binding of each biotinylated mAb in the presence of
excess unlabeled mAb. The results from the competitive binding
ELISA (shown in FIG. 2) determined that these five mAbs could
detect four different epitopes on IFNAR1. mAbs 2E1 and 2E8 can
compete with each other, which indicates that they recognize the
same or an overlapping epitope.
Ability of mAbs to Block Type I IFN Activity.
[0288] The blocking activities of mAbs to hIFNAR1 were determined
using an ISGF3 electrophoretic mobility shift assay (EMSA) as well
as an anti-viral assay. Type 1 IFNs induce the transcription of
interferon-stimulated genes through the formation and activation of
IFN-stimulating response element (ISRE) binding proteins. One of
these binding proteins is ISGF3 which is a multi-subunit protein
complex formed in the cytoplasm within minutes of type 1 IFN
treatment (Schindler et al., Proc. Natl. Acad. Sci. (USA), 89: 7836
(1997); Fu et al., Proc. Natl. Acad. Sci. (USA), 89: 7840 (1997)).
By investigating ISGF3 formation in Hela cells induced by the
addition of 25 ng/ml of several human type 1 IFNs (IFN-.alpha.2/1,
-.alpha.1, -.alpha.2, -.alpha.5, -.alpha.8 and IFN-.beta.), the
blocking activities of mAbs were detected in the range of 5-500
.mu.g mAb/ml. FIG. 3 contains representative autoradiographs
depicting. ISGF3, formation induced by hIFN-.alpha.8
(IFN-.alpha.D). mAbs 2E1 and 4A7 inhibited ISGF3 formation induced
by IFN-.alpha.8 at a concentration of 5 .mu.g mAb/ml; mAb 5A8
completely inhibited the activity of IFN-.alpha.8 at a
concentration of 500 .mu.g mAb/ml and partially inhibited the
activity of IFN-.alpha.8 at a concentration of 50 .mu.g mAb/ml;
mAbs 2E8 and 2H6 were unable to block the activity of IFN-.alpha.8.
Results obtained with all type 1 IFNs tested are summarized in
Table II below. Although there is some variation in the potency of
blocking activities of mAbs 2E1 and 4A7 depending upon the
subspecies of IFN-.alpha., mAbs 2E1 and 4A7 inhibited the
activities of all IFN-.alpha.s tested and mAb 2E1 was a more potent
inhibitor. At a concentration of 500 .mu.g mAb/ml, mAb 5A8 showed
blocking activity on IFN-.alpha.8 and partial blocking activities
on -.alpha.2/1 and -.alpha.2. mAbs 2E8 and 2H6 showed no blocking
activity on any of these hIFN-.alpha.s. None of these mAbs to
hIFNAR1 were able to block ISGF3 formation induced by IFN-.beta..
TABLE-US-00002 TABLE II Effects of anti-hIFNAR1 mAbs on ISGF3
formation induced by type 1 IFNs Ab .mu.g/ml IFN.alpha.2/1
IFN.alpha.1 IFN.alpha.2 IFN.alpha.5 IFN.alpha.8 IFN.beta. 2E1 5 - -
+ - + - 50 + + + +/- + - 500 + + + + + - 2E8 5 - - - - - - 50 - - -
- - - 500 - - - - - - 2H6 5 - - - - - - 10 - - - - - - 100 - - - -
- - 4A7 5 - - +/- - + - 50 + +/- +/- - + - 500 + + + +/- + - 5A8 5
- - - - - - 50 - - - - +/- - 500 +/- - +/- - + - IgG 5 - - - - - -
ISGF3 EMSA was carried out using Hela cells treated with 25 ng/ml
of IFNs plus 5-500 .mu.g/ml of mAbs for 30 min. Results were
expressed as complete blocking (+), partial blocking (+/-) and no
blocking (-). A typical autoradiograph is shown in FIG. 2.
[0289] The neutralizing effect of these mAbs was also characterized
by anti-viral assays (Table III below). Assays were done using
serial dilutions of mAbs in the range of 0.1 to 30 .mu.g mAb/ml and
10 units/ml of type 1 IFNs. The units of these IFNs were determined
using NIH IFN-.alpha.2 (IFN-.alpha.A) as a standard. mAb 2E1 and
mAb 4A7 blocked the activity of all IFN-.alpha.s. Abs 2E8m 2H6 and
5A8 showed no neutralizing activities in the anti-viral assay. None
of these mAbs were able to neutralize the effect of IFN-.beta..
Similar results were obtained using 100 units/ml of type 1 IFNs.
Overall, the results obtained in the anti-viral assay correlated
well with the results obtained in the EMSA assay. TABLE-US-00003
TABLE III Effects of anti-hIFNAR1 mAbs on the anti-viral effects of
type 1 IFNs EC50 of mAb (.mu.g/ml) mAb IFN.alpha.2/1 IFN.alpha.1
IFN.alpha.2 IFN.alpha.5 IFN.alpha.8 IFN.beta. 2E1 3 3 1 1 1 NB 2E8
NB NB NB NB NB NB 2H6 NB NB NB NB NB NB 4A7 20 10 10 6 3 NB 5A8 NB
NB NB NB NB NB The neutralizing antibody titer (EC50) was defined
as the concentration of antibody which neutralizes 50% of the
anti-viral cytopathic effects induced by 10 units/ml of type 1 IFNs
on A549 cells. The experiment was done using serial dilutions of
mAbs in the range of 0.1-30 .mu.g/ml in duplicate. mAbs found to
exhibit no blocking effect at a concentration of 30 .mu.g/ml in
this assay were designated as nonblocking mAb (NB).
[0290] From the results of the ISGF3 formation assays (Table II)
and the anti-viral assay (Table III), it was determined that mAbs
2E1 and 4A7 are blocking mAbs against all the IFN-.alpha.s tested,
mAb 5A8 is a very weak blocking mAb, and mAbs 2E8 and 2H6 are
nonblocking mAbs. None of these mAbs was able to block the activity
of hIFN-.beta..
Both Domain 1 and 2 of the IFNAR1 may be Required for IFN
Signaling.
[0291] Domain 1 (residues 1-200) and domain 2 (residues 204-404) of
IFNAR1 were expressed separately as immunoadhesins, as shown in
FIG. 4, and the binding capacity of the blocking mAbs was
determined against the domain 1 and domain 2 adhesin molecules in a
capture ELISA. The concentrations of domain 1-IgG and domain 2-IgG
in the culture supernatant were determined by comparison to the
known concentrations of CD4-IgG in an ELISA. mAbs 2H6 and 4A7 bound
only to domain 1-IgG. mAb 5A8 bound to both domain 1-IgG and domain
2-IgG, while mAbs 2E1 and 2E8 were unable to bind to either of
these domain-IgGs as shown in FIG. 5. These results indicate that
three out of five mAbs bound to domain 1, and implicate the
participation of domain 1 in IFN signaling. Also, mAbs 2E1 and 2E8
were determined to recognize conformational epitopes composed of
regions in both domains 1 and 2, implicating the participation of
both domains in the IFN signaling.
Determination of mAb Binding to Alanine Substitution Mutants of the
hIFNAR1.
[0292] To define areas of IFNAR1 which play an important role in
mAb binding, multiple alanine substitution mutants in the
hydrophilic regions of IFNAR1 were generated. Residues 19-25,
69-74, 76-80, 103-111, 148-152, 157-162, 244-249, 291-298, 352-359,
and 383-388 were selected for mutagenesis as shown in FIG. 4. After
adjusting the concentrations (30-100 ng/ml) of the IFNAR1-IgG
mutants in the culture supernatants of 293 transfectants to be
equivalent, the binding abilities of the mAbs to these mutants were
determined in a capture ELISA. The results shown in Table IV below
were obtained using mAbs at a concentration of 10 .mu.g/ml in the
capture ELISA. The binding capacity of the most potent blocking
mAb, 2E1, was significantly reduced or almost undetectable when the
hydrophilic amino acids in residues 69-74 (domain 1), 244-249
(domain 2) or 291-298 (domain 2) were substituted with alanines as
shown in Table 2 below. The binding to the alanine mutant of
residues 69-74 was significantly reduced with all mAbs except mAb
5A8. The binding of mAb 5A8 to this mutant was 67% of binding to
the wild type. Since mAb 5A8 was shown to bind to domain 1-IgG and
domain 2-IgG separately (FIG. 6), some of the 67% binding to this
69-74 mutant by mAb 5A8 is believed to be due to binding with
domain 2. Thus, the alanine substitution of residues 69-74 affected
the binding of all mAbs, indicating that some structural change
occurs in this portion of the receptor which interferes with the
interaction between mAbs 2E1 and 2E8 and IFNAR1. TABLE-US-00004
TABLE IV The binding of mAbs to IFNAR1 multiple alanine mutants %
wild type binding of mAbs Mutant Alanine substitution 2E1 2E8 2H6
4A7 5A8 1 19-25 (RWNRSDE (SEQ ID NO. 1)-AWNASAA 101 84 77 95 110
(SEQ ID NO. 2)) 2 69-74 (EEIKLR (SEQ ID NO. 3)-AAIALA 21 18 0 0 67
(SEQ ID NO. 4)) 3 76-80 (RAEKE (SEQ ID NO. 5)-AAAAA 97 69 48 92 109
(SEQ ID NO. 6)) 4 103-111 (EVHLEAEDK (SEQ ID NO.7)-AVALAAAAA 66 33
39 80 34 (SEQ ID NO. 8)) 5 148-152 (EERIE (SEQ ID NO. 9)-AAAIA 87
43 68 90 80 (SEQ ID NO. 10)) 6 157-162 (RHKIYK (SEQ ID NO.
11)-AAAIYA 84 77 90 100 100 (SEQ ID NO. 12)) 7 244-249 (HLYKWK (SEQ
ID NO. 13)-ALYAWA 0 77 105 106 110 (SEQ ID NO. 14)) 8 291-298
(EEIKFDTE (SEQ ID NO. 15)-AAIAFATA 6 0 64 96 75 (SEQ ID NO. 16)) 9
352-359 (ERKIIEKK (SEQ ID NO. 17)-AAAIIAAA 105 81 101 101 81 (SEQ
ID NO. 18)) 10 383-388 (DEKLNK (SEQ ID NO. 19)-AAALNA 105 116 93
103 83 (SEQ ID NO. 20)) The level of binding was determined in a
capture ELISA. The % binding was calculated by dividing the binding
O.D. to each mutant-IgG by the binding O.D to the wild type
IFNAR1-IgG.
[0293] To determine which residues were important for the mAb
binding in residues 69-74, 244-249, and 291-298, single alanine
mutants were generated and examined for their ability to bind to
mAbs in capture ELISA as described above. The results of these
binding studies are shown in Table V below. In domain 1, Arg74 was
determined to be the crucial residue for the binding of mAb 2H6. In
domain 2, residues Glu291 and Asp296 were determined to play
important roles in the binding of mAbs 2E1 and 2E8. In addition,
Lys249 was also found to be important for the binding of mAb 2E1.
TABLE-US-00005 TABLE V mAb binding to IFNAR1 single alanine mutants
% Wild type binding of mAbs area mutant 2E1 2E8 2H6 4A7 5A8 AA
69-74 E69A 72 72 64 69 91 E70A 81 80 79 85 83 K72A 90 89 89 91 110
R74A 57 53 0 30 84 AA 244-249 H244A 92 96 94 96 99 K247A 74 66 88
86 93 K249A 5 54 69 73 71 AA 291-298 E291A 7 3 49 58 61 E292A 34 29
55 58 66 K294A 54 54 65 69 82 D296A 5 3 49 53 70 E298A 36 31 53 60
72
Inhibition of mAb Binding to Membrane hIFNAR1 by Soluble
hIFNAR1-IgG.
[0294] The above-described epitope mapping studies were performed
with soluble receptor proteins. In order to demonstrate that the
binding of these mAbs to a soluble hIFNAR1-IgG reflects the
behavior of the ECD displayed by a membrane associated hIFNAR1, the
ability of mAbs to bind membrane hIFNAR in the presence of soluble
hIFNAR-IgGs was determined. Fluoresceinated (F-) mAbs were
incubated with wild type or mutant soluble hIFNAR1-IgGs at room
temperature for 30 minutes. These mixtures were then added to U266
human myeloma cells. After incubation at 4.degree. C. for 30
minutes, cells were washed and analyzed by FACS. In the presence of
wild type hIFNAR1-IgG, the binding of F-2E1 to U266 cells was
completely inhibited as shown in Table VI below. TABLE-US-00006
TABLE VI Inhibition of mAb binding to U266 cells by soluble
hIFNAR1-IgG mutants as determined by Flow Cytometry Mean
Fluorescence Intensity soluble hIFNAR1 F-2E1 F-2E8 F-4A7 F-IgG None
7.60 7.96 9.49 2.99 wild type 2.83 3.16 2.45 -- Mutant #7 7.82 3.27
2.95 -- Mutant #8 7.65 7.60 3.16 -- Fluoresceinated mAbs (1
.mu.g/100 .mu.l) were incubated with 10 .mu.g of soluble
hIFNAR1-IgGs for 30 minutes at room temperature. These mixtures
were then added to U266 cells (10.sup.5 cells/25 .mu.l) and
incubated for 30 minutes at 4.degree. C. After washing, cells were
analyzed by FACScan. Mutant #7 and mutant #8 have multiple alanine
substitutions at residues 244-249 (HLYKWK-ALYAWA) and residues
291-298 (EEIKFDTE-AAIAFATA) as shown in Table IV.
The same results were obtained with mAbs F-2E8 and F4A7. These
results demonstrated that wild type soluble hIFNAR1 can effectively
inhibit the mAb binding to membrane hIFNAR1 on U266 cells and
indicated that the structure of the soluble hIFNAR1-IgG indeed
mimics the structure of the ECD of membrane hIFNAR1. In addition,
inhibition experiments were performed with soluble hIFNAR1-IgG
mutants (designated as Mutants #7 and #8 in Table IV). As expected,
soluble mutant # 7 (alanine substitutions in residues 244-249)
inhibited the binding of mAbs F-2E8 and F4A7 but did not inhibit
the binding of F-2E1 while soluble mutant #8 (alanine substitutions
in residues 291-298) inhibited the binding of mAbs F4A7 but did not
inhibit the binding of F-2E1 and F-2E8. From these results, it was
determined that the soluble and membrane bound IFNAR1 epitopes
recognized by mAb 2E1 include residues 244-249 and 291-298 and the
soluble and membrane bound IFNAR1 epitopes recognized by mAb 2E8
include residues 291-298. Discussion
[0295] The results obtained in these studies demonstrated that both
domain 1 and domain 2 of hIFNAR1 are necessary to mediate an
IFN-.alpha. signal. First, the blocking mAb 4A7 bound to the domain
1-IgG, which indicated the participation of domain 1 in IFN
signaling. Second, the presence of domains 1 and 2 of hIFNAR1 and
amino acid residue K249 in domain 2 was required for the binding of
the most potent blocking mAb 2E1.
[0296] It was found that wild type and mutant soluble receptors
effectively inhibited mAb binding to membrane hIFNAR1 in a specific
manner. This result indicated that soluble hIFNAR1 retains the
structure of the ECD of membrane hIFNAR1, at least in the antibody
binding region.
[0297] The angle between the two subdomains is significantly
different between members of class 1 and class 2 of the cytokine
receptor family reported in Kossiakoff et al., Protein Sci. 3:
1697-1705 (1994). In class 1, the structures of the hGH receptor
(reported in de Vos et al., Science 255: 306-312 (1992)) and the
prolactin receptor (reported in Somers et al., Nature 372: 478-481
(1994)) display an angle of about 85.degree., whereas in class 2,
the structures of tissue factor (reported in Muller et al., J. Mol.
Biol. 256: 144-159 (1996)) and the IFN-.gamma. receptor (reported
in Walter et al., Nature 376: 230-235 (1995)) display an angle of
about 120.degree.. A model of the IFNAR1 structure was constructed
by displaying the IFNAR1 sequence on the backbone of tissue factor;
the orientation between domains 1 and 2 was modeled on that
observed between subdomains. FIG. 6 shows a space-filling rendering
of this model, with residues involved in the binding of mAbs
depicted in red. Residues 69-74 and 103-111 are located in domain
1, in subdomains SD100A and SD100B, respectively, and residues
244-249 and 291-298 in SD100A' of domain 2. Residues 69-74 are
situated far away from the other three, on top of the FIG. 6 model.
Since substitutions in this region significantly affect binding of
all mAbs except 5A8 (which was shown to bind both the domain 1 and
domain 2 of hIFNAR1-IgG), it was determined that they cause a major
structural change. The remaining three regions are clustered near
each other in space and were determined to constitute part of the
binding sites of the blocking mAbs 2E1 and 4A7.
[0298] mAbs 2E1 (Kd.sup.-1=66 pM) and 2E8 (Kd.sup.-1=97 pM) have
been shown to exhibit similar high affinities to hIFNAR1-IgG and
bind to the same epitope or overlapping epitopes according to the
competitive binding ELISA results. However, mAb 2E1 is a potent
blocking mAb while mAb 2E8 is a nonblocking mAb. The different
blocking activity of these two mAbs is explained by the results of
the mutant analysis as shown in Tables IV and V. The binding areas
are indeed overlapping but different.
[0299] The following hybridomas have been deposited with the
American Type Culture Collection, 12301 Parklawn Drive, Rockville,
Md., USA (ATCC): TABLE-US-00007 Cell Lines ATCC Accession No.
Deposit Date 5A8 HB 12129 Jun. 12, 1996 2E8 HB 12130 Jun. 12, 1996
2H6 HB 12131 Jun. 12, 1996 4A7 HB 12132 Jun. 12, 1996 2E1 HB 12133
Jun. 12, 1996
[0300] These deposits were made under the provisions of the
Budapest Treaty on the International Recognition of the Deposit of
Microorganisms for the Purpose of Patent Procedure and the
Regulations thereunder (Budapest Treaty). This assures maintenance
of a viable deposit for 30 years from the date of deposit. These
cell lines will be made available by ATCC under the terms of the
Budapest Treaty, and subject to an agreement between Genentech,
Inc. and ATCC, which assures permanent and unrestricted
availability of the cell lines to the public upon issuance of the
pertinent U.S. patent or upon laying open to the public of any U.S.
or foreign patent application, whichever comes first, and assures
availability of the cell lines to one determined by the U.S.
Commissioner of Patents and Trademarks to be entitled thereto
according to 35 USC .sctn.122 and the Commissioner's rules pursuant
thereto (including 37 CFR .sctn.1.14 with particular reference to
8860G 638).
[0301] The assignee of the present application has agreed that if
the deposited cell lines should be lost or destroyed when
cultivated under suitable conditions, they will be promptly
replaced on notification with a specimen of the same cell line.
Availability of the deposited cell lines is not to be construed as
a license to practice the invention in contravention of the rights
granted under the authority of any government in accordance with
its patent laws.
Sequence CWU 1
1
* * * * *