U.S. patent application number 13/692952 was filed with the patent office on 2013-04-04 for anti-interferon-alpha antibodies.
This patent application is currently assigned to GENENTECH, INC.. The applicant listed for this patent is GENENTECH, INC.. Invention is credited to ANAN CHUNTHARAPAI, JIN K. KIM, LEONARD G. PRESTA, TIMOTHY STEWART.
Application Number | 20130084295 13/692952 |
Document ID | / |
Family ID | 26722123 |
Filed Date | 2013-04-04 |
United States Patent
Application |
20130084295 |
Kind Code |
A1 |
CHUNTHARAPAI; ANAN ; et
al. |
April 4, 2013 |
ANTI-INTERFERON-ALPHA ANTIBODIES
Abstract
The present invention relates generally to the generation and
characterization of neutralizing anti-IFN-.alpha. monoclonal
antibodies with broad reactivity against various IFN-.alpha.
subtypes. The invention further relates to the use of such
anti-IFN-.alpha. antibodies in the diagnosis and treatment of
disorders associated with increased expression of IFN-.alpha., in
particular, autoimmune disorders such as insulin-dependent diabetes
mellitus (IDDM) and systemic lupus erythematosus (SLE).
Inventors: |
CHUNTHARAPAI; ANAN; (SOUTH
SAN FRANCISCO, CA) ; KIM; JIN K.; (SOUTH SAN
FRANCISCO, CA) ; PRESTA; LEONARD G.; (SOUTH SAN
FRANCISCO, CA) ; STEWART; TIMOTHY; (SOUTH SAN
FRANCISCO, CA) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
GENENTECH, INC.; |
SOUTH SAN FRANCISCO |
CA |
US |
|
|
Assignee: |
GENENTECH, INC.
SOUTH SAN FRANCISCO
CA
|
Family ID: |
26722123 |
Appl. No.: |
13/692952 |
Filed: |
December 3, 2012 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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13034551 |
Feb 24, 2011 |
8349331 |
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13692952 |
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11423588 |
Jun 12, 2006 |
7910707 |
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13034551 |
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10044896 |
Jan 9, 2002 |
7087726 |
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11423588 |
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60270775 |
Feb 22, 2001 |
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Current U.S.
Class: |
424/136.1 ;
435/320.1; 435/328; 435/69.6; 435/7.92; 530/387.3; 536/23.53 |
Current CPC
Class: |
G01N 2333/56 20130101;
A61P 5/50 20180101; A61P 7/06 20180101; G01N 2800/042 20130101;
A61P 3/10 20180101; A61P 37/02 20180101; C07K 2317/565 20130101;
C07K 2317/55 20130101; G01N 33/6866 20130101; C07K 2317/567
20130101; G01N 2800/24 20130101; G01N 33/564 20130101; C07K 2319/00
20130101; C07K 2317/24 20130101; A61K 2039/505 20130101; A61P 37/06
20180101; A61P 37/00 20180101; A61P 19/04 20180101; A61P 5/14
20180101; A61P 17/00 20180101; C07K 2317/92 20130101; C07K 16/249
20130101; C07K 2317/76 20130101 |
Class at
Publication: |
424/136.1 ;
530/387.3; 536/23.53; 435/320.1; 435/328; 435/69.6; 435/7.92 |
International
Class: |
C07K 16/24 20060101
C07K016/24 |
Claims
1. An anti-IFN-.alpha. monoclonal antibody which binds to and
neutralizes a biological activity of at least IFN-.alpha. subtypes,
IFN-.alpha.1, IFN-.alpha.2, IFN-.alpha.4, IFN-.alpha.5,
IFN-.alpha.8, IFN-.alpha.10, and IFN-.alpha.21.
2-19. (canceled)
20. An anti-IFN-.alpha. antibody light chain or a fragment thereof,
comprising the following CDR's: TABLE-US-00006 (SEQ ID NO: 7) (a)
L1 of the formula RASQSVSTSSYSYMH; (SEQ ID NO: 8) (b) L2 of the
formula YASNLES; and (SEQ ID NO: 9) (c) L3 of the formula
QHSWGIPRTF.
21. (canceled)
22. An anti-IFN-.alpha. antibody heavy chain or a fragment thereof,
comprising the following CDR's: TABLE-US-00007 (SEQ ID NO: 10) (a)
H1 of the formula GYTFTEYIIH; (SEQ ID NO: 11) (b) H2 of the formula
SINPDYDITNYNQRFKG; and (SEQ ID NO: 12) (c) H3 of the formula
WISDFFDY.
23. (canceled)
24. An anti-IFN-.alpha. antibody comprising (A) at least one light
chain or a fragment thereof, comprising the following CDR's:
TABLE-US-00008 (SEQ ID NO: 7) (a) L1 of the formula
RASQSVSTSSYSYMH; (SEQ ID NO: 8) (b) L2 of the formula YASNLES; and
(SEQ ID NO: 9) (c) L3 of the formula QHSWGIPRTF; and
(B) at least one heavy chain or a fragment thereof, comprising the
following CDR's: TABLE-US-00009 (SEQ ID NO: 10) (a) H1 of the
formula GYTFTEYIIH; (SEQ ID NO: 11) (b) H2 of the formula
SINPDYDITNYNQRFKG; and (SEQ ID NO: 12) (c) H3 of the formula
WISDFFDY.
25-30. (canceled)
31. An isolated nucleic acid molecule encoding an antibody of claim
1.
32-33. (canceled)
34. An isolated nucleic acid molecule encoding an antibody of claim
24.
35. An isolated nucleic acid molecule encoding an antibody light
chain or light chain fragment of claim 20.
36. An isolated nucleic acid molecule encoding an antibody heavy
chain or heavy chain fragment of claim 22.
37. An isolated nucleic acid molecule comprising the light chain
polypeptide-encoding nucleic acid sequence of the vector deposited
with ATCC on Jan. 9, 2001 and having accession No. PTA-2882.
38. An isolated nucleic acid molecule comprising the heavy chain
polypeptide-encoding nucleic acid sequence of the vector deposited
with ATCC on Jan. 9, 2001 and having accession No. PTA-2881.
39. A vector comprising a nucleic acid molecule according to claim
31.
40. A host cell transformed with a nucleic acid molecule according
to claim 31.
41. A method of producing the antibody of claim 1 comprising
culturing a host cell comprising a nucleic acid sequence encoding
the antibody under conditions wherein the nucleic acid sequence is
expressed to produce the antibody.
42. A hybridoma cell line comprising a nucleic acid molecule
according to claim 31.
43. A hybridoma cell line deposited with ATCC on Jan. 18, 2001 and
having accession No. PTA-2917.
44. An antibody produced by the hybridoma cell line of claim
42.
45. A pharmaceutical composition comprising an effective amount of
the antibody of claim 1 in admixture with a pharmaceutically
acceptable carrier.
46-47. (canceled)
48. A pharmaceutical composition comprising an effective amount of
the antibody of claim 24 in admixture with a pharmaceutically
acceptable carrier.
49. A method for diagnosing a condition associated with the
expression of IFN-.alpha. in a cell, comprising contacting said
cell with an anti-IFN-.alpha. antibody of claim 1, and detecting
the presence of IFN-.alpha..
50. A method for the treatment of a disease or condition associated
with the expression of IFN-.alpha. in a patient, comprising
administering to said patient an effective amount of an
anti-IFN-.alpha. antibody of claim 1.
51-54. (canceled)
Description
[0001] This application claims the benefit under Title 35, United
States Codes .sctn.119(e) of the U.S. provisional Application No.
60/270,775, filed on Feb. 22, 2001.
BACKGROUND OF THE INVENTION
[0002] 1. Field of the Invention
[0003] The present invention relates generally to the generation
and characterization of neutralizing anti-IFN-.alpha. monoclonal
antibodies with broad reactivity against various IFN-.alpha.
subtypes. The invention further relates to the use of such
anti-IFN-.alpha. antibodies in the diagnosis and treatment of
disorders associated with increased expression of IFN-.alpha., in
particular, autoimmune disorders such as insulin-dependent diabetes
mellitus (IDDM) and systemic lupus erythematosus (SLE).
[0004] 2. Description of the Related Art
Interferon-.alpha. (IFN-.alpha.)
[0005] Although interferons were initially discovered for their
anti-viral activities, subsequent research has unraveled a plethora
of regulatory activities associated with these powerful cytokines.
Type I interferons form an ancient family of cytokines that
includes IFN-.alpha., IFN-.beta., IFN-.delta., IFN-.omega. and
IFN-.tau. (Roberts et al., J. Interferon Cytokine Res. 18: 805-816
[1998]). They are coded by intronless genes and are widely
distributed amongst vertebrates. Whereas IFN-13 is coded by a
single gene in primates and rodents, more than 10 and 15 different
subtypes of IFN-.alpha. have been found in mice and man
respectively. Other interferons of type I are more restricted, e.g.
IFN-.delta. in the pig, IFN-.tau. in cattle and sheep, and
IFN-.omega. in cattle and humans. Thus, human type I interferons
comprise multiple members of the IFN-.alpha. family, and single
members of the IFN-.beta. and IFN-.omega. families. All type I IFNs
appear to bind to a single receptor that is comprised of at least
two membrane spanning proteins. Type II interferons on the other
hand are represented by a single member, IFN-.gamma., and bind to a
distinct receptor.
[0006] Although all type I IFNs, including IFN-.alpha., exhibit
anti-viral and anti-proliferative activities and thereby help to
control viral infections and tumors (Lefevre et al., Biochimie 80:
779-788 [1998]; Horton et al., Cancer Res. 59: 4064-4068 [1999];
Alexenko et al., J. Interferon Cytokine Res. 17: 769-779 [1997];
Gresser, J. Leukoc, Biol. 61: 567-574 [1997]), there are also
several autoimmune diseases that are associated with increased
expression of IFN.alpha., most notably insulin-dependent diabetes
mellitus (IDDM) and systemic lupus erythematosus (SLE).
[0007] Type I diabetes, also known as autoimmune diabetes or
insulin-dependent diabetes mellitus (IDDM), is an autoimmune
disease characterized by the selective destruction of pancreatic 13
cells by autoreactive T lymphocytes (Bach, Endocr. Rev. 15: 516-542
[1994]; Castano and Eisenbarth, Annu. Rev. Immunol. 8: 647-679
[1990]; Shehadeh and Lafferty, Diabetes Rev. 1: 141-151 [1993]).
The pathology of IDDM is very complex involving an interaction
between an epigenetic event (possibly a viral infection), the
pancreatic 13 cells and the immune system in a genetically
susceptible host. A number of cytokines, including IFN-.alpha. and
IFN-.gamma., have been implicated in the pathogenesis of IDDM in
humans and in animal models of the disease (Campbell et al., J.
Clin. Invest. 87: 739-742 [1991]; Huang et al., Diabetes 44:
658-664 [1995]; Rhodes and Taylor, Diabetologia 27: 601-603
[1984]). For example, pancreatic Ifn-.alpha. mRNA expression and
the presence of immunoreactive IFN-.alpha. in .beta. cells of
patients with IDDM have been reported (Foulis et al., Lancet 2:
1423-1427 [1987]; Huang et al., [1995] supra; Somoza et al., J.
Immunol. 153: 1360-1377 [1994]). IFN-.alpha. expression has been
associated with hyperexpression of major histocompatibility complex
(MHC) class I.sub.A antigens in human islets (Foulis et al., [1987]
supra; Somoza et al., [1994] supra). In two rodent models of
autoimmune diabetes, the diabetes-prone DP-BB rat and
streptozotocin-treated mice, Ifn-.alpha. mRNA expression in islets
precedes insulitis and diabetes (Huang et al., Immunity 1: 469-478
[1994]). Furthermore, transgenic mice harboring a hybrid human
insulin promoter-Ifn-.alpha. construct develop hypoinsulinemic
diabetes accompanied by insulitis (Stewart et al., Science 260:
1942-1946 [1993]).
[0008] It appears that local expression of IFN-.alpha. by
pancreatic islet cells in response to potential diabetogenic
stimuli such as viruses may trigger the insulitic process.
Consistent with its role as an initiating agent, IFN-.alpha. has
been shown to induce intercellular adhesion molecule-1 (ICAM-1) and
HLA class I.sub.A on endothelial cells from human islets, which may
contribute to leukocyte infiltration during insulitis (Chakrabarti
et al., J. Immunol. 157: 522-528 [1996]). Furthermore, IFN-.alpha.
facilitates T cell stimulation by the induction of the
co-stimulatory molecules ICAM-I and B7.2 on antigen-presenting
cells in islets (Chakrabarti et al., Diabetes 45: 1336-1343
[1996]). These studies collectively indicate that early IFN-.alpha.
expression by .beta. cells may be a critical event in the
initiation of autoimmune diabetes. Although there are a number of
reports implicating IFN-.gamma. in the development of IDDM in
rodent models, there is a poor correlation between the expression
of this cytokine and human IDDM. Thus, cells expressing IFN-.gamma.
can be found in the islets of a subset of human patients selected
for significant lymphocytic infiltration into the islets. In a
group of patients that were not selected by this criterion there
was no obvious association between IFN-.gamma. expression and human
IDDM.
[0009] Based on the increased level of IFN-.alpha. expression in
patients with systemic lupus erythematosus (SLE), IFN-.alpha. has
also been implicated in the pathogenesis of SLE (Ytterberg and
Schnitzer, Arthritis Rheum. 25: 401-406 [1982]; Shi et al., Br. J.
Dermatol. 117: 155-159 [1987]). It is interesting to note that
IFN-.alpha. is currently used for the treatment of cancer as well
as viral infection such as chronic hepatitis due to hepatitis B or
hepatitis C virus infection. Consistent with the observations of
increased levels of IFN-.alpha. triggering autoimmunity,
significant increase in the appearance of autoimmune disorders such
as IDDM, SLE and autoimmune thyroiditis has been reported in the
patients undergoing IFN-.alpha. therapy. For example, prolonged use
of IFN-.alpha. as an anti-viral therapy has been shown to induce
IDDM (Waguri et al., Diabetes Res. Clin. Pract. 23: 33-36 [1994];
Fabris et al., J. Hepatol. 28: 514-517 [1998]) or SLE
(Garcia-Porrua et al., Clin. Exp. Rheumatol. 16: 107-108 [1998]).
The treatment of coxsackievirus B (CBV) infection with IFN-.alpha.
therapy is also associated with the induction of IDDM (Chehadeh et
al., J. Infect. Dis. 181: 1929-1939 [2000]). Similarly, there are
multiple case reports documenting IDDM or SLE in IFN-.alpha.
treated cancer patients (Ronnblom et al., J. Intern. Med. 227:
207-210 [1990]).
Antibody Therapy
[0010] The use of monoclonal antibodies as therapeutics has gained
increased acceptance with several monoclonal antibodies (mAbs)
either approved for human use or in late stage clinical trials. The
first mAb approved by the US Food and Drug Administration (FDA) for
the treatment of allograft rejection was anti-CD3 (OKT3) in 1986.
Since then the pace of progress in the field of mAbs has been
considerably accelerated, particularly from 1994 onwards which led
to approval of additional seven mAbs for human treatment. These
include ReoPro.RTM. for the management of complications of coronary
angioplasty in 1994, Zenapax.RTM. (anti-CD25) for the prevention of
allograft rejection in 1997, Rituxan.RTM. (anti-CD20) for the
treatment of B cell non-Hodgkin's lymphoma in 1997, Infliximab.RTM.
(anti-TNF-.alpha.) initially for the treatment of Crohn's disease
in 1998 and subsequently for the treatment of rheumatoid arthritis
in 1999, Simulect.RTM. (anti-CD25) for the prevention of allograft
rejection in 1998, Synagis.RTM. (anti-F protein of respiratory
syncitial virus) for the treatment of respiratory infections in
1998, and Herceptin.RTM. (anti-HER2/neu) for the treatment of HER2
overexpressing metastatic breast tumors in 1998 (Glennie and
Johnson, Immunol. Today 21: 403-410 [2000]).
Anti-IFN-.alpha. Antibodies
[0011] Disease states that are amenable to intervention with mAbs
include all those in which there is a pathological level of a
target antigen. For, example, an antibody that neutralizes
IFN-.alpha. present in the sera of patients with SLE, and expressed
by the pancreatic islets in IDDM, is a potential candidate for
therapeutic intervention in these diseases. It could also be used
for therapeutic intervention in other autoimmune diseases with
underlying increase in and causative role of IFN-.alpha.
expression. In both human IDDM (Foulis, et al., Lancet 2: 1423-1427
[1987]; Huang, at al., Diabetes 44: 658-664 [1995]; Somoza, et al.,
J. Immunol. 153: 1360-1377 [1994]) and human SLE (Hooks, et al.,
Arthritis & Rheumatism 25: 396-400 [1982]; Kim, et al., Clin.
Exp. Immunol. 70: 562-569 [1987]; Lacki, et at, J. Med. 28: 99-107
[1997]; Robak, et al., Archivum Immunologiae et Therapiae
Experimentalis 46: 375-380 [1998]; Shiozawa, et al., Arthritis
& Rheumatism 35: 417-422 [1992]; von Wussow, et al.,
Rheumatology International 8: 225-230 [1988]) there appears to be
correlation between disease and IFN-.alpha. but not with either
IFN-.beta. or IFN-.gamma.. Thus, anti-interferon mAb intervention
in IDDM or SLE would require specific neutralization of most, if
not all, of the IFN-.alpha. subtypes, without any significant
neutralization of IFN-.beta. or IFN-.gamma.. Leaving the activity
of these last two interferons intact may also have an advantage in
allowing the retention of significant anti-viral activity.
[0012] While a few mAbs that show reactivity with a range of
recombinant human IFN-.alpha. subtypes have been described, these
were found to neutralize only a limited subset of the recombinant
IFN-.alpha. subtypes analyzed or were not capable of neutralizing
the mixture of IFN-.alpha. subtypes that are produced by stimulated
peripheral blood leukocytes (Tsukui et al., Microbiol. Immunol. 30:
1129-1139 [1986]; Berg, J. Interferon Res. 4: 481-491 [1984];
Meager and Berg, J. Interferon Res. 6: 729-736 [1986]; U.S. Pat.
No. 4,902,618; and EP publication No. 0,139,676 B1).
[0013] Accordingly, there is a great need for anti-IFN-.alpha.
antibodies that not only bind to most, preferably all, subtypes of
IFN-.alpha. but also neutralize such subtypes while do not
interfere with the biological function of other interferons.
SUMMARY OF THE INVENTION
[0014] The present invention is based on the development of a
monoclonal antibody that was experimentally found to neutralize all
seven of different recombinant human IFN-.alpha. subtypes tested
and two independent pools of natural human IFN-.alpha.
subtypes.
[0015] In one aspect, the invention provides an anti-human
IFN-.alpha. monoclonal antibody which binds to and neutralizes a
biological activity of at least human IFN-.alpha. subtypes
IFN-.alpha.1, IFN-.alpha.2, IFN-.alpha.4, IFN-.alpha.5,
IFN-.alpha.8, IFN-.alpha.10, and IFN-.alpha.21. In a further
aspect, the invention provides an anti-human IFN-.alpha. monoclonal
antibody which binds to and neutralizes a biological activity of
all human IFN-.alpha. subtypes. The antibody of the invention can
significantly reduce or eliminate a biological activity of the
human IFN-.alpha. in question. In one embodiment, the antibody of
the invention is capable of neutralizing at least 60%, or at least
70%, preferably at least 75%, more preferably at least 80%, even
more preferably at least 85%, still more preferably at least 90%,
still more preferably at least 95%, most preferably at least 99% of
a biological activity of the subject human IFN-.alpha.. In another
embodiment, the human IFN-.alpha. biological activity-neutralizing
monoclonal antibody does not neutralize the corresponding
biological activity of human IFN-.beta..
[0016] The biological activity of the subject human IFN-.alpha.'s
may be IFNAR2-binding activity. In a particular embodiment, the
invention provides an anti-human IFN-.alpha.monoclonal antibody is
capable of binding to and blocking at least 60%, or at least 70%,
preferably at least 75%, more preferably at least 80%, even more
preferably at least 85%, still more preferably at least 90%, still
more preferably at least 95%, most preferably at least 99% of the
IFNAR2-binding activity of all, or substantially all human
IFN-.alpha. subtypes. In another embodiment, the invention provides
an anti-human IFN-.alpha. monoclonal antibody that is capable of
binding to and blocking at least 60%, or at least 70%, preferably
at least 75%, more preferably at least 80%, even more preferably at
least 85%, still more preferably at least 90%, still more
preferably at least 95%, most preferably at least 99% of the
IFNAR2-binding activity of each of human IFN-.alpha. subtypes 1, 2,
4, 5, 8, 10 and 21. In another embodiment, the anti-human
IFN-.alpha. monoclonal antibody does not cross-react with human
IFN-.beta..
[0017] The biological activity of the subject human IFN-.alpha.'s
may be an antiviral activity. In one embodiment, the anti-human
IFN-.alpha. monoclonal antibody is capable of binding to and
neutralizing the antiviral activity of all, or substantially all
human IFN-.alpha. subtypes. In another embodiment, the anti-human
IFN-.alpha. monoclonal antibody is capable of binding to and
neutralizing the antiviral activity of each of human IFN-.alpha.
subtypes 1, 2, 4, 5, 8, 10 and 21. In a particular embodiment, the
invention provides an anti-human IFN-.alpha. monoclonal antibody
that is capable of binding to and neutralizing at least 60%, or at
least 70%, preferably at least 75%, more preferably at least 80%,
even more preferably at least 85%, still more preferably at least
90%, still more preferably at least 95%, most preferably at least
99% of the antiviral activity of all, or substantially all human
IFN-.alpha. subtypes. In yet another embodiment, the invention
provides an anti-human IFN-.alpha. monoclonal antibody which hinds
to and neutralizes at least 60%, or at least 70%, or at least 75%,
or at least 80%, or at least 85%, or at least 90%, or at least 95%,
or at least 99% of the antiviral activity of each of human
IFN-.alpha. subtypes 1, 2, 4, 5, 8, 10 and 21. In still another
embodiment, the human IFN-.alpha. antiviral activity-neutralizing
monoclonal antibody does not neutralize the antiviral activity of
human IFN-.beta..
[0018] The antibody may be a murine, humanized or human antibody.
The antibody may be the murine anti-human IFN-.alpha. monoclonal
antibody 9F3 or a humanized version of it such as version 13 (V13)
or chimeric form thereof. The scope of the invention also covers an
antibody that binds essentially the same IFN-.alpha. epitope as
murine anti-human IFN-.alpha. monoclonal antibody 9F3 or a
humanized or chimeric form thereof. For example, a reference
antibody for this purpose is an anti-IFN-.alpha. antibody produced
by the murine hybridoma cell line 9F3.18.5 deposited with ATCC on
Jan. 18, 2001 and having accession No. PTA-2917. In another
embodiment, the invention provides a murine or murine/human
chimeric anti-human IFN-.alpha. monoclonal antibody comprising the
murine light chain variable domain amino acid sequence shown in
FIG. 5A (SEQ ID NO:1) and/or the murine heavy chain variable domain
amino acid sequence shown in FIG. 5B (SEQ ID NO:2). In yet another
embodiment, the invention provides a humanized anti-human
IFN-.alpha. monoclonal antibody comprising the humanized light
chain variable domain amino acid sequence shown in FIG. 5A (SEQ ID
NO:3) and/or the humanized heavy chain variable domain amino acid
sequence shown in FIG. 5B (SEQ ID NO:5).
[0019] Additionally provided is an anti-human IFN-.alpha.
monoclonal antibody that binds essentially the same epitopes on
human IFN-.alpha. subtypes 1, 2, 4, 5, 8, 10 and 21 that are bound
by murine anti-human IFN-.alpha. monoclonal antibody 9F3 or a
humanized or chimeric form thereof. Further provided herein is an
anti-human IFN-.alpha. monoclonal antibody that competes with
murine anti-human IFN-.alpha. monoclonal antibody 9F3 for binding
to each of human IFN-.alpha. subtypes 1, 2, 4, 5, 8, 10 and 21.
[0020] Also provided is an isolated nucleic acid molecule encoding
any of the antibodies described herein, a vector comprising the
isolated nucleic acid molecule, a host cell transformed with the
nucleic acid molecule, and a method of producing the antibody
comprising culturing the host cell under conditions wherein the
nucleic acid molecule is expressed to produce the antibody and
optionally recovering the antibody from the host cell. The antibody
may be of the IgG class and isotypes such as IgG.sub.1, IgG.sub.2,
IgG.sub.3, or IgG.sub.4. The scope of the invention also covers
antibody fragments such as Fv, scFv, Fab, F(ab').sub.2, and Fab'
fragments.
[0021] In another aspect, the present invention provides an
anti-human IFN-.alpha.monoclonal antibody light chain or a fragment
thereof, comprising the following CDR's (as defined by Kabat, et
al., Sequences of Proteins of Immunological Interest, Fifth
Edition, NIH Publication 91-3242, Bethesda Md. [1991], vols. 1-3):
(a) L1 of the formula RASQSVSTSSYSYMH (SEQ ID NO: 7); (b) L2 of the
formula YASNLES (SEQ ID NO: 8); and (c) L3 of the formula
QHSWGIPRTF (SEQ ID NO: 9). The scope of the invention also covers
the light chain variable domain of such anti-human IFN-.alpha.
monoclonal antibody light chain fragment. The scope of the
invention further includes an anti-human IFN-.alpha. monoclonal
antibody light chain polypeptide comprising the mouse/human
chimeric light chain variable domain amino acid sequence, or the
entire chimeric light chain polypeptide amino acid sequence,
encoded by the XAIFN-ChLpDR1 vector deposited with the ATCC on Jan.
9, 2001 and having accession No. PTA-2880. The scope of the
invention additionally includes an anti-human IFN-.alpha.
monoclonal antibody light chain polypeptide comprising the
humanized light chain variable domain amino acid sequence, or the
entire humanized light chain polypeptide amino acid sequence,
encoded by the VLV30-IgG vector deposited with the ATCC on Jan. 9,
2001 and having accession No. PTA-2882.
[0022] In yet another aspect, the invention provides an anti-human
IFN-.alpha. monoclonal antibody heavy chain or a fragment thereof,
comprising the following CDR's: (a) H1 of the formula GYTFT EYIIH
(SEQ ID NO: 10); (b) H2 of the formula SINPDYDITNYNQRFKG (SEQ ID
NO: 11); and (c) H3 of the formula WISDFFDY (SEQ ID NO: 12). The
scope of the invention also covers the heavy chain variable domain
of such anti-human IFN-.alpha. monoclonal antibody heavy chain
fragment. The scope of the invention further includes an anti-human
IFN-.alpha. monoclonal antibody heavy chain polypeptide comprising
the mouse/human chimeric heavy chain variable domain amino acid
sequence, or the entire chimeric heavy chain polypeptide amino acid
sequence, encoded by the XAIFN-ChHpDR2 vector deposited with the
ATCC on Jan. 9, 2001 and having accession No. PTA-2883.
Additionally included is an anti-human IFN-.alpha. monoclonal
antibody heavy chain polypeptide comprising the humanized heavy
chain variable domain amino acid sequence, or the entire humanized
heavy chain polypeptide amino acid sequence, encoded by the vector
VHV30-IgG2 deposited with the ATCC on Jan. 9, 2001 and having
accession No. PTA-2881.
[0023] In a further aspect, the invention provides an anti-human
IFN-.alpha. monoclonal antibody comprising (A) at least one light
chain or a fragment thereof, comprising the following CDR's: (a) L1
of the formula RASQSVSTSSYSYMH (SEQ ID NO: 7); (b) L2 of the
formula YASNLES (SEQ ID NO: 8); and (c) L3 of the formula
QHSWGIPRTF (SEQ ID NO: 9); and (B) at least one heavy chain or a
fragment thereof, comprising the following CDR's: (a) H1 of the
formula GYTFTEYIIH (SEQ ID NO: 10); (b) H2 of the formula
SINPDYDITNYNQRFKG (SEQ ID NO: 11); and (c) H3 of the formula
WISDFFDY (SEQ ID NO: 12). The antibody may be a homo-tetrameric
structure composed of two disulfide-bonded antibody heavy
chain-light chain pairs. The scope of the invention specifically
includes a linear antibody, a murine antibody, a chimeric antibody,
a humanized antibody, or a human antibody. Further provided is a
chimeric antibody comprising (1) the mouse/human chimeric light
chain variable domain amino acid sequence, or the entire chimeric
light chain polypeptide amino acid sequence, encoded by the
XAIFN-ChLpDR1 vector deposited with the ATCC on Jan. 9, 2001 and
having accession No. PTA-2880; and (2) the mouse/human chimeric
heavy chain variable domain amino acid sequence, or the entire
chimeric heavy chain polypeptide amino acid sequence, encoded by
the XAIFN-ChHpDR2 vector deposited with the ATCC on Jan. 9, 2001
and having accession No. PTA-2883. Additionally provided herein is
a humanized antibody comprising (1) the humanized light chain
variable domain amino acid sequence, or the entire humanized light
chain polypeptide amino acid sequence, encoded by the VLV30-IgG
vector deposited with the ATCC on Jan. 9, 2001 and having accession
No. PTA-2882; and (2) the humanized heavy chain variable domain
amino acid sequence, or the entire humanized heavy chain
polypeptide amino acid sequence, encoded by the vector VHV30-IgG2
deposited with the ATCC on Jan. 9, 2001 and having accession No.
PTA-2881.
[0024] In yet another aspect, the invention provides a
pharmaceutical composition comprising an effective amount of the
antibody of the invention in admixture with a pharmaceutically
acceptable carrier.
[0025] In a different aspect, the invention provides a method for
diagnosing a condition associated with the expression of
IFN-.alpha. in a cell, comprising contacting the cell with an
anti-IFN-.alpha. antibody, and detecting the presence of
IFN-.alpha..
[0026] In yet another aspect, the invention provides a method for
the treatment of a disease or condition associated with the
expression of IFN-.alpha. in a patient, comprising administering to
the patient an effective amount of an anti-IFN-.alpha. antibody.
The patient is a mammalian patient, preferably a human patient. The
disease is an autoimmune disease, such as insulin-dependent
diabetes mellitus (IDDM); systemic lupus erythematosus (SLE); or
autoimmune thyroiditis.
BRIEF DESCRIPTION OF THE DRAWINGS
[0027] FIG. 1 shows a schematic diagram of the strategy used for
the development of the anti-human IFN-.alpha. monoclonal
antibodies.
[0028] FIG. 2 shows that a murine anti-human IFN-.alpha. mAb (9F3)
is able to neutralize a spectrum of recombinant IFN-.alpha.
subtypes but not recombinant IFN-13. The indicated IFN's were
assayed for inhibition of encephalomyocarditis (EMC) viral growth
in A549 cells in the presence of increasing concentrations of the
mAb 9F3. Data are presented as the percentage of the viral growth
inhibition activity obtained with the indicated IFN in the absence
of mAb 9F3.
[0029] FIGS. 3A-3B show the neutralization of leukocyte interferon
(Sigma) (FIG. 3A) and lymphoblastoid interferon (NIH reference
Ga23-901-532) (FIG. 3B). In FIG. 3A, 20,000 IU/ml (filled bars) or
5,000 IU/ml (open bars) of leukocyte interferon (Sigma Product No.
1-2396) were incubated with blank control (buffer only) (denoted as
"-"), 10 .mu.g/ml control mouse IgG (denoted as "mIgG"), or 10
.mu.g/ml mAb 9F3 (denoted as "9F3"). Dilutions were assayed and the
amount of remaining activity shown. The results shown are means of
duplicate determinations. In FIG. 3B, lymphoblastoid interferon
(NIH reference Ga23-901-532) was assayed at 10 (filled columns) or
3 (open columns) IU/ml in the presence or absence of the indicated
concentrations of mAb 9F3. A higher cytopathic effect is indicative
of a decrease in interferon activity. The results shown are the
means of duplicate determinations.
[0030] FIG. 4 depicts results of an electrophoretic mobility shift
assay (EMSA) showing the induction of an ISGF3/ISRE complex by
IFN-.alpha. and the ability of 9F3 mAb to prevent the formation of
the complex. EMSA was performed in the presence or absence of
either human IFN-.alpha.2 (denoted as ".alpha.2") or IFN-.beta.
(denoted as ".beta.") at a concentration of 25 ng/ml with 9F3 mAb
(denoted as "9F3") or murine IgG control antibody (denoted as
"IgG") at a concentration of 10 .mu.g/1.
[0031] FIG. 5A shows the alignment of light chain variable domain
amino acid sequences of murine 9F3 (murine, SEQ ID NO: 1),
humanized 9F3 version 13 (V13, SEQ ID NO: 3), and the consensus
human variable domain light .kappa. subgroup I (hu.kappa.I, SEQ ID
NO: 4). The CDRs (L1, SEQ ID NO: 7; L2, SEQ ID NO: 8; and L3, SEQ
ID NO: 9) are highlighted by underlining. The residue numbering is
according to Kabat et al., (1991) supra. The differences between
the murine 9F3 and V13 sequences and the differences between 9F3
and hu.kappa.I sequences are indicated by asterisks.
[0032] FIG. 5B shows the alignment of heavy chain variable domain
amino acid sequences of murine 9F3 (murine, SEQ ID NO: 2),
humanized 9F3 version 13 (V13, SEQ ID NO: 5), and the consensus
human variable domain heavy subgroup III (huIII, SEQ ID NO: 6). The
CDRs (H1, SEQ ID NO: 10; H2, SEQ ID NO: 11; and H3, SEQ ID NO: 12)
are highlighted by underlining. The residue numbering is according
to Kabat et al. (1991) supra. The differences between the murine
9F3 and V13 sequences and the differences between 9F3 and huh
sequences are indicated by asterisks.
[0033] FIG. 6 shows neutralization activity of the starting mAb 9F3
(left panel) and the chimeric protein CH8-2 (right panel) toward
the viral growth inhibition exhibited by recombinant IFN-.alpha.
subtypes in A549 cells challenged with encephalomyocarditis (EMC)
virus.
[0034] FIG. 7 depicts a model of humanized 9F3 version 13. Backbone
of VL and VH domains is shown as a ribbon. CDRs are shown in white
and are labeled (L1, L2, L3, H1, H2, H3). Framework side chains
altered from human to murine are shown in white and are labeled by
residue number.
DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENT
A. Definitions
[0035] Unless defined otherwise, technical and scientific terms
used herein have the same meaning as commonly understood by one of
ordinary skill in the art to which this invention belongs. See,
e.g. Singleton et al., Dictionary of Microbiology and Molecular
Biology 2nd ed., J. Wiley & Sons (New York, N.Y. 1994);
Sambrook et al., Molecular Cloning, A Laboratory Manual, Cold
Springs Harbor Press (Cold Springs Harbor, N.Y. 1989). For purposes
of the present invention, the following terms are defined
below.
[0036] As used herein, the term "type I interferon" is defined to
include all subtypes of native sequence type I interferons of any
mammalian species, including interferon-.alpha., interferon-.beta.,
interferon-.delta., interferon-.omega. and interferon-.tau..
Similarly, the term "human type I interferon" is defined to include
all subtypes of native sequence type I human interferons, including
human interferon-.alpha., interferon-.beta. and interferon-.omega.
classes and which bind to a common cellular receptor.
[0037] Unless otherwise expressly provided, the terms
"interferon-.alpha.," "IFN-.alpha.," and "human
interferon-.alpha.", "human IFN-.alpha." and "hIFN-.alpha." are
used herein to refer to all species of native sequence human alpha
interferons, including all subtypes of native sequence human
interferons-.alpha.. Natural (native sequence) 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: 443-444 [1993]; Roberts et al., J.
Interferon Cytokine Res. 18: 805-816 [1998]). 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.11 or .omega., contains at least 5 pseudogenes and one
functional gene (denoted herein as "IFN-.alpha..sub.111" or
"IFN-.omega.") which exhibits 70% homology with the IFN-.alpha.
genes (Weissmann and Weber [1986] supra).
[0038] As used herein, the terms "first human interferon-.alpha.
(hIFN-.alpha.) receptor", "IFN-.alpha.R", "hIFNAR1", "IFNAR1", and
"Uzc 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.
[0039] As used herein, the terms "second human interferon-.alpha.
(hIFN-.alpha.) receptor", "IFN-.alpha..beta.R", "hIFNAR2",
"IFNAR2", and "Novick chain" are defined as the 515 amino acid
receptor protein cloned by Domanski et al., J. Biol. Chem., 37:
21606-21611 (1995), including an extracellular domain of 217
residues, a transmembrane domain of 21 residues, and an
intracellular domain of 250 residues, as shown in FIG. 1 on page
21608 of Domanski et al. Also encompassed by the foregoing teens
are fragments of IFNAR2 that contain the extracellular domain (ECD)
(or fragments of the ECD) of IFNAR2, and soluble forms of IFNAR2,
such as IFNAR2 ECD fused to an immunoglobulin sequence, e.g. IFNAR2
ECD-IgG Fc as described below.
[0040] The term "native sequence" in connection with type I
interferon, IFN-.alpha.; or any other polypeptide refers to a
polypeptide that has the same amino acid sequence as a
corresponding polypeptide derived from nature, regardless of its
mode of preparation. Such native sequence polypeptide can be
isolated from nature or can be produced by recombinant and/or
synthetic means or any combinations thereof. The term "native
sequence" specifically encompasses naturally-occurring truncated or
secreted forms (e.g., an extracellular domain sequence),
naturally-occurring variant forms (e.g., alternatively spliced
forms) and naturally-occurring allelic variants of the full length
polypeptides.
"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.
[0041] "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 which 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.
[0042] "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
(Chothia et al., J. Mol. Biol. 186:651 [1985]; Novotny and Haber,
Proc. Natl. Acad. Sci. U.S.A. 82:4592 [1985]; Chothia et al.,
Nature 342: 877-883 [1989]).
[0043] 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. (1991) supra). 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.
[0044] 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.
[0045] "Fv" is the minimum antibody fragment which 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.
[0046] 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.
[0047] The "light chains" of antibodies (immunoglobulins) from any
vertebrate species can be assigned to one of two clearly distinct
types, called .kappa. and .lamda., based on the amino acid
sequences of their constant domains.
[0048] 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..di-elect cons. .gamma., and
.mu. respectively. The subunit structures and three-dimensional
configurations of different classes of immunoglobulins are well
known.
[0049] The term "antibody" includes all classes and subclasses of
intact immunoglobulins. The term "antibody" also covers antibody
fragments. The term "antibody" specifically covers monoclonal
antibodies, including antibody fragment clones.
[0050] "Antibody fragments" comprise a portion of an intact
antibody that contains 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
multi specific antibodies formed from antibody fragments.
[0051] 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 which typically
include different antibodies directed against different
determinants (cpitopes), 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, and are not
contaminated 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.
[0052] 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]).
[0053] "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 part or all of 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 which 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);
Presta, Curr. Op. Struct. Biol., 2:593-596 (1992); and Clark,
Immunol. Today 21: 397-402 (2000). 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.
[0054] "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), Dall'Acqua and
Carter, Curr. Opin. Struct. Biol. 8: 443-450 (1998), and Hudson,
Curr. Opin. Immunol. 11: 548-557 (1999).
[0055] 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).
[0056] An "isolated" antibody is one which has been identified and
separated and/or recovered from a component of its natural
environment. Contaminant components of its natural environment are
materials which 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.
[0057] By "neutralizing antibody" is meant an antibody molecule
which is able to eliminate or significantly reduce an effector
function of a target antigen to which it binds. Accordingly, a
"neutralizing" anti-IFN-.alpha. antibody is capable of eliminating
or significantly reducing an effector function, such as receptor
binding and/or elicitation of a cellular response, of
IFN-.alpha..
[0058] For the purpose of the present invention, the ability of an
anti-IFN-.alpha.antibody to neutralize the receptor activation
activity of IFN-.alpha. can be monitored, for example, in a Kinase
Receptor Activation (KIRA) Assay as described in WO 95/14930,
published Jun. 1, 1995, by measuring the ability of a candidate
antibody to reduce tyrosine phosphorylation (resulting from ligand
binding) of the IFNAR1/R2 receptor complex.
[0059] For the purpose of the present invention, the ability of the
anti-IFN-.alpha.antibodies to neutralize the elicitation of a
cellular response by IFN-.alpha. is preferably tested by monitoring
the neutralization of the antiviral activity of IFN-.alpha., as
described by Kawade, J. Interferon Res. 1:61-70 (1980), or Kawade
and Watanabe, J. Interferon Res. 4:571-584 (1984), or Yousefi, et
al., Am. J. Clin. Pathol. 83: 735-740 (1985), or by testing the
ability of an anti-IFN-.alpha. antibody to neutralize the ability
of IFN-.alpha. to activate the binding of the signaling molecule,
interferon-stimulated factor 3 (ISGF3), to an oligonucleotide
derived from the interferon-stimulated response element (ISRE), in
an electrophoretic mobility shift assay, as described by
Kurabayashi et al., Mol. Cell Biol., 15: 6386 (1995).
[0060] "Significant" reduction means at least about 60%, or at
least about 70%, preferably at least about 75%, more preferably at
least about 80%, even more preferably at least about 85%, still
more preferably at least about 90%, still more preferably at least
about 95%, most preferably at least about 99% reduction of an
effector function of the target antigen (e.g. IFN-.alpha.), such as
receptor (e.g. IFNAR2) binding and/or elicitation of a cellular
response. Preferably, the "neutralizing" antibodies as defined
herein will be capable of neutralizing at least about 60%, or at
least about 70%, preferably at least about 75%, more preferably at
least about 80%, even more preferably at least about 85%, still
more preferably at least about 90%, still more preferably at least
about 95%, most preferably at least about 99% of the anti-viral
activity of IFN-.alpha., as determined by the anti-viral assay of
Kawade (1980), supra, or Yousefi (1985), supra. In another
preferred embodiment, the "neutralizing" antibodies herein will be
capable of reducing tyrosine phosphorylation, due to IFN-.alpha.
binding, of the IFNAR1/IFNAR2 receptor complex, by at least about
60%, or at least about 70%, preferably at least about 75%, more
preferably at least about 80%; even more preferably at least about
85%, still more preferably at least about 90%, still more
preferably at least about 95%, most preferably at least about 99%,
as determined in the KIRA assay referenced above. In a particularly
preferred embodiment, the neutralizing anti-IFN-.alpha. antibodies
herein will be able to neutralize all, or substantially all,
subtypes of IFN-.alpha. and will not be able to neutralize
IFN-.beta.. In this context, the term "substantially all" means
that the neutralizing anti-IFN-.alpha.antibody will neutralize at
least IFN-.alpha.1, IFN-.alpha.2, IFN-.alpha.4, IFN-.alpha.5,
IFN-.alpha.8, IFN-.alpha.10, and IFN-.alpha.21.
[0061] For the purpose of the present invention, the ability of an
anti-IFN-.alpha.antibody to block the binding of an IFN-.alpha. to
receptor is defined as the property or capacity of a certain
concentration of the antibody to reduce or eliminate the binding of
IFN-.alpha. to IFNAR2 in a competition binding assay, as compared
to the effect of an equivalent concentration of irrelevant control
antibody on IFN-.alpha. binding to IFNAR2 in the assay. Preferably,
the blocking anti-IFN-.alpha. antibody reduces the binding of
IFN-.alpha. to IFNAR2 by at least about 50%, or at least about 55%,
or at least about 60%, or at least about 65%, or at least about
70%, or at least about 75%, or at least about 80%, or at least
about 85%, or at least about 90%, or at least about 95%, or at
least about 99%, as compared to the irrelevant control
antibody.
[0062] For the purpose of the present invention, the ability of an
anti-IFN-.alpha.antibody to block the binding of IFN-.alpha. to
IFNAR2 can be determined by a routine competition assay such as
that described in Antibodies: A Laboratory Manual, Cold Spring
Harbor Laboratory, Ed Harlow and David Lane (1988). For example,
the IFN-.alpha.-binding ELISA assay described in Example 2 below
could be modified to employ competition binding between an
anti-IFN-.alpha. antibody and a soluble IFNAR2. Such an assay could
be performed by layering the IFN-.alpha. on microtiter plates,
incubating the layered plates with serial dilutions of unlabeled
anti-IFN-.alpha. antibody or unlabeled control antibody admixed
with a selected concentration of labeled IFNAR2 ECD-human IgG Fc
fusion protein, detecting and measuring the signal in each
incubation mixture, and then comparing the signal measurements
exhibited by the various dilutions of antibody.
[0063] In a particularly preferred embodiment, the blocking
anti-IFN-.alpha. antibodies herein will be able to block the
IFNAR2-binding of all, or substantially all, subtypes of
IFN-.alpha. and will not cross-react with IFN-.beta.. In this
context, the term "substantially all" means that the blocking
anti-IFN-.alpha. antibody will block the IFNAR2-binding of at least
IFN-.alpha.1, IFN-.alpha.2, IFN-.alpha.4, IFN-.alpha.5,
IFN-.alpha.10, and IFN-.alpha.21. In a particularly preferred
embodiment, the blocking anti-IFN-.alpha. antibodies of the present
invention will block the IFNAR2-binding of all known subtypes of
IFN-.alpha..
[0064] The term "epitope" is used to refer to binding sites for
(monoclonal or polyclonal) antibodies on protein antigens.
[0065] Antibodies which bind to a particular epitope can be
identified by "epitope mapping." There are many methods known in
the art for mapping and characterizing the location of epitopes on
proteins, including solving the crystal structure of an
antibody-antigen complex, competition assays, gene fragment
expression assays, and synthetic peptide-based assays, as
described, for example, in Chapter 11 of Harlow and Lane, Using
Antibodies, a Laboratory Manual, Cold Spring Harbor Laboratory
Press, Cold Spring Harbor, N.Y., 1999. Competition assays are
discussed above and below. According to the gene fragment
expression assays, the open reading frame encoding the protein is
fragmented either randomly or by specific genetic constructions and
the reactivity of the expressed fragments of the protein with the
antibody to be tested is determined. The gene fragments may, for
example, be produced by PCR and then transcribed and translated
into protein in vitro, in the presence of radioactive amino acids.
The binding of the antibody to the radioactively labeled protein
fragments is then determined by immunoprecipitation and gel
electrophoresis. Certain epitopes can also be identified by using
large libraries of random peptide sequences displayed on the
surface of phage particles (phage libraries). Alternatively, a
defined library of overlapping peptide fragments can be tested for
binding to the test antibody in simple binding assays. The latter
approach is suitable to define linear epitopes of about 5 to 15
amino acids.
[0066] An antibody binds "essentially the same epitope" as a
reference antibody, when the two antibodies recognize identical or
sterically overlapping epitopes. The most widely used and rapid
methods for determining whether two epitopes bind to identical or
sterically overlapping epitopes are competition assays, which can
be configured in all number of different formats, using either
labeled antigen or labeled antibody. Usually, the antigen is
immobilized on a 96-well plate, and the ability of unlabeled
antibodies to block the binding of labeled antibodies is measured
using radioactive or enzyme labels.
[0067] The term amino acid or amino acid residue, as used herein,
refers to naturally occurring L amino acids or to D amino acids as
described further below with respect to variants. The commonly used
one- and three-letter abbreviations for amino acids are used herein
(Bruce Alberts et al., Molecular Biology of the Cell, Garland
Publishing, Inc., New York (3d ed. 1994)).
[0068] "Percent (%) amino acid sequence identity" with respect to
the polypeptide sequences referred to herein is defined as the
percentage of amino acid residues in a candidate sequence that are
identical with the amino acid residues in a sequence, after
aligning the sequences and introducing gaps, if necessary, to
achieve the maximum percent sequence identity, and not considering
any conservative substitutions as part of the sequence identity.
Alignment for purposes of determining percent amino acid sequence
identity can be achieved in various ways that are within the skill
in the art, for instance, using publicly available computer
software such as BLAST, BLAST-2, ALIGN, ALIGN-2 or Megalign
(DNASTAR) software. Those skilled in the art can determine
appropriate parameters for measuring alignment, including any
algorithms needed to achieve maximal alignment over the full-length
of the sequences being compared. For purposes herein, however, %
amino acid sequence identity values are obtained as described below
by using the sequence comparison computer program ALIGN-2. The
ALIGN-2 sequence comparison computer program was authored by
Genentech, Inc. and its source code has been filed with user
documentation in the U.S. Copyright Office, Washington D.C., 20559,
where it is registered under U.S. Copyright Registration No.
TXU510087. The ALIGN-2 program is publicly available through
Genentech, Inc., South San Francisco, Calif., and the source code
for the ALIGN-2 program and instructions for its use are disclosed
in International Application Publication No. WO2000/39297 published
Jul. 6, 2000. The ALIGN-2 program should be compiled for use on a
UNIX operating system, preferably digital UNIX V4.0D. All sequence
comparison parameters are set by the ALIGN-2 program and do not
vary.
[0069] For purposes herein, the % amino acid sequence identity of a
given amino acid sequence A to, with, or against a given amino acid
sequence B (which can alternatively be phrased as a given amino
acid sequence A that has or comprises a certain % amino acid
sequence identity to, with, or against a given amino acid sequence
B) is calculated as follows:
100 times the fraction X/Y
[0070] where X is the number of amino acid residues scored as
identical matches by the sequence alignment program ALIGN-2 in that
program's alignment of A and B, and where Y is the total number of
amino acid residues in B. It will be appreciated that where the
length of amino acid sequence A is not equal to the length of amino
acid sequence B, the % amino acid sequence identity of A to B will
not equal the % amino acid sequence identity of B to A. Unless
specifically stated otherwise, all % amino acid sequence identity
values used herein are obtained as described above using the
ALIGN-2 sequence comparison computer program. However, % amino acid
sequence identity may also be determined using the sequence
comparison program NCBI-BLAST2 (Altschul et al., Nucleic Acids Res.
25:3389-3402 (1997)). The NCBI-BLAST2 sequence comparison program
may be downloaded from http://www.ncbi.nlm.nih.gov. NCBI-BLAST2
uses several search parameters, wherein all of those search
parameters are set to default values including, for example,
unmask=yes, strand=all, expected occurrences=10, minimum low
complexity length=15/5, multi-pass e-value=0.01, constant for
multi-pass=25, dropoff for final gapped alignment=25 and scoring
matrix=BLOSUM62.
[0071] In situations where NCBI-BLAST2 is employed for amino acid
sequence comparisons, the % amino acid sequence identity of a given
amino acid sequence A to, with, or against a given amino acid
sequence B (which can alternatively be phrased as a given amino
acid sequence A that has or comprises a certain % amino acid
sequence identity to, with, or against a given amino acid sequence
B) is calculated as follows:
100 times the fraction X/Y
[0072] where X is the number of amino acid residues scored as
identical matches by the sequence alignment program NCBI-BLAST2 in
that program's alignment of A and B, and where Y is the total
number of amino acid residues in B. It will be appreciated that
where the length of amino acid sequence A is not equal to the
length of amino acid sequence B, the % amino acid sequence identity
of A to B will not equal the % amino acid sequence identity of B to
A.
[0073] Nucleic acid is "operably linked" when it is placed into a
functional relationship with another nucleic acid sequence. For
example, DNA for a presequence or secretory leader is operably
linked to DNA for a polypeptide if it is expressed as a preprotein
that participates in the secretion of the polypeptide; a promoter
or enhancer is operably linked to a coding sequence if it affects
the transcription of the sequence; or a ribosome binding site is
operably linked to a coding sequence if it is positioned so as to
facilitate translation. Generally, "operably linked" means that the
DNA sequences being linked are contiguous, and, in the case of a
secretory leader, contiguous and in reading phase. However,
enhancers do not have to be contiguous. Linking is accomplished by
ligation at convenient restriction sites. If such sites do not
exist, the synthetic oligonucleotide adaptors or linkers are used
in accordance with conventional practice.
[0074] The term "disease state" refers to a physiological state of
a cell or of a whole mammal in which an interruption, cessation, or
disorder of cellular or body functions, systems, or organs has
occurred.
[0075] The term "effective amount" refers to an amount of a drug
effective to treat (including prevention) of a disease, disorder or
unwanted physiological conditions in a mammal. In the present
invention, an "effective amount" of an anti-IFN-.alpha. antibody
may reduce, slow down or delay an autoimmune disorder such as IDDM
or SLE; reduce, prevent or inhibit slow to some extent and
preferably stop) the development of an autoimmune disorder such as
IDDM or SLE; and/or relieve to some extent one or more of the
symptoms associated with autoimmune disorders such as IDDM or
SLE.
[0076] In the methods of the present invention, the term "control"
and grammatical variants thereof, are used to refer to the
prevention, partial or complete inhibition, reduction, delay or
slowing down of an unwanted event, e.g. physiological condition,
such as the generation of autoreactive T cells and development of
autoimmunity.
[0077] "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 prone to
have the disorder or those in which the disorder is to be
prevented. For purposes of this invention, beneficial or desired
clinical results include, but are not limited to, alleviation of
symptoms, diminishment of extent of disease, stabilized (i.e., not
worsening) state of disease, delay or slowing of disease
progression, amelioration or palliation of the disease state, and
remission (whether partial or total), whether detectable or
undetectable. "Treatment" can also mean prolonging survival as
compared to expected survival if not receiving treatment. Those in
need of treatment include those already with the condition or
disorder as well as those prone to have the condition or disorder
or those in which the condition or disorder is to be prevented.
[0078] "Pharmaceutically acceptable" carriers, excipients, or
stabilizers are ones which are nontoxic to the cell or mammal being
exposed thereto at the dosages and concentrations employed. Often
the physiologically acceptable carrier is an aqueous pH buffered
solution. Examples of physiologically acceptable carriers 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.TM., polyethylene glycol (PEG), and
Pluronics.TM..
[0079] "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.
B. Methods for Carrying Out the Invention
[0080] 1. Generation of Antibodies
[0081] (i) Polyclonal Antibodies
[0082] Methods of preparing polyclonal antibodies are known in the
art. Polyclonal antibodies can be raised in a mammal, for example,
by one or more injections of an immunizing agent and, if desired,
an adjuvant. Typically, the immunizing agent and/or adjuvant will
be injected in the mammal by multiple subcutaneous or
intraperitoneal injections. It may be useful to conjugate the
immunizing agent to a protein known to be immunogenic in the mammal
being immunized, such as serum albumin, or soybean trypsin
inhibitor. Examples of adjuvants which may be employed include
Freund's complete adjuvant and MPL-TDM.
[0083] In another preferred embodiment, animals are immunized with
a mixture of various, preferably all, IFN-.alpha. subtypes in order
to generate anti-IFN-.alpha. antibodies with broad reactivity
against IFN-.alpha. subtypes. In another preferred embodiment,
animals are immunized with the mixture of human IFN-.alpha.
subtypes that is present in the human lymphoblastoid interferons
secreted by Burkitt lymphoma cells (Namalva cells) induced with
Sendai virus, as described in Example 1 below. A suitable
preparation of such human lymphoblastoid interferons can be
obtained commercially (Product No. 1-9887) from Sigma Chemical
Company, St. Louis, Mo.
[0084] (ii) Monoclonal Antibodies
[0085] Monoclonal antibodies may 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).
[0086] In the hybridoma method, a mouse or other appropriate host
animal, such as a hamster or macaque monkey, is immunized as
hereinabove described to elicit lymphocytes that produce or are
capable of producing antibodies that will specifically bind to the
protein used for immunization. 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 (Coding, Monoclonal Antibodies: Principles and
Practice, pp. 59-103, [Academic Press, 1996]).
[0087] 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.
[0088] 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 MOP-21 and MC-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]).
[0089] Culture medium in which hybridoma cells are growing is
assayed for production of monoclonal antibodies directed against
the antigen. 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 immunosorbent assay
(ELISA).
[0090] 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).
[0091] After hybridoma cells are identified that produce antibodies
of the desired specificity, affinity, and/or activity, the cells
may be subcloned by limiting dilution procedures and grown by
standard methods (Goding, Monoclonal Antibodies: Principles and
Practice, pp. 59-103, Academic Press, 1996). Suitable culture media
for this purpose include, for example, DMEM or RPMI-1640 medium. In
addition, the hybridoma cells may be grown in vivo as ascites
tumors in an animal.
[0092] 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.
[0093] DNA encoding the monoclonal antibodies is readily isolated
and sequenced using conventional procedures (e.g., by using
oligonucleotide probes that are capable of binding specifically to
genes encoding the heavy and light chains of the monoclonal
antibodies). The hybridoma cells serve as a preferred source of
such DNA. Once isolated, the DNA may 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 monoclonal antibodies in the recombinant
host cells. The DNA also may be modified, for example, by
substituting the coding sequence for human heavy and light chain
constant domains in place of the homologous murine sequences
(Morrison, et al., Proc. Nat. Acad. Sci. 81: 6851 [1984]), or by
covalently joining to the immunoglobulin coding sequence all or
part of the coding sequence for a non-immunoglobulin polypeptide.
In that manner, "chimeric" or "hybrid" antibodies are prepared that
have the binding specificity of an anti-IFN-.alpha. monoclonal
antibody herein.
[0094] 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 an IFN-.alpha. and another antigen-combining
site having specificity for a different antigen.
[0095] Chimeric or hybrid antibodies also may be prepared in vitro
using known methods in synthetic protein chemistry, including those
involving crosslinking agents. For example, immunotoxins may 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.
[0096] Recombinant production of antibodies will be described in
more detail below.
[0097] (iii) Humanized Antibodies
[0098] Generally, a humanized antibody has one or more amino acid
residues introduced into it from a non-human source. These
non-human amino acid residues are often referred to as "import"
residues, which are typically taken from an "import" variable
domain. Humanization can be essentially performed following the
method of Winter and co-workers by substituting rodent CDRs or CDR
sequences for the corresponding sequences of a human antibody
(Jones et al., Nature 321: 522-525 [1986]; Riechmann et al., Nature
332: 323-327 [1988]; Verhoeyen et al., Science 239: 1534-1536
[1988)]; reviewed in Clark, Immunol. Today 21: 397-402 [2000]).
[0099] Accordingly, such "humanized" antibodies are chimeric
antibodies (Cahilly, 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 rodent antibodies.
[0100] It is 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
conformational 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 its
antigen. In this way, FR residues can be selected and combined from
the consensus and import sequence 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. For
further details, see U.S. Pat. No. 5,821,337.
[0101] (iv) Human Antibodies
[0102] Attempts to use the same technology for generating human
mAbs have been hampered by the lack of a suitable human myeloma
cell line. The best results were obtained using heteromyelomas
(mouse.times.human hybrid myelomas) as fusion partners (Kozbor, J.
Immunol. 133: 3001 (1984); Brodeur, et al., Monoclonal Antibody
Production Techniques and Applications, pp. 51-63, Marcel Dekker,
Inc., New York, 1987). Alternatively, human antibody-secreting
cells can be immortalized by infection with the Epstein-Barr virus
(EBV). However, EBV-infected cells are difficult to clone and
usually produce only relatively low yields of immunoglobulin (James
and Bell, J. Immunol. Methods 100: 5-40 [1987]). In future, the
immortalization of human B cells might possibly be achieved by
introducing a defined combination of transforming genes. Such a
possibility is highlighted by a recent demonstration that the
expression of the telomerase catalytic subunit together with the
SV40 large T oncoprotein and an oncogenic allele of H-ras resulted
in the tumorigenic conversion of normal human epithelial and
fibroblast cells (Hahn et al., Nature 400: 464-468 [1999]).
[0103] It is now possible to produce transgenic animals (e.g. mice)
that are capable, upon immunization, of producing a repertoire of
human antibodies in the absence of endogenous immunoglobulin
production (Jakobovits et al., Nature 362: 255-258 [1993]; Lonberg
and Huszar, Int. Rev. Immunol. 13: 65-93 [1995]; Fishwild et al.,
Nat. Biotechnol. 14: 845-851 [1996]; Mendez et al., Nat. Genet. 15:
146-156 [1997]; Green, J. Immunol. Methods 231: 11-23 [1999];
Tomizuka et al., Proc. Natl. Acad. Sci. USA 97: 722-727 [2000];
reviewed in Little et al., Immunol. Today 21: 364-370 [2000]). For
example, it has been described that the homozygous deletion of the
antibody heavy chain joining region (J.sub.H) gene in chimeric and
germ-line mutant mice results in complete inhibition of endogenous
antibody production (Jakobovits et al., Proc. Natl. Acad. Sci. USA
90: 2551-2555 [1993]). Transfer of the human germ-line
immunoglobulin gene array in such germ-line mutant mice results in
the production of human antibodies upon antigen challenge
(Jakobovits et al., Nature 362: 255-258 [1993]).
[0104] Mendez et al. (Nature Genetics 15: 146-156 [1997]) have
generated a line of transgenic mice designated as "XenoMouse.RTM.
II" that, when challenged with an antigen, generates high affinity
fully human antibodies. This was achieved by germ-line integration
of megabase human heavy chain and light chain loci into mice with
deletion into endogenous J.sub.H segment as described above. The
XenoMouse.RTM. II harbors 1,020 kb of human heavy chain locus
containing approximately 66 V.sub.H genes, complete D.sub.H and
J.sub.H regions and three different constant regions (.mu., .delta.
and .gamma.), and also harbors 800 kb of human .kappa. locus
containing 32 V.kappa. genes, J.kappa. segments and C.kappa. genes.
The antibodies produced in these mice closely resemble that seen in
humans in all respects, including gene rearrangement, assembly, and
repertoire. The human antibodies are preferentially expressed over
endogenous antibodies due to deletion in endogenous J.sub.H segment
that prevents gene rearrangement in the murine locus.
[0105] Tomizuka et al. (Proc. Natl. Acad. Sci. USA 97: 722-727
[2000]) have recently described generation of a double
trans-chromosomic (Tc) mice by introducing two individual human
chromosome fragments (hCFs), one containing the entire Ig heavy
chain locus (IgH, .about.1.5 Mb) and the other the entire .kappa.
light chain locus (Ig.kappa., .about.2 Mb) into a mouse strain
whose endogenous IgH and Ig.kappa. loci were inactivated. These
mice mounted antigen-specific human antibody response in the
absence of mouse antibodies. The Tc technology may allow for the
humanization of over megabase-sized, complex loci or gene clusters
(such as those encoding T-cell receptors, major histocompatibility
complex, P450 cluster etc) in mice or other animals. Another
advantage of the method is the elimination of a need of cloning the
large loci. This is a significant advantage since the cloning of
over megabase-sized DNA fragments encompassing whole Ig loci
remains difficult even with the use of yeast artificial chromosomes
(Peterson et al., Trends Genet. 13: 61-66 [1997]; Jacobovits, Curr.
Biol. 4: 761-763 [1994]). Moreover, the constant region of the
human IgH locus is known to contain sequences difficult to clone
(Kang and Cox, Genomics 35: 189-195 [1996]).
[0106] Alternatively, the phage display technology can be used to
produce human antibodies and antibody fragments in vitro, from
immunoglobulin variable (V) domain gene repertoires from
unimmunized donors (McCafferty et al., Nature 348: 552-553 [1990];
reviewed in Kipriyanov and Little, Mol. Biotechnol. 12: 173-201
[1999]; Hoogenboom and Chames, Immunol. Today 21: 371-378 [2000]).
According to this technique, antibody V domain genes are cloned
in-frame into either a major or minor coat protein gene of a
filamentous bacteriophage, such as M13 or fd, and displayed as
functional antibody fragments on the surface of the phage particle.
Because the filamentous particle contains a single-stranded DNA
copy of the phage genome, selections based on the functional
properties of the antibody also result in selection of the gene
encoding the antibody exhibiting those properties. Thus, the phage
mimics some of the properties of the B-cell. Phage display can be
performed in a variety of formats (reviewed in Johnson and
Chiswell, Current Opinion in Structural Biology 3: 564-571 [1993)];
Winter et al., Annu. Rev. Immunol. 12: 433-455 [1994]; Dall'Acqua
and Carter, Curr. Opin. Struct. Biol. 8: 443-450 [1998]; Hoogenboom
and Chames, Immunol. Today 21: 371-378 [2000]). Several sources of
V-gene segments can be used for phage display. Clackson et al.,
(Nature 352: 624-628 [1991]) isolated a diverse array of
anti-oxazolone antibodies from a small random combinatorial library
of V genes derived from the spleens of immunized mice. A repertoire
of V genes from unimmunized human donors can be constructed and
antibodies to a diverse array of antigens (including self-antigens)
can be isolated essentially following the techniques described by
Marks et al., J. Mol. Biol. 222: 581-597 (1991), or Griffiths et
al., EMBO J. 12: 725-734 (1993). In a natural immune response,
antibody genes accumulate mutations at a high rate (somatic
hypermutation). Some of the changes introduced will confer higher
affinity, and B cells displaying high-affinity surface
immunoglobulin are preferentially replicated and differentiated
during subsequent antigen challenge. This natural process can be
mimicked by employing the technique known as "chain shuffling"
(Marks et al., Bio/Technol. 10: 779-783 [1992]). In this method,
the affinity of "primary" human antibodies obtained by phage
display can be improved by sequentially replacing the heavy and
light chain V region genes with repertoires of naturally occurring
variants (repertoires) of V domain genes obtained from unimmunized
donors. This technique allows the production of antibodies and
antibody fragments with affinities in the nM range. A strategy for
making very large phage antibody repertoires (also known as "the
mother-of-all libraries") has been described by Waterhouse et al.,
Nucl. Acids Res. 21: 2265-2266 (1993), and the isolation of a high
affinity human antibody directly from such large phage library is
reported by Griffiths et al., EMBO J. 13: 3245-3260 (1994). Gene
shuffling can also be used to derive human antibodies from rodent
antibodies, where the human antibody has similar affinities and
specificities to the starting rodent antibody. According to this
method, which is also referred to as "epitope imprinting", the
heavy or light chain V domain gene of rodent antibodies obtained by
phage display technique is replaced with a repertoire of human V
domain genes, creating rodent-human chimeras. Selection on antigen
results in isolation of human variable capable of restoring a
functional antigen-binding site, i.e. the epitope governs
(imprints) the choice of partner. When the process is repeated in
order to replace the remaining rodent V domain, a human antibody is
obtained (see PCT patent application WO 93/06213, published 1 Apr.
1993). Unlike traditional humanization of rodent antibodies by CDR
grafting, this technique provides completely human antibodies,
which have no framework or CDR residues of rodent origin.
[0107] (v) Bispecific Antibodies
[0108] 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 an IFN-.alpha. to provide a neutralizing
antibody, the other one is for any other antigen.
[0109] 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 (Millstein and Cuello, Nature 305: 537-539
[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 PCT application publication No. WO
93/08829 (published 13 May 1993), and in Traunecker et al., EMBO J.
10: 3655-3659 (1991).
[0110] 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. 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.
[0111] For further details of generating bispecific antibodies see,
for example, Suresh et al., Methods in Enzymology 121, 210
(1986).
[0112] (vi) Heteroconjugate Antibodies
[0113] Heteroconjugate antibodies are also within the scope of the
present invention. Heteroconjugate antibodies are composed of two
covalently joined antibodies. 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 (PCT
application publication Nos. WO 91/00360 and WO 92/200373; 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.
[0114] (vii) Antibody Fragments
[0115] In certain embodiments, the neutralizing anti-IFN-.alpha.
antibody (including murine, human and humanized antibodies, and
antibody variants) is an antibody fragment. Various techniques have
been developed for the production of antibody fragments.
Traditionally, these fragments were derived via proteolytic
digestion of intact antibodies (see, e.g., Morimoto et al., J.
Biochem. Biophys. Methods 24:107-117 [1992] and Brennan et al.,
Science 229:81 [1985]). However, these fragments can now be
produced directly by recombinant host cells (reviewed in Hudson,
Curr. Opin. Immunol. 11: 548-557 [1999]; Little et al., Immunol.
Today 21: 364-370 [2000]). For example, Fab'-SH fragments can be
directly recovered from E. coli and chemically coupled to form
F(ab').sub.2 fragments (Carter et al., Bio/Technology 10:163-167
[1992]). In another embodiment, the F(ab').sub.2 is formed using
the leucine zipper GCN4 to promote assembly of the F(ab').sub.2
molecule. According to another approach, Fv, Fab or F(ab').sub.2
fragments can be isolated directly from recombinant host cell
culture. Other techniques for the production of antibody fragments
will be apparent to the skilled practitioner.
[0116] (viii) Amino Acid Sequence Variants of Antibodies
[0117] Amino acid sequence modification(s) of the anti-IFN-.alpha.
antibodies described herein are contemplated. For example, it may
be desirable to improve the binding affinity and/or other
biological properties of the antibody. Amino acid sequence variants
of the anti-IFN-.alpha. antibodies are prepared by introducing
appropriate nucleotide changes into the nucleic acid encoding the
anti-IFN-.alpha. antibody chains, or by peptide synthesis. Such
modifications include, for example, deletions from, and/or
insertions into and/or substitutions of, residues within the amino
acid sequences of the anti-IFN-.alpha. antibody. Any combination of
deletion, insertion, and substitution is made to arrive at the
final construct, provided that the final construct possesses the
desired characteristics. The amino acid changes also may alter
post-translational processes of the anti-IFN-.alpha. antibody, such
as changing the number or position of glycosylation sites.
[0118] A useful method for identification of certain residues or
regions of the neutralizing anti-IFN-.alpha.antibody that are
preferred locations for mutagenesis is called "alanine scanning
mutagenesis," as described by Cunningham and Wells Science,
244:1081-1085 (1989). Here, a residue or group of target residues
are identified (e.g., charged residues such as arg, asp, his, lys,
and gln) and replaced by a neutral or negatively charged amino acid
(most preferably alanine or polyalanine) to affect the interaction
of the amino acids with the antigen. Those amino acid locations
demonstrating functional sensitivity to the substitutions then are
refined by introducing further or other variants at, or for, the
sites of substitution. Thus, while the site for introducing an
amino acid sequence variation is predetermined, the nature of the
mutation per se need not be predetermined. For example, to analyze
the performance of a mutation at a given site, ala scanning or
random mutagenesis is conducted at the target codon or region and
the expressed anti-IFN-.alpha. antibody variants are screened for
the desired activity.
[0119] Amino acid sequence insertions include amino- and/or
carboxyl-terminal fusions ranging in length from one residue to
polypeptides containing a hundred or more residues, as well as
intra-sequence insertions of single or multiple amino acid
residues. Examples of terminal insertions include an
anti-IFN-.alpha. neutralizing antibody with an N-terminal methionyl
residue or the antibody fused to an epitope tag. Other insertional
variants of the anti-IFN-.alpha. antibody molecule include the
fusion to the N- or C-terminus of the antibody of an enzyme or a
polypeptide which increases the serum half-life of the
antibody.
[0120] Another type of variant is an amino acid substitution
variant. These variants have at least one amino acid residue in the
neutralizing anti-IFN-.alpha. antibody molecule removed and a
different residue inserted in its place. The sites of greatest
interest for substitution mutagenesis include the hypervariable
regions, but FR alterations are also contemplated. Conservative
substitutions are shown in Table 1 under the heading of "preferred
substitutions". If such substitutions result in a change in
biological activity, then more substantial changes, denominated
"exemplary substitutions" in Table 1, or as further described below
in reference to amino acid classes, may be introduced and the
products screened.
TABLE-US-00001 TABLE 1 Exemplary Preferred Original Residue
Substitutions Substitutions Ala (A) val; leu; ile val Arg (R) lys;
gln; asn lys Asn (N) gln; his; asp, lys; arg gln Asp (D) glu; asn
glu Cys (C) ser; ala ser Gln (Q) asn; glu asn Glu (E) asp; gln asp
Gly (G) ala ala His (H) asn; gln; lys; arg arg Ile (I) leu; val;
met; ala; leu phe; norleucine Leu (L) norleucine; ile; val; ile
met; ala; phe Lys (K) arg; gln; asn arg Met (M) leu; phe; ile leu
Phe (F) leu; val; ile; ala; tyr tyr Pro (P) ala ala Ser (S) thr thr
Thr (T) ser ser Trp (W) tyr; phe tyr Tyr (Y) trp; phe; thr; ser phe
Val (V) ile; leu; met; phe; leu ala; norleucine
[0121] Substantial modifications in the biological properties of
the antibody are accomplished by selecting substitutions that
differ significantly in their effect on maintaining (a) the
structure of the polypeptide backbone in the area of the
substitution, for example, as a sheet or helical conformation, (b)
the charge or hydrophobicity of the molecule at the target site, or
(c) the bulk of the side chain. Naturally occurring residues are
divided into groups based on common side-chain properties:
[0122] (1) hydrophobic: norleucine, met, ala, val, leu, ile;
[0123] (2) neutral hydrophilic: cys, ser, thr;
[0124] (3) acidic: asp, glu;
[0125] (4) basic: asn, gln, his, lys, arg;
[0126] (5) residues that influence chain orientation: gly, pro;
and
[0127] (6) aromatic: trp, tyr, phe.
[0128] Non-conservative substitutions will entail exchanging a
member of one of these classes for another class.
[0129] Any cysteine residue not involved in maintaining the proper
conformation of the neutralizing anti-IFN-.alpha. antibody also may
be substituted, generally with serine, to improve the oxidative
stability of the molecule and prevent aberrant crosslinking.
Conversely, cysteine bond(s) may be added to the antibody to
improve its stability (particularly where the antibody is an
antibody fragment such as a Fv fragment).
[0130] A particularly preferred type of substitution variant
involves substituting one or more hypervariable region residues of
a parent antibody (e.g. a humanized or human antibody). Generally,
the resulting variant(s) selected for further development will have
improved biological properties relative to the parent antibody from
which they are generated. A convenient way for generating such
substitution variants is affinity maturation using phage display.
Briefly, several hypervariable region sites (e.g. 6-7 sites) are
mutated to generate all possible amino substitutions at each site.
The antibody variants thus generated are displayed in a monovalent
fashion from filamentous phage particles as fusions to the gene III
product of M13 packaged within each particle. The phage-displayed
variants are then screened for their biological activity (e.g.
antagonist activity) as herein disclosed. In order to identify
candidate hypervariable region sites for modification, alanine
scanning mutagenesis can be performed to identify hypervariable
region residues contributing significantly to antigen binding.
Alternatively, or in addition, it may be beneficial to analyze a
crystal structure of the antigen-antibody complex to identify
contact points between the antibody and IFN-.alpha.. Such contact
residues and neighboring residues are candidates for substitution
according to the techniques elaborated herein. Once such variants
are generated, the panel of variants is subjected to screening as
described herein and antibodies with superior properties in one or
more relevant assays may be selected for further development.
[0131] (ix) Glycosylation Variants
[0132] Antibodies are glycosylated at conserved positions in their
constant regions (Jefferis and Lund, Chem. Immunol. 65:111-128
[1997]; Wright and Morrison, Trends Biotechnol. 15:26-32 [1997]).
The oligosaccharide side chains of the immunoglobulins affect the
protein's function (Boyd et al., Mol. Immunol. 32:1311-1318 [1996];
Wittwe and Howard, Biochem. 29:4175-4180 [1990]), and the
intramolecular interaction between portions of the glycoprotein
which can affect the conformation and presented three-dimensional
surface of the glycoprotein (Jefferis and Lund, supra; Wyss and
Wagner, Current Opin. Biotech. 7:409-416 [1996]). Oligosaccharides
may also serve to target a given glycoprotein to certain molecules
based upon specific recognition structures. For example, it has
been reported that in agalactosylated IgG, the oligosaccharide
moiety `flips` out of the inter-CH2 space and terminal
N-acetylglucosamine residues become available to bind mannose
binding protein (Malhotra et al., Nature Med. 1:237-243 [1995]).
Removal by glycopeptidase of the oligosaccharides from CAMPATH-1H
(a recombinant humanized murine monoclonal IgG1 antibody which
recognizes the CDw52 antigen of human lymphocytes) produced in
Chinese Hamster Ovary (CHO) cells resulted in a complete reduction
in complement mediated lysis (CMCL) (Boyd et al., Mol. Immunol.
32:1311-1318 [1996]), while selective removal of sialic acid
residues using neuraminidase resulted in no loss of DMCL.
Glycosylation of antibodies has also been reported to affect
antibody-dependent cellular cytotoxicity (ADCC). In particular, CHO
cells with tetracycline-regulated expression of
.beta.(1,4)--N-acetylglucosaminyltransferase III (GnTIII), a
glycosyltransferase catalyzing formation of bisecting GlcNAc, was
reported to have improved ADCC activity (Umana et al., Mature
Biotech. 17:176-180 [1999]).
[0133] Glycosylation of antibodies is typically either N-linked or
O-linked. N-linked refers to the attachment of the carbohydrate
moiety to the side chain of an asparagine residue. The tripeptide
sequences asparagine-X-serine and asparagine-X-threonine, where X
is any amino acid except proline, are the recognition sequences for
enzymatic attachment of the carbohydrate moiety to the asparagine
side chain. Thus, the presence of either of these tripeptide
sequences in a polypeptide creates a potential glycosylation site.
O-linked glycosylation refers to the attachment of one of the
sugars N-aceylgalactosamine, galactose, or xylose to a hydroxyamino
acid, most commonly serine or threonine, although 5-hydroxyproline
or 5-hydroxylysine may also be used.
[0134] Glycosylation variants of antibodies are variants in which
the glycosylation pattern of an antibody is altered. By altering is
meant deleting one or more carbohydrate moieties found in the
antibody, adding one or more carbohydrate moieties to the antibody,
changing the composition of glycosylation (glycosylation pattern),
the extent of glycosylation, etc.
[0135] Addition of glycosylation sites to the antibody is
conveniently accomplished by altering the amino acid sequence such
that it contains one or more of the above-described tripeptide
sequences (for N-linked glycosylation sites). The alteration may
also be made by the addition of or substitution by, one or more
serine or threonine residues to the sequence of the original
antibody (for O-linked glycosylation sites). Similarly, removal of
glycosylation sites can be accomplished by amino acid alteration
within the native glycosylation sites of the antibody.
[0136] The amino acid sequence is usually altered by altering the
underlying nucleic acid sequence. Nucleic acid molecules encoding
amino acid sequence variants of the anti-IFN-.alpha. antibody are
prepared by a variety of methods known in the art. These methods
include, but are not limited to, isolation from a natural source
(in the case of naturally occurring amino acid sequence variants)
or preparation by oligonucleotide-mediated (or site-directed)
mutagenesis, PCR mutagenesis, and cassette mutagenesis of an
earlier prepared variant or a non-variant version of the
anti-IFN-.alpha. antibody.
[0137] The glycosylation (including glycosylation pattern) of
antibodies may also be altered without altering the amino acid
sequence or the underlying nucleotide sequence. Glycosylation
largely depends on the host cell used to express the antibody.
Since the cell type used for expression of recombinant
glycoproteins, e.g. antibodies, as potential therapeutics is rarely
the native cell, significant variations in the glycosylation
pattern of the antibodies can be expected (see, e.g. Hse et al., J.
Biol. Chem. 272:9062-9070 [1997]). In addition to the choice of
host cells, factors which affect glycosylation during recombinant
production of antibodies include growth mode, media formulation,
culture density, oxygenation, pH, purification schemes and the
like. Various methods have been proposed to alter the glycosylation
pattern achieved in a particular host organism including
introducing or overexpressing certain enzymes involved in
oligosaccharide production (U.S. Pat. Nos. 5,047,335; 5,510,261 and
5,278,299). Glycosylation, or certain types of glycosylation, can
be enzymatically removed from the glycoprotein, for example using
endoglycosidase H (Endo H). In addition, the recombinant host cell
can be genetically engineered, e.g. make defective in processing
certain types of polysaccharides. These and similar techniques are
well known in the art.
[0138] The glycosylation structure of antibodies can be readily
analyzed by conventional techniques of carbohydrate analysis,
including lectin chromatography, NMR, Mass spectrometry, HPLC, GPC,
monosaccharide compositional analysis, sequential enzymatic
digestion, and HPAEC-PAD, which uses high pH anion exchange
chromatography to separate oligosaccharides based on charge.
Methods for releasing oligosaccharides for analytical purposes are
also known, and include, without limitation, enzymatic treatment
(commonly performed using peptide-N-glycosidase
F/endo-.beta.-galactosidase), elimination using harsh alkaline
environment to release mainly O-linked structures, and chemical
methods using anhydrous hydrazine to release both N- and O-linked
oligosaccharides.
[0139] (x) Other Modifications of Antibodies
[0140] The neutralizing anti-IFN-.alpha. antibodies disclosed
herein may also be formulated as immunoliposomes. Liposomes
containing the antibody are prepared by methods known in the art,
such as described in Epstein et al., Proc. Natl. Acad. Sci. USA
82:3688 (1985); Hwang et al., Proc. Natl. Acad. Sci. USA 77:4030
(1980); and U.S. Pat. Nos. 4,485,045 and 4,544,545. Liposomes with
enhanced circulation time are disclosed in U.S. Pat. No.
5,013,556.
[0141] Particularly useful liposomes can be generated by the
reverse phase evaporation method with a lipid composition
comprising phosphatidylcholine, cholesterol and PEG-derivatized
phosphatidylethanolamine (PEG-PE). Liposomes are extruded through
filters of defined pore size to yield liposomes with the desired
diameter. Fab' fragments of the antibody of the present invention
can be conjugated to the liposomes as described in Martin et al.,
J. Biol. Chem. 257:286-288 (1982) via a disulfide interchange
reaction. A chemotherapeutic agent (such as Doxorubicin) is
optionally contained within the liposome. See Gabizon et al., J.
National Cancer Inst. 81(19):1484 (1989).
[0142] The antibody of the present invention may also be used in
ADEPT by conjugating the antibody to a prodrug-activating enzyme
which converts a prodrug (e.g., a peptidyl chemotherapeutic agent,
see WO81/01145) to an active drug. See, for example, WO 88/07378
and U.S. Pat. No. 4,975,278.
[0143] The enzyme component of the immunoconjugate useful for ADEPT
includes any enzyme capable of acting on a prodrug in such a way so
as to covert it into its more active form exhibiting the desired
biological properties.
[0144] Enzymes that are useful in the method of this invention
include, but are not limited to, alkaline phosphatase useful for
converting phosphate-containing prodrugs into free drugs;
arylsulfatase useful for converting sulfate-containing prodrugs
into free drugs; cytosine deaminase useful for converting non-toxic
5-fluorocytosine into the anti-cancer drug, 5-fluorouracil;
proteases, such as serratia protease, thermolysin, subtilisin,
carboxypeptidases and cathepsins (such as cathepsins B and L), that
are useful for converting peptide-containing prodrugs into free
drugs; D-alanylcarboxypeptidases, useful for converting prodrugs
that contain D-amino acid substituents; carbohydrate-cleaving
enzymes such as .beta.-galactosidase and neuraminidase useful for
converting glycosylated prodrugs into free drugs; .beta.-lactamase
useful for converting drugs derivatized with .beta.-lactams into
free drugs; and penicillin amidases, such as penicillin V amidase
or penicillin G amidase, useful for converting drugs derivatized at
their amine nitrogens with phenoxyacetyl or phenylacetyl groups,
respectively, into free drugs. Alternatively, antibodies with
enzymatic activity, also known in the art as "abzymes", can be used
to convert the prodrugs of the invention into free active drugs
(see, e.g., Massey, Nature 328:457-458 (1987)). Antibody-abzyme
conjugates can be prepared as described herein for delivery of the
abzyme to a desired cell population.
[0145] The enzymes can be covalently bound to the neutralizing
anti-IFN-.alpha. antibodies by techniques well known in the art
such as the use of the heterobifunctional crosslinking reagents
discussed above. Alternatively, fusion proteins comprising at least
the antigen binding region of an antibody of the invention linked
to at least a functionally active portion of an enzyme of the
invention can be constructed using recombinant DNA techniques well
known in the art (see, e.g., Neuberger et al., Nature 312:604-608
[1984]).
[0146] In certain embodiments of the invention, it may be desirable
to use an antibody fragment, rather than an intact antibody. In
this case, it may be desirable to modify the antibody fragment in
order to increase its serum half-life. This may be achieved, for
example, by incorporation of a salvage receptor binding epitope
into the antibody fragment (e.g., by mutation of the appropriate
region in the antibody fragment or by incorporating the epitope
into a peptide tag that is then fused to the antibody fragment at
either end or in the middle, e.g., by DNA or peptide synthesis).
See WO96/32478 published Oct. 17, 1996.
[0147] The salvage receptor binding epitope generally constitutes a
region wherein any one or more amino acid residues from one or two
loops of a Fc domain are transferred to an analogous position of
the antibody fragment. Even more preferably, three or more residues
from one or two loops of the Fc domain are transferred. Still more
preferred, the epitope is taken from the CH2 domain of the Fc
region (e.g., of an IgG) and transferred to the CH1, CH3, or
V.sub.H region, or more than one such region, of the antibody.
Alternatively, the epitope is taken from the CH2 domain of the Fe
region and transferred to the C.sub.L region or V.sub.L region, or
both, of the antibody fragment.
[0148] Covalent modifications of the neutralizing anti-IFN-.alpha.
antibodies are also included within the scope of this invention.
They may be made by chemical synthesis or by enzymatic or chemical
cleavage of the antibody, if applicable. Other types of covalent
modifications of the antibody are introduced into the molecule by
reacting targeted amino acid residues of the antibody with an
organic derivatizing agent that is capable of reacting with
selected side chains or the N- or C-terminal residues. Exemplary
covalent modifications of polypeptides are described in U.S. Pat.
No. 5,534,615, specifically incorporated herein by reference. A
preferred type of covalent modification of the antibody comprises
linking the antibody to one of a variety of nonproteinaceous
polymers, e.g., polyethylene glycol, polypropylene glycol, or
polyoxyalkylenes, in the manner set forth in U.S. Pat. No.
4,640,835; 4,496,689; 4,301,144; 4,670,417; 4,791,192 or
4,179,337.
[0149] 2. Screening for Antibodies with the Desired Properties
[0150] Techniques for generating antibodies have been described
above. Anti-IFN-.alpha. antibodies with the desired, broad range
neutralizing properties can then be identified by methods known in
the art.
[0151] (i) Binding Assays
[0152] Thus, for example, the neutralizing anti-IFN-.alpha.
antibodies of the present invention can be identified in
IFN-.alpha. binding assays, by incubating a candidate antibody with
one or more individual IFN-.alpha. subtypes, or an array or mixture
of various IFN-.alpha. subtypes, and monitoring binding and
neutralization of a biological activity of IFN-.alpha.. The binding
assay may be performed with purified IFN-.alpha. polypeptide(s). In
one embodiment, the binding assay is a competitive binding assay,
where the ability of a candidate antibody to compete with a known
anti-IFN-.alpha. antibody for IFN-.alpha. binding is evaluated. The
assay may be performed in various formats, including the ELISA
format, also illustrated in the Examples below. IFN-.alpha. binding
of a candidate antibody may also be monitored in a BIAcore.TM.
Biosensor assay, as described below.
[0153] Any suitable competition binding assay known in the art can
be used to characterize the ability of a candidate anti-IFN-.alpha.
monoclonal antibody to compete with murine anti-human IFN-.alpha.
monoclonal antibody 9F3 for binding to a particular IFN-.alpha.
species. A routine competition assay is described in Antibodies: A
Laboratory Manual, Cold Spring Harbor Laboratory, Ed Harlow and
David Lane (1988). In another embodiment, the IFN-.alpha.-binding
ELISA assay described in Example 2 below could be modified to
employ IFN-.alpha. binding competition between a candidate antibody
and the 9F3 antibody. Such an assay could be performed by layering
the IFN-.alpha. on microtiter plates, incubating the layered plates
with serial dilutions of unlabeled anti-IFN-.alpha. antibody or
unlabeled control antibody admixed with a selected concentration of
labeled 9F3 antibody, detecting and measuring the signal from the
9F3 antibody label, and then comparing the signal measurements
exhibited by the various dilutions of antibody.
[0154] (ii) Antiviral Assays
[0155] The ability of a candidate antibody to neutralize a
biological activity of IFN-.alpha. can, for example, be carried out
by monitoring the neutralization of the antiviral activity of
IFN-.alpha. as described by Kawade, J. Interferon Res. 1:61-70
(1980), or Kawade and Watanabe, J. Interferon Res. 4:571-584
(1984). Briefly, a fixed concentration of IFN-.alpha.premixed with
various dilutions of a candidate antibody is added to human
amnion-derived FL cells, and the ability of the candidate antibody
to neutralize the antiviral activity of IFN-.alpha. is determined,
using an appropriate virus, e.g. Sindbis virus. The titers are
expressed in international units (IU), as determined with the
international reference human IFN-.alpha. (NIH Ga23-901-527).
[0156] The candidate anti-IFN-.alpha. antibody is considered able
to inhibit the anti-viral activity of a selected IFN-.alpha.
subtype if a certain concentration of the antibody inhibits 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 certain concentration of the
candidate anti-IFN-.alpha. antibody will inhibit at least or about
60%, or at least or about 70%, preferably at least or about 75%, or
more preferably at least or about 80%, or even more preferably at
least or about 85%, or still more preferably at least or about 90%,
or still more preferably at least or about 95%, or most preferably
at least or about 99% of the anti-viral activity of the selected
IFN-.alpha. subtype in the anti-viral activity assay as compared to
baseline activity measured in the presence of an equivalent
concentration of control antibody. The candidate anti-IFN-.alpha.
antibody is considered unable to inhibit the anti-viral activity of
a selected IFN-.alpha. subtype if there is no concentration of the
antibody that exhibits more anti-viral activity inhibition than the
baseline level of anti-viral activity inhibition measured in the
presence of an equivalent concentration of control antibody.
[0157] In a preferred embodiment, each interferon species used in
the viral infectivity assay is titrated to a concentration that
provides the same level of inhibition of viral growth 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 interferon species. In order to assess the ability
of an anti-IFN-.alpha. antibody to inhibit the anti-viral activity
of various IFN-.alpha. subtypes, the effective concentration (EC50)
of anti-IFN-.alpha. antibody for inhibiting 50% of a particular
IFN-.alpha. subtype's anti-viral activity (at the concentration
titrated to provide the normalized units of activity) is determined
for each IFN-.alpha. subtype to be tested.
[0158] In one embodiment, the antiviral activity neutralization
assay is performed as described in Example 1 below. Briefly, A549
cells are grown to a density of 5.times.10.sup.5 A549 cells/well on
96-well microliter plates. Serial dilutions of candidate
anti-IFN-.alpha. antibody are incubated with 0.2 units/.mu.l of a
selected IFN-.alpha. subtype (normalized to 0.2 units/.mu.l of NIH
reference standard recombinant human IFN-.alpha.2) in a total
volume of 100 .mu.l at 37.degree. C. for one hour. Each 100 .mu.l
volume of antibody/interferon incubation mixture is then added to
5.times.10.sup.5 A549 cells and 100 .mu.l of culture medium in an
individual well on the microliter plate, yielding a final
IFN-.alpha. concentration of 100 units/ml in each well. The
resulting cell culture mixtures are incubated for 24 hours at
37.degree. C. Cells are then challenged with 2.times.10.sup.5 pfu
of encephalomyocarditis (EMC) virus and incubated for an additional
24 hours at 37.degree. C. At the end of the incubation, cell
viability is determined by visual microscopic examination or
crystal violet staining. The effective concentration (EC50) of a
candidate IFN-.alpha. antibody for inhibiting 50% of a particular
IFN-.alpha. subtype's anti-viral activity in the assay is
determined for each IFN-.alpha. subtype to be tested.
[0159] In one aspect of the invention, the anti-IFN-.alpha.
antibody that exhibits anti-viral activity neutralization against
the subject IFN-.alpha. subtypes will exhibit an EC50 of up to or
about 20 .mu.g/ml, or up to or about 10 .mu.g/ml, or up to or about
5 .mu.g/ml, or up to or about 4 .mu.g/ml, or up to or about 3
.mu.g/ml, or up to or about 2 .mu.g/ml, or up to or about 1
.mu.g/ml with respect to each of the subject IFN-.alpha. subtypes
in the A549 cell EMC viral inhibition assay described above.
[0160] In another aspect of the invention, the anti-IFN-.alpha.
antibody that exhibits anti-viral activity neutralization against
the subject IFN-.alpha. subtypes will exhibit an EC50 from or about
0.1 .mu.g/ml to or about 20 .mu.g/ml, from or about 0.1 .mu.g/ml to
or about 10 .mu.g/ml, from or about 0.1 .mu.g/ml to or about 5
.mu.g/ml, or from or about 0.1 .mu.g/ml to or about 4 .mu.g/ml,
from or about 0.1 .mu.g/ml to or about 3 .mu.g/ml, from or about
0.1 .mu.g/ml to or about 2 .mu.g/ml, or from or about 0.1 .mu.g/ml
to or about 1 .mu.g/ml with respect to each of the subject
IFN-.alpha. subtypes in the A549 cell EMC viral inhibition assay
described above.
[0161] In another aspect of the invention, the anti-IFN-.alpha.
antibody that anti-viral activity neutralization against the
subject IFN-.alpha. subtypes will exhibit an EC50 from or about 0.2
.mu.g/ml to or about 20 .mu.g/ml, from or about 0.2 .mu.g/ml to or
about 10 .mu.g/ml, from or about 0.2 .mu.g/ml to or about 5
.mu.g/ml, or from or about 0.2 .mu.g/ml to or about 4 .mu.g/ml,
from or about 0.2 .mu.g/ml to or about 3 .mu.g/ml, from or about
0.2 .mu.g/ml to or about 2 .mu.g/ml, or from or about 0.2 .mu.g/ml
to or about 1 .mu.g/ml with respect to each of the subject
IFN-.alpha. subtypes in the A549 cell EMC viral inhibition assay
described above.
[0162] In another aspect of the invention, the anti-IFN-.alpha.
antibody that neutralizes the anti-viral activity of the subject
IFN-.alpha. subtypes will exhibit an EC50 from or about 0.3
.mu.g/ml to or about 20 .mu.g/ml, from or about 0.3 .mu.g/ml to or
about 10 .mu.g/ml, from or about 0.3 .mu.g/ml to or about 5
.mu.g/ml, or from or about 0.3 .mu.g/ml to or about 4 .mu.g/ml,
from or about 0.3 .mu.g/ml to or about 3 .mu.g/ml, from or about
0.3 .mu.g/ml to or about 2 .mu.g/ml, or from or about 0.3 .mu.g/ml
to or about 1 .mu.g/ml with respect to each of the subject
IFN-.alpha. subtypes in the A549 cell EMC viral inhibition assay
described above.
[0163] In another aspect of the invention, the anti-IFN-.alpha.
antibody that neutralizes the anti-viral activity of the subject
IFN-.alpha. subtypes will exhibit an EC50 from or about 0.4
.mu.g/ml to or about 20 .mu.g/ml, from or about 0.4 .mu.g/ml to or
about 10 .mu.g/ml, from or about 0.4 .mu.g/ml to or about 5
.mu.g/ml, or from or about 0.4 .mu.g/ml to or about 4 .mu.g/ml,
from or about 0.4 .mu.g/ml to or about 3 .mu.g/ml, from or about
0.4 .mu.g/ml to or about 2 .mu.g/ml, or from or about 0.4 .mu.g/ml
to or about 1 .mu.g/ml with respect to each of the subject
IFN-.alpha. subtypes in the A549 cell EMC viral inhibition assay
described above.
[0164] In another aspect of the invention, the anti-IFN-.alpha.
antibody that neutralizes the anti-viral activity of the subject
IFN-.alpha. subtypes will exhibit an EC50 from or about 0.5
.mu.g/ml to or about 20 .mu.g/ml, from or about 0.5 .mu.g/ml to or
about 10 .mu.g/ml, from or about 0.5 .mu.g/ml to or about 5
.mu.g/ml, or from or about 0.5 .mu.g/ml to or about 4 .mu.g/ml,
from or about 0.5 .mu.g/ml to or about 3 .mu.g/ml, from or about
0.5 .mu.g/ml to or about 2 .mu.g/ml, or from or about 0.5 .mu.g/ml
to or about 1 .mu.g/ml with respect to each of the subject
IFN-.alpha. subtypes in the A549 cell EMC viral inhibition assay
described above.
[0165] In another aspect of the invention, the anti-IFN-.alpha.
antibody that neutralizes the anti-viral activity of the subject
IFN-.alpha. subtypes will exhibit an EC50 from or about 0.6
.mu.g/ml to or about 20 .mu.g/ml, from or about 0.6 .mu.g/ml to or
about 10 .mu.g/ml, from or about 0.6 .mu.g/ml to or about 5
.mu.g/ml, or from or about 0.6 .mu.g/ml to or about 4 .mu.g/ml,
from or about 0.6 .mu.g/ml to or about 3 .mu.g/ml, from or about
0.6 .mu.g/ml to or about 2 .mu.g/ml, or from or about 0.6 .mu.g/ml
to or about 1 .mu.g/ml with respect to each of the subject
IFN-.alpha. subtypes in the A549 cell EMC viral inhibition assay
described above.
[0166] In another aspect of the invention, the anti-IFN-.alpha.
antibody that neutralizes the anti-viral activity of the subject
IFN-.alpha. subtypes will exhibit an EC50 from or about 0.7
.mu.g/ml to or about 20 .mu.g/ml, from or about 0.7 .mu.g/ml to or
about 10 .mu.g/ml, from or about 0.7 .mu.g/ml to or about 5
.mu.g/ml, or from or about 0.7 .mu.g/ml to or about 4 .mu.g/ml,
from or about 0.7 .mu.g/ml to or about 3 .mu.g/ml, from or about
0.7 .mu.g/ml to or about 2 .mu.g/ml, or from or about 0.7 .mu.g/ml
to or about 1 .mu.g/ml with respect to each of the subject
IFN-.alpha. subtypes in the A549 cell EMC viral inhibition assay
described above.
[0167] In another aspect of the invention, the anti-IFN-.alpha.
antibody that neutralizes the anti-viral activity of the subject
IFN-.alpha. subtypes will exhibit an EC50 from or about 0.8
.mu.g/ml to or about 20 .mu.g/ml, from or about 0.8 .mu.g/ml to or
about 10 .mu.g/ml, from or about 0.8 .mu.g/ml to or about 5
.mu.g/ml, or from or about 0.8 .mu.g/ml to or about 4 .mu.g/ml,
from or about 0.8 .mu.g/ml to or about 3 .mu.g/ml, from or about
0.8 .mu.g/ml to or about 2 .mu.g/ml, or from or about 0.8 .mu.g/ml
to or about 1 .mu.g/ml with respect to each of the subject
IFN-.alpha. subtypes in the A549 cell EMC viral inhibition assay
described above.
[0168] In another aspect of the invention, the anti-IFN-.alpha.
antibody that neutralizes the anti-viral activity of the subject
IFN-.alpha. subtypes will exhibit an EC50 from or about 0.9
.mu.g/ml to or about 20 .mu.g/ml, from or about 0.9 .mu.g/ml to or
about 10 .mu.g/ml, from or about 0.9 .mu.g/ml to or about 5
.mu.g/ml, or from or about 0.9 .mu.g/ml to or about 4 .mu.g/ml,
from or about 0.9 .mu.g/ml to or about 3 .mu.g/ml, from or about
0.9 .mu.g/ml to or about 2 .mu.g/ml, or from or about 0.9 .mu.g/ml
to or about 1 .mu.g/ml with respect to each of the subject
IFN-.alpha. subtypes in the A549 cell EMC viral inhibition assay
described above.
[0169] In another aspect of the invention, the anti-IFN-.alpha.
antibody that neutralizes the anti-viral activity of the subject
IFN-.alpha. subtypes will exhibit an EC50 from or about 1 .mu.g/ml
to or about 20 .mu.g/ml, from or about 1 .mu.g/ml to or about 10
.mu.g/ml, from or about 1 .mu.g/ml to or about 5 .mu.g/ml, or from
or about 1 .mu.g/ml to or about 4 .mu.g/ml, from or about 1
.mu.g/ml to or about 3 .mu.g/ml, or from or about 1 .mu.g/ml to or
about 2 .mu.g/ml with respect to each of the subject IFN-.alpha.
subtypes in the A549 cell EMC viral inhibition assay described
above.
[0170] (iii) Cross-Blocking Assays
[0171] To screen for antibodies which bind to an epitope on
IFN-.alpha. bound by an antibody of interest, a routine
cross-blocking assay such as that described in Antibodies, A
Laboratory Manual, Cold Spring Harbor Laboratory, Ed Harlow and
David Lane (1988), can be performed. Alternatively, or
additionally, epitope mapping can be performed by methods known in
the art. For example, the IFN-.alpha. epitope bound by a monoclonal
antibody of the present invention can be determined by competitive
binding analysis as described in Fendly et al. Cancer Research
50:1550-1558 (1990). In another example, cross-blocking studies can
be done with direct fluorescence on microtiter plates. In this
method, the monoclonal antibody of interest is conjugated with
fluorescein isothiocyanate (FITC), using established procedures
(Wofsy et al. Selected Methods in Cellular Immunology, p. 287,
Mishel and Schiigi (eds.) San Francisco: W.J. Freeman Co. (1980)).
The selected IFN-.alpha. is layered onto the wells of microtiter
plates, the layered wells are incubated with mixtures of (1)
FITC-labeled monoclonal antibody of interest and (2) unlabeled test
monoclonal antibody, and the fluorescence in each well is
quantitated to determine the level of cross-blocking exhibited by
the antibodies. Monoclonal antibodies are considered to share an
epitope if each blocks binding of the other by 50% or greater in
comparison to an irrelevant monoclonal antibody control.
[0172] The results obtained in the cell-based biological assays can
then be followed by testing in animal, e.g. murine, models, and
human clinical trials. If desired, murine monoclonal antibodies
identified as having the desired properties can be converted into
chimeric antibodies, or humanized by techniques well known in the
art, including the "gene conversion mutagenesis" strategy, as
described in U.S. Pat. No. 5,821,337, the entire disclosure of
which is hereby expressly incorporated by reference. Humanization
of a particular anti-IFN-.alpha. antibody herein is also described
in the Examples below.
[0173] (iv) Phage Display Method
[0174] Anti-IFN-.alpha. antibodies of the invention can be
identified 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
(e.g. Fab, F(ab').sub.2, etc.) of antibody variable region (Fv)
fused to phage coat protein. Such phage libraries are panned by
affinity chromatography against the desired antigen. Clones
expressing Fv fragments capable of binding to the desired antigen
are adsorbed to the antigen and thus separated from the non-binding
clones in the library. The binding clones are then eluted from the
antigen, and can be further enriched by additional cycles of
antigen adsorption/elution. Any of the anti-IFN-.alpha. antibodies
of the invention can be obtained by designing a suitable antigen
screening procedure to select for the phage clone of interest
followed by construction of a full length anti-IFN-.alpha. antibody
clone using the Fv sequences from the phage clone of interest and
suitable constant region (Fc) sequences described in Kabat et al.
(1991), supra.
[0175] Construction of Phage Display Libraries
[0176] The antigen-binding domain of an antibody is formed from two
variable (V) regions of about 110 amino acids 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 or D(ab').sub.2
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: 433-455 (1994). As used herein, scFv encoding phage
clones and Fab or F(ab').sub.2 encoding phage clones are
collectively referred to as "Fv phage clones" or "Fv clones".
[0177] The naive repertoire of an animal (the repertoire before
antigen challenge) provides it with antibodies that can bind with
moderate affinity (K.sub.d.sup.-1 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. In human 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).
[0178] 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 cells making a
combination that binds the immunogen to proliferate (clonal
expansion) and to secrete the corresponding antibody. These naive
antibodies are then matured to high affinity (K.sub.d.sup.-1 of
10.sup.9-10.sup.10 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.
[0179] 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- or VIII-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).
[0180] 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 (including F(ab').sub.2) 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: 4133-4137 (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 pIII must
be provided by helper phage.
[0181] 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).
[0182] 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-IFN-.alpha. clones is desired,
the subject is immunized with IFN-.alpha. 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 IFN-.alpha. clones
is obtained by generating an anti-human IFN-.alpha. antibody
response in transgenic mice carrying a functional human
immunoglobulin gene array (and lacking a functional endogenous
antibody production system) such that IFN-.alpha. immunization
gives rise to B cells producing human-sequence antibodies against
IFN-.alpha..
[0183] In another preferred embodiment, animals are immunized with
a mixture of various, preferably all, IFN-.alpha. subtypes in order
to generate an antibody response that includes B cells producing
anti-IFN-.alpha. antibodies with broad reactivity against
IFN-.alpha. subtypes. In another preferred embodiment, animals are
immunized with the mixture of human IFN-.alpha. subtypes that is
present in the human lymphoblastoid interferons secreted by Burkitt
lymphoma cells (Namalva cells) induced with Sendai virus, as
described in Example 1 below. A suitable preparation of such human
lymphoblastoid interferons can be obtained commercially (Product
No. 1-9887) from Sigma Chemical Company, St. Louis, Mo.
[0184] Additional enrichment for anti-IFN-.alpha. reactive cell
populations can be obtained by using a suitable screening procedure
to isolate B cells expressing IFN-.alpha.-specific membrane bound
antibody, e.g., by cell separation with IFN-.alpha. affinity
chromatography or adsorption of cells to fluorochrome-labeled
IFN-.alpha. followed by fluorescence-activated cell sorting
(FACS).
[0185] 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 IFN-.alpha. 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, lagomorphs, luprine, canine, feline, porcine,
bovine, equine, and avian species, etc.
[0186] 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).
[0187] 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).
[0188] 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.d.sup.-1 of
about 10.sup.-8 M).
[0189] 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).
[0190] The antibodies produced by naive libraries (either natural
or synthetic) can be of moderate affinity (K.sub.d.sup.-1 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., 226: 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. WO 9607754 (published 14 Mar. 1996) described a method for
inducing mutagenesis in a complementarity determining region of an
immunoglobulin light chain to create a library of light chain
genes. 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 10.sup.-9 M range.
[0191] Panning Phage Display Libraries for Anti-IFN-.alpha.
Clones
[0192] a. Synthesis of IFN-.alpha..
[0193] Nucleic acid sequence encoding the IFN-.alpha. subtypes used
herein can be designed using published amino acid and nucleic acid
sequences of interferons, e.g. see the J. Interferon Res., 13:
443-444 (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.2), IFN-.alpha.B
(IFN-.alpha.8), IFN-.alpha. (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) 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.7
(IFN-.alpha.J), see Cohen et al., Dev. Biol. Standard, 60: 111-122
(1985). DNAs encoding the 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
interferon-encoding DNA. Alternatively, DNA encoding the interferon
can be isolated from a genomic or cDNA library.
[0194] Following construction of the DNA molecule encoding the
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 which 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.
[0195] 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.sup.r) (U.S. Pat. No.
5,288,931, ATCC No. 55,244), Bacillus subtilis, Salmonella
typhimurium, Serratia marcesans, and Pseudomonas species.
[0196] 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.
[0197] However, interest has been greatest in vertebrate host
cells. Examples of useful mammalian host cells include monkey
kidney CV1 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 (CV1 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 (W138, ATCC CCL 75); human liver cells (Hep G2, HB
8065); mouse mammary tumor (MMT 060562, ATCC CCL51); TRI 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).
[0198] Optionally, the DNA encoding the 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
stII, ecotin, lamB, herpes GD, 1pp, 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)).
[0199] 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.
[0200] 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.
[0201] 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 Ilsiao 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.
[0202] Prokaryotic host cells used to produce the interferon of
interest can be cultured as described generally in Sambrook et al.,
supra.
[0203] The mammalian host cells used to produce the interferon of
interest can be cultured in a variety of media. Commercially
available media such as Ham's NO (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.
[0204] The host cells referred to in this disclosure encompass
cells in in vitro culture as well as cells that are within a host
animal.
[0205] In an intracellular expression system or periplasmic space
secretion system, the recombinantly expressed 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
interferon is purified from the soluble protein fraction. If the
IFN-.alpha. 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 DEAF; chromatofocusing; SDS-PAGE; ammonium
sulfate precipitation; gel filtration using, for example, Sephadex
G-75; hydrophobic affinity resins and ligand affinity using
interferon receptor immobilized on a matrix.
[0206] Many of the human IFN-.alpha. used herein can be obtained
from commercial sources, e.g. from Sigma (St. Louis, Mo.),
Calbiochem-Novabiochem Corporation (San Diego, Calif.) or ACCURATE
Chemical & Scientific Corporation (Westbury, N.Y.).
[0207] Standard cloning procedures described in Maniatis et al.,
Molecular Cloning: A Laboratory Manual, Cold Spring Harbor
Laboratory Press, Cold Spring Harbor, N.Y. (1989) are used to
construct plasmids that direct the translocation of the various
species of hIFN-.alpha. into the periplasmic space of E. coli. PCR
reactions are 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 are then subcloned into the corresponding sites of the
expression vector pB0720 described in Cunningham et al., Science
243:1330-1336 (1989). The resulting plasmids place 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 is confirmed using the United States
Biochemical Sequenase Kit version 2.0. Each plasmid is 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 are purified from E. coli paste
containing each IFN-.alpha. by affinity chromatography. Bacterial
cells are lysed, and the lysate is centrifuged at 10,000.times.g to
remove debris. The supernatant is applied to an immunoaffinity
column containing a mouse anti-hIFN-.alpha.B antibody (LI-1) that
is obtained as described in Staehelin et al., Proc. Natl. Acad.
Sci. 78:1848-1852 (1981). LI-1 is 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 is eluted
from the column with 0.1 M citrate, pH 3.0, containing 20% (w/v)
glycerol. The purified IFN is analyzed by SDS-PAGE and
immunoblotting, and is assayed for bioactivity by the hIFN-induced
anti-viral assay as described herein.
[0208] Human IFN-.alpha.2/1 hybrid molecule
(IFN-.alpha.2.sub.1-62/.alpha..sub.64-166) 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).
[0209] b. Immobilization of IFN-.alpha.
[0210] The purified IFN-.alpha. 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 IFN-.alpha. 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.
[0211] Alternatively, IFN-.alpha. 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.
[0212] c. Panning Procedures
[0213] The phage library samples are contacted with immobilized
IFN-.alpha.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 phage 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 IFN-.alpha. antigen, e.g.
in a procedure similar to the antigen competition method of
Clackson et al., Nature, 352: 624-628 (1991). Phage can be enriched
20-1.000-fold in a single round of selection. Moreover, the
enriched phage can be grown in bacterial culture and subjected to
further rounds of selection.
[0214] In a preferred embodiment, phage are serially incubated with
various IFN-.alpha. subtypes immobilized in order to identify and
further characterize phage clones that exhibit appreciable binding
to a majority, preferably all, of IFN-.alpha. subtypes. In this
method, phage are first incubated with one specific IFN-.alpha.
subtype. The phage bound to this subtype are eluted and subjected
to selection with another IFN-.alpha. subtype. The process of
binding and elution is thus repeated with all IFN-.alpha. subtypes.
At the end, the procedure yields a population of phage displaying
antibodies with broad reactivity against all IFN-.alpha. subtypes.
These phage can then be tested against other IFN species, i.e.
other than IFN-.alpha., in order to select those clones which do
not show appreciable binding to other species of IFNs. Finally, the
selected phage clones can be examined for their ability to
neutralize biological activity, e.g. anti-viral activity, of
various IFN-.alpha. subtypes, and clones representing antibodies
with broad neutralization activity against a majority, preferably
all, of IFN-.alpha. subtypes are finally selected.
[0215] 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 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).
[0216] It is possible to select between phage antibodies of
different affinities, even with affinities that differ slightly,
for IFN-.alpha.. 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
IFN-.alpha., rare high affinity phage could be competed out. To
retain all the higher affinity mutants, phage can be incubated with
excess biotinylated IFN-.alpha., but with the biotinylated
IFN-.alpha. at a concentration of lower molarity than the target
molar affinity constant for IFN-.alpha.. The high affinity-binding
phage 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 phage with lower affinity.
Conditions used in washing phage bound to a solid phase can also be
manipulated to discriminate on the basis of dissociation
kinetics.
[0217] In one embodiment, phage are serially incubated with various
IFN-.alpha.subtypes immobilized on a solid support, such as
chromatographic polymer matrix beads described above. In this
method, the phage are first incubated with one specific
IFN-.alpha.subtype. The phage bound to this subtype are eluted from
solid phase with a suitable eluent, such as any salt or acid buffer
capable of releasing the bound phage into solution. Next, the
eluted phage clones are subjected to selection with another
IFN-.alpha. subtype. In order to enrich the population for clones
that compete with soluble IFNAR2 for binding to IFN-.alpha., the
phage clones recovered from the series of IFN-.alpha. subtype
chromatographic separations are incubated with a complex of
immobilized IFNAR2 preadsorbed to IFN-.alpha., and the non-adsorbed
phage clones are recovered from the incubation reaction
mixture.
[0218] The selection procedures can be designed to utilize any
suitable batch chromatographic technique. In one embodiment, the
phage clones are adsorbed to IFN-.alpha.-derivatized polymer matrix
beads in suspension, the adsorbed beads are recovered by
centrifugation, the recovered beads are resuspended and incubated
in a suitable elution buffer, such as any salt or acid buffer
capable of releasing the hound phage into solution, the elution
mixture is centrifuged, the eluted phage clones are recovered from
the supernatant, and then the adsorption/elution procedure is
repeated for every additional IFN-.alpha. subtype. In order to
enrich the population for clones that compete with soluble IFNAR2
for binding to IFN-.alpha., the phage clones recovered from the
IFN-.alpha. subtype chromatographic separations are incubated with
a suspension of IFNAR2-derivatized polymer matrix beads preadsorbed
to IFN-.alpha., the incubation mixture is centrifuged, and the
non-adsorbed phage clones are recovered from the supernatant.
[0219] In another embodiment, the selection procedure is designed
to enrich the phage population for the property of inhibiting
IFN-.alpha. binding to IFNAR2 during each of the affinity
chromatographic separations. In this method, the phage are serially
incubated with each of the specific IFN-.alpha. subtypes
immobilized on a solid support and then eluted from solid phase
with an eluent comprising an excess of soluble IFNAR2, such as
IFNAR2 ECD-IgG Fc, under conditions wherein soluble IFNAR2 is
capable of displacing any phage clone that competes with IFNAR2 for
binding to the immobilized IFN-.alpha.. The process of binding and
elution is thus repeated with each of the specific IFN-.alpha.
subtypes.
[0220] In another embodiment, the phage clones are adsorbed to
IFN-.alpha.-derivatized polymer matrix beads in suspension, the
adsorbed beads are recovered by centrifugation, the recovered beads
are resuspended and incubated in a suitable elution buffer
comprising an excess of soluble IFNAR2 (such as IFNAR2 ECD-IgG Fc)
under conditions wherein the soluble IFNAR2 is capable of
displacing any phage clone that competes with IFNAR2 for binding to
the immobilized IFN-.alpha. and releasing the bound phage into
solution, the elution mixture is centrifuged, the eluted phage
clones are recovered from the supernatant, and then the
adsorption/elution procedure is repeated for every additional
IFN-.alpha. subtype.
[0221] At the end, the procedure yields a population of phage
displaying antibodies with IFNAR2-binding inhibition activity
against a broad range of IFN-.alpha. subtypes. These phage can then
be tested against other IFN species (other than IFN-.alpha.
species), such as IFN-.beta., in order to select those clones which
do not show appreciable binding to other species of IFNs. Finally,
the selected phage clones can be examined for their ability to
neutralize biological activity, e.g. anti-viral activity, of
various IFN-.alpha. subtypes, and clones representing antibodies
with broad neutralization activity against a majority, preferably
all, of IFN-.alpha.subtypes are finally selected.
[0222] Activity Selection of Anti-IFN-.alpha. Clones
[0223] In one embodiment, the invention provides anti-IFN-.alpha.
antibodies that bind to as well as neutralize the activity of a
majority, preferably all, of IFN-.alpha. subtypes, but do not
significantly bind to or neutralize the activity of any other
interferon species. For example, the ability of various phage
clones to neutralize the anti-viral activities of various
IFN-.alpha. subtypes can be tested, essentially in the same manner
as described earlier for the antibodies.
[0224] 4. Preparation of Soluble LFNAR2-IgG
[0225] A cDNA encoding the human immunoglobulin fusion proteins
(immunoadhesins) based on the extracellular domain (ECD) of the
hIFNAR2 (pRK5 htFNAR2-IgG clone) can be generated using methods
similar to those described by Haak-Frendscho et al., Immunology 79:
594-599 (1993) for the construction of a murine receptor
immunoadhesin. Briefly, the plasmid pRKCD4.sub.2Fc.sub.1 is
constructed as described in Example 4 of WO 89/02922
(PCT/US88/03414 published Apr. 6, 1989). The cDNA coding sequence
for the first 216 residues of the mature hIFNAR2 ECD is obtained
from the published sequence (Novick et al., Cell, 77: 391-400
[1994]). The CD4 coding sequence in the pRKCD4.sub.2Fc.sub.1 is
replaced with the hIFNAR2 ECD encoding cDNA to form the
pRK5hIFNAR2-IgG clone. hIFNAR2-IgG is expressed in human embryonic
kidney 293 cells by transient transfection using a calcium
phosphate precipitation technique. The immunoadhesin is 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 hIFNAR2-IgG
is eluted with 0.1 M citrate buffer, pH 3.0, containing 20% (w/v)
glycerol. The hIFNAR2-IgG purified is over 95% pure, as judged by
SDS-PAGE.
[0226] 5. Diagnostic Uses of Anti-IFN-.alpha. Antibodies
[0227] The anti-IFN-.alpha. antibodies of the invention are unique
research reagents in diagnostic assays for IFN-.alpha. expression.
As discussed earlier, IFN-.alpha. expression is increased in
certain autoimmune diseases such as IDDM, SLE, and autoimmune
thyroiditis. Increased expression of various IFN-.alpha. subtypes
in such disorders can be detected and quantitated using
anti-IFN-.alpha. antibodies of the instant invention with broad
reactivity against a majority of IFN-.alpha. subtypes.
Anti-IFN-.alpha. antibodies are also useful for the affinity
purification of various IFN-.alpha. subtypes from recombinant cell
culture or natural sources.
[0228] Anti-IFN-.alpha. antibodies can be used for the detection of
IFN-.alpha. in any one of a number of well known diagnostic assay
methods. For example, a biological sample may be assayed for
IFN-.alpha. by obtaining the sample from a desired source, admixing
the sample with anti-IFN-.alpha. antibody to allow the antibody to
form antibody/IFN-.alpha. complex with any IFN-.alpha. subtype
present in the mixture, and detecting any antibody/IFN-.alpha.
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/IFN-.alpha.
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.
[0229] Analytical methods for IFN-.alpha. all use one or more of
the following reagents: labeled IFN-.alpha. analogue, immobilized
IFN-.alpha. analogue, labeled anti-IFN-.alpha. antibody,
immobilized anti-IFN-.alpha. antibody and steric conjugates. The
labeled reagents also are known as "tracers."
[0230] The label used is any detectable functionality that does not
interfere with the binding of IFN-.alpha. and anti-IFN-.alpha.
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, .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.
[0231] 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.
Nos. 3,940,475 (fluorimetry) and 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.
[0232] 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.
[0233] Immobilization of reagents is required for certain assay
methods. Immobilization entails separating the anti-IFN-.alpha.
antibody from any IFN-.alpha. that remains free in solution. This
conventionally is accomplished by either insolubilizing the
anti-IFN-.alpha.antibody or IFN-.alpha. 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-IFN-.alpha. antibody or IFN-.alpha.
analogue afterward, e.g., by immunoprecipitation.
[0234] Other assay methods, known as competitive or sandwich
assays, are well established and widely used in the commercial
diagnostics industry.
[0235] Competitive assays rely on the ability of a tracer
IFN-.alpha. analogue to compete with the test sample IFN-.alpha.
for a limited number of anti-IFN-.alpha. antibody antigen-binding
sites. The anti-IFN-.alpha. antibody generally is insolubilized
before or after the competition and then the tracer and IFN-.alpha.
bound to the anti-IFN-.alpha. antibody are separated from the
unbound tracer and IFN-.alpha.. This separation is accomplished by
decanting (where the binding partner was pre-insolubilized) or by
centrifuging (where the binding partner was precipitated after the
competitive reaction). The amount of test sample IFN-.alpha. is
inversely proportional to the amount of bound tracer as measured by
the amount of marker substance. Dose-response curves with known
amounts of IFN-.alpha. are prepared and compared with the test
results to quantitatively determine the amount of IFN-.alpha.
present in the test sample. These assays are called ELISA systems
when enzymes are used as the detectable markers.
[0236] Another species of competitive assay, called a "homogeneous"
assay, does not require a phase separation. Here, a conjugate of an
enzyme with the IFN-.alpha. is prepared and used such that when
anti-IFN-.alpha. antibody binds to the IFN-.alpha. the presence of
the anti-IFN-.alpha. antibody modifies the enzyme activity. In this
case, the IFN-.alpha. 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-IFN-.alpha.
antibody so that binding of the anti-IFN-.alpha. antibody inhibits
or potentiates the enzyme activity of the label. This method per se
is widely practiced under the name of EMIT.
[0237] 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 IFN-.alpha.
fragment so that antibody to hapten is substantially unable to bind
the conjugate at the same time as anti-IFN-.alpha. antibody. Under
this assay procedure the IFN-.alpha. present in the test sample
will bind anti-IFN-.alpha. 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.
[0238] Sandwich assays particularly are useful for the
determination of IFN-.alpha. or anti-IFN-.alpha. antibodies. In
sequential sandwich assays an immobilized anti-IFN-.alpha. antibody
is used to adsorb test sample IFN-.alpha., the test sample is
removed as by washing, the bound IFN-.alpha. is used to adsorb a
second, labeled anti-IFN-.alpha. antibody and bound material is
then separated from residual tracer. The amount of bound tracer is
directly proportional to test sample IFN-.alpha.. In "simultaneous"
sandwich assays the test sample is not separated before adding the
labeled anti-IFN-.alpha.. A sequential sandwich assay using an
anti-IFN-.alpha. monoclonal antibody as one antibody and a
polyclonal anti-IFN-.alpha. antibody as the other is useful in
testing samples for IFN-.alpha..
[0239] The foregoing are merely exemplary diagnostic assays for
IFN-.alpha.. Other methods now or hereafter developed that use
anti-IFN-.alpha. antibody for the determination of IFN-.alpha. are
included within the scope hereof, including the bioassays described
above.
[0240] 6. Therapeutic Compositions and Administration of
Anti-IFN-.alpha. Antibodies
[0241] Therapeutic formulations of the anti-IFN-.alpha. 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).
[0242] The anti-IFN-.alpha. 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-IFN-.alpha.
antibody ordinarily will be stored in lyophilized form or in
solution.
[0243] Therapeutic anti-IFN-.alpha. 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.
[0244] The route of anti-IFN-.alpha. 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.
[0245] 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-IFNAR2 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: 4030-4034 (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.
[0246] Anti-IFN-.alpha. 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-IFN-.alpha. antibody can be
aerosolized using a fluorocarbon formulation and a metered dose
inhaler, or inhaled as a lyophilized and milled powder.
[0247] An "effective amount" of anti-IFN-.alpha. antibody to be
employed therapeutically will depend, for example, upon the
therapeutic objectives, the route of administration, the type of
anti-IFN-.alpha. 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-IFN-.alpha. antibody until a dosage is
reached that achieves the desired effect. The progress of this
therapy is easily monitored by conventional assays.
[0248] The patients to be treated with the anti-IFN-.alpha.
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-IFN-.alpha. antibody of the
invention until the patient's pancreatic islet cells are no longer
viable. It is desirable to administer an anti-IFN-.alpha. 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-IFN-.alpha. antibody treatment for a
trial period during which insulin response and the level of
anti-islet antibodies are monitored for relapse.
[0249] In the treatment and prevention of an immune-mediated or
autoimmune disorder by an anti-IFN-.alpha. 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.
[0250] 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.
[0251] 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.
[0252] 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-IFN-.alpha. 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.
[0253] 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.
EXAMPLES
[0254] The following examples are offered by way of illustration
and not by way of limitation. The examples are provided so as to
provide those of ordinary skill in the art with a complete
disclosure and description of how to make and use the compounds,
compositions, and methods of the invention and are not intended to
limit the scope of what the inventors regard as their invention.
Efforts have been made to insure accuracy with respect to numbers
used (e.g. amounts, temperature, etc.) but some experimental errors
and deviation should be accounted for. Unless indicated otherwise,
parts are parts by weight, temperature is in degrees C., and
pressure is at or near atmospheric. The disclosures of all
citations in the specification are expressly incorporated herein by
reference.
Example 1
Generation and Characterization of a Broad Reactive Mouse
Anti-IFN-.alpha. Monoclonal Antibody
Materials and Methods
[0255] A Murine Monoclonal Antibody with Broad Reactivity Against
IFN-.alpha. Subtypes
[0256] A pan-IFN-.alpha. neutralizing antibody was developed by
sequentially immunizing mice with the mixture of human IFN-.alpha.
subtypes, generating a large number of candidate mAbs, and then
screening for binding and activity. In particular, Balb/c mice were
immunized into each hind footpad 9 times (at two week intervals)
with 2.5 .mu.g of lymphoblastoid hIFN-.alpha. (Product No. 1-9887
of Sigma, St. Louis, Mo.) resuspended in MPL-TDM (Ribi
Immunochemical Research, Inc., Hamilton, Mont.). Three days after
the final boost, popliteal lymph node cells were fused with murine
myeloma cells P3X63Ag8.U.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 various species of hIFN-.alpha. in an ELISA. The
selected hybridoma culture supernatants were then tested for their
ability to inhibit the anti-viral cytophathic effect of IFN on
human lung carcinoma cell line A549 cells as described below. As
indicated in FIG. 1, three mAbs obtained from 1794 fusion wells
were able to neutralize a diverse set of IFN-.alpha. subtypes.
These three mAbs were subcloned and re-analyzed.
Neutralization of Antiviral Activity of IFN-.alpha.
[0257] The ability of a candidate antibody to neutralize the
antiviral activity of IFN-.alpha. was assayed as described by
Yousefi, S., et al., Am. J. Clin. Pathol., 83: 735-740 (1985).
Briefly, the assay was performed using human lung carcinoma A549
cells challenged with encephalomyocarditis (EMC) virus. Serial
dilutions of mAbs were incubated with various units of type I
interferons for one hour at 37.degree. C. in a total volume of 100
.mu.l. These mixtures were then incubated with 5.times.10.sup.5
A549 cells in 100 .mu.l of cell culture medium for 24 hours. Cells
were then challenged with 2.times.10.sup.5 pfu of EMC virus for an
additional 24 hours. At the end of the incubation, cell viability
was determined by visual microscopic examination or crystal violet
staining. The neutralizing antibody titer (EC50) was defined as the
concentration of antibody which neutralizes 50% of the anti-viral
cytopathic effect by 100 units/ml of type I IFNs. The units of type
I IFNs used in this study were determined using NIH reference
recombinant human IFN-.alpha.2 as a standard. The specific
activities of the various type I IFNs tested were as follows:
IFN-.alpha.2/-.alpha.1 (IFN-.alpha.2 residues 1-62/-.alpha.1
residues 64-166) (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-.alpha.10 (1.5.times.10.sup.5
IU/mg). The leukocyte IFN tested was Sigma Product No. 1-2396. The
lymphoblastoid IFN tested was NIH reference standard Ga23-901-532.
The data shown in FIG. 3B was obtained using the above-described
assay format in experiments performed by Access Biomedical (San
Diego, Calif.) at the behest of applicant.
Electrophoretic Mobility Shift Assay
[0258] Most of the immediate actions of IFN have been linked to
activation of latent cytoplasmic signal transducers and activators
of transcription (STAT) proteins to produce a multiprotein complex,
interferon-stimulated gene factor-3 (ISGF3), which induces
transcription from target promoter interferon-stimulated response
element (ISRE). ISGF3 is composed of three protein subunits: STAT1,
STAT2 and p48/ISGF3.gamma.. The p48 protein belongs to the
interferon regulatory factor (IRF) family, and is a DNA-binding
protein that directly interacts with ISRE. Thus, monitoring ISRE
specific cellular DNA-binding complex in response to IFN treatment
provides a simple, rapid and convenient method to assess the effect
of IFN on target cells. One of the convenient formats to carry out
such an analysis is electrophoretic mobility shift assay (EMSA),
wherein the induction of an ISRE-binding activity by IFN treatment
results in the shift in the electrophoretic mobility of a
radiolabeled double-stranded oligonucleotide probe corresponding to
the consensus sequence of ISRE.
[0259] The assay was carried out essentially as described by
Kurabayashi et al., Mol. Cell. Biol., 15: 6386 (1995). Briefly, 5
ng of a specific IFN-.alpha. subtype plus various concentrations
(5-100 .mu.g/ml) of anti-IFN-.alpha. 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 preincubated with antibody for 15 minutes
at 4.degree. C. before the addition of the hIFN-.alpha.. Cells were
washed in PBS and resuspended in 125 .mu.l 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). 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 buffer B (20 mM HEPES, pH 7.9, 400 mM
NaCl, 0.1 mM EDTA, I mM DTT, 1 mM phenylmethylsulfonyl fluoride, 10
.mu.g/ml leupeptin, 10 .mu.g/ml aprotinin) and kept on ice for 30
min. The nuclear fraction was cleared by centrifugation and the
supernatant stored at -70.degree. C. until use. Double-stranded
probes were prepared from single-stranded oligonucleotides (ISG15
top: 5'-GATCGGGAAAGGGAAACCGAAACTGAAGCC-3' [SEQ ID NO. 13], ISG15
bottom: 5'-GATCGGCTTCAGTTTCGGTTTCCCTTTC CC-3' [SEQ ID NO. 14])
utilizing a DNA polymerase I Klenow filling 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
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
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 RT for 30 minutes. DNA-protein complexes were resolved
in 6% non-denaturing polyacrylamide gels and analyzed by
autoradiograph. 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-STAT1 antibody.
Cloning of a Gene Encoding 9F3 Anti-IFN-.alpha. Monoclonal
Antibody
[0260] The murine anti-human IFN-.alpha. mAb 9F3 was generated,
cloned and sequenced. The plasmid pEMX1 used for expression and
mutagenesis of F(ab)s in E. coli has been described previously
(Werther et al., J. Immunol. 157: 4986-4995 [1996]). Briefly, the
plasmid contains a DNA fragment encoding a consensus human .kappa.
subgroup 1 light chain (VL.kappa.1-CL) and a consensus human
subgroup III heavy chain (VHIII-CH1) and an alkaline phosphatase
promoter. The use of the consensus sequences for VL and VH has been
described previously (Carter et al., Proc. Natl. Acad. Sci. USA 89:
4285-4289 [1992]).
Results
[0261] We have previously shown that there is a wide spectrum of
IFN-.alpha. subtypes expressed by the islets of patients with IDDM
(Huang et al., Diabetes 44: 658-664 [1995]). We also demonstrated
that there is no obvious association between IDDM and the
expression of either IFN-.beta. or IFN-.gamma. (Huang et al.,
[1995] supra). While the specific IFN-.alpha. subtypes expressed as
part of the SLE pathology have not been defined, as with IDDM, the
association is with IFN-.alpha. and not with either of IFN-.beta.
or IFN-.gamma. (Hooks, et al., Arthritis & Rheumatism 25:
396-400 [1982]; Kim, et al., Clin. Exp. Immunol. 70: 562-569
[1987]; Lacki, et al., J. Med. 28: 99-107 [1997]; Robak, et al.,
Archivum Immunologiae et Therapiae Experimentalis 46: 375-380
[1998]; Shiozawa, et al., Arthritis & Rheumatism 35: 417-422
[1992]; von Wussow, et al., Rheumatology International 8: 225-230
[1988]). These observations led us to propose that a candidate
antibody for therapeutic intervention in IDDM or SLE would need to
neutralize a majority of the IFN-.alpha. subtypes while leaving
intact the activities of other interferons (.beta., .gamma. and
.omega.) and interleukins that may be required for host
defense.
[0262] One of them (9F3) was able to neutralize a wide spectrum of
recombinant interferon .alpha. subtypes and was further
characterized. As shown in FIG. 2A, 9F3 was able to neutralize the
anti-viral activity of seven recombinant interferons,
IFN-.alpha.-2, 4, 5, 8 and 10 (FIG. 2) and IFN-.alpha. 1 and 21
(Table 2 and FIG. 6). These IFN-.alpha. subtypes cover the full
spectrum of sequences as projected in a type I interferon sequence
dendrogram. More importantly, the 9F3 mAb that neutralized the
IFN-.alpha. subtypes was unable to neutralize IFN-.beta. (FIG. 2,
Table 2) or IFN-.gamma.. The small increase in activity shown in
FIG. 2 for IFN-.beta. was not reproducible in other assays and
appears to be the result of assay variation.
[0263] Other mAbs that are neutralizing toward IFN-.alpha. have
been developed (Tsukui et al., Microbiol. Immunol. 30: 1129-1139
[1986]; Berg, J. Interferon Res. 4: 481-491 [1984]; Meager and
Berg, J. Interferon Res. 6:729-736 [1986]; U.S. Pat. No. 4,902,618;
and EP publication No. 0,139,676 B1). However, these antibodies
neutralize only a limited number of recombinant IFN-.alpha.
subtypes and are unable to neutralize a wide spectrum of
IFN-.alpha. subtypes such as those produced by activated
leukocytes. In contrast, 9F3 Mab was able to neutralize at least
95% of the anti-viral activity in the heterogeneous collection of
IFN-.alpha. subtypes produced by activated leukocytes (FIG. 3A).
Similarly, 9F3 mAb was also able to block the anti-viral activity
of an independent preparation of lymphoblastoid IFN(NIH reference
standard) as determined in an independent experiment (FIG. 3B).
[0264] The ability of 9F3 mAb to neutralize IFN-.alpha. was also
tested using an alternative bioassay. The assay was based on the
ability of IFN-.alpha. to activate the binding of the signaling
molecule, interferon-stimulated gene factor 3 (ISGF3), to an
oligonucleotide derived from the interferon-stimulated response
element (ISRE) in a DNA binding assay known as electrophoretic
mobility shift assay (Horvath et al., Genes Dev. 9: 984-994
[1995]). The transduction of type I interferon signals to the
nucleus relies on activation of a protein complex, ISGF3, involving
two signal transducers and activators of transcription (STAT)
proteins, STAT1 and STAT2, and the interferon regulatory factor
(IRE) protein, p48/ISGF3.gamma. (Wathelet et al., Mol. Cell. 1:
507-518 [1998]). The latter is a DNA sequence recognition subunit
of ISGF3 and directly interacts with ISRE (McKendry et al., Proc.
Natl. Acad. Sci. USA 88: 11455-11459 [1991]; John et al., Mol.
Cell. Biol. 11: 4189-4195 [1991]). The treatment of COS cells with
either IFN-.alpha. or IFN-.beta. led to the appearance of a complex
corresponding to the binding of ISGF3 to the ISRE derived probe.
The appearance of the IFN-.alpha.-induced but not the
IFN-.beta.-induced complex was blocked by 9F3 mAb (FIG. 4).
Furthermore, 9F3 mAb was able to neutralize the activity of six
recombinant IFN-.alpha. subtypes that were tested in this assay
(Table 2).
TABLE-US-00002 TABLE 2 Inhibition of ISGF3 formation induced by
type I IFNs by mAb 9F3 mAb IFN-.alpha.2/1 IFN-.alpha.1 IFN-.alpha.2
IFN-.alpha.5 IFN-.alpha.8 IFN-.alpha.21 IFN-.beta. 9F3.18.5 +++ +++
+++ + +++ +++ - IgG1 - - - - - - -
The extent of inhibition of the IFN induced complex by 9F3 is
indicated where - indicates that the induced band was not altered;
+ indicates that the band was partially lost and +++ indicates that
the induced band was largely abolished. mAb was used at 10
.mu.g/ml; IFN-.alpha. was used at 25 ng/ml
[0265] Having established that 9F3 was able to neutralize both a
wide variety of recombinant IFN-.alpha. subtypes and the mixture of
IFN-.alpha. subtypes produced by activated leukocytes, we cloned
and sequenced the cDNAs encoding both the heavy and light chains of
9F3 mAb. The heavy and light chains were purified and the N
terminal amino acid sequences derived were used to design
degenerate 5' primers corresponding to the N terminus, and the 3'
primers were designed corresponding to the constant domain of mouse
.kappa. light chain and IgG2 heavy chain. The corresponding cDNAs
were cloned using conventional PCR technique and the nucleotide
sequence of the inserts was determined. FIG. 5 shows sequence
alignment of VL (5A) and VH (5B) domains of a murine 9F3 monoclonal
antibody, a humanized version (V13) and consensus sequence of the
human heavy chain subgroup III and the human .kappa. light chain
subgroup III. In order to ensure that the cDNAs that were cloned
encoded the correct Mab reflecting the specificity and
characteristics of 9F3 mAb, recombinant chimeric proteins were
generated that utilized the mouse cDNA sequences shown in FIG. 5
and a human CH1 domain. The resultant chimera (CH8-2) was able to
fully neutralize various recombinant IFN-.alpha. subtypes (FIG. 6).
The amino acid sequences for the heavy and light chains were then
used to generate a humanized antibody.
Example 2
Humanization of 9F3 pan-IFN-.alpha. Neutralizing Monoclonal
Antibody
Materials and Methods
Construction of Humanized F(ab)s
[0266] To construct the first F(ab) variant of humanized 9F3,
site-directed mutagenesis (Kunkel, Proc. Natl. Acad. Sci. USA 82:
488-492 [1985]) was performed on a deoxyuridine-containing template
of pEMX1. The six CDRs were changed to the murine 9F3 sequence
(FIG. 5); the residues included in each CDR were from the
sequence-based CDR definitions (Kabat et al., (1991) supra). F-1
therefore consisted of a complete human framework (VL .kappa.
subgroup 1 and VH subgroup III) with the six complete murine CDR
sequences. Plasmids for all other F(ab) variants were constructed
from the plasmid template of F-1. Plasmids were transformed into E.
coli strain XL-1 Blue (Stratagene, San Diego, Calif.) for
preparation of double- and single-stranded DNA using commercial
kits (Qiagen, Valencia, Calif.). For each variant, DNA coding for
light and heavy chains was completely sequenced using the
dideoxynucleotide chain termination method (Sequenase, U.S.
Biochemical Corp., Cleveland, Ohio). Plasmids were transformed into
E. coli strain 16C9, a derivative of MM294, plated onto Luria broth
plates containing 50 .mu.g/ml carbenicillin, and a single colony
selected for protein expression. The single colony was grown in 5
ml Luria broth-100 .mu.g/ml carbenicillin for 5-8 h at 37.degree.
C. The 5 ml culture was added to 500 ml AP5 medium containing 50
.mu.g/ml carbenicillin and allowed to grow for 20 h in a 4 L
baffled shake flask at 30.degree. C. AP5 medium consists of: 1.5 g
glucose, 11.0 g Hycase SF, 0.6 g yeast extract (certified), 0.19 g
MgSO.sub.4 (anhydrous), 1.07 g NH.sub.4Cl, 3.73 g KCI, 1.2 g NaCl,
120 ml 1 M triethanolamine, pH 7.4, to 1 L water and then sterile
filtered through 0.1 .mu.m Sealkeen filter. Cells were harvested by
centrifugation in a 1 L centrifuge bottle at 3000.times.g and the
supernatant removed. After freezing for 1 h, the pellet was
resuspended in 25 ml cold 10 mM Tris-1 mM EDTA-20% sucrose, pH 7.5,
250 .mu.l of 0.1 M benzamidine (Sigma, St. Louis, Mo.) was added to
inhibit proteolysis. After gentle stirring on ice for 3 h, the
sample was centrifuged at 40,000.times.g for 15 min. The
supernatant was then applied to a Protein G-Sepharose CL-4B
(Pharmacia, Uppsala, Sweden) column (0.5 ml bed volume)
equilibrated with 10 mM Tris-1 mM EDTA, pH 7.5. The column was
washed with 10 ml of 10 mM Tris-1 mM EDTA, pH 7.5, and eluted with
3 ml 0.3 M glycine, pH 3.0, into 1.25 ml 1 M Tris, pH 8.0. The
F(ab) was then buffer exchanged into PBS using a Centricon-30
(Amicon, Beverly, Mass.) and concentrated to a final volume of 0.5
ml. SDS-PAGE gels of all F(ab)s were run to ascertain purity and
the molecular weight of each variant was verified by electrospray
mass spectrometry. F(ab) concentrations were determined using
quantitative amino acid analysis.
Construction of Chimeric and Humanized IgG
[0267] For generation of human IgG2 versions of chimeric and
humanized 9F3, the appropriate murine or humanized VL and VH (F-13,
Table 3) domains were subcloned into separate previously described
pRK vectors (Eaton et al., Biochemistry 25: 8343-8347 [1986]) that
contained DNA coding for human IgG2 CH1-Fc or human light chain CL
domain. The DNA coding for the entire light and the entire heavy
chain of each variant was verified by dideoxynucleotide sequencing.
The chimeric IgG consists of the entire murine 9F3 VH domain fused
to a human CH1 domain at amino acid SerH113 and the entire murine
9F3 VL domain fused to a human CL domain at amino acid LysL
107.
[0268] Heavy and light chain plasmids were co-transfected into an
adenovirus-transformed human embryonic kidney cell line, 293
(Graham et al., J. Gen. Virol. 36: 59-74 [1977]), using a high
efficiency procedure (Gorman et al., DNA Prot. Eng. Tech. 2: 3-10
[1990]). Media was changed to serum-free and harvested daily for up
to five days. Antibodies were purified from the pooled supernatants
using Protein A-Sepharose CL-4B (Pharmacia). The eluted antibody
was buffer exchanged into PBS using a Centricon-30 (Amicon),
concentrated to 0.5 ml, sterile filtered using a Millex-GV
(Millipore, Bedford, Mass.) and stored at 4.degree. C. IgG2
concentrations were determined using quantitative amino acid
analysis.
IFN-.alpha. Binding Assay
[0269] In the ELISA, 96 well microtiter plates (Nunc) were coated
by adding 50 .mu.l of 0.1 .mu.g/ml IFN-.alpha. in PBS to each well
and incubated at 4.degree. C. overnight. The plates were then
washed three times with wash buffer (PBS plus 0.05% Tween 20). The
wells in microtiter plates were then blocked with 200 .mu.l of
SuperBlock (Pierce) and incubated at room temperature for 1 hour.
The plates were then washed again three times with wash buffer.
After washing step, 100 .mu.l of serial dilutions of humanized mAb
starting at 10 .mu.g/ml were added to designated wells. The plates
were incubated at room temperature for 1 hour on a shaker apparatus
and then washed three times with wash buffer. Next, 100 .mu.l of
horseradish peroxidase (HRP)-conjugated goat anti-human Fab
specific (Cappel), diluted at 1:1000 in assay buffer (0.5% bovine
serum albumin, 0.05% Tween 20 in PBS), was added to each well. The
plates were incubated at room temperature on a shaker apparatus and
then washed three times with wash buffer, followed by addition of
100 .mu.l of substrate (TMB, 3,3',5,5'-tetramethylbenzidine;
Kirkegaard & Perry) to each well and incubated at room
temperature for 10 minutes. The reaction was stopped by adding 100
.mu.l of stop solution (from Kirkegaard & Perry) to each well,
and absorbance at 450 nm was read in an automated microtiter plate
reader.
BIAcore.TM. Biosensor Assay
[0270] IFN-.alpha. binding of the humanized F(ab)s, chimeric and
humanized IgG2 antibodies were measured using a BIACore.TM.
biosensor (Karlsson et al., Methods: A companion to Methods in
Enzymology 6: 97-108 [1994]). The IFN-.alpha. was immobilized on
the sensor chip at 60 .mu.g/ml in 50 mM MES buffer, pH 6.3.
Antibodies were exposed to the chip at 75 .mu.g/ml (500 nM) in
phosphate-buffered saline/1% Tween-20. The antibody on-rate
(k.sub.on) was measured.
Computer Graphics Modes of Murine and Humanized F(ab)s
[0271] Sequences of the VL and VH domains (FIGS. 5A and B) were
used to construct a computer graphics model of the murine 9F3 VL-VH
domains (FIG. 7). This model was used to determine which framework
residues should be incorporated into the humanized antibody. A
model of the humanized F(ab) was also constructed to verify correct
selection of murine framework residues. Construction of models was
performed as described previously (Carter et al., [1992] supra;
Werther et al., [1996] supra).
Results
[0272] The consensus sequence for the human heavy chain subgroup
III and the light chain subgroup I were used as the framework for
the humanization as shown in FIG. 5 (Kabat et al., (1991), supra).
This framework has been successfully used in the humanization of
other murine antibodies (Carter et al., Proc. Natl. Acad. Sci. USA
89: 4285-4289 [1992]; Presta et al., J. Immunol. 151: 2623-2632
[1993]; Eigenbrot et al., Proteins 18: 49-62 [1994]; Werther et
al., J. Immunol. 157: 4986-4995 [1996]). All humanized variants
were initially made and screened for binding as F(ab)s expressed in
E. coli. Typical yields from 500 ml shake flasks were 0.1-0.4 mg
F(ab).
[0273] The complementarity determining region (CDR) residues have
been defined either based on sequence hypervariability (Kabat et
al., (1991) supra) or crystal structure of F(ab)-antigen complexes
(Chothia et al., Nature 342: 877-883 [1989]). Although the
sequence-based CDRs are larger than the structure-based CDRs, the
two definitions are generally in agreement except for CDR-H1.
According to the sequence-based definition, CDR-H1 includes
residues H31-1135, whereas the structure-based system defines
residues H26-H32 as CDR-H1 (light chain residue numbers are
prefixed with L; heavy chain residue numbers are prefixed with H).
For the present study, CDR-H1 was defined as a combination of the
two, i.e. including residues H26-H35. The other CDRs were defined
using the sequence-based definition (Kabat et al., (1991)
supra).
[0274] In the initial variant, F-1, the CDR residues were
transferred from the murine antibody to the human framework. In
addition, F(ab)s which consisted of the chimeric heavy chain with
F-1 light chain (Ch-1) and F-1 heavy chain with chimeric light
chain (Ch-2) were generated and tested for binding. F-1 bound
IFN-.alpha. poorly (Table 3). Comparing the binding affinities of
Ch-1 and Ch-2 (Table 3) suggested that framework residues in the
F-1 VH domain needed to be altered in order to increase
binding.
TABLE-US-00003 TABLE 3 Humanized Anti-IFN-.alpha. Versions
OD.sub.450 nm at 10 .mu.g/ml Version Template Changes.sup.a Mean SD
N Ch-1 F-1 VL/ 1.45 0.11 3 Murine VH Ch-2 Murine VL/ .024 0.04 3
F-2 VH F-1 Human FR/ 0.06 0.00 3 CDR swap F-2 F-1 ArgH71Leu; 0.08
0.01 3 AsnH73Lys F-3 F-2 PheH67Ala; 0.14 0.02 3 IleH69Leu;
LeuH78Ala F-4 F-3 ArgH94Ser 0.495 0.02 3 F-5 F-4 AlaH24Thr 0.545
0.03 3 F-6 F-5 ValH48Ile; 0.527 0.02 2 AlaH49Gly F-7 F-5 H78Leu
0.259 0.02 2 F-8 F-5 H69Ile 0.523 0.05 3 F-9 F-5 H67Phe 0.675 0.09
3 F-10 F-9 H69Ile 0.690 0.03 3 F-11 F-10 LysH75Ser 0.642 0.06 3
F-12 F-10 AsnH76Arg 0.912 0.05 3 F-13 F-12 LeuL46Val 1.050 0.16 3
TyrL49Ser F-14 F-13 H71Arg 0.472 0.06 3 F-15 F-13 H73Asn 0.868 0.32
3 .sup.aMurine residues are in bold; residue numbers are according
to Kabat et al. (1991). Standard text indicates a change from a
human framework residue to mouse. Italic text indicates a change
from a mouse framework residue to human. Fab binding to IFN-.alpha.
was assayed by ELISA and results are provided as OD.sub.450 nm at
10 .mu.g/ml. SD, standard deviation; n, number of experimental
replicates.
[0275] Previous humanizations (Xiang et al., J. Mol. Biol. 253:
385-390 [1995]; Werther et al., [1996] supra) as well as studies of
F(ab)-antigen crystal structures (Chothia et al., [1989] supra;
Tramontano et al., J. Mol. Biol. 215: 175-182 [1990]) have shown
that residues H71 and H73 can have a profound effect on binding,
possibly by influencing the conformations of CDR-H1 and CDR-H2.
Changing the human residues at positions H71 and 1173 to their
murine counterparts improved binding only slightly (version F-2,
Table 3). Further simultaneous changes at positions H67, H69 and
H78 (version F-3) followed by changes ArgH94Ser (version F-4) and
AlaH24Thr (version F-5) significantly improved binding (Table 3).
Since positions H67, H69 and H78 had been changed simultaneously,
each was individually altered back to the human consensus framework
residue; versions F-7, F-8, F-9, and F-10 show that the human
residue is preferred at position H67, position H69 does not show
any preference for the human or murine residue, and the murine
residue is preferred at position H78.
[0276] We have found during previous humanizations that residues in
a framework loop, FR-3 (Kabat et al., (1991) supra), adjacent to
CDR-H1 and CDR-H2 can affect binding (Eigenbrot et al., (1994)
supra). Accordingly, two residues in this loop were changed to
their murine counterparts: LysH75 to murine Ser (version F-11) and
AsnH76 to murine Arg (version F-12). Only the AsnH76Arg change
effected an improvement in binding (Table 3).
[0277] Inspection of the models of the murine and humanized F(ab)s
suggested that residue L46, buried at the VL-VH interface and
interacting with CDR-H3, might also play a role either in
determining the conformation of CDR-H3 and/or affecting the
interactions between the VL and VH domains. Similarly, L49 position
which is adjacent to CDR-L2 differs between the human consensus
(Tyr) and the 9F3 (Ser) sequence. Therefore, LeuL46Val and
TyrL49Ser residues were simultaneously substituted, which resulted
in a variant (F-13) with further improvement in the binding (Table
3). Based on its best binding among all the variants generated,
F-13 was chosen as the final humanized version.
[0278] A humanized recombinant anti-IFN-.alpha. monoclonal antibody
(V13IgG2) was generated by fusing VH and VL domains derived from
F-13 to human IgG2 CHI-Fc and human CL domains respectively. The
K.sub.ON rates and K.sub.D values of V13IgG2 were then compared
with a chimeric IgG2 or murine 9F3. BIACore.TM. measurement of
V13IgG2 and chimeric IgG2 binding to immobilized IFN-.alpha. showed
that their K.sub.ON rates were similar (Table 4). Affinity
measurement using Kinexa.TM. technology showed that the affinity of
V13IgG2 for IFN.alpha. was reduced by 2-fold compared to the
parental murine 9F3 antibody (Table 4).
TABLE-US-00004 TABLE 4 BIACore .TM. and Kinexa .TM. Data for
Anti-IFN.alpha. Antibodies Antibody.sup.a K.sub.on(.mu.M/sec) Kd
(nM).sup.b Method 0.14 BIACore .TM. ChIgG2 3.9 BIACore .TM. V13IgG2
3.3 BIACore .TM. V13Fab 4.1 BIACore .TM. Antibody.sup.a K.sub.D(pM)
murine 9F3 1.5 Kinexa .TM. V13Fab 3.4 Kinexa .TM. .sup.aV13IgG2 is
F-13 VH domain joined to human IgG2 CH1-Fc and F-13 VL domain
joined to a human CL domain; ChIgG2 is mouse 9F3 VH domain joined
to human IgG2 CH1-Fc and mouse 9F3 VL domain joined to human CL
domain. .sup.bKoff/Kon.
Deposit of Material
[0279] The following materials have been deposited with the
American Type Culture Collection, 10801 University Blvd., Manassas,
Va. 20110-2209, USA (ATCC):
TABLE-US-00005 Material ATCC Dep. No. Deposit Date 1. A hybridoma
cell line PTA-2917 Jan. 18, 2001 secreting 9F3 murine anti-
IFN-.alpha. monoclonal antibodies (Id. Ref.: 9F3.18.5) 2. pRK-based
vector for the PTA-2883 Jan. 9, 2001 expression of heavy chain of
chimeric CH8-2 full-length IgG (Id. Ref: XAIFN-ChHpDR2) 3.
pRK-based vector for the PTA-2880 Jan. 9, 2001 expression of light
chain of chimeric CH8-2 full-length IgG (Id. Ref.: XAIFN-ChLpDR1)
4. pRK-based vector for the PTA-2881 Jan. 9, 2001 expression of
heavy chain of humanized V13 full-length IgG.sub.2 (Id. Ref.:
VHV30-IgG2) 5. pRK-based vector for the PTA-2882 Jan. 9, 2001
expression of light chain of humanized V13 full-length IgG.sub.2
(Id. Ref.: VLV30-IgG)
[0280] This deposit was 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 culture of the deposit for 30 years from the date of
deposit. The deposit 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 progeny of the culture of the deposit 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 progeny to
one determined by the U.S. Commissioner of Patents and Trademarks
to be entitled thereto according to 35 U.S.C. .sctn.122 and the
Commissioner's rules pursuant thereto (including 37 C.F.R.
.sctn.1.14 with particular reference to 886 OG 638).
[0281] The assignee of the present application has agreed that if a
culture of the materials on deposit should die or be lost or
destroyed when cultivated under suitable conditions, the materials
will be promptly replaced on notification with another of the same.
Availability of the deposited material 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.
[0282] The foregoing written specification is considered to be
sufficient to enable one skilled in the art to practice the
invention. The present invention is not to be limited in scope by
the construct deposited, since the deposited embodiment is intended
as a single illustration of certain aspects of the invention and
any constructs that are functionally equivalent are within the
scope of this invention. The deposit of material herein does not
constitute an admission that the written description herein
contained is inadequate to enable the practice of any aspect of the
invention, including the best mode thereof, nor is it to be
construed as limiting the scope of the claims to the specific
illustrations that it represents. Indeed, various modifications of
the invention in addition to those shown and described herein will
become apparent to those skilled in the art from the foregoing
description and fall within the scope of the appended claims.
Sequence CWU 1
1
141114PRTMurine 1Asp Ile Val Leu Thr Gln Ser Pro Ala Ser Leu Ala
Val Ser Leu Gly1 5 10 15 Gln Arg Ala Thr Ile Ser Cys Arg Ala Ser
Gln Ser Val Ser Thr Ser 20 25 30 Ser Tyr Ser Tyr Met His Trp Tyr
Gln Gln Lys Pro Gly Gln Pro Pro 35 40 45 Lys Val Leu Ile Ser Tyr
Ala Ser Asn Leu Glu Ser Gly Val Pro Ala 50 55 60 Arg Phe Ser Gly
Ser Gly Ser Gly Thr Asp Phe Thr Leu Asn Ile His65 70 75 80 Pro Val
Glu Glu Gly Asp Thr Ala Thr Tyr Phe Cys Gln His Ser Trp 85 90 95
Gly Ile Pro Arg Thr Phe Gly Ala Gly Thr Lys Leu Glu Leu Arg Arg 100
105 110 Ala Val2119PRTMurine 2Glu Val Gln Leu Gln Gln Ser Gly Pro
Glu Leu Val Lys Pro Gly Ala1 5 10 15 Ser Val Lys Ile Ser Cys Lys
Thr Ser Gly Tyr Thr Phe Thr Glu Tyr 20 25 30 Ile Ile His Trp Val
Lys Gln Gly His Gly Arg Ser Leu Glu Trp Ile 35 40 45 Gly Ser Ile
Asn Pro Asp Tyr Asp Ile Thr Asn Tyr Asn Gln Arg Phe 50 55 60 Lys
Gly Lys Ala Thr Leu Thr Leu Asp Lys Ser Ser Arg Thr Ala Tyr65 70 75
80 Leu Glu Leu Arg Ser Leu Thr Ser Glu Asp Ser Ala Val Tyr Tyr Cys
85 90 95 Ala Ser Trp Ile Ser Asp Phe Phe Asp Tyr Trp Gly Gln Gly
Thr Thr 100 105 110 Leu Met Val Ser Ala Ala Ser 115
3114PRTArtificial SequenceThis sequence represents a humanized
chimeric antibody comprising human and non-human sequences. 3Asp
Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly1 5 10
15 Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Gln Ser Val Ser Thr Ser
20 25 30 Ser Tyr Ser Tyr Met His Trp Tyr Gln Gln Lys Pro Gly Lys
Ala Pro 35 40 45 Lys Val Leu Ile Ser Tyr Ala Ser Asn Leu Glu Ser
Gly Val Pro Ser 50 55 60 Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp
Phe Thr Leu Thr Ile Ser65 70 75 80 Ser Leu Gln Pro Glu Asp Phe Ala
Thr Tyr Tyr Cys Gln His Ser Trp 85 90 95 Gly Ile Pro Arg Thr Phe
Gly Gln Gly Thr Lys Val Glu Ile Lys Arg 100 105 110 Thr
Val4110PRTHomo sapiens 4Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu
Ser Ala Ser Val Gly1 5 10 15 Asp Arg Val Thr Ile Thr Cys Arg Ala
Ser Gln Ser Ile Ser Asn Tyr 20 25 30 Leu Ala Trp Tyr Gln Gln Lys
Pro Gly Lys Ala Pro Lys Leu Leu Ile 35 40 45 Tyr Ala Ala Ser Ser
Leu Glu Ser Gly Val Pro Ser Arg Phe Ser Gly 50 55 60 Ser Gly Ser
Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro65 70 75 80 Glu
Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Tyr Asn Ser Leu Pro Trp 85 90
95 Thr Phe Gly Gln Gly Thr Lys Val Glu Ile Lys Arg Thr Val 100 105
110 5119PRTArtificial SequenceThis sequence represents a humanized
chimeric antibody comprising human and non-human sequences. 5Glu
Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly1 5 10
15 Ser Leu Arg Leu Ser Cys Ala Thr Ser Gly Tyr Thr Phe Thr Glu Tyr
20 25 30 Ile Ile His Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu
Trp Val 35 40 45 Ala Ser Ile Asn Pro Asp Tyr Asp Ile Thr Asn Tyr
Asn Gln Arg Phe 50 55 60 Lys Gly Arg Phe Thr Ile Ser Leu Asp Lys
Ser Lys Arg Thr Ala Tyr65 70 75 80 Leu Gln Met Asn Ser Leu Arg Ala
Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Ser Trp Ile Ser Asp
Phe Phe Asp Tyr Trp Gly Gln Gly Thr Leu 100 105 110 Val Thr Val Ser
Ser Ala Ser 115 6119PRTHomo sapiens 6Glu Val Gln Leu Val Glu Ser
Gly Gly Gly Leu Val Gln Pro Gly Gly1 5 10 15 Ser Leu Arg Leu Ser
Cys Ala Ala Ser Gly Phe Thr Phe Ser Ser Tyr 20 25 30 Ala Met Ser
Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45 Ala
Val Ile Ser Gly Asp Gly Gly Ser Thr Tyr Tyr Ala Asp Ser Val 50 55
60 Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu
Tyr65 70 75 80 Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val
Tyr Tyr Cys 85 90 95 Ala Arg Gly Arg Val Gly Tyr Tyr Asp Tyr Trp
Gly Gln Gly Thr Leu 100 105 110 Val Thr Val Ser Ser Ala Ser 115
715PRTHomo sapiens 7Arg Ala Ser Gln Ser Val Ser Thr Ser Ser Tyr Ser
Tyr Met His1 5 10 15 87PRTHomo sapiens 8Tyr Ala Ser Asn Leu Glu
Ser1 5 910PRTHomo sapiens 9Gln His Ser Trp Gly Ile Pro Arg Thr Phe1
5 10 1010PRTHomo sapiens 10Gly Tyr Thr Phe Thr Glu Tyr Ile Ile His1
5 10 1117PRTHomo sapiens 11Ser Ile Asn Pro Asp Tyr Asp Ile Thr Asn
Tyr Asn Gln Arg Phe Lys1 5 10 15 Gly128PRTHomo sapiens 12Trp Ile
Ser Asp Phe Phe Asp Tyr1 5 1330DNAHomo sapiens 13gatcgggaaa
gggaaaccga aactgaagcc 301430DNAHomo sapiens 14gatcggcttc agtttcggtt
tccctttccc 30
* * * * *
References