U.S. patent application number 10/762629 was filed with the patent office on 2004-07-22 for recombinant il-18 antagonists useful in treatment of il-18 mediated disorders.
This patent application is currently assigned to SmithKline Beecham Corporation and SmithKline Beecham p.l.c.. Invention is credited to Abdel-Meguid, Sherin S., Ho, Yen Sen, Holmes, Stephen D., Taylor, Alexander H..
Application Number | 20040141964 10/762629 |
Document ID | / |
Family ID | 22419067 |
Filed Date | 2004-07-22 |
United States Patent
Application |
20040141964 |
Kind Code |
A1 |
Abdel-Meguid, Sherin S. ; et
al. |
July 22, 2004 |
Recombinant IL-18 antagonists useful in treatment of IL-18 mediated
disorders
Abstract
Chimeric, humanized and other IL-18 mAbs, derived from high
affinity neutralizing mAbs, pharmaceutical compositions containing
same, methods of treatment and diagnostics are provided.
Inventors: |
Abdel-Meguid, Sherin S.;
(Exton, PA) ; Ho, Yen Sen; (Berwyn, PA) ;
Holmes, Stephen D.; (Harlow, GB) ; Taylor, Alexander
H.; (Exton, PA) |
Correspondence
Address: |
GLAXOSMITHKLINE
Corporate Intellectual Property - UW2220
P.O. Box 1539
King of Prussia
PA
19406-0939
US
|
Assignee: |
SmithKline Beecham Corporation and
SmithKline Beecham p.l.c.
|
Family ID: |
22419067 |
Appl. No.: |
10/762629 |
Filed: |
January 22, 2004 |
Related U.S. Patent Documents
|
|
|
|
|
|
Application
Number |
Filing Date |
Patent Number |
|
|
10762629 |
Jan 22, 2004 |
|
|
|
09914695 |
Aug 31, 2001 |
|
|
|
6706487 |
|
|
|
|
09914695 |
Aug 31, 2001 |
|
|
|
PCT/US00/07349 |
Mar 17, 2000 |
|
|
|
60125299 |
Mar 19, 1999 |
|
|
|
Current U.S.
Class: |
424/131.1 |
Current CPC
Class: |
A61P 17/06 20180101;
A61K 2039/505 20130101; C07K 16/244 20130101; A61P 37/02 20180101;
A61P 19/02 20180101; A61P 37/00 20180101; A61P 25/00 20180101; A61P
3/10 20180101; A61P 1/04 20180101; A61P 1/00 20180101 |
Class at
Publication: |
424/131.1 |
International
Class: |
A61K 039/395 |
Claims
What is claimed is:
1. A rodent neutralizing monoclonal antibody specific for human
interleukin-18 and having a binding affinity characterized by a
dissociation constant equal to or less than about
3.9.times.10.sup.-11 M.
2. The monoclonal antibody according to claim 1 which is a rat
monoclonal antibody.
3. The monoclonal antibody according to claim 1 which is a murine
monoclonal antibody.
4. The monoclonal antibody according to claim 2 which comprises the
light chain amino acid sequence of SEQ ID NO: 1, and the heavy
chain amino acid sequence of SEQ ID NO: 9.
5. The monoclonal antibody according to claim 3 which comprises the
light chain amino acid sequence of SEQ ID NO: 17 and the heavy
chain amino acid sequence of SEQ ID NO: 25.
6. The monoclonal antibody according to claim 2 which comprises the
light chain amino acid sequence of SEQ ID NO: 33 and the heavy
chain amino acid sequence of SEQ ID NO: 41.
7. The monoclonal antibody according to claim 1 having the
identifying characteristics of 2C10, 14B7 or 13G9.
8. A hybridoma which produces the monoclonal antibody of claim
4.
9. A hybridoma which produces the monoclonal antibody of claim
5.
10. A hybridoma which produces the monoclonal antibody of claim
6.
11. A hybridoma having the identifying characteristics of cell line
19522C10(2)F2(1)A1, 195214B7(1)H10 and 187413G9(3)F12
12. A neutralizing Fab fragment or F(ab').sub.2 fragment thereof,
produced by deleting the Fc region of the monoclonal antibody of
claim 1.
13. An altered antibody comprising a heavy chain and a light chain,
wherein the framework regions of said heavy and light chains are
derived from at least one selected antibody and the amino acid
sequences of the complementarity determining regions of each said
chain are derived from the monoclonal antibody of claim 1.
14. An immunoglobulin light chain complementarity determining
region (CDR), the amino acid sequence of which is selected from the
group consisting of:
2 (a) SEQ ID NO: 3 (b) SEQ ID NO: 5 (c) SEQ ID NO: 7 (d) SEQ ID NO:
19 (e) SEQ ID NO: 21 (f) SEQ ID NO: 23 (g) SEQ ID NO: 35 (h) SEQ ID
NO: 37 (i) SEQ ID NO: 39
15. An immunoglobulin heavy chain complementarity determining
region (CDR), the amino acid sequence of which is selected from the
group consisting of:
3 (a) SEQ ID NO: 11 (b) SEQ ID NO: 13 (c) SEQ ID NO: 15 (d) SEQ ID
NO: 27 (e) SEQ ID NO: 29 (f) SEQ ID NO: 31 (g) SEQ ID NO: 43 (h)
SEQ ID NO: 45 (i) SEQ ID NO: 47
16. A nucleic acid molecule encoding the immunoglobulin
complementarity determining region (CDR) of claim 14.
17. A nucleic acid molecule encoding the immunoglobulin
complementarity determining region (CDR) of claim 15.
18. A pharmaceutical composition comprising the altered antibody of
claim 13 and a pharmaceutically acceptable carrier.
19. A method of treating conditions associated with autoimmune
disease comprising the step of administering to said human in need
thereof an effective amount of the altered antibody of claim
13.
20. The method of claim 19 where said disease is multiple
sclerosis.
21. The method of claim 19 where said disease is rheumatoid
arthritis type l or insulin dependent diabetes.
22. The method of claim 19 where said disease is inflammatory bowel
disease.
23. The method of claim 19 where said disease is psoriasis.
24. An isolated nucleic acid sequence which is selected from the
group consisting of: (a) a nucleic acid sequence encoding the
altered antibody of claim 13 (b) a nucleic acid sequence
complementary to (a); and (c) a fragment or analog of (a) or (b),
which encodes a protein, characterized by having a specificity for
human interleukin-18; wherein said sequence optionally contains a
restriction site.
25. A method to assess the presence or absence of human IL-18 in a
human which comprises obtaining a sample of biological fluid from a
patient and allowing the monoclonal antibody of claim 1 to come in
contact with such sample under conditions such that an
IL-18/monoclonal antibody complex can form and detecting the
presence or absence of said IL-18/monoclonal antibody complex.
26. A method for aiding in the diagnosis of autoimmune disease
associated with comprising the steps of determining the amount of
human IL-18 in a sample of a patient according to the method of
claim 25 and comparing that to the mean amount of human IL-18 in
the normal population, whereby the presence of significantly
elevated amount of human IL-18 in the patient is an indication of
autoimmune disease.
Description
FIELD OF THE INVENTION
[0001] The present invention relates generally to the field of
antibodies and altered antibodies, useful in the treatment and
diagnosis of conditions mediated by IL-18, and more specifically to
mAbs, Fabs. chimeric and humanized antibodies.
BACKGROUND OF THE INVENTION
[0002] Human interleukin-18 is a recently identified cytokine that
is synthesized as a biologically inactive 193 amino acid precursor
protein (Ushio et al., J. Immunol. 156:4274, 1996). Cleavage of the
precursor protein, for example by caspase-1 or caspase-4, liberates
the 156 amino acid mature protein (Gu et al., Science 275:206,
1997; Ghayur et al., Nature 386:619, 1997), which exhibits
biological activities that include the costimulation of T cell
proliferation, the enhancement of NK cell cytotoxicity, the
induction of IFN-.gamma. production by T cells and NK cells, and
the potentiation of T helper type 1 (Th 1) differentiation (Okamura
et al., Nature 378:88, 1995; Ushio et al., J. Immunol. 156:4274,
1996; Micallef et al., Eur. J. Immunol. 26:1647, 1996; Kohno et
al., J. Immunol. 158:1541, 1997; Zhang et al., Infect. Immunol.
65:3594, 1997; Robinson et al., Immunity 7:571, 1997). In addition,
IL-18 is an efficacious inducer of human monocyte proinflammatory
mediators, including IL-8, tumor necrosis factor-.alpha.
(TNF-.alpha.), and prostaglandin E.sub.2 (PGE.sub.2) (Ushio, S. et
al., J. Immunol. 156:4274-4279, 1996; Puren, A. J. et al., J. Clin.
Invest. 10:711-721, 1997; Podolin et al., J. Immunol. submitted,
1999).
[0003] The previously cloned IL-1 receptor-related protein
(IL-1Rrp) (Parnet et al., J. Biol. Chem. 271:3967, 1996) was
recently identified as a subunit of the IL-18 receptor (Kd=18 nM)
(Torigoe et al., J. Biol. Chem. 272:25737, 1997). A second subunit
of the IL-18 receptor exhibits homology to the IL-1 receptor
accessory protein, and has been termed AcPL (for accessory
protein-like). Expression of both IL-1Rrp and AcPL are required for
IL-18-induced NF-.kappa.B and JNK activation (Born et al., J. Biol.
Chem. 273:29445, 1998). In addition to NF-.kappa.B and JNK, IL-18
signals through IL-1 receptor-associated kinase (IRAK), p561ck
(LCK), and mitogen-activated protein kinase (MAPK) (Micallef et
al., Eur. J. Immunol. 26:1647, 1996; Matsumoto et al., Biophys
Biochem. Res. Comm. 234:454, 1997; Tsuji-Takayama et al., Biochem.
Biophys. Res. Comm. 237:126, 1997).
[0004] Th1 cells, which produce proinflammatory cytokines such as
IFN-.gamma., IL-2 and TNF-.beta. (Mosmann et al., J. Immunol.
136:2348. 1986), have been implicated in mediating many of
autoimmune diseases, including multiple sclerosis (MS), rheumatoid
arthritis (RA), type 1, or insulin dependent, diabetes (IDDM).
inflammatory bowel disease (IBD), and psoriasis (Mosmann and Sad,
Immunol. Today 17:138. 1996). Thus, antagonism of a Th1-promoting
cytokine such as IL-18 would be expected to inhibit disease
development. Il-18 specific mAbs could be used as an
antagonist.
[0005] The role of IL-18 in the development of autoimmune diseases
has been demonstrated. Accordingly, it has been demonstrated that
IL-18 expression is significantly increased in the pancreas and
spleen of the nonobese diabetic (NOD) mouse immediately prior to
the onset of disease (Rothe et al., J. Clin. Invest. 99:469, 1997).
Similarly, IL-18 levels have been shown to be markedly elevated in
the synovial fluid of rheumatoid arthritis patients (Kawashima et
al., Arthritis and Rheumatism 39:598, 1996). Furthermore, it has
been demonstrated that IL-18 administration increases the clinical
severity of murine experimental allergic encephalomyelitis (EAE), a
Th1-mediated autoimmune disease that is a model for multiple
sclerosis. In addition, it has been shown that neutralizing
anti-rat IL-18 antiserum prevents the development of EAE in female
Lewis rats (Wildbaum et al., J. Immunol. 161:6368, 1998).
Accordingly, IL-18 is a desirable target for the development of a
novel therapeutic for autoimmunity.
[0006] Taniguchi et al., J. Immunol. Methods 206:107, describe
seven murine and six rat anti-human IL-18 mAbs, which bind to four
distinct antigenic sites. One of the murine mAbs (#125-2H), and the
six rat mAbs inhibit IL-18-induced IFN-.gamma. production by KG-1
cells, with the rat mAbs exhibiting neutralizing activities 10-fold
lower than that of #125-2H. As demonstrated by Western blot
analysis, three of the murine mAbs, but none of the rat mAbs, are
strongly reactive with membrane-bound human IL-18. In addition, an
enzyme-linked immunosorbent assay (ELISA) to detect human IL-18 is
described, utilizing #125-2H and a rat mAb. The limit of detection
of this ELISA is 10 pg/ml.
[0007] European patent application EP 0 712 931 discloses two mouse
anti-human IL-18 mAbs, H1 (IgG1) and H2 (IgM). As demonstrated by
Western blot analysis, both mAbs react with membrane-bound human
IL-18, but not with membrane-bound human IL-12. H1 is utilized in
an immunoaffinity chromatography protocol to purify human IL-18,
and in an ELISA to measure human IL-18. H2 is utilized in a
radioimmunoassay to measure human IL-18.
[0008] Neutralizing IL-18 antibodies may potentially be useful in
relieving autoimmune diseases and related symptoms in man. Hence
there is a need in the art for a high affinity IL-18 antagonist,
such as a neutralizing monoclonal antibody to human interleukin 18.
which would reduce Th1 differentiation and proliferation and thus
autoimmune diseases and related symptoms.
SUMMARY OF THE INVENTION
[0009] In a first aspect, the present invention provides rodent
(e.g. rat and murine) neutralizing monoclonal antibodies specific
for human interleukin-18 and having a binding affinity
characterized by a dissociation constant equal to or less than
about 3.9.times.10.sup.-11 M as described in the detailed
description. Exemplary of such monoclonal antibodies are the rat
monoclonal antibody 2C10 and rat and murine monoclonal antibodies
such as 14B7 and 13G9. Another aspect of the invention are
hybridomas such as 19522C10(2)F2(1)A1, 195214B7(1)H10 and
187413G9(3)F12
[0010] In a related aspect, the present invention provides
neutralizing Fab fragments or F(ab').sub.2 fragments thereof
specific for human interleukin-18 produced by deleting the Fc
region of the rodent neutralizing monoclonal antibodies of the
present invention.
[0011] In still another related aspect, the present invention
provides an altered antibody specific for human interleukin-18
which comprises complementarity determining regions (CDRs) derived
from a non-human neutralizing monoclonal antibody (mAb)
characterized by a dissociation constant equal to or less than
about 3.9.times.10.sup.-11 M for human interleukin-18 and nucleic
acid molecules encoding the same. When the altered antibody is a
humanized antibody, the sequences that encode complementarity
determining regions (CDRs) from a non-human immunoglobulin are
inserted into a first immunoglobulin partner in which at least one,
and preferably all complementarity determining regions (CDRs) of
the first immunoglobulin partner are replaced by CDRs from the
non-human monoclonal antibody. Preferably, the first immunoglobulin
partner is operatively linked to a second immunoglobulin partner as
well, which comprises all or a part of an immunoglobulin constant
chain.
[0012] In a related aspect, the present invention provides CDRs
derived from non-human neutralizing monoclonal antibodies (mAbs)
characterized by a dissociation constant equal to or less than
about 3.9.times.10.sup.-11 M for human interleukin-18, and nucleic
acid molecules encoding such CDRs.
[0013] In still another aspect, there is provided a chimeric
antibody containing human heavy and light chain constant regions
and heavy and light chain variable regions derived from non-human
neutralizing monoclonal antibodies characterized by a dissociation
constant equal to or less than about 3.9.times.10.sup.-11 M for
human interleukin-18.
[0014] In yet another aspect, the present invention provides a
pharmaceutical composition which contains one (or more) of the
above described altered antibodies and a pharmaceutically
acceptable carrier.
[0015] In a further aspect, the present invention provides a method
for treating conditions in humans associated with excess Th1
production, for example autoimmune diseases, by administering to
said human an effective amount of the pharmaceutical composition of
the invention.
[0016] In yet another aspect, the present invention provides
methods for, and components useful in, the recombinant production
of altered antibodies (e.g., engineered antibodies, CDRs, Fab or
F(ab).sub.2 fragments, or analogs thereof) which are derived from
non-human neutralizing monoclonal antibodies (mAbs) characterized
by a dissociation constant equal to or less than
3.9.times.10.sup.-11 M for human IL-18. These components include
isolated nucleic acid sequences encoding same, recombinant plasmids
containing the nucleic acid sequences under the control of selected
regulatory sequences which are capable of directing the expression
thereof in host cells (preferably mammalian) transfected with the
recombinant plasmids. The production method involves culturing a
transfected host cell line of the present invention under
conditions such that an altered antibody, preferably a humanized
antibody, is expressed in said cells and isolating the expressed
product therefrom.
[0017] In yet another aspect of the invention is a method to
diagnose conditions associated with excess Th1 production in a
human which comprises obtaining a sample of biological fluid from a
patient and allowing the antibodies and altered antibodies of the
instant invention to come in contact with such sample under
conditions such that an IL-18/antibody (monoclonal or altered)
complex is formed and detecting the presence or absence of said
IL-18/antibody complex.
[0018] Other aspects and advantages of the present invention are
described further in the detailed description and the preferred
embodiments thereof.
BRIEF DESCRIPTION OF THE DRAWINGS
[0019] FIG. 1 [SEQ ID NOS: 1 and 2] illustrates the light chain
variable region for the rat antibody 2C10. FIG. 1 includes sequence
data for both strands. The boxed areas indicate the CDR's [SEQ ID
NOS: 3-8]. The bolded area indicates the degenerate primer
sequence.
[0020] FIG. 2. [SEQ ID NOS: 9 and 10] illustrates the heavy chain
variable region for the rat antibody 2C10. FIG. 2 includes sequence
data for both strands. The boxed areas indicate the CDR's [SEQ ID
NOS: 11-16]. The bolded area indicates the degenerate primer
sequence.
[0021] FIG. 3 [SEQ ID NOS: 17 and 18] illustrates the light chain
variable region for the murine antibody 13G9. FIG. 3 includes
sequence data for both strands. The boxed areas indicate the CDR's
[SEQ ID NOS: 19-24]. The bolded area indicates the degenerate
primer sequence.
[0022] FIG. 4 [SEQ ID NOS: 25 and 26] illustrates the heavy chain
variable region for the murine antibody 13G9. FIG. 4 includes
sequence data for both strands. The boxed areas indicate the CDR's
[SEQ ID NOS: 27-32]. The bolded area indicates the degenerate
primer sequence.
[0023] FIG. 5 [SEQ ID NOS: 33 and 34] illustrates the light chain
variable region for the rat antibody 14B7. FIG. 5 includes sequence
data for both strands. The boxed areas indicate the CDR's [SEQ ID
NOS: 35-40]. The bolded area indicates the degenerate primer
sequence.
[0024] FIG. 6 [SEQ ID NOS: 41 and 42] illustrates the heavy chain
variable region for the rat antibody 14B7. FIG. 6 includes sequence
data for both strands. The boxed areas indicate the CDR's [SEQ ID
NOS: 43-48]. The bolded area indicates the degenerate primer
sequence.
DETAILED DESCRIPTION OF THE INVENTION
[0025] The present invention provides a variety of antibodies,
altered antibodies and fragments thereof, which are characterized
by human IL-18 binding specificity, neutralizing activity, and high
affinity for human IL-18 as exemplified in rat monoclonal antibody
2C10, murine monoclonal antibody 13G9 and rat monoclonal antibody
14B7. The antibodies of the present invention were prepared by
conventional hybridoma techniques to generate novel neutralizing
antibodies. These products are useful in therapeutic and
pharmaceutical compositions for treating IL-18-mediated disorders,
e.g. autoimmune diseases, including multiple sclerosis (MS),
rheumatoid arthritis (RA), type 1, or insulin dependent, diabetes
(IDDM), inflammatory bowel disease (IBD), and psoriasis (Mosmann
and Sad, Immunol. Today 17:138, 1996). These products are also
useful in the diagnosis of IL-18-mediated conditions by measurement
(e.g., enzyme linked immunosorbent assay (ELISA)) of endogenous
IL-18 levels in humans or IL-18 released ex vivo from activated
cells.
[0026] I. Definitions.
[0027] "Altered antibody" refers to a protein encoded by an altered
immunoglobulin coding region, which may be obtained by expression
in a selected host cell. Such altered antibodies are engineered
antibodies (e.g., chimeric or humanized antibodies) or antibody
fragments lacking all or part of an immunoglobulin constant region,
e.g., Fv, Fab, or F(ab).sub.2 and the like.
[0028] "Altered immunoglobulin coding region" refers to a nucleic
acid sequence encoding altered antibody of the invention. When the
altered antibody is a CDR-grafted or humanized antibody, the
sequences that encode the complementarity determining regions
(CDRs) from a non-human immunoglobulin are inserted into a first
immunoglobulin partner comprising human variable framework
sequences. Optionally, the first immunoglobulin partner is
operatively linked to a second immunoglobulin partner.
[0029] "First immunoglobulin partner" refers to a nucleic acid
sequence encoding a human framework or human immunoglobulin
variable region in which the native (or naturally-occurring)
CDR-encoding regions are replaced by the CDR-encoding regions of a
donor antibody. The human variable region can be an immunoglobulin
heavy chain, a light chain (or both chains), an analog or
functional fragments thereof. Such CDR regions, located within the
variable region of antibodies (immunoglobulins) can be determined
by known methods in the art. For example Kabat et al. (Sequences of
Proteins of Immunological Interest, 4th Ed., U.S. Department of
Health and Human Services, National Institutes of Health (1987))
disclose rules for locating CDRs. In addition, computer programs
are known which are useful for identifying CDR
regions/structures.
[0030] "Neutralizing" refers to an antibody that inhibits IL-18
activity by preventing the binding of human IL-18 to its specific
receptor or by inhibiting the signaling of IL-18 through its
receptor, should binding occur. A mAb is neutralizing if it is 90%
effective, preferably 95% effective and most preferably 100%
effective in inhibiting IL-18 activity as measured in the IL-18
neutralization assay, see Example 1 and Table I.
[0031] The term "high affinity" refers to an antibody having a
binding affinity characterized by a K.sub.d equal to or less than
3.9.times.10.sup.-11 M for human IL-18 as determined by optical
biosensor analysis (see Example 2 and Table I).
[0032] By "binding specificity for human IL-18" is meant a higher
affinity for human IL-18 than murine, or other IL-18.
[0033] "Second immunoglobulin partner" refers to another nucleotide
sequence encoding a protein or peptide to which the first
immunoglobulin partner is fused in frame or by means of an optional
conventional linker sequence (i.e. operatively linked). Preferably
it is an immunoglobulin gene. The second immunoglobulin partner may
include a nucleic acid sequence encoding the entire constant region
for the same (i.e. homologous--the first and second altered
antibodies are derived from the same source) or an additional
(i.e., heterologous) antibody of interest. It may be an
immunoglobulin heavy chain or light chain (or both chains as part
of a single polypeptide). The second immunoglobulin partner is not
limited to a particular immunoglobulin class or isotype. In
addition, the second immunoglobulin partner may comprise part of an
immunoglobulin constant region, such as found in a Fab, or
F(ab).sub.2 (i.e., a discrete part of an appropriate human constant
region or framework region). Such second immunoglobulin partner may
also comprise a sequence encoding an integral membrane protein
exposed on the outer surface of a host cell, e.g., as part of a
phage display library, or a sequence encoding a protein for
analytical or diagnostic detection, e.g., horseradish peroxidase,
.beta.-galactosidase, etc.
[0034] The terms Fv, Fc, Fd, Fab, or F(ab).sub.2 are used with
their standard meanings (see, e.g., Harlow et al., Antibodies A
Laboratory Manual, Cold Spring Harbor Laboratory, (1988)).
[0035] As used herein, an "engineered antibody" describes a type of
altered antibody, i.e., a full-length synthetic antibody (e.g., a
chimeric or humanized antibody as opposed to an antibody fragment)
in which a portion of the light and/or heavy chain variable domains
of a selected acceptor antibody are replaced by analogous parts
from one or more donor antibodies which have specificity for the
selected epitope. For example, such molecules may include
antibodies characterized by a humanized heavy chain associated with
an unmodified light chain (or chimeric light chain), or vice versa.
Engineered antibodies may also be characterized by alteration of
the nucleic acid sequences encoding the acceptor antibody light
and/or heavy variable domain framework regions in order to retain
donor antibody binding specificity. These antibodies can comprise
replacement of one or more CDRs (preferably all) from the acceptor
antibody with CDRs from a donor antibody described herein.
[0036] A "chimeric antibody" refers to a type of engineered
antibody which contains naturally-occurring variable region (light
chain and heavy chains) derived from a donor antibody in
association with light and heavy chain constant regions derived
from an acceptor antibody.
[0037] A "humanized antibody" refers to a type of engineered
antibody having its . CDRs derived from a non-human donor
immunoglobulin, the remaining immunoglobulin-derived parts of the
molecule being derived from one (or more) human immunoglobulin(s).
In addition, framework support residues may be altered to preserve
binding affinity (see, e.g. Queen et al. Proc. Natl Acad Sci USA,
86:10029-10032 (1989). Hodgson et al. Bio/Technology. 9:421
(1991)).
[0038] The term "donor antibody" refers to an antibody (monoclonal,
or recombinant) which contributes the nucleic acid sequences of its
variable regions, CDRs, or other functional fragments or analogs
thereof to a first immunoglobulin partner, so as to provide the
altered immunoglobulin coding region and resulting expressed
altered antibody with the antigenic specificity and neutralizing
activity characteristic of the donor antibody. One donor antibody
suitable for use in this invention is a non-human neutralizing
monoclonal antibody (i.e., rat) designated as 2C10. The antibody
2C10 is defined as a high affinity, human-IL-18 specific (i.e.,
does not recognize murine IL-18), neutralizing antibody of isotype
IgG.sub.1.K having the variable light chain DNA and amino acid
sequences of SEQ ID NOs: 1 and 2 and respectively, the variable
heavy chain DNA and amino acid sequences of SEQ ID NOs: 9 and 10 on
a suitable murine IgG constant region.
[0039] The term "acceptor antibody" refers to an antibody
(monoclonal, or recombinant) heterologous to the donor antibody,
which contributes all (or any portion, but preferably all) of the
nucleic acid sequences encoding its heavy and/or light chain
framework regions and/or its heavy and/or light chain constant
regions to the first immunoglobulin partner. Preferably a human
antibody is the acceptor antibody.
[0040] "CDRs" are defined as the complementarity determining region
amino acid sequences of an antibody which are the hypervariable
regions of immunoglobulin heavy and light chains. See, e.g., Kabat
et al., Sequences of Proteins of Immunological Interest, 4th Ed.,
U.S. Department of Health and Human Services, National Institutes
of Health (1987). There are three heavy chain and three light chain
CDRs (or CDR regions) in the variable portion of an immunoglobulin.
Thus, "CDRs" as used herein refers to all three heavy chain CDRs,
or all three light chain CDRs (or both all heavy and all light
chain CDRs, if appropriate).
[0041] CDRs provide the majority of contact residues for the
binding of the antibody to the antigen or epitope. CDRs of interest
in this invention are derived from donor antibody variable heavy
and light chain sequences, and include analogs of the naturally
occurring CDRs, which analogs also share or retain the same antigen
binding specificity and/or neutralizing ability as the donor
antibody from which they were derived.
[0042] By sharing the antigen binding specificity or neutralizing
ability is meant, for example, that although mAb 2C10 may be
characterized by a certain level of antigen affinity, a CDR encoded
by a nucleic acid sequence of 2C10 in an appropriate structural
environment may have a lower, or higher affinity. It is expected
that CDRs of 2C10 in such environments will nevertheless recognize
the same epitope(s) as 2C10. Exemplary light chain CDRs of 2C10
include
[0043] SEQ ID NO: 3;
[0044] SEQ ID NO: 5:
[0045] SEQ ID NO: 7:
[0046] and exemplary heavy chain CDRs of 2C10 include
[0047] SEQ ID NO: 11:
[0048] SEQ ID NO: 13;
[0049] and SEQ ID NO: 15.
[0050] A "functional fragment" is a partial heavy or light chain
variable sequence (e.g., minor deletions at the amino or carboxy
terminus of the immunoglobulin variable region) which retains the
same antigen binding specificity and/or neutralizing ability as the
antibody from which the fragment was derived.
[0051] An "analog" is an amino acid sequence modified by at least
one amino acid, wherein said modification can be chemical or a
substitution or a rearrangement of a few amino acids (i.e., no more
than 10), which modification permits the amino acid sequence to
retain the biological characteristics, e.g., antigen specificity
and high affinity, of the unmodified sequence. For example,
(silent) mutations can be constructed, via substitutions, when
certain endonuclease restriction sites are created within or
surrounding CDR-encoding regions.
[0052] Analogs may also arise as allelic variations. An "allelic
variation or modification" is an alteration in the nucleic acid
sequence encoding the amino acid or peptide sequences of the
invention. Such variations or modifications may be due to
degeneracy in the genetic code or may be deliberately engineered to
provide desired characteristics. These variations or modifications
may or may not result in alterations in any encoded amino acid
sequence.
[0053] The term "effector agents" refers to non-protein carrier
molecules to which the altered antibodies, and/or natural or
synthetic light or heavy chains of the donor antibody or other
fragments of the donor antibody may be associated by conventional
means. Such non-protein carriers can include conventional carriers
used in the diagnostic field, e.g., polystyrene or other plastic
beads, polysaccharides, e.g., as used in the BIAcore [Pharmacia]
system, or other non-protein substances useful in the medical field
and safe for administration to humans and animals. Other effector
agents may include a macrocycle, for chelating a heavy metal atom,
or radioisotopes. Such effector agents may also be useful to
increase the half-life of the altered antibodies. e.g.,
polyethylene glycol.
[0054] II. High Affinity IL-18 Monoclonal Antibodies
[0055] For use in constructing the antibodies, altered antibodies
and fragments of this invention, a non-human species (for example,
bovine, ovine, monkey, chicken, rodent (e.g., murine and rat),
etc.) may be employed to generate a desirable immunoglobulin upon
presentment with native human IL-18 or a peptide epitope therefrom.
Conventional hybridoma techniques are employed to provide a
hybridoma cell line secreting a non-human mAb to IL-18. Such
hybridomas are then screened for binding using IL-18 coated to
96-well plates, as described in the Examples section, or
alternatively with biotinylated IL-18 bound to a streptavidin
coated plate.
[0056] One exemplary, high affinity, neutralizing mAb of this
instant invention is mAb 2C10, a rat antibody which can be used for
the development of a chimeric or humanized antibody, described in
more detail in examples below. The 2C10 mAb is characterized by an
antigen binding specificity for human IL-18 of about K.sub.d
3.9.times.10.sup.-11 M. This mAB is characterized by being isotype
IgG.sub.1.K.
[0057] Another desirable donor antibody is the murine mAb 13G9.
This mAb is characterized by being isotype IgG.sub.1.K. The mAb has
a dissociation constant for IL-18 of about 12.times.10.sup.-9
M.
[0058] Yet, another desirable donor antibody is the rat mAb, 14B7.
This mAb is characterized by having a dissociation constant for
IL-18 about 1.5.times.10.sup.-10M. 14B7 is also characterized by
being isotype IgG.sub.1.K.
[0059] This invention is not limited to the use of the 13G9, 2C10,
14B7, or their hypervariable (i.e., CDR) sequences. Any other
appropriate high affinity IL-18 antibodies characterized by a
dissociation constant equal or less than about 3.9.times.10.sup.-11
M for human IL-18 and corresponding anti-IL-18 CDRs may be
substituted therefor. Wherever in the following description the
donor antibody is identified as 13G9, 2C10, 14B7, this designation
is made for illustration and simplicity of description only.
[0060] III. Antibody Fragments
[0061] The present invention also includes the use of Fab fragments
or F(ab').sub.2 fragments derived from mAbs directed against human
IL-18. These fragments are useful as agents protective in vivo
against IL-18 and Th1-mediated conditions or in vitro as part of an
IL-18 diagnostic. A Fab fragment contains the entire light chain
and amino terminal portion of the heavy chain; and an F(ab').sub.2
fragment is the fragment formed by two Fab fragments bound by
disulfide bonds. MAbs 13G9, 2C10, 14B7, and other similar high
affinity, IL-18 binding antibodies, provide sources of Fab
fragments and F(ab').sub.2 fragments which can be obtained by
conventional means, e.g., cleavage of the mAb with the appropriate
proteolytic enzymes, papain and/or pepsin, or by recombinant
methods. These Fab and F(ab').sub.2 fragments are useful themselves
as therapeutic, prophylactic or diagnostic agents, and as donors of
sequences including the variable regions and CDR sequences useful
in the formation of recombinant or humanized antibodies as
described herein.
[0062] The Fab and F(ab').sub.2 fragments can be constructed via a
combinatorial phage library (see, e.g., Winter et al., Ann. Rev.
Immunol., 12:433-455 (1994)) or via immunoglobulin chain shuffling
(see, e.g., Marks et al., Bio/Technology, 10:779-783 (1992), which
are both hereby incorporated by reference in their entirety)
wherein the Fd or v.sub.H immunoglobulin from a selected antibody
(e.g., 13G9) is allowed to associate with a repertoire of light
chain immunoglobulins, v.sub.L (or v.sub.K), to form novel Fabs.
Conversely, the light chain immunoglobulin from a selected antibody
may be allowed to associate with a repertoire of heavy chain
immunoglobulins, v.sub.H (or Fd), to form novel Fabs.
[0063] IV. Anti-IL-18 Amino Acid and Nucleotide Sequences of
Interest
[0064] The mAb 2C10 or other antibodies described above may
contribute sequences, such as variable heavy and/or light chain
peptide sequences, framework sequences, CDR sequences, functional
fragments, and analogs thereof, and the nucleic acid sequences
encoding them, useful in designing and obtaining various altered
antibodies which are characterized by the antigen binding
specificity of the donor antibody.
[0065] As one example, the present invention provides variable
light chain and variable heavy chain sequences from the IL-18 mAb
2C10 and sequences derived therefrom.
[0066] The nucleic acid sequences of this invention, or fragments
thereof, encoding the variable light chain and heavy chain peptide
sequences are also useful for mutagenic introduction of specific
changes within the nucleic acid sequences encoding the CDRs or
framework regions. and for incorporation of the resulting modified
or fusion nucleic acid sequence into a plasmid for expression.
[0067] Taking into account the degeneracy of the genetic code,
various coding sequences may be constructed which encode the
variable heavy and light chain amino acid sequences, and CDR
sequences of the invention as well as functional fragments and
analogs thereof which share the antigen specificity of the donor
antibody. The isolated nucleic acid sequences of this invention, or
fragments thereof. encoding the variable chain peptide sequences or
CDRs can be used to produce altered antibodies, e.g., chimeric or
humanized antibodies, or other engineered antibodies of this
invention when operatively combined with a second immunoglobulin
partner.
[0068] It should be noted that in addition to isolated nucleic acid
sequences encoding portions of the altered antibody and antibodies
described herein, other such nucleic acid sequences are encompassed
by the present invention, such as those complementary to the native
CDR-encoding sequences or complementary to the modified human
framework regions surrounding the CDR-encoding regions. Useful DNA
sequences include those sequences which hybridize under stringent
hybridization conditions [see, T. Maniatis et al, Molecular Cloning
(A Laboratory Manual), Cold Spring Harbor Laboratory (1982), pages
387 to 389] to the DNA sequences. An example of one such stringent
hybridization condition is hybridization at 4.times.SSC at
65.degree. C., followed by a washing in 0.1.times.SSC at 65.degree.
C. for an hour. Alternatively an exemplary stringent hybridization
condition is in 50% formamide, 4.times.SSC at 42.degree. C.
Preferably, these hybridizing DNA sequences are at least about 18
nucleotides in length, i.e., about the size of a CDR.
[0069] V. Altered Immunoglobulin Molecules and Altered
Antibodies
[0070] Altered immunoglobulin molecules can encode altered
antibodies which include engineered antibodies such as chimeric
antibodies and humanized antibodies. A desired altered
immunoglobulin coding region contains CDR-encoding regions that
encode peptides having the antigen specificity of an IL-18
antibody, preferably a high affinity antibody such as provided by
the present invention, inserted into a first immunoglobulin partner
(a human framework or human immunoglobulin variable region).
[0071] Preferably, the first immunoglobulin partner is operatively
linked to a second immunoglobulin partner. The second
immunoglobulin partner is defined above, and may include a sequence
encoding a second antibody region of interest, for example an Fc
region. Second immunoglobulin partners may also include sequences
encoding another immunoglobulin to which the light or heavy chain
constant region is fused in frame or by means of a linker sequence.
Engineered antibodies directed against functional fragments or
analogs of IL-18 may be designed to elicit enhanced binding with
the same antibody.
[0072] The second immunoglobulin partner may also be associated
with effector agents as defined above, including non-protein
carrier molecules, to which the second immunoglobulin partner may
be operatively linked by conventional means.
[0073] Fusion or linkage between the second immunoglobulin
partners, e.g., antibody sequences, and the effector agent may be
by any suitable means, e.g., by conventional covalent or ionic
bonds, protein fusions, or hetero-bifunctional cross-linkers, e.g.,
carbodiimide, glutaraldehyde, and the like. Such techniques are
known in the art and readily described in conventional chemistry
and biochemistry texts.
[0074] Additionally, conventional linker sequences which simply
provide for a desired amount of space between the second
immunoglobulin partner and the effector agent may also be
constructed into the altered immunoglobulin coding region. The
design of such linkers is well known to those of skill in the
art.
[0075] In addition, signal sequences for the molecules of the
invention may be modified to enhance expression.
[0076] An exemplary altered antibody contains a variable heavy
and/or light chain peptide or protein sequence having the antigen
specificity of mAb 2C10, e.g., the V.sub.H and V.sub.L chains.
Still another desirable altered antibody of this invention is
characterized by the amino acid sequence containing at least one,
and preferably all of the CDRs of the variable region of the heavy
and/or light chains of the rat antibody molecule 2C10 with the
remaining sequences being derived from a human source, or a
functional fragment or analog thereof.
[0077] In still a further embodiment, the engineered antibody of
the invention may have attached to it an additional agent. For
example, the procedure of recombinant DNA technology may be used to
produce an engineered antibody of the invention in which the Fc
fragment or CH2 CH3 domain of a complete antibody molecule has been
replaced by an enzyme or other detectable molecule (i.e., a
polypeptide effector or reporter molecule).
[0078] The second immunoglobulin partner may also be operatively
linked to a non-immunoglobulin peptide, protein or fragment thereof
heterologous to the CDR-containing sequence, for example, having
the antigen specificity of rat 2C 10. The resulting protein may
exhibit both anti-IL-18 antigen specificity and characteristics, of
the non-immunoglobulin upon expression. That fusion partner
characteristic may be, e.g., a functional characteristic such as
another binding or receptor domain, or a therapeutic characteristic
if the fusion partner is itself a therapeutic protein, or
additional antigenic characteristics.
[0079] Another desirable protein of this invention may comprise a
complete antibody molecule, having full length heavy and light
chains, or any discrete fragment thereof, such as the Fab or
F(ab').sub.2 fragments, a heavy chain dimer, or any minimal
recombinant fragments thereof such as an F.sub.v or a single-chain
antibody (SCA) or any other molecule with the same specificity as
the selected donor mAb, e.g., mAb 2C10. Such protein may be used in
the form of an altered antibody, or may be used in its unfused
form.
[0080] Whenever the second immunoglobulin partner is derived from
an antibody different from the donor antibody, e.g., any isotype or
class of immunoglobulin framework or constant regions, an
engineered antibody results. Engineered antibodies can comprise
immunoglobulin (Ig) constant regions and variable framework regions
from one source, e.g., the acceptor antibody, and one or more
(preferably all) CDRs from the donor antibody, e.g., the anti-IL-18
antibody described herein. In addition, alterations, e.g.,
deletions, substitutions, or additions, of the acceptor mAb light
and/or heavy variable domain framework region at the nucleic acid
or amino acid levels, or the donor CDR regions may be made in order
to retain donor antibody antigen binding specificity.
[0081] Such engineered antibodies are designed to employ one (or
both) of the variable heavy and/or light chains of the IL-18 mAb
(optionally modified as described) or one or more of the
below-identified heavy or light chain CDRs. The engineered
antibodies would be expected to be are neutralizing, i.e. they
desirably block binding to the receptor of the IL-18 protein and
they also block or prevent proliferation of IL-18 dependent
cells.
[0082] Such engineered antibodies may include a humanized antibody
containing the framework regions of a selected human immunoglobulin
or subtype, or a chimeric antibody containing the human heavy and
light chain constant regions fused to the IL-18 antibody functional
fragments. A suitable human (or other animal) acceptor antibody may
be one selected from a conventional database, e.g., the KABAT.RTM.
database, Los Alamos database, and Swiss Protein database, by
homology to the nucleotide and amino acid sequences of the donor
antibody. A human antibody characterized by a homology to the
framework regions of the donor antibody (on an amino acid basis)
may be suitable to provide a heavy chain constant region and/or a
heavy chain variable framework region for insertion of the donor E
CDRs. A suitable acceptor antibody capable of donating light chain
constant or variable framework regions may be selected in a similar
manner. It should be noted that the acceptor antibody heavy and
light chains are not required to originate from the same acceptor
antibody.
[0083] Desirably the heterologous framework and constant regions
are selected from human immunoglobulin classes and isotypes, such
as IgG (subtypes 1 through 4). IgM, IgA, and IgE. However, the
acceptor antibody need not comprise only human immunoglobulin
protein sequences. For instance a gene may be constructed in which
a DNA sequence encoding part of a human immunoglobulin chain is
fused to a DNA sequence encoding a non-immunoglobulin amino acid
sequence such as a polypeptide effector or reporter molecule.
[0084] One example of a particularly desirable humanized antibody
would contain CDRs of 2C10 inserted onto the framework regions of a
selected human antibody sequence. For neutralizing humanized
antibodies, one, two or preferably three CDRs from the IL-18
antibody heavy chain and/or light chain variable regions are
inserted into the framework regions of the selected human antibody
sequence, replacing the native CDRs of the latter antibody.
[0085] Preferably, in a humanized antibody, the variable domains in
both human heavy and light chains have been engineered by one or
more CDR replacements. It is possible to use all six CDRs, or
various combinations of less than the six CDRs. Preferably all six
CDRs are replaced. It is possible to replace the CDRs only in the
human heavy chain, using as light chain the unmodified light chain
from the human acceptor antibody. Still alternatively, a compatible
light chain may be selected from another human antibody by recourse
to the conventional antibody databases. The remainder of the
engineered antibody may be derived from any suitable acceptor human
immunoglobulin.
[0086] The engineered humanized antibody thus preferably has the
structure of a natural human antibody or a fragment thereof, and
possesses the combination of properties required for effective
therapeutic use, e.g., treatment of IL-18 mediated inflammatory
diseases in man, or for diagnostic uses.
[0087] As another example, an engineered antibody may contain CDRs
of the variable light chain region of 2C10 and CDRs of the variable
heavy chain region of 13G9. The resulting humanized antibody should
be characterized by the same antigen binding specificity and high
affinity of mAb 2C10.
[0088] It will be understood by those skilled in the art that an
engineered antibody may be further modified by changes in variable
domain amino acids without necessarily affecting the specificity
and high affinity of the donor antibody (i.e., an analog). It is
anticipated that heavy and light chain amino acids may be
substituted by other amino acids either in the variable domain
frameworks or CDRs or both.
[0089] In addition, the constant region may be altered to enhance
or decrease selective properties of the molecules of the instant
invention. For example, dimerization, binding to Fc receptors, or
the ability to bind and activate complement (see, e.g., Angal et
al., Mol. Immunol. 30:105-108 (1993), Xu et al., J. Biol. Chem,
269:3469-3474 (1994). Winter et al., EP 307,434-B).
[0090] An altered antibody which is a chimeric antibody differs
from the humanized antibodies described above by providing the
entire non-human donor antibody heavy chain and light chain
variable regions, including framework regions, in association with
human immunoglobulin constant regions for both chains. It is
anticipated that chimeric antibodies which retain additional
non-human sequence relative to humanized antibodies of this
invention may elicit a significant immune response in humans.
[0091] Such antibodies could be useful in the prevention and
treatment of IL-18 mediated disorders, as discussed below.
[0092] VI. Production of Altered Antibodies and Engineered
Antibodies
[0093] Preferably, the variable light and/or heavy chain sequences
and the CDRs of mAb 2C10 or other suitable donor mAbs, and their
encoding nucleic acid sequences, are utilized in the construction
of altered antibodies, preferably humanized antibodies, of this
invention, by the following process. The same or similar techniques
may also be employed to generate other embodiments of this
invention.
[0094] A hybridoma producing a selected donor mAb, e.g., the rat
antibody 2C10, is conventionally cloned, and the DNA of its heavy
and light chain variable regions obtained by techniques known to
one of skill in the art, e.g., the techniques described in Sambrook
et al., (Molecular Cloning (A Laboratory Manual), 2nd edition, Cold
Spring Harbor Laboratory (1989)). The variable heavy and light
regions of 2C10 containing at least the CDR-encoding regions and
those portions of the acceptor mAb light and/or heavy variable
domain framework regions required in order to retain donor mAb
binding specificity, as well as the remaining
immunoglobulin-derived parts of the antibody chain derived from a
human immunoglobulin can be obtained using polynucleotide primers
and reverse transcriptase. The CDR-encoding regions are identified
using a known database and by comparison to other antibodies.
[0095] A rat/human chimeric antibody may then be prepared and
assayed for binding ability. Such a chimeric antibody contains the
entire non-human donor antibody V.sub.H and V.sub.L regions, in
association with human Ig constant regions for both chains.
[0096] A humanized antibody may be derived from the chimeric
antibody, or preferably, made synthetically by inserting the donor
mAb CDR-encoding regions from the heavy and light chains
appropriately within the selected heavy and light chain framework.
Alternatively, a humanized antibody of the invention may be
prepared using standard mutagenesis techniques. Thus, the resulting
humanized antibody contains human framework regions and donor mAb
CDR-encoding regions. There may be subsequent manipulation of
framework residues. The resulting humanized antibody can be
expressed in recombinant host cells, e.g., COS, CHO or myeloma
cells. Other humanized antibodies may be prepared using this
technique on other suitable IL-18-specific, neutralizing, high
affinity, non-human antibodies.
[0097] A conventional expression vector or recombinant plasmid can
be produced by placing these coding sequences for the altered
antibody in operative association with conventional regulatory
control sequences capable of controlling the replication and
expression in, and/or secretion from, a host cell. Regulatory
sequences include promoter sequences, e.g., CMV promoter, and
signal sequences, which can be derived from other known antibodies.
Similarly, a second expression vector can be produced having a DNA
sequence which encodes a complementary antibody light or heavy
chain. Preferably this second expression vector is identical to the
first except insofar as the coding sequences and selectable markers
are concerned, so to ensure as far as possible that each
polypeptide chain is functionally expressed. Alternatively, the
heavy and light chain coding sequences for the altered antibody may
reside on a single vector.
[0098] A selected host cell is co-transfected by conventional
techniques with both the first and second vectors (or simply
transfected by a single vector) to create the transfected host cell
of the invention comprising both the recombinant or synthetic light
and heavy chains. The transfected cell is then cultured by
conventional techniques to produce the engineered antibody of the
invention. The humanized antibody which includes the association of
both the recombinant heavy chain and/or light chain is screened
from culture by appropriate assay, such as ELISA or RIA. Similar
conventional techniques may be employed to construct other altered
antibodies and molecules of this invention.
[0099] Suitable vectors for the cloning and subcloning steps
employed in the methods and construction of the compositions of
this invention may be selected by one of skill in the art. For
example, the conventional pUC series of cloning vectors, may be
used. One vector used is pUC19, which is commercially available
from supply houses, such as Amersham (Buckinghamshire, United
Kingdom) or Pharmacia (Uppsala. Sweden). Additionally, any vector
which is capable of replicating readily, has an abundance of
cloning sites and selectable genes (e.g., antibiotic resistance),
and is easily manipulated may be used for cloning. Thus, the
selection of the cloning vector is not a limiting factor in this
invention.
[0100] Similarly, the vectors employed for expression of the
engineered antibodies according to this invention may be selected
by one of skill in the art from any conventional vector. The
vectors also contain selected regulatory sequences (such as CMV
promoters) which direct the replication and expression of
heterologous DNA sequences in selected host cells. These vectors
contain the above described DNA sequences which code for the
engineered antibody or altered immunoglobulin coding region. In
addition, the vectors may incorporate the selected immunoglobulin
sequences modified by the insertion of desirable restriction sites
for ready manipulation.
[0101] The expression vectors may also be characterized by genes
suitable for amplifying expression of the heterologous DNA
sequences, e.g., the mammalian dihydrofolate reductase gene (DHFR).
Other preferable vector sequences include a poly A signal sequence,
such as from bovine growth hormone (BGH) and the betaglobin
promoter sequence (betaglopro). The expression vectors useful
herein may be synthesized by techniques well known to those skilled
in this art.
[0102] The components of such vectors, e.g. replicons, selection
genes, enhancers, promoters, signal sequences and the like, may be
obtained from commercial or natural sources or synthesized by known
procedures for use in directing the expression and/or secretion of
the product of the recombinant DNA in a selected host. Other
appropriate expression vectors of which numerous types are known in
the art for mammalian, bacterial, insect, yeast, and fungal
expression may also be selected for this purpose.
[0103] The present invention also encompasses a cell line
transfected with a recombinant plasmid containing the coding
sequences of the engineered antibodies or altered immunoglobulin
molecules thereof. Host cells useful for the cloning and other
manipulations of these cloning vectors are also conventional.
However, most desirably, cells from various strains of E. coli are
used for replication of the cloning vectors and other steps in the
construction of altered antibodies of this invention.
[0104] Suitable host cells or cell lines for the expression of the
engineered antibody or altered antibody of the invention are
preferably mammalian cells such as CHO, COS, a fibroblast cell
(e.g., 3T3), and myeloid cells, and more preferably a CHO or a
myeloid cell. Human cells may be used, thus enabling the molecule
to be modified with human glycosylation patterns. Alternatively,
other eukaryotic cell lines may be employed. The selection of
suitable mammalian host cells and methods for transformation,
culture, amplification, screening and product production and
purification are known in the art. See. e.g. Sambrook et al., cited
above.
[0105] Bacterial cells may prove useful as host cells suitable for
the expression of the recombinant Fabs of the present invention
(see, e.g., Pluckthun. A., Immunol. Rev., 130:151-188 (1992)).
However, due to the tendency of proteins expressed in bacterial
cells to be in an unfolded or improperly folded form or in a
non-glycosylated form, any recombinant Fab produced in a bacterial
cell would have to be screened for retention of antigen binding
ability. If the molecule expressed by the bacterial cell was
produced in a properly folded form, that bacterial cell would be a
desirable host. For example, various strains of E. coli used for
expression are well-known as host cells in the field of
biotechnology. Various strains of B. subtilis, Streptomyces, other
bacilli and the like may also be employed in this method.
[0106] Where desired, strains of yeast cells known to those skilled
in the art are also available as host cells, as well as insect
cells, e.g. Drosophila and Lepidoptera and viral expression
systems. See, e.g. Miller et al., Genetic Engineering. 8:277-298,
Plenum Press (1986) and references cited therein.
[0107] The general methods by which the vectors of the invention
may be constructed, the transfection methods required to produce
the host cells of the invention, and culture methods necessary to
produce the altered antibody of the invention from such host cell
are all conventional techniques. Likewise, once produced, the
altered antibodies of the invention may be purified from the cell
culture contents according to standard procedures of the art,
including ammonium sulfate precipitation, affinity columns, column
chromatography, gel electrophoresis and the like. Such techniques
are within the skill of the art and do not limit this
invention.
[0108] Yet another method of expression of the humanized antibodies
may utilize expression in a transgenic animal, such as described in
U.S. Pat. No. 4,873,316. This relates to an expression system using
the animal's casein promoter which when transgenically incorporated
into a mammal permits the female to produce the desired recombinant
protein in its milk.
[0109] Once expressed by the desired method, the engineered
antibody is then examined for in vitro activity by use of an
appropriate assay. Presently conventional ELISA assay formats are
employed to assess qualitative and quantitative binding of the
engineered antibody to IL-18. Additionally, other in vitro assays
may also be used to verify neutralizing efficacy prior to
subsequent human clinical studies performed to evaluate the
persistence of the engineered antibody in the body despite the
usual clearance mechanisms.
[0110] Following the general procedures described for preparing
humanized antibodies, one of skill in the art may also construct
humanized antibodies from other donor IL-18 antibodies, variable
region sequences and CDR peptides described herein. Engineered
antibodies can be produced with variable region frameworks
potentially recognized as "self" by recipients of the engineered
antibody. Minor modifications to the variable region frameworks can
be implemented to effect large increases in antigen binding without
appreciable increased immunogenicity for the recipient. Such
engineered antibodies may effectively treat a human for IL-18
mediated conditions. Such antibodies may also be useful in the
diagnosis of such conditions.
[0111] VII. Therapeutic/Prophylactic Uses
[0112] This invention also relates to a method of treating humans
experiencing autoimmune related symptoms, such as MS, which
comprises administering an effective dose of antibodies including
one or more of the engineered antibodies or altered antibodies
described herein, or fragments thereof.
[0113] The therapeutic response induced by the use of the molecules
of this invention is produced by the binding to human IL-18 and
thus subsequently blocking Th1 stimulation. Thus, the molecules of
the present invention, when in preparations and formulations
appropriate for therapeutic use, are highly desirable for those
persons experiencing autoimmune disease, such as but not limited to
MS, RA, IDDM, IBD and psoriasis.
[0114] The altered antibodies, antibodies and fragments thereof of
this invention may also be used in conjunction with other
antibodies, particularly human mAbs reactive with other markers
(epitopes) responsible for the condition against which the
engineered antibody of the invention is directed.
[0115] The therapeutic agents of this invention are believed to be
desirable for treatment of autoimmune conditions from about 2 days
to 6 months or as needed. For example, longer treatments may be
desirable when treating MS or the like. The dose and duration of
treatment relates to the relative duration of the molecules of the
present invention in the human circulation, and can be adjusted by
one of skill in the art depending upon the condition being treated
and the general health of the patient.
[0116] The mode of administration of the therapeutic agent of the
invention may be any suitable route which delivers the agent to the
host. The altered antibodies, antibodies, engineered antibodies,
and fragments thereof, and pharmaceutical compositions of the
invention are particularly useful for parenteral administration,
i.e. subcutaneously, intramuscularly, intravenously, or
intranasaly.
[0117] Therapeutic agents of the invention may be prepared as
pharmaceutical compositions containing an effective amount of the
engineered (e.g. humanized) antibody of the invention as an active
ingredient in a pharmaceutically acceptable carrier. In the
prophylactic agent of the invention. an aqueous suspension or
solution containing the engineered antibody, preferably buffered at
physiological pH, in a form ready for injection is preferred. The
compositions for parenteral administration will commonly comprise a
solution of the engineered antibody of the invention or a cocktail
thereof dissolved in an pharmaceutically acceptable carrier,
preferably an aqueous carrier. A variety of aqueous carriers may be
employed, e.g., 0.4% saline, 0.3% glycine, and the like. These
solutions are sterile and generally free of particulate matter.
These solutions may be sterilized by conventional, well known
sterilization techniques (e.g. filtration). The compositions may
contain pharmaceutically acceptable auxiliary substances as
required to approximate physiological conditions such as pH
adjusting and buffering agents, etc. The concentration of the
antibody of the invention in such pharmaceutical formulation can
vary widely, i.e., from less than about 0.5%, usually at or at
least about 1% to as much as 15 or 20% by weight and will be
selected primarily based on fluid volumes, viscosities, etc.,
according to the particular mode of administration selected.
[0118] Thus, a pharmaceutical composition of the invention for
intramuscular injection could be prepared to contain 1 mL sterile
buffered water, and between about 1 ng to about 100 mg, e.g. about
50 ng to about 30 mg or more preferably, about 5 mg to about 25 mg,
of an engineered antibody of the invention. Similarly, a
pharmaceutical composition of the invention for intravenous
infusion could be made up to contain about 250 ml of sterile
Ringer's solution, and about 1 to about 30 and preferably 5 mg to
about 25 mg of an engineered antibody of the invention. Actual
methods for preparing parenterally administrable compositions are
well known or will be apparent to those skilled in the art and are
described in more detail in, for example, Remington's
Pharmaceutical Science, 15th ed., Mack Publishing Company, Easton,
Pa.
[0119] It is preferred that the therapeutic agent of the invention,
when in a pharmaceutical preparation, be present in unit dose
forms. The appropriate therapeutically effective dose can be
determined readily by those of skill in the art. To effectively
treat an inflammatory disorder in a human or other animal, one dose
of approximately 0.1 mg to approximately 20 mg per 70 kg body
weight of a protein or an antibody of this invention should be
administered parenterally, preferably i.v. or i.m.
(intramuscularly). Such dose may, if necessary, be repeated at
appropriate time intervals selected as appropriate by a physician
during the disease.
[0120] The altered antibodies and engineered antibodies of this
invention may also be used in diagnostic regimens, such as for the
determination of IL-18 mediated disorders or tracking progress of
treatment of such disorders. As diagnostic reagents, these altered
antibodies may be conventionally labeled for use in ELISA's and
other conventional assay formats for the measurement of IL-18
levels in serum, plasma or other appropriate tissue, or the release
by human cells in culture. The nature of the assay in which the
altered antibodies are used are conventional and do not limit this
disclosure.
[0121] Thus, one embodiment of the present invention relates to a
method for aiding the diagnosis of autoimmune disease and other
conditions associated with excess Th1 T cell production in a
patient which comprises the steps of determining the amount of
human IL-18 in sample (plasma or tissue) obtained from said patient
and comparing said determined amount to the mean amount of human
IL-18 in the normal population, whereby the presence of a
significantly elevated amount of IL-18 in the patient's sample is
an indication autoimmune disease and other conditions associated
with excess Th1 T cell production.
[0122] The antibodies, altered antibodies or fragments thereof
described herein can be lyophilized for storage and reconstituted
in a suitable carrier prior to use. This technique has been shown
to be effective with conventional immunoglobulins and art-known
lyophilization and reconstitution techniques can be employed.
[0123] The following examples illustrate various aspects of this
invention including the construction of exemplary engineered
antibodies and expression thereof in suitable vectors and host
cells, and are not to be construed as limiting the scope of this
invention. All amino acids are identified by conventional three
letter or single letter codes. All necessary restriction enzymes,
plasmids, and other reagents and materials were obtained from
commercial sources unless otherwise indicated. All general cloning
legation and other recombinant DNA methodology were as performed in
T. Maniatis et al., cited above, or the second edition thereof
(1989), eds. Sambrook et al., by the same publisher ("Sambrook et
al.").
EXAMPLE 1
Production of MAbs to IL-18
[0124] A. Monoclonal Antibody Generation
[0125] Mice (F1 hybrids of Balb/c and C57BL/6) or rats (Sprague
Dawley) were immunised with 30 .mu.g recombinant IL-18 in adjuvant
and 4 weeks later with 30 .mu.g IL-18 in adjuvant. On the basis of
a good serum antibody titre to IL-18 animals received a further
immunization of 10-30 .mu.g IL-18 (i.p. in saline). Three days
following the final immunization a splenectomy was performed. Mouse
or rat spleen cells were used to prepare hybridomas by standard
procedures. (Zola, H.Ed., Monoclonal Antibodies. CRC Press Inc.
1987). Positive hybridomas were cloned by the limiting dilution
method.
[0126] B. Purification of Mabs
[0127] Mabs were purified by ProsepA (Bio Processing, Consett, UK)
chromatography respectively using the manufacturer's instructions.
Mabs were >95% pure by SDS-PAGE.
[0128] C. Isotyping of Mabs
[0129] All rat and mouse Mabs were isotyped by commercially
available kits (Zymed, Amersham) and found to be IgG1 kappa.
EXAMPLE 2
Assays
[0130] A. Biotinylation of Il-18
[0131] IL-18 was biotinylated using a kit purchased from Molecular
Probes Inc. using a 10:1 ratio of biotinylation reagent.
Biotinylation had no effect on the biological activity of IL-18
[0132] B. Hybridoma Screening Assay
[0133] 96-well plates were coated with streptavidin (2 ug/ml, 100
ul/well in PBS) by incubation overnight at 4.degree. C. The
solution was then aspirated and non-specific binding sites were
blocked with 250 .mu.l/well of 1% bovine serum albumin (BSA) in TBS
buffer (50 mM Tris, 150 mM NaCl, 0.02% Kathon, pH 7.4) for 5-60
minutes at RT. Following this and each of the following steps, the
plate was washed 4 times in wash buffer (10 mM Tris, 150 mM NaCl,
0.05% Tween 20, 0.02% Kathon, pH 7.4). To each well, 100 .mu.L
biotin IL-18 (100 ng/ml) in assay buffer (0.5% BSA, 0.05% bovine
gamma globulin, 0.01% Tween 40, 20 .mu.M diethylenetriaminepentaac-
etic in TBS buffer) was added and the plates were incubated for 30
min at RT in a shaker-incubator. To each well 50 .mu.l hybridoma
medium and 50 .mu.l assay buffer was then added and incubated for
60 min at RT in a shaker-incubator. To each well was then added 100
ul 0.5 .mu.g/ml Eu.sup.3+ labelled anti-mouse or anti-rat antibody
in assay buffer. Finally 200 .mu.l /well of enhancer (Wallac) was
added and incubated for 5 min at RT and the time-resolved
fluorescence measured. Hybridomas having counts >100K were
expanded into 24-well plates.
[0134] C. Immunoassay
[0135] To determine the specificity of the anti-IL-18 Mabs)
generated 96-well plates were coated, blocked and incubated with
biotin IL-18 as above. All the following incubations were performed
in a shaker-incubator at RT. After washing the wells 50 .mu.l IL-18
(3 .mu.g/ml) or assay buffer and 50 .mu.l Mab were added and
incubated for 60 min. After washing the wells 100 ul 0.5 .mu.g/ml
Eu.sup.3+ labelled anti-mouse or anti-rat antibody in assay buffer
was added for 60 min, the wells washed and then 100 .mu.l /well of
enhancer (Wallac) was added and incubated for 5 min at RT and the
time-resolved fluorescence measured. All positive hybridomas showed
displacement of binding with IL-18.
[0136] D. Neutralization Assay
[0137] PBMC from healthy donors were isolated by Ficol-Paque
(Pharmacia) gradient and cultured in 96 well plates in 10% FBS
DMEM/F12 media with 1 .mu.g/ml ConA (Sigma) in the presence of
IL-18 (5 ng/ml) and/or Mabs. After 18 h culture at 37.degree. C.,
5% CO.sub.2 in air, 90% humidity 25 .mu.l media was removed and
interferon gamma (IFNg) concentration measured by immunoassay. The
results, obtained from an average of three experiments, are
summarized in Table I.
[0138] E. Affinity Measurements of Monoclonal Antibody
[0139] The affinity of the purified mAbs was measured in the
BIAcore optical biosensor (Pharmacia Biosensor, Uppsala, Sweden)
using a flow rate of 30 ul/min. Kinetic data was evaluated using
relationships described previously (Karlsson et al, J. Immunol.
Meth., 145:229-240 (1991) and which is incorporated by reference in
its entirely. The mAb (diluted in HBS buffer, 10 mM HEPES, 150 mM
NaCl, 0.01% Tween-20. pH 7.4) was injected over a rabbit anti-mouse
IgG Fc or goat anti-rat IgG Fc surface, followed by buffer flow and
the RU was recorded. IL-18 (diluted in HBS buffer) was then
injected for 180 seconds followed by a buffer flow for 500 seconds
and the RU was recorded. The sensor chip surface was regenerated by
an injection of 0.1 M phosphoric acid. The on-rates (Kass) and
off-rates (Kdiss) of binding were calculated using BIAcore software
and together these yield a calculated equilibrium constant
(K.sub.D) of 12.times.10.sup.-9 M for mAb 13G9,
3.9.times.10.sup.-11 M for mAb 2C10 and 1.5.times.10.sup.-10 M for
mAb 14B7. See Table I.
[0140] F. Epitope Analysis of Monoclonal Antibody
[0141] The epitope analysis of the purified Mabs was measured in
the BIAcore. Using a flow rate of 10 .mu.l/min, the first Mab
(diluted in HBS buffer) was injected over a rabbit anti-mouse IgG
Fc or goat anti-rat IgG Fc surface, followed by an injection of
IL-18 for 240s, an injection of blocking Mabs for 48s and an
injection of the second Mab for 240s. The surface was regenerated
by an injection of 0.1M phosphoric acid and the RU was recorded
after each injection. It was found that Mabs 13G9, 2C10 and 14B7
have similar or overlapping epitopes.
1TABLE I Affinity and neutralizing activity of mAbs reactive with
human IL-18 Neutralisation mAb Kd (pM).sup.a IC50 (nM).sup.b 2C10
(rat) 39 0.1 14B7 (rat) 150 0.2 13G9 (mouse) 12000 3.0
.sup.aDetermined by optical biosensor (BIAcore) analysis
(25.degree. C.) .sup.bInhibition of IFN gamma production (in nM) of
PBMC in response to 5 ng/ml human IL-18
EXAMPLE 3
CDR Sequences
[0142] Gene Cloning and Sequence Analysis
[0143] The variable heavy and light genes were cloned from
hybridoma cells using standard molecular biological methods
described briefly as follows. Total RNA was isolated from the
hybridoma cells using TRIzol Reagent (Life Technologies Cat. #
15596-026) according to manufacturer's protocol. The RNA was
reverse transcribed with a RT-PCR kit per the manufacturer's
instructions (Boehringer Mannheim Cat. No. 1483-188) using a
poly-dT oligonucleotide for priming. Following first strand cDNA
synthesis, the heavy and light V regions were PCR amplified using
3' constant region specific primers and degenerate 5' primers. The
degenerate 5' primer sequences were designed to encode the
previously determined N terminal amino acid sequences of the
variable heavy or light chain regions. Full length sequences from
multiple clones were obtained from each PCR amplification and
aligned to provide consensus. Accordingly, the first 17 bases of
DNA sequence for both the heavy and light chains are PCR primer
generated, however the translated protein sequence is native.
[0144] The nucleotide and deduced amino acid sequences for
hybridoma antibodies 2C10, 13G9, and 14B7 are shown FIGS. 1-6. In
each case the CDR s and the nucleotide sequences encoding them are
boxed. The degenerate primer sequences are bolded.
Sequence CWU 1
1
48 1 324 DNA Rattus norvegicus CDS (1)...(324) Light Chain V Region
1 gac att caa atg acc cag tct cca gct tcc ctg tct gca tct ctg gga
48 Asp Ile Gln Met Thr Gln Ser Pro Ala Ser Leu Ser Ala Ser Leu Gly
1 5 10 15 gaa act gtc tcc atc gaa tgt ctg gca agt gag gac ata tac
act tat 96 Glu Thr Val Ser Ile Glu Cys Leu Ala Ser Glu Asp Ile Tyr
Thr Tyr 20 25 30 tta aca tgg tat cag cag aaa cca ggg aaa tct cct
caa ctc ctg atc 144 Leu Thr Trp Tyr Gln Gln Lys Pro Gly Lys Ser Pro
Gln Leu Leu Ile 35 40 45 tat ggt gca aat aag ttg caa gat ggg gtc
cca tca cgg ttc agt ggc 192 Tyr Gly Ala Asn Lys Leu Gln Asp Gly Val
Pro Ser Arg Phe Ser Gly 50 55 60 agt gga tct ggc aca cag tat tct
ctc aag atc agc ggc ata caa cct 240 Ser Gly Ser Gly Thr Gln Tyr Ser
Leu Lys Ile Ser Gly Ile Gln Pro 65 70 75 80 gaa gat gaa ggg gat tat
ttc tgt cta cag ggt tcc aag ttt ccg ctc 288 Glu Asp Glu Gly Asp Tyr
Phe Cys Leu Gln Gly Ser Lys Phe Pro Leu 85 90 95 acg ttc ggt tct
ggg acc aag ctg gag atc aaa cgg 324 Thr Phe Gly Ser Gly Thr Lys Leu
Glu Ile Lys Arg 100 105 2 108 PRT Rattus norvegicus 2 Asp Ile Gln
Met Thr Gln Ser Pro Ala Ser Leu Ser Ala Ser Leu Gly 1 5 10 15 Glu
Thr Val Ser Ile Glu Cys Leu Ala Ser Glu Asp Ile Tyr Thr Tyr 20 25
30 Leu Thr Trp Tyr Gln Gln Lys Pro Gly Lys Ser Pro Gln Leu Leu Ile
35 40 45 Tyr Gly Ala Asn Lys Leu Gln Asp Gly Val Pro Ser Arg Phe
Ser Gly 50 55 60 Ser Gly Ser Gly Thr Gln Tyr Ser Leu Lys Ile Ser
Gly Ile Gln Pro 65 70 75 80 Glu Asp Glu Gly Asp Tyr Phe Cys Leu Gln
Gly Ser Lys Phe Pro Leu 85 90 95 Thr Phe Gly Ser Gly Thr Lys Leu
Glu Ile Lys Arg 100 105 3 33 DNA Rattus norvegicus CDS (1)...(33)
VK2C10 Light Chain CDR I 3 ctg gca agt gag gac ata tac act tat tta
aca 33 Leu Ala Ser Glu Asp Ile Tyr Thr Tyr Leu Thr 1 5 10 4 11 PRT
Rattus norvegicus 4 Leu Ala Ser Glu Asp Ile Tyr Thr Tyr Leu Thr 1 5
10 5 21 DNA Rattus norvegicus CDS (1)...(21) VK2C10 Light Chain CDR
II 5 ggt gca aat aag ttg caa gat 21 Gly Ala Asn Lys Leu Gln Asp 1 5
6 7 PRT Rattus norvegicus 6 Gly Ala Asn Lys Leu Gln Asp 1 5 7 27
DNA Rattus norvegicus CDS (1)...(27) VK2C10 Light Chain CDR III 7
cta cag ggt tcc aag ttt ccg ctc acg 27 Leu Gln Gly Ser Lys Phe Pro
Leu Thr 1 5 8 9 PRT Rattus norvegicus 8 Leu Gln Gly Ser Lys Phe Pro
Leu Thr 1 5 9 378 DNA Rattus norvegicus CDS (1)...(378) Heavy Chain
V Region 9 gag gtc cag cta cag cag tct ggg gct gag ctt gtg aga cct
ggg acc 48 Glu Val Gln Leu Gln Gln Ser Gly Ala Glu Leu Val Arg Pro
Gly Thr 1 5 10 15 tct gtg aag tta tct tgc aaa gtt tct ggc gaa ata
agt aca gga tac 96 Ser Val Lys Leu Ser Cys Lys Val Ser Gly Glu Ile
Ser Thr Gly Tyr 20 25 30 tat ttc cac ttt gtg agg cga agg cct gga
cag ggt ctg gaa tgg ata 144 Tyr Phe His Phe Val Arg Arg Arg Pro Gly
Gln Gly Leu Glu Trp Ile 35 40 45 gga agg att gat cct gag gat gat
agt act aaa tat gct gag agg ttc 192 Gly Arg Ile Asp Pro Glu Asp Asp
Ser Thr Lys Tyr Ala Glu Arg Phe 50 55 60 aaa gac agg gcg acg ctc
act gca caa aca tcc tcc aac aca gcc tac 240 Lys Asp Arg Ala Thr Leu
Thr Ala Gln Thr Ser Ser Asn Thr Ala Tyr 65 70 75 80 ctg aac ctc agc
agc ctg acc tct gag gac act gca act tat ttt tgt 288 Leu Asn Leu Ser
Ser Leu Thr Ser Glu Asp Thr Ala Thr Tyr Phe Cys 85 90 95 acc aca
tgg cgg ata tac cga gat agt tct ggc cgc ccc ttc tat gtt 336 Thr Thr
Trp Arg Ile Tyr Arg Asp Ser Ser Gly Arg Pro Phe Tyr Val 100 105 110
atg gat gcc tgg ggt caa gga gct tca gtc act gtc tcc tca 378 Met Asp
Ala Trp Gly Gln Gly Ala Ser Val Thr Val Ser Ser 115 120 125 10 126
PRT Rattus norvegicus 10 Glu Val Gln Leu Gln Gln Ser Gly Ala Glu
Leu Val Arg Pro Gly Thr 1 5 10 15 Ser Val Lys Leu Ser Cys Lys Val
Ser Gly Glu Ile Ser Thr Gly Tyr 20 25 30 Tyr Phe His Phe Val Arg
Arg Arg Pro Gly Gln Gly Leu Glu Trp Ile 35 40 45 Gly Arg Ile Asp
Pro Glu Asp Asp Ser Thr Lys Tyr Ala Glu Arg Phe 50 55 60 Lys Asp
Arg Ala Thr Leu Thr Ala Gln Thr Ser Ser Asn Thr Ala Tyr 65 70 75 80
Leu Asn Leu Ser Ser Leu Thr Ser Glu Asp Thr Ala Thr Tyr Phe Cys 85
90 95 Thr Thr Trp Arg Ile Tyr Arg Asp Ser Ser Gly Arg Pro Phe Tyr
Val 100 105 110 Met Asp Ala Trp Gly Gln Gly Ala Ser Val Thr Val Ser
Ser 115 120 125 11 15 DNA Rattus norvegicus CDS (1)...(15) VH2C10
Heavy Chain CDR I 11 gga tac tat ttc cac 15 Gly Tyr Tyr Phe His 1 5
12 5 PRT Rattus norvegicus 12 Gly Tyr Tyr Phe His 1 5 13 51 DNA
Rattus norvegicus CDS (1)...(51) VH2C10 Heavy Chain CDR II 13 agg
att gat cct gag gat gat agt act aaa tat gct gag agg ttc aaa 48 Arg
Ile Asp Pro Glu Asp Asp Ser Thr Lys Tyr Ala Glu Arg Phe Lys 1 5 10
15 gac 51 Asp 14 17 PRT Rattus norvegicus 14 Arg Ile Asp Pro Glu
Asp Asp Ser Thr Lys Tyr Ala Glu Arg Phe Lys 1 5 10 15 Asp 15 51 DNA
Rattus norvegicus CDS (1)...(51) VH2C10 Heavy Chain CDR III 15 tgg
cgg ata tac cga gat agt tct ggc cgc ccc ttc tat gtt atg gat 48 Trp
Arg Ile Tyr Arg Asp Ser Ser Gly Arg Pro Phe Tyr Val Met Asp 1 5 10
15 gcc 51 Ala 16 17 PRT Rattus norvegicus 16 Trp Arg Ile Tyr Arg
Asp Ser Ser Gly Arg Pro Phe Tyr Val Met Asp 1 5 10 15 Ala 17 342
DNA Mus musculus CDS (1)...(342) Light Chain V region 17 gac gtt
gtt atg act caa act cct ctc tcc ctg cct gtc agt ctt gga 48 Asp Val
Val Met Thr Gln Thr Pro Leu Ser Leu Pro Val Ser Leu Gly 1 5 10 15
gat caa gcc tcc atc tct tgc aga tct agt cag agc ctt gta cac agt 96
Asp Gln Ala Ser Ile Ser Cys Arg Ser Ser Gln Ser Leu Val His Ser 20
25 30 aat gga aac acc tat tta cat tgg tac ctg cag aag cca ggc cag
tct 144 Asn Gly Asn Thr Tyr Leu His Trp Tyr Leu Gln Lys Pro Gly Gln
Ser 35 40 45 cca aag ctc ctg atc tac aaa gtt tcc aac cga ttt tct
ggg gtc cca 192 Pro Lys Leu Leu Ile Tyr Lys Val Ser Asn Arg Phe Ser
Gly Val Pro 50 55 60 gac agg ttc agt ggc agt gga tca ggt aca gat
ttc aca ctc aag atc 240 Asp Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp
Phe Thr Leu Lys Ile 65 70 75 80 agc aga gtg gag gct gag gat ctg gga
gtt tat ttc tgc tct caa agt 288 Ser Arg Val Glu Ala Glu Asp Leu Gly
Val Tyr Phe Cys Ser Gln Ser 85 90 95 aca cat gtt cct ccg tac acg
ttc gga ggg ggg acc aag ctg gaa ata 336 Thr His Val Pro Pro Tyr Thr
Phe Gly Gly Gly Thr Lys Leu Glu Ile 100 105 110 aaa cgg 342 Lys Arg
18 114 PRT Mus musculus 18 Asp Val Val Met Thr Gln Thr Pro Leu Ser
Leu Pro Val Ser Leu Gly 1 5 10 15 Asp Gln Ala Ser Ile Ser Cys Arg
Ser Ser Gln Ser Leu Val His Ser 20 25 30 Asn Gly Asn Thr Tyr Leu
His Trp Tyr Leu Gln Lys Pro Gly Gln Ser 35 40 45 Pro Lys Leu Leu
Ile Tyr Lys Val Ser Asn Arg Phe Ser Gly Val Pro 50 55 60 Asp Arg
Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Lys Ile 65 70 75 80
Ser Arg Val Glu Ala Glu Asp Leu Gly Val Tyr Phe Cys Ser Gln Ser 85
90 95 Thr His Val Pro Pro Tyr Thr Phe Gly Gly Gly Thr Lys Leu Glu
Ile 100 105 110 Lys Arg 19 48 DNA Mus musculus CDS (1)...(48)
VK13G9 Light Chain CDR I 19 aga tct agt cag agc ctt gta cac agt aat
gga aac acc tat tta cat 48 Arg Ser Ser Gln Ser Leu Val His Ser Asn
Gly Asn Thr Tyr Leu His 1 5 10 15 20 16 PRT Mus musculus 20 Arg Ser
Ser Gln Ser Leu Val His Ser Asn Gly Asn Thr Tyr Leu His 1 5 10 15
21 21 DNA Mus musculus CDS (1)...(21) VK13G9 Light Chain CDR II 21
aaa gtt tcc aac cga ttt tct 21 Lys Val Ser Asn Arg Phe Ser 1 5 22 7
PRT Mus musculus 22 Lys Val Ser Asn Arg Phe Ser 1 5 23 30 DNA Mus
musculus CDS (1)...(30) VK13G9 Light Chain CDR III 23 tct caa agt
aca cat gtt cct ccg tac acg 30 Ser Gln Ser Thr His Val Pro Pro Tyr
Thr 1 5 10 24 10 PRT Mus musculus 24 Ser Gln Ser Thr His Val Pro
Pro Tyr Thr 1 5 10 25 369 DNA Mus musculus CDS (1)...(369) Heavy
Chain V Region 25 caa gtt act ctt aag gag tct ggc cct ggg ata ttg
aag ccc tca cag 48 Gln Val Thr Leu Lys Glu Ser Gly Pro Gly Ile Leu
Lys Pro Ser Gln 1 5 10 15 acc ctc agt ctg act tgt tct ttc tct ggg
ttt tct ctg agc act tct 96 Thr Leu Ser Leu Thr Cys Ser Phe Ser Gly
Phe Ser Leu Ser Thr Ser 20 25 30 ggt atg ggt att gcc tgg gtt cgt
cag cct tca ggg aag ggt ctg gag 144 Gly Met Gly Ile Ala Trp Val Arg
Gln Pro Ser Gly Lys Gly Leu Glu 35 40 45 tgg ctg gca gac att tgg
tgg gat gat aat aag tat tat aat cca tcc 192 Trp Leu Ala Asp Ile Trp
Trp Asp Asp Asn Lys Tyr Tyr Asn Pro Ser 50 55 60 ctg gag agc cag
ctc aca atc tcc aag gat acc tcc aga aac cag gta 240 Leu Glu Ser Gln
Leu Thr Ile Ser Lys Asp Thr Ser Arg Asn Gln Val 65 70 75 80 ttc ctc
acg atc acc agt gtg gac act gca gat tct gcc act tat tac 288 Phe Leu
Thr Ile Thr Ser Val Asp Thr Ala Asp Ser Ala Thr Tyr Tyr 85 90 95
tgt gct cgt cat cat tac gac ggt agt agc ctc ctg cct atg gac tac 336
Cys Ala Arg His His Tyr Asp Gly Ser Ser Leu Leu Pro Met Asp Tyr 100
105 110 tgg ggt caa gga acc tca gtc acc gtc tcc tca 369 Trp Gly Gln
Gly Thr Ser Val Thr Val Ser Ser 115 120 26 123 PRT Mus musculus 26
Gln Val Thr Leu Lys Glu Ser Gly Pro Gly Ile Leu Lys Pro Ser Gln 1 5
10 15 Thr Leu Ser Leu Thr Cys Ser Phe Ser Gly Phe Ser Leu Ser Thr
Ser 20 25 30 Gly Met Gly Ile Ala Trp Val Arg Gln Pro Ser Gly Lys
Gly Leu Glu 35 40 45 Trp Leu Ala Asp Ile Trp Trp Asp Asp Asn Lys
Tyr Tyr Asn Pro Ser 50 55 60 Leu Glu Ser Gln Leu Thr Ile Ser Lys
Asp Thr Ser Arg Asn Gln Val 65 70 75 80 Phe Leu Thr Ile Thr Ser Val
Asp Thr Ala Asp Ser Ala Thr Tyr Tyr 85 90 95 Cys Ala Arg His His
Tyr Asp Gly Ser Ser Leu Leu Pro Met Asp Tyr 100 105 110 Trp Gly Gln
Gly Thr Ser Val Thr Val Ser Ser 115 120 27 21 DNA Mus musculus CDS
(1)...(21) VH13G9 Heavy Chain CDR I 27 act tct ggt atg ggt att gcc
21 Thr Ser Gly Met Gly Ile Ala 1 5 28 7 PRT Mus musculus 28 Thr Ser
Gly Met Gly Ile Ala 1 5 29 48 DNA Mus musculus CDS (1)...(48)
VH13G9 Heavy Chain CDR II 29 gac att tgg tgg gat gat aat aag tat
tat aat cca tcc ctg gag agc 48 Asp Ile Trp Trp Asp Asp Asn Lys Tyr
Tyr Asn Pro Ser Leu Glu Ser 1 5 10 15 30 16 PRT Mus musculus 30 Asp
Ile Trp Trp Asp Asp Asn Lys Tyr Tyr Asn Pro Ser Leu Glu Ser 1 5 10
15 31 39 DNA Mus musculus CDS (1)...(39) VH13G9 Heavy Chain CDR III
31 cat cat tac gac ggt agt agc ctc ctg cct atg gac tac 39 His His
Tyr Asp Gly Ser Ser Leu Leu Pro Met Asp Tyr 1 5 10 32 13 PRT Mus
musculus 32 His His Tyr Asp Gly Ser Ser Leu Leu Pro Met Asp Tyr 1 5
10 33 324 DNA Rattus norvegicus CDS (1)...(324) Light Chain V
Region 33 gat att caa atg acg cag tct cca gct tcc ctg tct gca tct
ctg gga 48 Asp Ile Gln Met Thr Gln Ser Pro Ala Ser Leu Ser Ala Ser
Leu Gly 1 5 10 15 gaa act gtc tcc atc gaa tgt cta gca agt gag gac
ata tac agt tat 96 Glu Thr Val Ser Ile Glu Cys Leu Ala Ser Glu Asp
Ile Tyr Ser Tyr 20 25 30 tta gca tgg tat caa cag aag cca ggg aaa
tct cct cag ctc ctg atc 144 Leu Ala Trp Tyr Gln Gln Lys Pro Gly Lys
Ser Pro Gln Leu Leu Ile 35 40 45 tat gcc aca aaa agg ttg caa gat
ggg gtc cca tca cgg ttc agt ggc 192 Tyr Ala Thr Lys Arg Leu Gln Asp
Gly Val Pro Ser Arg Phe Ser Gly 50 55 60 agt gga tct ggc aca cag
tat tct ctc aaa ata agc gac atg caa cct 240 Ser Gly Ser Gly Thr Gln
Tyr Ser Leu Lys Ile Ser Asp Met Gln Pro 65 70 75 80 gaa gat gaa ggg
gat tat ttc tgt cta cag aat tcc aag ttt ccg gtc 288 Glu Asp Glu Gly
Asp Tyr Phe Cys Leu Gln Asn Ser Lys Phe Pro Val 85 90 95 acg ttc
ggt tct ggg acc aag ctg gag atc aaa cgg 324 Thr Phe Gly Ser Gly Thr
Lys Leu Glu Ile Lys Arg 100 105 34 108 PRT Rattus norvegicus 34 Asp
Ile Gln Met Thr Gln Ser Pro Ala Ser Leu Ser Ala Ser Leu Gly 1 5 10
15 Glu Thr Val Ser Ile Glu Cys Leu Ala Ser Glu Asp Ile Tyr Ser Tyr
20 25 30 Leu Ala Trp Tyr Gln Gln Lys Pro Gly Lys Ser Pro Gln Leu
Leu Ile 35 40 45 Tyr Ala Thr Lys Arg Leu Gln Asp Gly Val Pro Ser
Arg Phe Ser Gly 50 55 60 Ser Gly Ser Gly Thr Gln Tyr Ser Leu Lys
Ile Ser Asp Met Gln Pro 65 70 75 80 Glu Asp Glu Gly Asp Tyr Phe Cys
Leu Gln Asn Ser Lys Phe Pro Val 85 90 95 Thr Phe Gly Ser Gly Thr
Lys Leu Glu Ile Lys Arg 100 105 35 33 DNA Rattus norvegicus CDS
(1)...(33) VK14B7 Light Chain CDR I 35 cta gca agt gag gac ata tac
agt tat tta gca 33 Leu Ala Ser Glu Asp Ile Tyr Ser Tyr Leu Ala 1 5
10 36 11 PRT Rattus norvegicus 36 Leu Ala Ser Glu Asp Ile Tyr Ser
Tyr Leu Ala 1 5 10 37 21 DNA Rattus norvegicus CDS (1)...(21)
VK14B7 Light Chain CDR II 37 gcc aca aaa agg ttg caa gat 21 Ala Thr
Lys Arg Leu Gln Asp 1 5 38 7 PRT Rattus norvegicus 38 Ala Thr Lys
Arg Leu Gln Asp 1 5 39 27 DNA Rattus norvegicus CDS (1)...(27)
VK14B7 Light Chain CDR III 39 cta cag aat tcc aag ttt ccg gtc acg
27 Leu Gln Asn Ser Lys Phe Pro Val Thr 1 5 40 9 PRT Rattus
norvegicus 40 Leu Gln Asn Ser Lys Phe Pro Val Thr 1 5 41 368 DNA
Rattus norvegicus CDS (1)...(368) Heavy Chain V Region 41 gag gtt
cag ctt cag cag tct ggg gct gag ctt gtg aga cct ggg acc 48 Glu Val
Gln Leu Gln Gln Ser Gly Ala Glu Leu Val Arg Pro Gly Thr 1 5 10 15
tct gtg aag ttt tct tgc aaa gtt tct ggc gat acc cct aca aca tac 96
Ser Val Lys Phe Ser Cys Lys Val Ser Gly Asp Thr Pro Thr Thr Tyr 20
25 30 tac gtg cac ttt gtg aga caa agg cct gga cag ggt ctg gaa tgg
ata 144 Tyr Val His Phe Val Arg Gln Arg Pro Gly Gln Gly Leu Glu Trp
Ile 35 40 45 gga agg att gat cct gag gat act agt act aaa tat gct
gag aag ttc 192 Gly Arg Ile Asp Pro Glu Asp Thr Ser Thr Lys Tyr Ala
Glu Lys Phe 50 55 60 aga aat aag gcg aca ttc act gca gat cca tcc
tcc aac aca gcc tac 240 Arg Asn Lys Ala Thr Phe Thr Ala Asp Pro Ser
Ser Asn Thr Ala Tyr 65 70 75 80 cta aac ctc agc agc ctg acc cct gag
gac act gca acc tat ttt tgt 288 Leu Asn Leu Ser Ser Leu Thr Pro Glu
Asp Thr Ala Thr Tyr Phe Cys 85 90 95 acc ata atg cgg tac cat agt
acc tat agg gtc tat gtt atg gat ttc 336 Thr Ile Met Arg
Tyr His Ser Thr Tyr Arg Val Tyr Val Met Asp Phe 100 105 110 tgg ggt
caa gga act gca gtc act gtc tcc tc 368 Trp Gly Gln Gly Thr Ala Val
Thr Val Ser 115 120 42 122 PRT Rattus norvegicus 42 Glu Val Gln Leu
Gln Gln Ser Gly Ala Glu Leu Val Arg Pro Gly Thr 1 5 10 15 Ser Val
Lys Phe Ser Cys Lys Val Ser Gly Asp Thr Pro Thr Thr Tyr 20 25 30
Tyr Val His Phe Val Arg Gln Arg Pro Gly Gln Gly Leu Glu Trp Ile 35
40 45 Gly Arg Ile Asp Pro Glu Asp Thr Ser Thr Lys Tyr Ala Glu Lys
Phe 50 55 60 Arg Asn Lys Ala Thr Phe Thr Ala Asp Pro Ser Ser Asn
Thr Ala Tyr 65 70 75 80 Leu Asn Leu Ser Ser Leu Thr Pro Glu Asp Thr
Ala Thr Tyr Phe Cys 85 90 95 Thr Ile Met Arg Tyr His Ser Thr Tyr
Arg Val Tyr Val Met Asp Phe 100 105 110 Trp Gly Gln Gly Thr Ala Val
Thr Val Ser 115 120 43 15 DNA Rattus norvegicus CDS (1)...(15)
VH14B7 Heavy Chain CDR I 43 aca tac tac gtg cac 15 Thr Tyr Tyr Val
His 1 5 44 5 PRT Rattus norvegicus 44 Thr Tyr Tyr Val His 1 5 45 51
DNA Rattus norvegicus CDS (1)...(51) VH14B7 Heavy Chain CDR II 45
agg att gat cct gag gat act agt act aaa tat gct gag aag ttc aga 48
Arg Ile Asp Pro Glu Asp Thr Ser Thr Lys Tyr Ala Glu Lys Phe Arg 1 5
10 15 aat 51 Asn 46 17 PRT Rattus norvegicus 46 Arg Ile Asp Pro Glu
Asp Thr Ser Thr Lys Tyr Ala Glu Lys Phe Arg 1 5 10 15 Asn 47 42 DNA
Rattus norvegicus CDS (1)...(42) VH14B7 Heavy Chain CDR III 47 atg
cgg tac cat agt acc tat agg gtc tat gtt atg gat ttc 42 Met Arg Tyr
His Ser Thr Tyr Arg Val Tyr Val Met Asp Phe 1 5 10 48 14 PRT Rattus
norvegicus 48 Met Arg Tyr His Ser Thr Tyr Arg Val Tyr Val Met Asp
Phe 1 5 10
* * * * *