U.S. patent application number 14/639985 was filed with the patent office on 2015-06-25 for dual specificity antibodies and methods of making and using.
This patent application is currently assigned to ABBVIE INC.. The applicant listed for this patent is AbbVie Inc.. Invention is credited to George Avgerinos, Albert COLLINSON, Richard DIXON, Tariq GHAYUR, Zehra KAYMAKCALAN.
Application Number | 20150175694 14/639985 |
Document ID | / |
Family ID | 22802758 |
Filed Date | 2015-06-25 |
United States Patent
Application |
20150175694 |
Kind Code |
A1 |
COLLINSON; Albert ; et
al. |
June 25, 2015 |
DUAL SPECIFICITY ANTIBODIES AND METHODS OF MAKING AND USING
Abstract
Antibodies having dual specificity for two different but
structurally related antigens are provided. The antibodies can be,
for example, entirely human antibodies, recombinant antibodies, or
monoclonal antibodies. Preferred antibodies have dual specificity
for IL-1.alpha. and IL-1.beta. and neutralize IL-1.alpha. and
IL-1.beta. activity in vitro and in vivo. An antibody of the
invention can be a full-length antibody or an antigen-binding
portion thereof. Methods of making and methods of using the
antibodies of the invention are also provided. The antibodies, or
antibody portions, of the invention are useful for detecting two
different but structurally related antigens (e.g., IL-1.alpha. and
IL-1.beta.) and for inhibiting the activity of the antigens, e.g.,
in a human subject suffering from a disorder in which IL-1.alpha.
and/or IL-1.beta. activity is detrimental.
Inventors: |
COLLINSON; Albert;
(Marlborough, MA) ; Avgerinos; George; (Sudbury,
MA) ; DIXON; Richard; (North Grafton, MA) ;
GHAYUR; Tariq; (Holliston, MA) ; KAYMAKCALAN;
Zehra; (Westborough, MA) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
AbbVie Inc. |
North Chicago |
IL |
US |
|
|
Assignee: |
ABBVIE INC.
North Chicago
IL
|
Family ID: |
22802758 |
Appl. No.: |
14/639985 |
Filed: |
March 5, 2015 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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14469122 |
Aug 26, 2014 |
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14639985 |
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13900841 |
May 23, 2013 |
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14469122 |
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12316340 |
Dec 11, 2008 |
8475766 |
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13900841 |
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09894550 |
Jun 28, 2001 |
7491516 |
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12316340 |
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60215379 |
Jun 29, 2000 |
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Current U.S.
Class: |
530/387.3 ;
506/18; 506/26; 506/9 |
Current CPC
Class: |
C07K 16/46 20130101;
A61P 29/00 20180101; A61P 37/06 20180101; C07K 14/545 20130101;
C07K 2317/56 20130101; A61P 17/06 20180101; A61P 1/04 20180101;
C07K 2317/34 20130101; C07K 2317/33 20130101; C07K 2317/76
20130101; A61K 2039/505 20130101; A61K 47/6845 20170801; G01N
33/6869 20130101; C07K 16/245 20130101; C07K 16/468 20130101; C07K
2317/10 20130101; A61P 25/00 20180101; C07K 2317/31 20130101 |
International
Class: |
C07K 16/24 20060101
C07K016/24 |
Claims
1-69. (canceled)
70. A method of making an antibody or an antigen binding portion
thereof library comprising the steps of: a) obtaining a recombinant
heavy chain or an antigen binding portion thereof library A from an
antibody repertoire resulting from exposure to a first antigen; b)
obtaining a recombinant light chain or an antigen binding portion
thereof library B from an antibody repertoire resulting from
exposure to the first antigen; c) obtaining a recombinant heavy
chain or an antigen binding portion thereof library C from an
antibody repertoire resulting from exposure to a second antigen; d)
obtaining a recombinant light chain or an antigen binding portion
thereof library D from an antibody repertoire resulting from
exposure to the second antigen; and e) combining the recombinant
heavy chain or an antigen binding portion thereof library A with
the recombinant light chain or an antigen binding portion thereof
library D to obtain an antibody or an antigen binding portion
thereof library X and/or combining the recombinant heavy chain or
an antigen binding portion thereof library C with the recombinant
light chain or an antigen binding portion thereof library to obtain
an antibody or an antigen binding portion thereof library Y.
71. A method of making an antibody or an antigen binding portion
thereof library according to claim 70 further comprising the step
of combining the antibody or an antigen binding portion thereof
library X with the antibody or an antigen binding portion thereof
library Y to obtain an antibody or an antigen binding portion
thereof library Z.
72. The antibody or an antigen binding portion thereof library X
made according to the method of claim 70.
73. The antibody or an antigen binding portion thereof library Y
made according to the method of claim 70.
74. The antibody or an antigen binding portion thereof library Z
made according to the method of claim 71.
75. A method of making a dual specific antibody or an antigen
binding portion thereof comprising the steps of: a) obtaining a
recombinant heavy chain or an antigen binding portion thereof
library A from an antibody repertoire resulting from exposure to a
first antigen; b) obtaining a recombinant light chain or an antigen
binding portion thereof library B from an antibody repertoire
resulting from exposure to the first antigen; c) obtaining a
recombinant heavy chain or an antigen binding portion thereof
library C from a antibody repertoire resulting from exposure to a
second antigen; d) obtaining a recombinant light chain or an
antigen binding portion thereof library D from an antibody
repertoire resulting from exposure to the second antigen; e)
combining the recombinant heavy chain or an antigen binding portion
thereof library A with the recombinant light chain or an antigen
binding portion thereof library D to obtain an antibody or an
antigen binding portion thereof library X and/or combining the
recombinant heavy chain or an antigen binding portion thereof
library C with the recombinant light chain or an antigen binding
portion thereof library B to obtain an antibody or an antigen
binding portion thereof library Y; and f) selecting from the
antibody or an antigen binding portion thereof library X and/or
antibody or an antigen binding portion thereof library Y an
antibody or an antigen binding portion thereof that binds both the
first and the second antigen.
76. The dual specific antibody made by the method of claim 75.
77. A method of making a dual specific antibody or an antigen
binding portion thereof comprising the steps of: a) obtaining a
recombinant heavy chain or an antigen binding portion thereof
library A from an antibody repertoire resulting from exposure to a
first antigen; b) obtaining a recombinant light chain or an antigen
binding portion thereof library B from an antibody repertoire
resulting from exposure to the first antigen; c) obtaining a
recombinant heavy chain or an antigen binding portion thereof
library C from an antibody repertoire resulting from exposure to a
second antigen; d) obtaining a recombinant light chain or an
antigen binding portion thereof library D from an antibody
repertoire resulting from exposure to the second antigen; e)
combining the recombinant heavy chain or an antigen binding portion
thereof library A with the recombinant light chain or an antigen
binding portion thereof library D to obtain an antibody library X
and/or combining the recombinant heavy chain or an antigen binding
portion thereof library C with the recombinant light chain or an
antigen binding portion thereof library B to obtain an antibody or
an antigen binding portion thereof library Y; f) combining the
antibody or an antigen binding portion thereof library X with the
antibody library or an antigen binding portion thereof Y to obtain
an antibody or an antigen binding portion thereof library Z; and g)
selecting from the antibody or an antigen binding portion thereof
library Zan antibody or an antigen binding portion thereof that
binds both the first and the second antigen.
78. The dual specific antibody or an antigen binding portion
thereof made by the method according to claim 77.
79. The method according to claim 70 wherein the first and second
antigen is each independently selected from the group consisting of
proteins, polypeptides and peptides provided that the first and
second antigens are not the same.
80. The method of claim 79, wherein the proteins, polypeptides and
peptides are secreted proteins or surface receptors.
81. The method of claim 80, wherein the secreted protein is
selected from the group consisting of an MN, a TNF, an interleukin,
PF4, a CRO, 9E3, EMAP-II, a CSF, an FGF, and a PDGF.
82. The method according to claim 81, therein the first antigen is
IL-1.alpha. and the second antigen is IL-1.beta..
83-88. (canceled)
89. A method of making a dual specific antibody or an antigen
binding portion thereof, the method comprising: a) obtaining a
first recombinant heavy chain or an antigen binding portion thereof
library by exposure of an antibody repertoire to a first antigen;
b) obtaining a second recombinant light chain or an antigen binding
portion thereof library by exposure of an antibody repertoire to a
first antigen; c) obtaining a third recombinant heavy chain or an
antigen binding portion thereof library by exposure of an antibody
repertoire to a second antigen; d) obtaining a forth recombinant
light chain or an antigen binding portion thereof library by
exposure of an antibody repertoire to a second antigen; e)
combining the first library with the forth library to obtain a
fifth antibody or an antigen binding portion thereof library and/or
combining the second library with the third library to obtain a
sixth antibody or an antigen binding portion thereof library; and
identifying from the fifth or sixth library an antibody or an
antigen binding portion thereof that specifically binds to the
first and second antigens.
90. The method of claim 89, wherein the first, second, third or
fourth libraries comprise human antibody chains or antigen binding
portions thereof.
91. The method of claim 89, wherein the first, second, third and
fourth libraries comprise human antibody chains or antigen binding
portions thereof.
92. The method of claim 89, wherein the first and second antigens
are structurally related antigens.
93. The method of claim 89, wherein the first and second antigens
are each independently selected from the group consisting of
caspases, cytokines, secreted proteinases, and cytokine receptor
families, TNF receptor family members, TGF.beta. receptor family
members, EGF receptor family members, FGF receptor family members,
PDGF receptor family members, VEGF receptor family members and
angiopoietin receptor family members.
94. The method of claim 93, wherein the cytokines are selected from
the group consisting of TNF family members IL-6 family members,
interferons, TGF.beta. family members, EGF family members, FGF
family members, PDGF family members, VEGF family members,
angiopoietins, bone morphogenetic proteins.
95. The method of claim 89, wherein the first and second antigens
are each independently selected from the group consisting of IP-10,
PF4, a GRO, 9E3, EMAP-II, a CSF, an FGF, and a PDGF.
96. The method of claim 89, wherein the first antigen and the
second antigen are IL-1.alpha. and IL-1.beta.; IL-1.alpha. and
IL-18; IL-1.beta. and IL-18; EGFR and IGFR; HER2 and HER3; and EGFR
and HER3, respectively.
Description
CROSS-REFERENCE TO RELATED APPLICATION
[0001] This application is a continuation of U.S. application Ser.
No. 09/894,550, filed Jun. 28, 2001. U.S. application Ser. No.
09/894,550 claims benefit of U.S. Provisional Application No.
60/215,379, filed Jun. 29, 2000.
BACKGROUND OF THE INVENTION
[0002] The mammalian immune system includes B lymphocytes that, in
totality, express an antibody repertoire composed of hundreds of
billions of different antibody specificities. A normal immune
response to a particular antigen involves the selection from this
repertoire of one or more antibodies that specifically bind the
antigen, and the success of an immune response is based, at least
in part, on the ability of these antibodies to specifically
recognize (and ultimately eliminate) the stimulating antigen and
"ignore" other molecules in the environment of the antibodies.
[0003] The usefulness of antibodies that specifically recognize one
particular target antigen led to the development of monoclonal
antibody technology. Standard hybridoma technology now allows for
the preparation of antibodies having a single specificity for an
antigen of interest. More recently, recombinant antibody
techniques, such as screening of in vitro antibody libraries, have
been developed. These techniques also allow for production of
antibodies with a single specificity for an antigen of
interest.
[0004] Antibodies having specificity for a single target antigen
may, at least under certain circumstances, display undesired
cross-reactivity or background binding to other antigens. This
cross-reactivity or background binding, however, is usually
unpredictable (i.e., it is not possible to predict which antigen
the antibody will cross-react with). Moreover, it is usually
distinguishable from specific antigen binding, since it typically
represents only a very minor portion of the binding ability of the
antibody (e.g., 1% or less of total antibody binding) and typically
is only observed at high antibody concentrations (e.g., 1000 fold,
or higher, concentrations than needed to observe specific antigen
binding). Although there are several antigens that may belong to a
structurally related family of proteins, the antibody response to a
particular family member is highly specific. In addition, there are
several examples of protein family members (e.g., members of the
IL-1 and TNF families) that bind to the same receptor, receptor
component or structurally related receptors, yet monoclonal
antibodies raised against one member of the family do not show high
cross reactivity towards other family members. There could be two
reasons for this lack of cross-reactivity of MAbs towards various
family members. First, in standard hybridoma production, one
searches for only a few antibodies of high specificity/affinity for
the target antigen and then checks for cross reactivity or
background binding of the few selected antibodies. Second, although
proteins within a family are structurally related, they may have
exclusive, non-overlapping immunodominant epitopes. Therefore, MAbs
raised by using full length protein may not cross react with other
structurally related proteins.
[0005] There are also examples of monoclonal antibodies raised
against an antigen of one species that will bind specifically to
the same functional antigen in another species. For example, an
anti-mouse X antibody may readily bind antigen X from human. This
is because they share significant sequence and structural
similarities though they are not identical. However, such
species-cross-reactive antibodies do not constitute "dual
specificity" antibodies, since they have specificity for the same
antigen from different species.
[0006] Thus, monoclonal antibodies having predictable dual or
multiple specificity, that is, antibodies having true specificity
for two or more different antigens, are still needed.
SUMMARY OF THE INVENTION
[0007] This invention provides methods for making antibodies having
dual specificity for at least two structurally-related, yet
different, antigens. The method generally involves providing an
antigen that comprises a common structural feature of the two
different but structurally related molecules; exposing an antibody
repertoire to the antigen; and selecting from the repertoire an
antibody that specifically binds the two different but structurally
related molecules to thereby obtain the dual specificity antibody.
In clinical settings, several members of the same family of
proteins may contribute to the various symptoms of a disease
process. Therefore, use of a dual specificity antibody of the
invention, which binds members of the same family of proteins, to
block the functions of more than one member of the protein family
can be beneficial for alleviating disease symptoms or for
interrupting the disease process itself. Moreover, such dual
specificity antibodies of the invention are useful to detect
structurally related antigens, to purify structurally related
antigens and in diagnostic assays involving structurally related
antigens.
[0008] In a preferred embodiment, the antigen is designed based on
a contiguous topological area of identity between the two different
but structurally related molecules. For example, the two different
but structurally related molecules can be proteins and the antigen
can be a peptide comprising an amino acid sequence of a contiguous
topological area of identity between the two proteins.
[0009] In another embodiment, the antigen is designed based on
structurally mimicking a loop of a common fold of the two different
but structurally related molecules. For example, the antigen can be
a cyclic peptide that structurally mimics a loop of a common fold
of two different but structurally related proteins.
[0010] In yet another embodiment, the antigen is designed based on
splicing together alternating and/or overlapping portions of the
two different but structurally related molecules to create a hybrid
molecule. For example, the antigen can be a hybrid peptide made by
splicing together alternating and/or overlapping amino acid
sequences of two different but structurally related proteins.
[0011] In still another embodiment, the antigen can comprise one of
the two different but structurally related molecules and the method
involves selecting antibodies that specifically recognize both
related molecules.
[0012] In the method of the invention, the antibody repertoire can
be exposed to the antigen of interest either in vivo or in vitro.
For example, in one embodiment, exposure of the repertoire to the
antigen involves immunizing an animal in vivo with the antigen.
This in vivo approach can further involve preparing a panel of
hybridomas from lymphocytes of the animal and selecting a hybridoma
that secretes an antibody that specifically binds the two different
but structurally related molecules. The animal that is immunized
can be, for example, a mouse, a rat, a rabbit, or a goat, or a
transgenic version of any of the foregoing animals, such as a mouse
that is transgenic for human immunoglobulin genes such that the
mouse makes human antibodies upon antigenic stimulation. Other
types of animals that can be immunized include mice with severe
combined immunodeficiency (SCID) that have been reconstituted with
human peripheral blood mononuclear cells (hu-PBMC-SCID chimeric
mice) or lymphoid cells or precursors thereof and mice that have
been treated with lethal total body irradiation, followed by
radioprotection with bone marrow cells of a severe combined
immunodeficiency (SCID) mouse, followed by engraftment with
functional human lymphocytes (the Trimera system). Still another
type of animal that can be immunized is an animal (e.g., mouse)
whose genome has been "knocked out" (e.g., by homologous
recombination) for an endogenous gene(s) encoding the antigen(s) of
interest, wherein upon immunization with the antigen(s) of interest
the KO animal recognizes the antigen(s) as foreign.
[0013] In another embodiment, the antibody repertoire is exposed to
the antigen in vitro by screening a recombinant antibody library
with the antigen. The recombinant antibody library can be, for
example, expressed on the surface of bacteriophage or on the
surface of yeast cells or on the surface of bacterial cells. In
various embodiments, the recombinant antibody library is, for
example, a scFv library or a Fab library. In yet another
embodiment, antibody libraries are expressed as RNA-protein
fusions.
[0014] Another approach to preparing the dual specificity
antibodies involves a combination of in vivo and in vitro
approaches, such as exposing the antibody repertoire to the antigen
by in vivo immunization of an animal with the antigen, followed by
in vitro screening of a recombinant antibody library prepared from
lymphoid cells of the animal with the antigen. Still another
approach involves exposing the antibody repertoire to the antigen
by in vivo immunization of an animal with the antigen, followed by
in vitro affinity maturation of a recombinant antibody library
prepared from lymphoid cells of the animal. Yet another approach
involves exposing the antibody repertoire to the antigen by in vivo
immunization of an animal with the antigen, followed by selection
of single antibody producing cells secreting an antibody of
interest, recovery of heavy and light chain variable region cDNAs
from these selected cells (e.g., by PCR) and expression of the
heavy- and light chain variable regions in mammalian host cells in
vitro (referred to as the selected lymphocyte antibody method, or
SLAM), thereby allowing for further selection and manipulation of
the selection antibody gene sequences. Still further, monoconal
antibodies can be selected by expression cloning by expressing
heavy and light chain antibody genes in mammalian cells and
selecting for mammalian cells secreting an antibody having the
requisite binding specificity.
[0015] The methods of the invention allow for the preparation of
various different types of dual specificity antibodies, including
fully human antibodies, chimeric antibodies and CDR-grafted
antibodies, and antigen-binding portions thereof. Dual specificity
antibodies prepared according to the methods of the invention are
also provided. A preferred dual specificity antibody of the
invention is one that specifically binds interleukin-1.alpha. and
interleukin-1.beta.. Such a dual specificity antibody can be used
in methods of detecting IL-1.alpha. or IL-1.beta. comprising
contacting IL-1.alpha. or IL-1.beta. with the dual-specificity
antibody, or antigen-binding portion thereof, such that IL-1.alpha.
or IL-1.beta. is detected. A neutralizing dual specificity antibody
also can be used in methods of inhibiting IL-1.alpha. or IL-1.beta.
activity comprising contacting IL-1.alpha. or IL-1.beta. with the
dual-specificity antibody, or antigen-binding portion thereof, such
that the activity of IL-1.alpha. or IL-1.beta. is inhibited. Such
dual specificity antibodies also can be used in methods of treating
an interleukin-1-related disorder comprising administering to a
subject suffering from an interleukin-1-related disorder the
dual-specificity antibody, or antigen-binding portion thereof.
[0016] In another embodiment, the invention provides a method of
making an antibody or an antigen binding portion thereof library by
performing the following steps:
a) obtaining a recombinant heavy chain or an antigen binding
portion thereof library A from an antibody repertoire resulting
from exposure to a first antigen; b) obtaining a recombinant light
chain or an antigen binding portion thereof library B from an
antibody repertoire resulting from exposure to the first antigen;
c) obtaining a recombinant heavy chain or an antigen binding
portion thereof library C from an antibody repertoire resulting
from exposure to a second antigen; d) obtaining a recombinant light
chain or an antigen binding portion thereof library D from an
antibody repertoire resulting from exposure to the second antigen;
and e) combining the recombinant heavy chain or an antigen binding
portion thereof library A with the recombinant light chain or an
antigen binding portion thereof library D to obtain an antibody or
an antigen binding portion thereof library X and/or combining the
recombinant heavy chain or an antigen binding portion thereof
library C with the recombinant light chain or an antigen binding
portion thereof library B to obtain an antibody or an antigen
binding portion thereof library Y.
[0017] In another embodiment of the present invention, the
immediately foregoing method of the invention can further comprise
the step of combining the antibody or an antigen binding portion
thereof library X with the antibody or an antigen binding portion
thereof library Y to obtain an antibody or an antigen binding
portion thereof library Z.
[0018] In a further embodiment, the present invention is directed
to the antibody or an antigen binding portion thereof libraries X,
Y and Z.
[0019] In another embodiment, the method of the present invention
allows for the identification of dual specific antibody or an
antigen binding portion thereof by selecting from the libraries X,
Y and/or Z an antibody or an antigen binding portion thereof that
binds both the first and the second antigen.
[0020] In a further embodiment, the present invention is directed
to the dual specific antibody made and/or selected by any of the
methods of the present invention.
[0021] In another embodiment, the present invention is also
directed to the nucleotide sequence encoding each member of the
antibody or an antigen binding portion thereof of libraries X, Y,
and Z; and the dual specific antibody or an antigen binding portion
thereof, a vector comprising the afore mentioned nucleotide
sequences and host cell transfected with the afore mentioned
vector.
[0022] In a preferred embodiment, the first and second antigen is
each independently selected from the group consisting of proteins,
polypeptides and peptides provided that the first and second
antigens are not the same. In a further embodiment, the proteins,
polypeptides and peptides are secreted proteins or surface
receptors and the secreted protein is selected from the group
consisting of an IFN, a TNF, an Interleukin, IP-10, PF4, a GRO,
9E3, EMAP-II, a CSF, an FGF, and a PDGF. In another preferred
embodiment the first antigen is IL-1.alpha. and the second antigen
is IL-1.beta..
DETAILED DESCRIPTION OF THE INVENTION
[0023] This invention pertains to the design and use of antigens
for generating dual specificity antibodies, ie., antibodies having
specificity for at least two different but structurally related
molecules, as well as the selection, preparation and use of such
dual specificity antibodies. The structural relatedness of the
antigens of the invention can be over the entire antigen (e.g.,
protein) or only in certain structurally-related regions. The
invention provides a method for obtaining a dual-specificity
antibody that specifically binds two different but structurally
related molecules, wherein the method involves: [0024] providing an
antigen that comprises a common structural feature of the two
different but structurally related molecules; [0025] exposing an
antibody repertoire to the antigen; and [0026] selecting from the
repertoire an antibody that specifically binds the two different
but structurally related molecules to thereby obtain the dual
specificity antibody.
[0027] It should be noted that while the invention is described
herein in terms of recognition of two different but related
antigens, it should be understood that the term "dual specificity
antibody" is intended to include antibodies that specifically
recognize even more than two different but related antigens, such
as antibodies that recognize three, four, five or more structurally
related but distinct antigens. Furthermore, the term "different but
structurally related antigens" is intended to include antigens
(e.g., proteins) whose overall structures are related as well as
antigens (e.g., proteins) which share one or more
structurally-related regions but that are otherwise unrelated.
Thus, "different but structurally related" antigens could be, for
example, two proteins that are members of the same protein family
having a common overall structure or could be, for example, two
proteins whose overall structure is disimilar (unrelated) but that
each contain a structurally-related domain.
[0028] Various types of antigens may be used to elicit the
antibodies of the invention and various methods of making
antibodies can be applied to obtain a dual specificity antibody of
the invention, as discussed in further detail below in the
following subsections.
I. Dual Specificity Antigens
[0029] To prepare a dual specificity antibody of the invention,
antibodies are raised against an antigen capable of eliciting dual
specificity antibodies. Such antigens generally are referred to
herein as dual specificity antigens. Various different types of
dual specificity antigens can be used in the invention and the
design of various types of dual specificity antigens is described
further in the following subsections.
[0030] A. Contiguous Topological Areas
[0031] In one embodiment, a dual specificity antigen of the
invention comprises a contiguous topological area of identity
and/or similarity between the two different but structurally
related molecules to which a dual specificity antibody is to be
raised. Preferably, the antigen comprises the largest (e.g.,
longest) contiguous topological area of identity and/or similarity
between the two different but structurally related molecules.
Preferably, the two different but structurally related molecules
are proteins and the dual specificity antigen comprises a linear
peptide corresponding to the largest (e.g., longest) contiguous
topological area of identity and/or similarity between the two
proteins. The appropriate region of identity/similarity that is
chosen is preferably a receptor or ligand binding region, although
other regions of identity/similarity can also be used.
[0032] To determine contiguous topological areas of identity
between two molecules (e.g., proteins), the two molecules (e.g.,
proteins) are compared (e.g., homology modeling, structural
information or aligned) and identical or similar regions are
identified. For proteins, an alignment algorithm can be used to
create optimal alignment and identify the largest (e.g., longest)
contiguous topological area of identity and/or similarity between
the two proteins. A preferred, non-limiting example of a
mathematical algorithm utilized for the comparison of two sequences
is the algorithm of Karlin and Altschul (1990) Proc. Natl. Acad.
Sci. USA 87:2264-68, modified as in Karlin and Altschul (1993)
Proc. Natl. Acad. Sci. USA 90:5873-77. Such an algorithm is
incorporated into the NBLAST and)(BLAST programs of Altschul, et
al. (1990) J. Mol. Biol. 215:403-10. To obtain gapped alignments
for comparison purposes, Gapped BLAST can be utilized as described
in Altschul et al., (1997) Nucleic Acids Research 25(17):3389-3402.
When utilizing BLAST and Gapped BLAST programs, the default
parameters of the respective programs (e.g.,)(BLAST and NBLAST) can
be used. See http://www.ncbi.nlm.nih.gov. An alternative
mathematical algorithm that can be used is that used in the ALIGN
program described in Myers and Miller (1988) Comput. Appl. Biosci.
4:11-17.
[0033] When an appropriate region of identity/similarity is chosen,
the dual specificity antigen corresponding to the region can be
chemically synthesized. For example, for peptide antigens, the
peptide can be synthesized by standard peptide synthesis methods.
In one embodiment, the peptide antigen comprises L amino acids. In
other embodiments, the peptide antigen may be partially or entirely
composed of D amino acids. An example of the design of a dual
specificity antigen based on a contiguous topological area of
identity and/or similarity between two different but structurally
related proteins is described in detail in Example 1.
[0034] B. Cyclic Peptides Mimicking a Structural Loop
[0035] In another embodiment, a dual specificity antigen of the
invention comprises a cyclic molecule, preferably a cyclic peptide,
that structurally mimics a key loop of a common fold of the two
different but structurally related molecules (e.g., proteins) to
which dual specificity antibodies are to be raised. To prepare this
type of antigen, the structures of the two related molecules are
compared and a loop of a common fold found in the two molecules is
identified. Standard molecular modeling and crystollographic
analysis can be used to aid in the identification of such loops and
common folds. Identical and similar regions (e.g., amino acid
sequences between two proteins) are identified and a consensus
sequence can be designed for similar but not identical regions. A
linear molecule, e.g, a linear peptide, is designed based on these
similar and identical regions and this linear molecule can then be
cyclized, by known chemical means, to create an antigen that mimics
the key loop. For example, a proline and a glycine can be added to
the end of a linear peptide to allow for cyclization of the
peptide. An example of the design of a dual specificity antigen
based on a cyclic peptide mimicking a structural loop shared by two
different but structurally related proteins is described in detail
in Example 2.
[0036] C. Hybrid Molecules
[0037] In another embodiment, a dual specificity antigen of the
invention comprises a hybrid molecule, preferably a hybrid peptide,
that includes alternating and/or overlapping regions of the two
different but structurally related molecules (e.g., proteins) to
which dual specificity antibodies are to be raised. To prepare this
type of antigen, the structures of the two molecules are compared,
and overlapping regions are identified (i.e., regions of identity),
as well as nonidentical regions between the two molecules. A hybrid
molecule (e.g., a hybrid peptide, when the two related molecules
are proteins) is prepared that preferably comprises alternating
regions (e.g, amino acid sequences) from each of the two molecules,
as well as an overlapping region that is common to both molecules.
Schematically, such a hybrid molecule can be described as: X--Y--Z,
wherein Y represents a region of identity or strong similarity
between the two related molecules (i.e, an overlapping region), X
represents a region from one of the related molecules and Z
represents a region from the other of the related molecules. An
example of the design of a dual specificity antigen based on a
hybrid peptide composed of sequences of two different but
structurally related proteins is described in detail in Example
3.
[0038] Another type of hybrid molecule is one in which a peptide
has been introduced into a full-length protein (referred to as a
"target" protein). Peptides are selected that represent functional
regions of two different but structurally related proteins, for
example, receptor interacting regions. Such peptides are referred
to herein as functional peptides. A functional peptide from one of
the related proteins is then introduced into the full-length
protein of the other related protein or, alternatively, an
unrelated protein. For example, a peptide of IL-1.alpha.
corresponding to a receptor interacting region of IL-1.alpha. is
identified and this functional peptide of IL-1.alpha. is introduced
into the full-length IL-1.beta. protein to create a hybrid
IL-1.alpha./IL-1.beta. molecule. Similarly, a peptide of IL-1.beta.
corresponding to a receptor interacting region of IL-1.beta. is
identified and this functional peptide of IL-1.beta. is introduced
into the full-length IL-1.alpha. protein to create a hybrid
IL-1.alpha./IL-1.beta. molecule. This introduction of the
functional peptide into the related full-length protein constrains
the functional peptide at both ends and maintains the
fold-structure of the functional peptide.
[0039] In case of an IL-1.alpha./IL-1.beta. hybrid, the functional
peptide preferably is inserted (replaces the natural amino acids)
in a target area representing the common fold structures of
IL-1.alpha. and IL-1.beta.. Such areas may be found over the entire
length of the protein (e.g., in the N-terminal region, in the
middle of the protein, in the C-terminal region). Furthermore,
functional peptides representing the common IL-1.alpha./IL-1.beta.
fold structures can also be inserted into an irrelevant protein,
such as albumin or some other naturally occurring protein. In this
instance, the preferred insertion site for the peptide is a region
that allows the peptide to maintain the desired fold structure.
Therefore, the insertion sites can be either at the N-terminus, the
middle, or the C-terminus of the protein. The positioning of the
peptide in any naturally occurring target protein is selected to
mimic the structural constraints placed upon it by the native
protein from which it is derived. While the functional peptide may
simply be inserted into the target protein such that the amino
acids of the functional peptide are added to the target protein,
preferably the amino acids of the functional peptide replace a
portion of the target protein into which it is inserted.
[0040] As a non-limiting example, a hybrid molecule is constructed
for use as a dual specificity antigen for raising dual specificity
antibodies to IL-1.alpha. and IL-1.beta. wherein a functional
peptide corresponding to a specific structural element of either
IL-1.alpha. or IL-1.beta. is introduced into the full length
IL-1.beta. or IL-1.alpha. in the equivalent structural position.
The chosen hybrid molecule replaces residues 160-176 of IL-1.beta.
with residues 168-184 of IL-1.alpha.. The resulting molecule
possesses the following amino acid sequence, in which the
substituted IL-1.alpha. sequences (residues 168-184) are
underlined:
TABLE-US-00001 (SEQ ID NO: 4)
APVRSLNCTLRDSQQKSLVMSGPYELKALHLQGQDMEQQVVFSMGAYKS
SKDDAKITVILGLKEKNLYLSCVLKDDKPTLQLESVDPKNYPKKKMEKR
FVFNKIEINNKLEFESAQFPNWYISTSQAENMPVFLGGTKGGQDITDFT MQFVSS
[0041] This molecule can be prepared using standard molecular
biology techniques (e.g., cloning, polymerase chain reaction) using
the publicly available IL-1.alpha. and IL-1.beta. cDNA sequences
and recombinant protein expression techniques. A hybrid cDNA can be
prepared, introduced into an appropriate expression vector and the
polypeptide can be expressed by introducing the expression vector
into an appropriate host cell.
[0042] D. Peptides Based on Hydrophobicity Plots
[0043] In another embodiment, a dual specificity antigen of the
invention is selected based on hydrophobicity plots to select
peptides predicted to be highly antigenic. For example, antigenic
indexes of peptides can be calculated using computer software as
described by Jameson and Wolf (CABIOS, 4(1), 181-186 (1988))
Regions of interest for antibody binding can be chosen to maximize
the probability of antigenicity.
[0044] E. Immunization with Antigen-Transfected Cells
[0045] In another embodiment, a dual specificity antibody of the
invention is prepared by immunization with antigen-transfected
cells (i.e., dual specificity antigens of the invention can be
antigen-transfected cells). Cell lines can be generated that stably
express the two different but structurally related antigens, or a
hybrid molecule thereof. For example, cell lines can be generated
that stably express IL-1.alpha. or IL-1.beta. or an
IL-1.alpha./IL-1.beta. hybrid molecule (e.g., SEQ ID NO: 4). The
molecules of interest can be secreted from the cells (in case of
soluble proteins) or can be expressed on the cell surface (in case
of receptors, enzymes). Gene delivery into the host cells to allow
for expression of the antigens by the host cells can be
accomplished by a number of conventional means, including but not
limited to transfection, electroporation, cell fusion, lipofection,
particle bombardment, microinjection, or viral infection. The cell
lines expressing the antigens of interest can then be transplanted
via one or more various routes (intraperitoneal, subcutaneous,
intramuscular, and the like) into an animal of interest for
antibody production. The cells then serve as a slow release source
of the antigen of interest. Preferably the cells express the full
length proteins. However, antigenic fragments can also be
expressed. For soluble proteins, the proteins preferably are
secreted by the cells. To generate a dual specificity antibody to
the extracellular domain of two closely related receptors, the
receptors preferably are expressed on the cell surface.
[0046] F. Immunization with One of the Structurally Related
Molecules
[0047] In another embodiment, the dual specificity antigen is
simply one of the two different but structurally related molecules
to which dual specificity antibodies are to be raised. One of the
two related molecules is used as the immunization agent and then
the resultant antibody repertoire is screened for antibodies that
bind, and more preferably neutralize, both of the two different but
structurally related molecules. For example, one can immunize with
either IL-1.alpha. or IL-1.beta. and then screen for .alpha./.beta.
binders, and more preferably neutralizers. As used in this
embodiment, the term "immunize" is intended to broadly encompass
the exposure of an antibody repertoire to the antigen, such as
IL-1.alpha. or IL-1.beta., either in vivo or in vitro. Thus, this
embodiment encompasses immunizing an animal with either IL-1.alpha.
or IL-1.beta., and screening the resultant antibodies raised to
select for those antibodies that bind both IL-1.alpha. and
IL-1.beta., as well as screening a recombinant antibody library in
vitro with either IL-1.alpha. or IL-1.beta. and then selecting for
recombinant antibodies that bind both IL-1.alpha. and
IL-1.beta..
II. Methods of Making Dual Specificity Antibodies
[0048] To prepare a dual specificity antibody of the invention, an
antibody repertoire (either in vivo or in vitro) is exposed to a
dual specificity antigen, prepared as described in the previous
section, and an appropriate dual specificity antibody is selected
from the repertoire. The two elements of antibody recognition of an
antigen are structural recognition and affinity maturation based on
specific molecular interactions. During a natural immune response,
low affinity antibodies that recognize structural motifs (for
example the recognition of an antigen by certain pattern
recognition receptors) are developed easily, and early on in the
natural immune response this is followed by somatic mutations to
increase the affinity of a few clones. Various in vivo and in vitro
processes have been developed to mimic this natural phenomenon. Low
affinity dual specificity antibodies can be generated by any of the
in vitro and in vivo methods described herein and higher affinity
dual specificity Mabs can be prepared by somatic mutagenesis
methods described herein. Moreover, to optimize high affinity dual
specificity MAbs, co-crystal structures of the low affinity MAbs
with the desired antigens can be made. The structural information
obtained can guide further affinity enhancements by altering
(mutating) specific contact residues of the MAbs to enhance
specific molecular interactions, as described herein.
[0049] Methods for making dual specificity antibodies using in vivo
approaches, in vitro approaches, or a combination of both, are
described in further detail in the following subsections.
[0050] A. In Vivo Approaches
[0051] A standard in vivo approach to preparing antibodies is by
immunizing an appropriate animal subject with an antigen to thereby
expose the in vivo antibody repertoire to the antigen, followed by
recovery of an antibody or antibodies of interest from the animal.
Such an approach can be adapted to the preparation of dual
specificity antibodies by use of a dual specificity antigen and
selection for antibodies that specifically recognize the two
structurally related molecules of interest. Dual specificity
antibodies can be prepared by immunizing a suitable subject, (e.g.,
rabbit, goat, mouse or other mammal, including transgenic and
knockout versions of such mammals) with an immunogenic preparation
of a dual specificity antigen. An appropriate immunogenic
preparation can contain, for example, a chemically synthesized or
recombinantly expressed dual specificity antigen. The preparation
can further include an adjuvant, such as Freund's complete or
incomplete adjuvant, or similar immunostimulatory compound.
Moreover, when used to raise antibodies, in particular by in vivo
immunization, a dual specificity antigen of the invention can be
used alone, or more preferably is used as a conjugate with a
carrier protein. Such an approach for enhancing antibody responses
is well known in the art. Examples of suitable carrier proteins to
which a dual specificity antigen can be conjugated include keyhole
limpet haemocyanin (KLH) and albumin.
[0052] Antibody-producing cells can be obtained from the subject
and used to prepare monoclonal antibodies by standard techniques,
such as the hybridoma technique originally described by Kohler and
Milstein (1975, Nature 256:495-497) (see also, Brown et al. (1981)
J. Immunol 127:539-46; Brown et al. (1980) J. Biol Chem
255:4980-83; Yeh et al. (1976) PNAS 76:2927-31; and Yeh et al.
(1982) Int. J. Cancer 29:269-75). The technology for producing
monoclonal antibody hybridomas is well known (see generally R. H.
Kenneth, in Monoclonal Antibodies: A New Dimension In Biological
Analyses, Plenum Publishing Corp., New York, N.Y. (1980); E. A.
Lerner (1981) Yale J Biol. Med., 54:387-402; M. L. Gefter et al.
(1977) Somatic Cell Genet., 3:231-36). Briefly, an immortal cell
line (typically a myeloma) is fused to lymphocytes (typically
splenocytes or lymph node cells or peripheral blood lymphocytes)
from a mammal immunized with a dual specificity immunogen as
described above, and the culture supernatants of the resulting
hybridoma cells are screened to identify a hybridoma producing a
monoclonal antibody with dual specificity for the two different but
structurally related molecules of interest. Any of the many well
known protocols used for fusing lymphocytes and immortalized cell
lines can be applied for the purpose of generating dual specificity
monoclonal antibodies (see, e.g., G. Galfre et al. (1977) Nature
266:550-52; Gefter et al. Somatic Cell Genet., cited supra; Lerner,
Yale J. Biol. Med, cited supra; Kenneth, Monoclonal Antibodies,
cited supra). Moreover, the ordinary skilled artisan will
appreciate that there are many variations of such methods, which
also would be useful. Typically, the immortal cell line (e.g., a
myeloma cell line) is derived from the same mammalian species as
the lymphocytes. For example, murine hybridomas can be made by
fusing lymphocytes from a mouse immunized with an immunogenic
preparation of the present invention with an immortalized mouse
cell line. Preferred immortal cell lines are mouse myeloma cell
lines that are sensitive to culture medium containing hypoxanthine,
aminopterin and thymidine ("HAT medium"). Any of a number of
myeloma cell lines may be used as a fusion partner according to
standard techniques, e.g., the P3-NS1/1-Ag4-1, P3-x63-Ag8.653 or
Sp2/O-Ag14 myeloma lines. These myeloma lines are available from
the American Type Culture Collection (ATCC), Rockville, Md.
Typically, HAT-sensitive mouse myeloma cells are fused to mouse
splenocytes using polyethylene glycol ("PEG"). Hybridoma cells
resulting from the fusion are then selected using HAT medium, which
kills unfused and unproductively fused myeloma cells (unfused
splenocytes die after several days because they are not
transformed). Hybridoma cells producing monoclonal antibodies that
specifically recognize the two structurally related molecules of
interest are identified by screening the hybridoma culture
supernatants for such antibodies, e.g., using a standard ELISA
assay, to select those antibodies that specifically can bind the
two related molecules.
[0053] Depending on the type of antibody desired, various animal
hosts may be used for in vivo immunization. A host that itself
expresses an endogenous version of the antigen(s) of interest can
be used or, alternatively, a host can be used that has been
rendered deficient in an endogenous version of the antigen(s) of
interest. For example, it has been shown that mice rendered
deficient for a particular endogenous protein via homologous
recombination at the corresponding endogenous gene (i.e.,
"knockout" mice) elicit a humoral response to the protein when
immunized with it and thus can be used for the production of high
affinity monoclonal antibodies to the protein (see e.g., Roes, J.
et al. (1995) J. Immunol. Methods 183:231-237; Lunn, M. P. et al.
(2000) J. Neurochem. 75:404-412).
[0054] For production of non-human antibodies (e.g., against a
human dual specificity antigen), various non-human mammals are
suitable as hosts for antibody production, including but not
limited to mice, rats, rabbits and goats (and knockout versions
thereof), although mice are preferred for hybridoma production.
Furthermore, for production of fully-human antibodies against a
human dual specificity antigen, a host non-human animal can be used
that expresses a human antibody repertoire. Such non-human animals
include transgenic animals (e.g., mice) carrying human
immunoglobulin transgenes, hu-PBMC-SCID chimeric mice, and
human/mouse radiation chimeras, each of which is discussed further
below.
[0055] Thus, in one embodiment, the animal that is immunized with a
dual specificity antigen is a non-human mammal, preferably a mouse,
that is transgenic for human immunoglobulin genes such that the
non-human mammal (e.g., mouse) makes human antibodies upon
antigenic stimulation. In such animals, typically, human germline
configuration heavy and light chain immunoglobulin transgenes are
introduced into animals that have been engineered so that their
endogenous heavy and light chain loci are inactive. Upon antigenic
stimulation of such animals (e.g., with a human antigen),
antibodies derived from the human immunoglobulin sequences (i.e.,
human antibodies) are produced, and human monoclonal antibodies can
be made from lymphocytes of such animals by standard hybridoma
technology. For further description of human immunoglobulin
transgenic mice and their use in the production of human antibodies
see for example, U.S. Pat. No. 5,939,598, PCT Publication No. WO
96/33735, PCT Publication No. WO 96/34096, PCT Publication WO
98/24893 and PCT Publication WO 99/53049 to Abgenix Inc., and U.S.
Pat. No. 5,545,806, No. 5,569,825, No. 5,625,126, No. 5,633,425,
No. 5,661,016, No. 5,770,429, No. 5,814,318, No. 5,877,397 and PCT
Publication WO 99/45962 to Genpharm Inc. See also MacQuitty, J. J.
and Kay, R. M. (1992) Science 257:1188; Taylor, L. D. et al. (1992)
Nucleic Acids Res. 20:6287-6295; Lonberg, N. et al. (1994) Nature
368:856-859; Lonberg, N. and Huszar, D. (1995) Int. Rev. Immunol.
13:65-93; Harding, F. A. and Lonberg, N. (1995) Ann. N.Y. Acad.
Sci. 764:536-546; Fishwild, D. M. et al. (1996) Nature
Biotechnology 14:845-851; Mendez, M. J. et al. (1997) Nature
Genetics 15:146-156; Green, L. L. and Jakobovits, A. (1998) J. Exp.
Med 188:483-495; Green, L. L. (1999) J. Immunol. Methods 231:11-23;
Yang, X. D. et al. (1999) J. Leukoc. Biol. 66:401-410; Gallo, M. L.
et al. (2000) Eur. J. Immunol. 30:534-540.
[0056] In another embodiment, the animal that is immunized with a
dual specificity antigen is a mouse with severe combined
immunodeficiency (SCID) that has been reconstituted with human
peripheral blood mononuclear cells or lymphoid cells or precursors
thereof. Such mice, referred to as hu-PBMC-SCID chimeric mice, have
been demonstrated to produce human immunoglobulin responses upon
antigenic stimulation. For further description of these mice and
their use in antibody generation, see for example Leader, K. A. et
al. (1992) Immunology 76:229-234; Bombil, F. et al. (1996)
Immunobiol. 195:360-375; Murphy, W. J. et al. (1996) Semin.
Immunol. 8:233-241; Herz, U. et al. (1997) Int. Arch. Allergy
Immunol. 113:150-152; Albert, S. E. et al. (1997) J. Immunol.
159:1393-1403; Nguyen, H. et al (1997) Microbiol. Immunol.
41:901-907; Arai, K. et al. (1998) J. Immunol. Methods 217:79-85;
Yoshinari, K. and Arai, K. (1998) Hybridoma 17:41-45; Hutchins, W.
A. et al. (1999) Hybridoma 18:121-129; Murphy, W. J. et al. (1999)
Clin. Immunol. 90:22-27; Smithson, S. L. et al. (1999) Mol.
Immunol. 36:113-124; Chamat, S. et al. (1999) J. Infect. Diseases
180:268-277; and Heard, C. et al. (1999) Molec. Med. 5:35-45.
[0057] In another embodiment, the animal that is immunized with a
dual specificity antigen is a mouse that has been treated with
lethal total body irradiation, followed by radioprotection with
bone marrow cells of a severe combined immunodeficiency (SCID)
mouse, followed by engraftment with functional human lymphocytes.
This type of chimera, referred to as the Trimera system, has been
used to produce human monoclonal antibodies by immunization of the
mice with an antigen of interest followed by preparation of
monoclonal antibodies using standard hybridoma technology. For
further description of these mice and their use in antibody
generation, see for example Eren, R. et al. (1998) Immunology
93:154-161; Reisner, Y and Dagan, S. (1998) Trends Biotechnol.
16:242-246; Ilan, E. et al. (1999) Hepatology 29:553-562; and
Bocher, W. O. et al. (1999) Immunology 96:634-641.
[0058] B. In Vitro Approaches
[0059] Alternative to preparing dual specificity antibodies by in
vivo immunization and selection, a dual specificity antibody of the
invention can be identified and isolated by screening a recombinant
combinatorial immunoglobulin library (e.g., an antibody phage
display library) with a dual specificity antigen, to thereby
isolate immunoglobulin library members that bind specifically to
the two structurally related, but different, molecules of interest.
Kits for generating and screening phage display libraries are
commercially available (e.g., the Pharmacia Recombinant Phage
Antibody System, Catalog No. 27-9400-01; and the Stratagene
SurfZAP.TM. Phage Display Kit, Catalog No. 240612). In various
embodiments, the phage display library is a scFv library or a Fab
library. The phage display technique for screening recombinant
antibody libraries has been described extensively in the art.
Examples of methods and compounds particularly amenable for use in
generating and screening antibody display library can be found in,
for example, McCafferty et al. International Publication No. WO
92/01047, U.S. Pat. No. 5,969,108 and EP 589,877 (describing in
particular display of scFv), Ladner et al. U.S. Pat. No. 5,223,409,
No. 5,403,484, No. 5,571,698, No. 5,837,500 and EP 436,597
(describing, for example, pIII fusion); Dower et al. International
Publication No. WO 91/17271, U.S. Pat. No. 5,427,908, U.S. Pat. No.
5,580,717 and EP 527,839 (describing in particular display of Fab);
Winter et al. International Publication WO 92/20791 and EP 368,684
(describing in particular cloning of immunoglobulin variable domain
sequences); Griffiths et al. U.S. Pat. No. 5,885,793 and EP 589,877
(describing in particular isolation of human antibodies to human
antigens using recombinant libraries); Garrard et al. International
Publication No. WO 92/09690 (describing in particular phage
expression techniques); Knappik et al. International Publication
No. WO 97/08320 (describing the human recombinant antibody library
HuCal); Salfeld et al. International Publication No. WO 97/29131,
describing the preparation of a recombinant human antibody to a
human antigen (human tumor necrosis factor alpha), as well as in
vitro affinity maturation of the recombinant antibody) and Salfeld
et al. U.S. Provisional Application No. 60/126,603, also describing
the preparation of a recombinant human antibody to a human antigen
(human interleukin-12), as well as in vitro affinity maturation of
the recombinant antibody)
[0060] Other descriptions of recombinant antibody library
screenings can be found in scientific publications such as Fuchs et
al. (1991) Bio/Technology 9:1370-1372; Hay et al. (1992) Hum
Antibod Hybridomas 3:81-85; Huse et al. (1989) Science
246:1275-1281; Griffiths et al. (1993) EMBO J 12:725-734; Hawkins
et al. (1992) J Mol Biol 226:889-896; Clarkson et al. (1991) Nature
352:624-628; Gram et al. (1992) PNAS 89:3576-3580; Garrad et al.
(1991) Bio/Technology 9:1373-1377; Hoogenboom et al. (1991) Nuc
Acid Res 19:4133-4137; Barbas et al. (1991) PNAS 88:7978-7982;
McCafferty et al. Nature (1990) 348:552-554; and Knappik et al.
(2000) J. Mol. Biol. 296:57-86.
[0061] Alternative to the use of bacteriophage display systems,
recombinant antibody libraries can be expressed on the surface of
yeast cells or bacterial cells. Methods for preparing and screening
libraries expressed on the surface of yeast cells are described
further in PCT Publication WO 99/36569. Methods for preparing and
screening libraries expressed on the surface of bacterial cells are
described further in PCT Publication WO 98/49286.
[0062] Once an antibody of interest has been identified from a
combinatorial library, DNAs encoding the light and heavy chains of
the antibody are isolated by standard molecular biology techniques,
such as by PCR amplification of DNA from the display package (e.g.,
phage) isolated during the library screening process. Nucleotide
sequences of antibody light and heavy chain genes from which PCR
primers can be prepared are known in the art. For example, many
such sequences are disclosed in Kabat, E. A., et al. (1991)
Sequences of Proteins of Immunological Interest, Fifth Edition,
U.S. Department of Health and Human Services, NIH Publication No.
91-3242 and in the "Vbase" human germline sequence database.
[0063] An antibody, or antibody portion, of the invention can be
prepared by recombinant expression of immunoglobulin light and
heavy chain genes in a host cell. To express an antibody
recombinantly, a host cell is transfected with one or more
recombinant expression vectors carrying DNA fragments encoding the
immunoglobulin light and heavy chains of the antibody such that the
light and heavy chains are expressed in the host cell and,
preferably, secreted into the medium in which the host cells are
cultured, from which medium the antibodies can be recovered.
Standard recombinant DNA methodologies are used obtain antibody
heavy and light chain genes, incorporate these genes into
recombinant expression vectors and introduce the vectors into host
cells, such as those described in Sambrook, Fritsch and Maniatis
(eds), Molecular Cloning; A Laboratory Manual, Second Edition, Cold
Spring Harbor, N.Y., (1989), Ausubel, F. M. et al. (eds.) Current
Protocols in Molecular Biology, Greene Publishing Associates,
(1989) and in U.S. Pat. No. 4,816,397 by Boss et al.
[0064] Once DNA fragments encoding the VH and VL segments of the
antibody of interest are obtained, these DNA fragments can be
further manipulated by standard recombinant DNA techniques, for
example to convert the variable region genes to full-length
antibody chain genes, to Fab fragment genes or to a scFv gene. In
these manipulations, a VL- or VH-encoding DNA fragment is
operatively linked to another DNA fragment encoding another
protein, such as an antibody constant region or a flexible linker.
The term "operatively linked", as used in this context, is intended
to mean that the two DNA fragments are joined such that the amino
acid sequences encoded by the two DNA fragments remain
in-frame.
[0065] The isolated DNA encoding the VH region can be converted to
a full-length heavy chain gene by operatively linking the
VH-encoding DNA to another DNA molecule encoding heavy chain
constant regions (CH1, CH2 and CH3). The sequences of human heavy
chain constant region genes are known in the art (see e.g., Kabat,
E. A., et al. (1991) Sequences of Proteins of Immunological
Interest, Fifth Edition, U.S. Department of Health and Human
Services, NIH Publication No. 91-3242) and DNA fragments
encompassing these regions can be obtained by standard PCR
amplification. The heavy chain constant region can be an IgG1,
IgG2, IgG3, IgG4, IgA, IgE, IgM or IgD constant region, but most
preferably is an IgG1 or IgG4 constant region. For a Fab fragment
heavy chain gene, the VH-encoding DNA can be operatively linked to
another DNA molecule encoding only the heavy chain CH1 constant
region.
[0066] The isolated DNA encoding the VL region can be converted to
a full-length light chain gene (as well as a Fab light chain gene)
by operatively linking the VL-encoding DNA to another DNA molecule
encoding the light chain constant region, CL. The sequences of
human light chain constant region genes are known in the art (see
e.g., Kabat, E. A., et al. (1991) Sequences of Proteins of
Immunological Interest, Fifth Edition, U.S. Department of Health
and Human Services, NIH Publication No. 91-3242) and DNA fragments
encompassing these regions can be obtained by standard PCR
amplification. The light chain constant region can be a kappa or
lambda constant region, but most preferably is a kappa constant
region.
[0067] To create a scFv gene, the VH- and VL-encoding DNA fragments
are operatively linked to another fragment encoding a flexible
linker, e.g., encoding the amino acid sequence
(Gly.sub.4-Ser).sub.3, such that the VH and VL sequences can be
expressed as a contiguous single-chain protein, with the VL and VH
regions joined by the flexible linker (see e.g., Bird et al. (1988)
Science 242:423-426; Huston et al. (1988) Proc. Natl. Acad. Sci.
USA 85:5879-5883; McCafferty et al., Nature (1990)
348:552-554).
[0068] To express the recombinant antibodies, or antibody portions
of the invention, DNAs encoding partial or full-length light and
heavy chains, obtained as described above, can be inserted into
expression vectors such that the genes are operatively linked to
transcriptional and translational control sequences. In this
context, the term "operatively linked" is intended to mean that an
antibody gene is ligated into a vector such that transcriptional
and translational control sequences within the vector serve their
intended function of regulating the transcription and translation
of the antibody gene. The expression vector and expression control
sequences are chosen to be compatible with the expression host cell
used. The antibody light chain gene and the antibody heavy chain
gene can be inserted into separate vector or, more typically, both
genes are inserted into the same expression vector. The antibody
genes are inserted into the expression vector by standard methods
(e.g., ligation of complementary restriction sites on the antibody
gene fragment and vector, or blunt end ligation if no restriction
sites are present). Prior to insertion of the light or heavy chain
sequences, the expression vector may already carry antibody
constant region sequences. For example, one approach to converting
the VH and VL sequences to full-length antibody genes is to insert
them into expression vectors already encoding heavy chain constant
and light chain constant regions, respectively, such that the VH
segment is operatively linked to the CH segment(s) within the
vector and the VL segment is operatively linked to the CL segment
within the vector. Additionally or alternatively, the recombinant
expression vector can encode a signal peptide that facilitates
secretion of the antibody chain from a host cell. The antibody
chain gene can be cloned into the vector such that the signal
peptide is linked in-frame to the amino terminus of the antibody
chain gene. The signal peptide can be an immunoglobulin signal
peptide or a heterologous signal peptide (i.e., a signal peptide
from a non-immunoglobulin protein).
[0069] In addition to the antibody chain genes, the recombinant
expression vectors of the invention carry regulatory sequences that
control the expression of the antibody chain genes in a host cell.
The term "regulatory sequence" is intended to includes promoters,
enhancers and other expression control elements (e.g.,
polyadenylation signals) that control the transcription or
translation of the antibody chain genes. Such regulatory sequences
are described, for example, in Goeddel; Gene Expression Technology:
Methods in Enzymology 185, Academic Press, San Diego, Calif.
(1990). It will be appreciated by those skilled in the art that the
design of the expression vector, including the selection of
regulatory sequences may depend on such factors as the choice of
the host cell to be transformed, the level of expression of protein
desired, etc. Preferred regulatory sequences for mammalian host
cell expression include viral elements that direct high levels of
protein expression in mammalian cells, such as promoters and/or
enhancers derived from cytomegalovirus (CMV) (such as the CMV
promoter/enhancer), Simian Virus 40 (SV40) (such as the SV40
promoter/enhancer), adenovirus, (e.g., the adenovirus major late
promoter (AdMLP)) and polyoma. For further description of viral
regulatory elements, and sequences thereof, see e.g., U.S. Pat. No.
5,168,062 by Stinski, U.S. Pat. No. 4,510,245 by Bell et al. and
U.S. Pat. No. 4,968,615 by Schaffner et al.
[0070] In addition to the antibody chain genes and regulatory
sequences, the recombinant expression vectors of the invention may
carry additional sequences, such as sequences that regulate
replication of the vector in host cells (e.g., origins of
replication) and selectable marker genes. The selectable marker
gene facilitates selection of host cells into which the vector has
been introduced (see e.g., U.S. Pat. Nos. 4,399,216, 4,634,665 and
5,179,017, all by Axel et al.). For example, typically the
selectable marker gene confers resistance to drugs, such as G418,
hygromycin or methotrexate, on a host cell into which the vector
has been introduced. Preferred selectable marker genes include the
dihydrofolate reductase (DHFR) gene (for use in dhfr host cells
with methotrexate selection/amplification) and the neo gene (for
G418 selection).
[0071] For expression of the light and heavy chains, the expression
vector(s) encoding the heavy and light chains is transfected into a
host cell by standard techniques. The various forms of the term
"transfection" are intended to encompass a wide variety of
techniques commonly used for the introduction of exogenous DNA into
a prokaryotic or eukaryotic host cell, e.g., electroporation,
calcium-phosphate precipitation, DEAE-dextran transfection and the
like. Although it is theoretically possible to express the
antibodies of the invention in either prokaryotic or eukaryotic
host cells, expression of antibodies in eukaryotic cells, and most
preferably mammalian host cells, is the most preferred because such
eukaryotic cells, and in particular mammalian cells, are more
likely than prokaryotic cells to assemble and secrete a properly
folded and immunologically active antibody. Prokaryotic expression
of antibody genes has been reported to be ineffective for
production of high yields of active antibody (Boss, M. A. and Wood,
C. R. (1985) Immunology Today 6:12-13).
[0072] Preferred mammalian host cells for expressing the
recombinant antibodies of the invention include Chinese Hamster
Ovary (CHO cells) (including dhfr- CHO cells, described in Urlaub
and Chasin, (1980) Proc. Natl. Acad. Sci. USA 77:4216-4220, used
with a DHFR selectable marker, e.g., as described in R. J. Kaufman
and P.A. Sharp (1982) Mol. Biol. 159:601-621), NS0 myeloma cells,
COS cells and SP2 cells. When recombinant expression vectors
encoding antibody genes are introduced into mammalian host cells,
the antibodies are produced by culturing the host cells for a
period of time sufficient to allow for expression of the antibody
in the host cells or, more preferably, secretion of the antibody
into the culture medium in which the host cells are grown.
Antibodies can be recovered from the culture medium using standard
protein purification methods.
[0073] Host cells can also be used to produce portions of intact
antibodies, such as Fab fragments or scFv molecules. It will be
understood that variations on the above procedure are within the
scope of the present invention. For example, it may be desirable to
transfect a host cell with DNA encoding either the light chain or
the heavy chain (but not both) of an antibody of this invention.
Recombinant DNA technology may also be used to remove some or all
of the DNA encoding either or both of the light and heavy chains
that is not necessary for binding to the antigens of interest The
molecules expressed from such truncated DNA molecules are also
encompassed by the antibodies of the invention. In addition,
bifunctional antibodies may be produced in which one heavy and one
light chain are an antibody of the invention and the other heavy
and light chain are specific for an antigen other than the antigens
of interest by crosslinking an antibody of the invention to a
second antibody by standard chemical crosslinking methods.
[0074] In a preferred system for recombinant expression of an
antibody, or antigen-binding portion thereof, of the invention, a
recombinant expression vector encoding both the antibody heavy
chain and the antibody light chain is introduced into dhfr- CHO
cells by calcium phosphate-mediated transfection. Within the
recombinant expression vector, the antibody heavy and light chain
genes are each operatively linked to CMV enhancer/AdMLP promoter
regulatory elements to drive high levels of transcription of the
genes. The recombinant expression vector also carries a DHFR gene,
which allows for selection of CHO cells that have been transfected
with the vector using methotrexate selection/amplification. The
selected transformant host cells are culture to allow for
expression of the antibody heavy and light chains and intact
antibody is recovered from the culture medium. Standard molecular
biology techniques are used to prepare the recombinant expression
vector, transfect the host cells, select for transformants, culture
the host cells and recover the antibody from the culture medium.
Still further the invention provides a method of synthesizing a
recombinant antibody of the invention by culturing a host cell of
the invention in a suitable culture medium until a recombinant
antibody of the invention is synthesized. The method can further
comprise isolating the recombinant antibody from the culture
medium.
[0075] Alternative to screening of recombinant antibody libraries
by phage display, other methodologies known in the art for
screening large combinatorial libraries can be applied to the
identification of dual specificity antibodies of the invention. One
type of alternative expression system is one in which the
recombinant antibody library is expressed as RNA-protein fusions,
as described in PCT Publication No. WO 98/31700 by Szostak and
Roberts, and in Roberts, R. W. and Szostak, J. W. (1997) Proc.
Natl. Acad Sci. USA 94:12297-12302. In this system, a covalent
fusion is created between an mRNA and the peptide or protein that
it encodes by in vitro translation of synthetic mRNAs that carry
puromycin, a peptidyl acceptor antibiotic, at their 3' end. Thus, a
specific mRNA can be enriched from a complex mixture of mRNAs
(e.g., a combinatorial library) based on the properties of the
encoded peptide or protein, e.g., antibody, or portion thereof,
such as binding of the antibody, or portion thereof, to the dual
specificity antigen. Nucleic acid sequences encoding antibodies, or
portions thereof, recovered from screening of such libraries can be
expressed by recombinant means as described above (e.g., in
mammalian host cells) and, moreover, can be subjected to further
affinity maturation by either additional rounds of screening of
mRNA-peptide fusions in which mutations have been introduced into
the originally selected sequence(s), or by other methods for
affinity maturation in vitro of recombinant antibodies, as
described above.
[0076] C. Combination Approaches
[0077] Dual specificity antibodies of the invention also can be
prepared using a combination of in vivo and in vitro approaches,
such as methods in which the dual specificity antigen is originally
exposed to an antibody repertoire in vivo in a host animal to
stimulate production of antibodies that bind the dual specificity
antigen but wherein further antibody selection and/or maturation
(i.e., improvement) is accomplished using one or more in vitro
techniques.
[0078] In one embodiment, such a combination method involves first
immunizing a non-human animal (e.g., a mouse, rat, rabbit, goat, or
transgenic version thereof, or a chimeric mouse) with the dual
specificity antigen to stimulate an antibody response against the
antigen, following by preparation and screening of a phage display
antibody library using immunoglobulin sequences from lymphocytes
stimulated in vivo by exposure to the dual specificity antigen. The
first step of this combination procedure can be conducted as
described in subsection IIA above, while the second step of this
procedure can be conducted as described in subsection IIB above.
Preferred methodologies for hyperimmunization of non-human animals
followed by in vitro screening of phage display libraries prepared
from the stimulated lymphocytes include those described by BioSite
Inc., see e.g., PCT Publication WO 98/47343, PCT Publication WO
91/17271, U.S. Pat. No. 5,427,908 and U.S. Pat. No. 5,580,717.
[0079] In another embodiment, a combination method involves first
immunizing a non-human animal (e.g., a mouse, rat, rabbit, goat, or
knockout and/or transgenic version thereof, or a chimeric mouse)
with the dual specificity antigen to stimulate an antibody response
against the antigen and selection of lymphocytes that are producing
antibodies having the desired dual specificity (e.g., by screening
hybridomas prepared from the immunized animals). The rearranged
antibody genes from the selected clones are then isolated (by
standard cloning methods, such as reverse transcriptase-polymerase
chain reaction) and subjected to in vitro affinity maturation, to
thereby enhance the binding properties of the selected antibody or
antibodies. The first step of this procedure can be conducted as
described in subsection IIA above, while the second step of this
procedure can be conducted as described in subsection JIB above, in
particular using in vitro affinity maturation methods such as those
described in PCT Publication WO 97/29131 and PCT Publication WO
00/56772.
[0080] In yet another combination method, recombinant antibodies
are generated from single, isolated lymphocytes using a procedure
referred to in the art as the selected lymphocyte antibody method
(SLAM), as described in U.S. Pat. No. 5,627,052, PCT Publication WO
92/02551 and Babcock, J. S. et al. (1996) Proc. Natl. Acad. Sci.
USA 93:7843-7848. In this method, as applied to the dual
specificity antibodies of the invention, a non-human animal (e.g.,
a mouse, rat, rabbit, goat, or transgenic version thereof, or a
chimeric mouse) first is immunized in vivo with the dual
specificity antigen to stimulate an antibody response against the
antigen and then single cells secreting antibodies of interest,
e.g., specific for the dual specificity antigen, are selected using
an antigen-specific hemolytic plaque assay (e.g., the dual
specificity antigen itself, or the structurally-related molecules
of interest, are coupled to sheep red blood cells using a linker,
such as biotin, thereby allowing for identification of single cells
that secrete antibodies with the appropriate specificity using the
hemolytic plaque assay). Following identification of
antibody-secreting cells of interest, heavy- and light-chain
variable region cDNAs are rescued from the cells by reverse
transcriptase-PCR and these variable regions can then be expressed,
in the context of appropriate immunoglobulin constant regions
(e.g., human constant regions), in mammalian host cells, such as
COS or CHO cells. The host cells transfected with the amplified
immunoglobulin sequences, derived from in vivo selected
lymphocytes, can then undergo further analysis and selection in
vitro, for example by panning the transfected cells to isolate
cells expressing antibodies having the desired dual specificity.
The amplified immunoglobulin sequences further can be manipulated
in vitro, such as by in vitro affinity maturation, as described
above.
[0081] In another embodiment, the combination method to produce a
dual specific antibody involves the following steps. A first
non-human animal is immunized with a first antigen and a second
non-human animal is immunized with a second different antigen,
wherein preferably the second antigen is structurally similar to
the first antigen, to stimulate an antibody response in vivo. A
recombinant heavy chain library and a recombinant light chain
library are constructed from antibody genes derived from the first
non-human animal and the second non-human animal, respectively, as
described in section IIB. The heavy chain library from the animal
immunized with the first antigen is combined with the light chain
library from the animal immunized with the second antigen to
generate an antibody library X. Similarly, the heavy chain library
from the animal immunized with the second antigen is combined with
the light chain library from the animal immunized with the first
antigen to generate an antibody library Y. Additionally, libraries
X and Y can be combined to generate library XY. Dual specific
antibodies that bind both first and second antigen can be
identified and isolated from X, Y and/or XY libraries.
III. Characteristics of Dual Specificity Antibodies
[0082] The invention provides dual specificity antibodies, as well
as antibody portions thereof, that can be prepared in accordance
with the methods of the invention. Preferably, the antibodies, or
portions thereof, are isolated antibodies. Preferably, the
antibodies, or portions thereof, are neutralizing antibodies. The
antibodies of the invention include monoclonal and recombinant
antibodies, and portions thereof. In various embodiments, the
antibody, or portion thereof, may comprise amino acid sequences
derived entirely from a single species, such as a fully human or
fully mouse antibody, or portion thereof. In other embodiments, the
antibody, or portion thereof, can be a chimeric antibody or a
CDR-grafted antibody or other form of humanized antibody.
[0083] The term "antibody", as used herein, is intended to refer to
immunoglobulin molecules comprised of four polypeptide chains, two
heavy (H) chains and two light (L) chains inter-connected by
disulfide bonds. Each heavy chain is comprised of a heavy chain
variable region (abbreviated herein as HCVR or VH) and a heavy
chain constant region. The heavy chain constant region is comprised
of three domains, CH1, CH2 and CH3. Each light chain is comprised
of a light chain variable region (abbreviated herein as LCVR or VL)
and a light chain constant region. The light chain constant region
is comprised of one domain, CL. The VH and VL regions can be
further subdivided into regions of hypervariability, termed
complementarity determining regions (CDR), interspersed with
regions that are more conserved, termed framework regions (FR).
Each VH and VL is composed of three CDRs and four FRs, arranged
from amino-terminus to carboxy-terminus in the following order:
FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4.
[0084] The term "antigen-binding portion" of an antibody (or simply
"antibody portion"), as used herein, refers to one or more
fragments of a dual specificity antibody that retain the ability to
specifically bind two different but structurally related antigens.
It has been shown that the antigen-binding function of an antibody
can be performed by fragments of a full-length antibody. Examples
of binding fragments encompassed within the term "antigen-binding
portion" of an antibody include (i) a Fab fragment, a monovalent
fragment consisting of the VL, VH, CL and CH1 domains; (ii) a
F(ab').sub.2 fragment, a bivalent fragment comprising two Fab
fragments linked by a disulfide bridge at the hinge region; (iii) a
Fd fragment consisting of the VH and CH1 domains; (iv) a Fv
fragment consisting of the VL and VH domains of a single arm of an
antibody, (v) a dAb fragment (Ward et al., (1989) Nature
341:544-546), which consists of a VH domain; and (vi) an isolated
complementarity determining region (CDR). Furthermore, although the
two domains of the Fv fragment, VL and VH, are coded for by
separate genes, they can be joined, using recombinant methods, by a
synthetic linker that enables them to be made as a single protein
chain in which the VL and VH regions pair to form monovalent
molecules (known as single chain Fv (scFv); see e.g., Bird et al.
(1988) Science 242:423-426; and Huston et al. (1988) Proc. Natl.
Acad. Sci. USA 85:5879-5883). Such single chain antibodies are also
intended to be encompassed within the term "antigen-binding
portion" of an antibody. Other forms of single chain antibodies,
such as diabodies are also encompassed. Diabodies are bivalent,
bispecific antibodies in which VH and VL domains are expressed on a
single polypeptide chain, but using a linker that is too short to
allow for pairing between the two domains on the same chain,
thereby forcing the domains to pair with complementary domains of
another chain and creating two antigen binding sites (see e.g.,
Holliger, P., et al. (1993) Proc. Natl. Acad. Sci. USA
90:6444-6448; Poljak, R. J., et al. (1994) Structure
2:1121-1123).
[0085] Still further, an antibody or antigen-binding portion
thereof may be part of a larger immunoadhesion molecules, formed by
covalent or noncovalent association of the antibody or antibody
portion with one or more other proteins or peptides. Examples of
such immunoadhesion molecules include use of the streptavidin core
region to make a tetrameric scFv molecule (Kipriyanov, S. M., et
al. (1995) Human Antibodies and Hybridomas 6:93-101) and use of a
cysteine residue, a marker peptide and a C-terminal polyhistidine
tag to make bivalent and biotinylated scFv molecules (Kipriyanov,
S. M., et al. (1994) Mol. Immunol. 31:1047-1058). Antibody
portions, such as Fab and F(ab').sub.2 fragments, can be prepared
from whole antibodies using conventional techniques, such as papain
or pepsin digestion, respectively, of whole antibodies. Moreover,
antibodies, antibody portions and immunoadhesion molecules can be
obtained using standard recombinant DNA techniques.
[0086] An "isolated dual specificity antibody", as used herein, is
intended to refer to an a dual specificity antibody that is
substantially free of other antibodies having different antigenic
specificities (e.g., an isolated antibody that specifically binds
two different but structurally related antigens, or
structurally-related regions of otherwise unrelated antigens, but
that is substantially free of antibodies that specifically bind
other unrelated antigens). Moreover, an isolated dual specificity
antibody may be substantially free of other cellular material
and/or chemicals.
[0087] A "neutralizing antibody", as used is intended to refer to
an antibody whose binding to a particular antigen results in
inhibition of the biological activity of the antigen. This
inhibition of the biological activity of the antigen can be
assessed by measuring one or more indicators of biological activity
of the antigen using an appropriate in vitro or in vivo assay.
[0088] A "monoclonal antibody" as used herein is intended to refer
to a hybridoma-derived antibody (e.g., an antibody secreted by a
hybridoma prepared by hybridoma technology, such as the standard
Kohler and Milstein hybridoma methodology). Thus, a
hybridoma-derived dual specificity antibody of the invention is
still referred to as a monoclonal antibody although it has
antigenic specificity for more than a single antigen.
[0089] The phrase "recombinant antibody" refers to antibodies that
are prepared, expressed, created or isolated by recombinant means,
such as antibodies expressed using a recombinant expression vector
transfected into a host cell, antibodies isolated from a
recombinant, combinatorial antibody library, antibodies isolated
from an animal (e.g., a mouse) that is transgenic for human
immunoglobulin genes (see e.g., Taylor, L. D., et al. (1992) Nucl.
Acids Res. 20:6287-6295) or antibodies prepared, expressed, created
or isolated by any other means that involves splicing of particular
immunoglobulin gene sequences (such as human immunoglobulin gene
sequences) to other DNA sequences. Examples of recombinant
antibodies include chimeric, CDR-grafted and humanized
antibodies.
[0090] The term "human antibody" refers to antibodies having
variable and constant regions corresponding to, or derived from,
human germline immunoglobulin sequences as described by, for
example, Kabat et al. (See Kabat, et al. (1991) Sequences of
Proteins of Immunological Interest, Fifth Edition, U.S. Department
of Health and Human Services, NIH Publication No. 91-3242). The
human antibodies of the invention, however, may include amino acid
residues not encoded by human germline immunoglobulin sequences
(e.g., mutations introduced by random or site-specific mutagenesis
in vitro or by somatic mutation in vivo), for example in the CDRs
and in particular CDR3.
[0091] Recombinant human antibodies of the invention have variable
regions, and may also include constant regions, derived from human
germline immunoglobulin sequences (See Kabat, E. A., et al. (1991)
Sequences of Proteins of Immunological Interest, Fifth Edition,
U.S. Department of Health and Human Services, NIH Publication No.
91-3242). In certain embodiments, however, such recombinant human
antibodies are subjected to in vitro mutagenesis (or, when an
animal transgenic for human Ig sequences is used, in vivo somatic
mutagenesis) and thus the amino acid sequences of the VH and VL
regions of the recombinant antibodies are sequences that, while
derived from and related to human germline VH and VL sequences, may
not naturally exist within the human antibody germline repertoire
in vivo. In certain embodiments, however, such recombinant
antibodies are the result of selective mutagenesis or backmutation
or both.
[0092] The term "backmutation" refers to a process in which some or
all of the somatically mutated amino acids of a human antibody are
replaced with the corresponding germline residues from a homologous
germline antibody sequence. The heavy and light chain sequences of
a human antibody of the invention are aligned separately with the
germline sequences in the VBASE database to identify the sequences
with the highest homology. Differences in the human antibody of the
invention are returned to the germline sequence by mutating defined
nucleotide positions encoding such different amino acid. The role
of each amino acid thus identified as candidate for backmutation
should be investigated for a direct or indirect role in antigen
binding and any amino acid found after mutation to affect any
desirable characteristic of the human antibody should not be
included in the final human antibody. To minimize the number of
amino acids subject to backmutation those amino acid positions
found to be different from the closest germline sequence but
identical to the corresponding amino acid in a second germline
sequence can remain, provided that the second germline sequence is
identical and colinear to the sequence of the human antibody of the
invention for at least 10, preferably 12 amino acids, on both sides
of the amino acid in question. Backmuation may occur at any stage
of antibody optimization.
[0093] The term "chimeric antibody" refers to antibodies which
comprise heavy and light chain variable region sequences from one
species and constant region sequences from another species, such as
antibodies having murine heavy and light chain variable regions
linked to human constant regions.
[0094] The term "CDR-grafted antibody" refers to antibodies which
comprise heavy and light chain variable region sequences from one
species but in which the sequences of one or more of the CDR
regions of V.sub.H and/or VL are replaced with CDR sequences of
another species, such as antibodies having murine heavy and light
chain variable regions in which one or more of the murine CDRs
(e.g., CDR3) has been replaced with human CDR sequences.
[0095] The term "humanized antibody" refers to antibodies which
comprise heavy and light chain variable region sequences from a
non-human species (e.g., a mouse) but in which at least a portion
of the VH and/or VL sequence has been altered to be more
"human-like", i.e., more similar to human germline variable
sequences. One type of humanized antibody is a CDR-grafted
antibody, in which human CDR sequences are introduced into
non-human VH and VL sequences to replace the corresponding nonhuman
CDR sequences.
[0096] One way of measuring the binding kinetics of an antibody is
by surface plasmon resonance. The term "surface plasmon resonance",
as used herein, refers to an optical phenomenon that allows for the
analysis of real-time biospecific interactions by detection of
alterations in protein concentrations within a biosensor matrix,
for example using the BIAcore system (Pharmacia Biosensor AB,
Uppsala, Sweden and Piscataway, N.J.). For further descriptions,
see Jonsson, U., et al. (1993) Ann. Biol. Clin. 51:19-26; Jonsson,
U., et al. (1991) Biotechniques 11:620-627; Johnsson, B., et al.
(1995) J. Mol. Recognit. 8:125-131; and Johnnson, B., et al. (1991)
Anal. Biochem. 198:268-277.
[0097] The term "K.sub.off", as used herein, is intended to refer
to the off rate constant for dissociation of an antibody from the
antibody/antigen complex.
[0098] The term "K.sub.d", as used herein, is intended to refer to
the dissociation constant of a particular antibody-antigen
interaction.
[0099] The dual specificity antibodies of the invention are
prepared using any of the various methods for preparing antibodies
described in subsection II above. The dual specificity antibodies
of the invention may be directed against essentially any
structurally related antigens, although preferred dual specificity
antibodies of the invention are those that specifically bind
IL-1.alpha. and IL-10, which can be prepared using a dual
specificity antigen such as those described in Examples 1-4. Other
structurally related antigens that can be applied to the current
invention include but are not limited to caspase family members,
cytokine families, such as IL-1 family members (e.g., IL-1/IL-18),
TNF family members (e.g., TNF.alpha./TNF.beta.), IL-6 family
members, Interferons, TGF family members, EGF family members, FGF
family members, PDGF family members, VEGF family members,
Angiopoietin family members, Bone morphogenic proteins, secreted
proteinases (metallo-proteinases), and cytokine receptor families,
such as IL-1-receptor family members, TNF-receptors family members
TGF.beta. receptor family members, EGF receptor family members, FGF
receptor family members, PDGF receptor family members, VEGF
receptor family members and Angiopoietin receptor family
members.
[0100] The dual specificity antibodies of the invention may display
equal binding activity toward the two different but structurally
related antigens to which it binds or, alternatively, the dual
specificity antibodies may bind more preferentially to one of the
two antigens, yet still have specificity towards the two related
antigens as compared to unrelated antigens. The binding activity of
the dual specificity antibodies toward the structurally related
antigens, as well as toward unrelated antigens, can be assessed
using standard in vitro immunoassays, such as ELISA or BIAcore
analysis. Preferably, the ratio of K.sub.d of antibody toward
structurally unrelated antigens to the K.sub.d of antibody toward
structurally related antigens should be at least 3, even more
preferably the ratio should be at least 5, even more preferably the
ratio should be at least 10, or even more preferably the ratio
should be at least 50, 100, 200, 300, 400, 500, 600, 700, 800, 900
or 1000.
[0101] In quantitative terms, the difference between background
binding and dual specificity is one of level or degree. For
example, background binding is at a low level, e.g., less than 5%,
more preferably less than 3% and most preferably, about 0.1-1%
whereas specific cross-reactivity or dual specificity binding is at
a higher level, e.g., greater than 1%, more preferably greater than
3%, even more preferably greater than 5% and even more preferably
greater than 10%. Additionally, preferably the IC.sub.50 of the
dual specificity antibody for the target antigens is close to the
ED.sub.50s of the antigens in a given bioassay.
[0102] A dual specificity antibody, or antigen-binding portion
thereof, of the invention is preferably selected to have desirable
binding kinetics (e.g., high affinity, low dissociation, slow
off-rate, strong neutralizing activity) for one, and more
preferably both, of the antigens to which it specifically binds.
For example, the dual specificity antibody, or portion thereof, may
bind one, and more preferably both, of the structurally related
antigens with a k.sub.off rate constant of 0.1 s.sup.-1 or less,
more preferably a k.sub.off rate constant of 1.times.10.sup.-2
s.sup.-1 or less, even more preferably a k.sub.off rate constant of
1.times.10.sup.-3 s.sup.-1 or less, even more preferably a
k.sub.off rate constant of 1.times.10.sup.-4 s.sup.-1 or less, or
even more preferably a k.sub.off rate constant of 1.times.10.sup.-5
s.sup.1 or less, as determined by surface plasmon resonance.
Alternatively or additionally, a dual specificity antibody, or
portion thereof, may inhibit the activity of one, and more
preferably both, of the structurally related antigens with an
IC.sub.50 of 1.times.10.sup.-6M or less, even more preferably with
an IC.sub.50 of 1.times.10.sup.-7M or less, even more preferably
with an IC.sub.50 of 1.times.10.sup.-8M or less, even more
preferably with an IC.sub.50 of 1.times.10.sup.-9M or less, even
more preferably with an IC.sub.50 of 1.times.10.sup.-10M or less,
or even more preferably with an IC.sub.50 of 1.times.10.sup.-11M or
less. Preferably, IC.sub.50 should be measured using a sensitive
bioassay where IC.sub.50 values should be close to the ED.sub.50
value of the antigen in that assay.
[0103] The invention also provides pharmaceutical compositions
comprising a dual specificity antibody, or antigen-binding portion
thereof, of the invention and a pharmaceutically acceptable
carrier. The pharmaceutical composition of the invention can
further comprise at least one additional therapeutic agent, e.g.,
one or more additional therapeutic agents for treating a disorder
in which use of the dual specificity antibody is beneficial to
amelioration of the disorder. For example, when the dual
specificity antibody specifically binds IL-1.alpha. and IL-1.beta.,
the pharmaceutical composition can further include one or more
additional therapeutic agents for treating disorders in which IL-1
activity is detrimental.
[0104] The antibodies and antibody-portions of the invention can be
incorporated into pharmaceutical compositions suitable for
administration to a subject. Typically, the pharmaceutical
composition comprises an antibody or antibody portion of the
invention and a pharmaceutically acceptable carrier. As used
herein, "pharmaceutically acceptable carrier" includes any and all
solvents, dispersion media, coatings, antibacterial and antifungal
agents, isotonic and absorption delaying agents, and the like that
are physiologically compatible. Examples of pharmaceutically
acceptable carriers include one or more of water, saline, phosphate
buffered saline, dextrose, glycerol, ethanol and the like, as well
as combinations thereof. In many cases, it will be preferable to
include isotonic agents, for example, sugars, polyalcohols such as
mannitol, sorbitol, or sodium chloride in the composition.
Pharmaceutically acceptable carriers may further comprise minor
amounts of auxiliary substances such as wetting or emulsifying
agents, preservatives or buffers, which enhance the shelf life or
effectiveness of the antibody or antibody portion.
[0105] The antibodies and antibody-portions of the invention can be
incorporated into a pharmaceutical composition suitable for
parenteral administration. Preferably, the antibody or
antibody-portions will be prepared as an injectable solution
containing 0.1-250 mg/ml antibody. The injectable solution can be
composed of either a liquid or lyophilized dosage form in a flint
or amber vial, ampule or pre-filled syringe. The buffer can be
L-histidine (1-50 mM), optimally 5-10 mM, at pH 5.0 to 7.0
(optimally pH 6.0). Other suitable buffers include but are not
limited to, sodium succinate, sodium citrate, sodium phosphate or
potassium phosphate. Sodium chloride can be used to modify the
toxicity of the solution at a concentration of 0-300 mM (optimally
150 mM for a liquid dosage form). Cryoprotectants can be included
for a lyophilized dosage form, principally 0-10% sucrose (optimally
0.5-1.0%). Other suitable cryoprotectants include trehalose and
lactose. Bulking agents can be included for a lyophilized dosage
form, principally 1-10% mannitol (optimally 2-4%). Stabilizers can
be used in both liquid and lyophilized dosage forms, principally
1-50 mM L-Methionine (optimally 5-10 mM). Other suitable bulking
agents include glycine, arginine, can be included as 0-0.05%
polysorbate-80 (optimally 0.005-0.01%). Additional surfactants
include but are not limited to polysorbate 20 and BRIJ
surfactants.
[0106] The compositions of this invention may be in a variety of
forms. These include, for example, liquid, semi-solid and solid
dosage forms, such as liquid solutions (e.g., injectable and
infusible solutions), dispersions or suspensions, tablets, pills,
powders, liposomes and suppositories. The preferred form depends on
the intended mode of administration and therapeutic application.
Typical preferred compositions are in the form of injectable or
infusible solutions, such as compositions similar to those used for
passive immunization of humans with other antibodies. The preferred
mode of administration is parenteral (e.g., intravenous,
subcutaneous, intraperitoneal, intramuscular). In a preferred
embodiment, the antibody is administered by intravenous infusion or
injection. In another preferred embodiment, the antibody is
administered by intramuscular or subcutaneous injection.
[0107] Therapeutic compositions typically must be sterile and
stable under the conditions of manufacture and storage. The
composition can be formulated as a solution, microemulsion,
dispersion, liposome, or other ordered structure suitable to high
drug concentration. Sterile injectable solutions can be prepared by
incorporating the active compound (i.e., antibody or antibody
portion) in the required amount in an appropriate solvent with one
or a combination of ingredients enumerated above, as required,
followed by filtered sterilization. Generally, dispersions are
prepared by incorporating the active compound into a sterile
vehicle that contains a basic dispersion medium and the required
other ingredients from those enumerated above. In the case of
sterile, lyophilized powders for the preparation of sterile
injectable solutions, the preferred methods of preparation are
vacuum drying and spray-drying that yields a powder of the active
ingredient plus any additional desired ingredient from a previously
sterile-filtered solution thereof. The proper fluidity of a
solution can be maintained, for example, by the use of a coating
such as lecithin, by the maintenance of the required particle size
in the case of dispersion and by the use of surfactants. Prolonged
absorption of injectable compositions can be brought about by
including in the composition an agent that delays absorption, for
example, monostearate salts and gelatin.
[0108] The antibodies and antibody-portions of the present
invention can be administered by a variety of methods known in the
art, although for many therapeutic applications, the preferred
route/mode of administration is subcutaneous injection, intravenous
injection or infusion. As will be appreciated by the skilled
artisan, the route and/or mode of administration will vary
depending upon the desired results. In certain embodiments, the
active compound may be prepared with a carrier that will protect
the compound against rapid release, such as a controlled release
formulation, including implants, transdermal patches, and
microencapsulated delivery systems. Biodegradable, biocompatible
polymers can be used, such as ethylene vinyl acetate,
polyanhydrides, polyglycolic acid, collagen, polyorthoesters, and
polylactic acid. Many methods for the preparation of such
formulations are patented or generally known to those skilled in
the art. See, e.g., Sustained and Controlled Release Drug Delivery
Systems, J. R. Robinson, ed., Marcel Dekker, Inc., New York,
1978.
[0109] In certain embodiments, an antibody or antibody portion of
the invention may be orally administered, for example, with an
inert diluent or an assimilable edible carrier. The compound (and
other ingredients, if desired) may also be enclosed in a hard or
soft shell gelatin capsule, compressed into tablets, or
incorporated directly into the subject's diet. For oral therapeutic
administration, the compounds may be incorporated with excipients
and used in the form of ingestible tablets, buccal tablets,
troches, capsules, elixirs, suspensions, syrups, wafers, and the
like. To administer a compound of the invention by other than
parenteral administration, it may be necessary to coat the compound
with, or co-administer the compound with, a material to prevent its
inactivation.
[0110] Supplementary active compounds can also be incorporated into
the compositions. In certain embodiments, an antibody or antibody
portion of the invention is coformulated with and/or coadministered
with one or more additional therapeutic agents that are useful for
treating disorders in which IL-1 activity is detrimental. For
example, an anti-IL-1.alpha./IL-1.beta. dual specificity
antibodies, or antibody portions, of the invention may be
coformulated and/or coadministered with one or more additional
antibodies that bind other targets (e.g., antibodies that bind
other cytokines or that bind cell surface molecules). Furthermore,
one or more antibodies of the invention may be used in combination
with two or more of the foregoing therapeutic agents. Such
combination therapies may advantageously utilize lower dosages of
the administered therapeutic agents, thus avoiding possible
toxicities or complications associated with the various
monotherapies.
IV. Uses of Dual Specificity Antibodies
[0111] Given their ability to bind two different but structurally
related antigens, the dual specificity antibodies, or portions
thereof, of the invention can be used to detect either or both of
these antigens (e.g., in a biological sample, such as serum or
plasma), using a conventional immunoassay, such as an enzyme linked
immunosorbent assays (ELISA), an radioimmunoassay (RIA) or tissue
immunohistochemistry. The invention provides a method for detecting
an antigen in a biological sample comprising contacting a
biological sample with a dual specificity antibody, or antibody
portion, of the invention that specifically recognizes the antigen
and detecting either the antibody (or antibody portion) bound to
antigen or unbound antibody (or antibody portion), to thereby
detect the antigen in the biological sample. The antibody is
directly or indirectly labeled with a detectable substance to
facilitate detection of the bound or unbound antibody. Suitable
detectable substances include various enzymes, prosthetic groups,
fluorescent materials, luminescent materials and radioactive
materials. Examples of suitable enzymes include horseradish
peroxidase, alkaline phosphatase, .beta.-galactosidase, or
acetylcholinesterase; examples of suitable prosthetic group
complexes include streptavidin/biotin and avidin/biotin; examples
of suitable fluorescent materials include umbelliferone,
fluorescein, fluorescein isothiocyanate, rhodamine,
dichlorotriazinylamine fluorescein, dansyl chloride or
phycoerythrin; an example of a luminescent material includes
luminol; and examples of suitable radioactive material include
.sup.125I, .sup.131I, .sup.35S or .sup.3H.
[0112] Alternative to labeling the antibody, the antigen(s) can be
assayed in biological fluids by a competition radioimmunoassay
utilizing antigen standards labeled with a detectable substance and
an unlabeled dual specificity antibody specific for the antigen(s).
In this assay, the biological sample, the labeled antigen standards
and the dual specificity antibody are combined and the amount of
labeled antigen standard bound to the unlabeled antibody is
determined. The amount of antigen in the biological sample is
inversely proportional to the amount of labeled antigen standard
bound to the unlabeled antibody.
[0113] In a preferred embodiment, the dual specificity antibody
specifically recognizes IL-1.alpha. and IL-1.beta. and the
foregoing detection methods are used to detect IL-1.alpha. and/or
IL-1.beta.. Accordingly, the invention further provides a method of
detecting IL-1.alpha. or IL-1.beta. in a biological sample or
tissue comprising contacting the biological sample or tissue
suspected of containing IL-1.alpha. or IL-1.beta. with a
dual-specificity antibody, or antigen-binding portion thereof, of
the invention and detecting IL-1.alpha. or IL-1.beta. in the
biological sample or tissue. The biological sample can be, for
example, an in vitro sample, such as a sample of cells, tissue or
bodily fluid (e.g., blood, plasma, urine, saliva etc.). Moreover,
the tissue detected can be tissue located in vivo in a subject,
e.g., tissue visualized by in vivo imaging of the tissue (e.g.,
using a labeled antibody)
[0114] The dual specificity antibodies of the invention also can be
used for diagnostic purposes. In one embodiment, an antibody of the
invention is used in a diagnostic assay in vitro, such as in a
laboratory test to detect the antigen(s) of interest or in a point
of care test to detect the antigen(s) of interest. Examples of
well-established in vitro assays utilizing antibodies include
ELISAs, RIAs, Western blots and the like. In another embodiment, an
antibody of the invention is used in a diagnostic assay in vivo,
such as an in vivo imaging test. For example, the antibody can be
labeled with a detectable substance capable of being detected in
vivo, the labeled antibody can be administered to a subject, and
the labeled antibody can be detected in vivo, thereby allowing for
in vivo imaging.
[0115] Dual specificity antibodies of the invention that
specifically recognize IL-1.alpha. and IL-1.beta. can be used in
diagnostic assays to detect IL-1.alpha. and/or IL-1.beta. for
diagnostic purposes, for example in a variety of inflammatory
diseases and disorders, as well as in spontaneous resorption of
fetuses. With regard to specific types of diseases and disorders,
the dual specificity anti-IL-1.alpha./IL-1.beta. antibodies of the
invention can be used for diagnostic purposes in any of the
diseases/disorders described herein with regard to the therapeutic
uses of such antibodies (see below), such as disorders in which
IL-1 activity is detrimental, discussed further below.
[0116] The dual specificity antibodies and antibody portions of the
invention preferably are capable of neutralizing, both in vitro and
in vivo, the activity of the antigens to which they bind.
Accordingly, such antibodies and antibody portions of the invention
can be used to inhibit the activity of the antigens, e.g., in a
cell culture containing the antigens or in human subjects or in
other mammalian subjects having the antigens with which the dual
specificity antibody of the invention reacts. In one embodiment,
the invention provides a method for inhibiting antigen activity
comprising contacting the antigen with a dual specificity antibody
or antibody portion of the invention such that antigen activity is
inhibited. In a preferred embodiment, the dual specificity antibody
binds IL-1.alpha. and IL-1.beta. and the method is a method for
inhibiting IL-1.alpha. and/or IL-1.beta. activity by contacting
IL-1.alpha. and/or IL-1.beta. with the dual specificity antibody,
or portion thereof. The IL-1.alpha. and/or IL-1.beta. activity can
be inhibited, for example, in vitro. For example, in a cell culture
containing, or suspected of containing, IL-1.alpha. and/or
IL-1.beta., an antibody or antibody portion of the invention can be
added to the culture medium to inhibit IL-1.alpha. and/or
IL-1.beta. activity in the culture. Alternatively, IL-1.alpha.
and/or IL-1.beta. activity can be inhibited in vivo in a
subject.
[0117] In another embodiment, the invention provides a method for
inhibiting antigen activity in a subject suffering from a disorder
in which that antigen activity is detrimental. The invention
provides methods for inhibiting antigen activity in a subject
suffering from such a disorder, which method comprises
administering to the subject a dual specificity antibody or
antibody portion of the invention such that antigen activity in the
subject is inhibited. Preferably, the antigen is a human antigen
and the subject is a human subject. An antibody of the invention
can be administered to a human subject for therapeutic purposes.
Moreover, an antibody of the invention can be administered to a
non-human mammal expressing an antigen with which the antibody
binds for veterinary purposes or as an animal model of human
disease. Regarding the latter, such animal models may be useful for
evaluating the therapeutic efficacy of antibodies of the invention
(e.g., testing of dosages and time courses of administration).
[0118] Preferably, the dual specificity antibody binds IL-1.alpha.
and IL-1.beta. and the method for inhibiting antigen activity in a
subject is a method for inhibiting IL-1 activity in a subject, for
example a subject suffering from a disorder in which IL-1 activity
is detrimental. As used herein, the term "a disorder in which IL-1
activity is detrimental" is intended to include diseases and other
disorders in which the presence of IL-1 (which encompasses both
IL-1.alpha. and IL-1.beta.) in a subject suffering from the
disorder has been shown to be or is suspected of being either
responsible for the pathophysiology of the disorder or a factor
that contributes to a worsening of the disorder. Accordingly, a
disorder in which IL-1 activity is detrimental is a disorder in
which inhibition of IL-1 activity (i.e., either or both of
IL-1.alpha. and IL-1.beta.) is expected to alleviate the symptoms
and/or progression of the disorder. Such disorders may be
evidenced, for example, by an increase in the concentration of IL-1
in a biological fluid of a subject suffering from the disorder
(e.g., an increase in the concentration of IL-1 in serum, plasma,
synovial fluid, etc. of the subject), which can be detected, for
example, using an anti-IL-1 antibody as described above.
[0119] Interleukin 1 plays a critical role in the pathology
associated with a variety of diseases involving immune and
inflammatory elements. These diseases include, but are not limited
to, rheumatoid arthritis, osteoarthritis, juvenile chronic
arthritis, Lyme arthritis, psoriatic arthritis, reactive arthritis,
spondyloarthropathy, systemic lupus erythematosus, Crohn's disease,
ulcerative colitis, inflammatory bowel disease, insulin dependent
diabetes mellitus, thyroiditis, asthma, allergic diseases,
psoriasis, dermatitis scleroderma, graft versus host disease, organ
transplant rejection, acute or chronic immune disease associated
with organ transplantation, sarcoidosis, atherosclerosis,
disseminated intravascular coagulation, Kawasaki's disease, Grave's
disease, nephrotic syndrome, chronic fatigue syndrome, Wegener's
granulomatosis, Henoch-Schoenlein purpurea, microscopic vasculitis
of the kidneys, chronic active hepatitis, uveitis, septic shock,
toxic shock syndrome, sepsis syndrome, cachexia, infectious
diseases, parasitic diseases, acquired immunodeficiency syndrome,
acute transverse myelitis, Huntington's chorea, Parkinson's
disease, Alzheimer's disease, stroke, primary biliary cirrhosis,
hemolytic anemia, malignancies, heart failure, myocardial
infarction, Addison's disease, sporadic, polyglandular deficiency
type I and polyglandular deficiency type II, Schmidt's syndrome,
adult (acute) respiratory distress syndrome, alopecia, alopecia
areata, seronegative arthopathy, arthropathy, Reiter's disease,
psoriatic arthropathy, ulcerative colitic arthropathy, enteropathic
synovitis, chlamydia, yeisinia and salmonella associated
arthropathy, spondyloarthopathy, atheromatous
disease/arteriosclerosis, atopic allergy, autoimmune bullous
disease, pemphigus vulgaris, pemphigus foliaceus, pemphigoid,
linear IgA disease, autoimmune haemolytic anaemia, Coombs positive
haemolytic anaemia, acquired pernicious anaemia, juvenile
pernicious anaemia, myalgic encephalitis/Royal Free Disease,
chronic mucocutaneous candidiasis, giant cell arteritis, primary
sclerosing hepatitis, cryptogenic autoimmune hepatitis, Acquired
Immunodeficiency Disease Syndrome, Acquired Immunodeficiency
Related Diseases, Hepatitis C, common varied immunodeficiency
(common variable hypogammaglobulinaemia), dilated cardiomyopathy,
female infertility, ovarian failure, premature ovarian failure,
fibrotic lung disease, cryptogenic fibrosing alveolitis,
post-inflammatory interstitial lung disease, interstitial
pneumonitis, connective tissue disease associated interstitial lung
disease, mixed connective tissue disease associated lung disease,
systemic sclerosis associated interstitial lung disease, rheumatoid
arthritis associated interstitial lung disease, systemic lupus
erythematosus associated lung disease, dermatomyositis/polymyositis
associated lung disease, Sjogren's disease associated lung disease,
ankylosing spondylitis associated lung disease, vasculitic diffuse
lung disease, haemosiderosis associated lung disease, drug-induced
interstitial lung disease, radiation fibrosis, bronchiolitis
obliterans, chronic eosinophilic pneumonia, lymphocytic
infiltrative lung disease, postinfectious interstitial lung
disease, gouty arthritis, autoimmune hepatitis, type-1 autoimmune
hepatitis (classical autoimmune or lupoid hepatitis), type-2
autoimmune hepatitis (anti-LKM antibody hepatitis), autoimmune
mediated hypoglycaemia, type B insulin resistance with acanthosis
nigricans, hypoparathyroidism, acute immune disease associated with
organ transplantation, chronic immune disease associated with organ
transplantation, osteoarthrosis, primary sclerosing cholangitis,
psoriasis type 1, psoriasis type 2, idiopathic leucopaenia,
autoimmune neutropaenia, renal disease NOS, glomerulonephritides,
microscopic vasulitis of the kidneys, lyme disease, discoid lupus
erythematosus, male infertility idiopathic or NOS, sperm
autoimmunity, multiple sclerosis (all subtypes), sympathetic
ophthalmia, pulmonary hypertension secondary to connective tissue
disease, Goodpasture's syndrome, pulmonary manifestation of
polyarteritis nodosa, acute rheumatic fever, rheumatoid
spondylitis, Still's disease, systemic sclerosis, Sjorgren's
syndrome, Takayasu's disease/arteritis, autoimmune
thrombocytopaenia, idiopathic thrombocytopaenia, autoimmune thyroid
disease, hyperthyroidism, goitrous autoimmune hypothyroidism
(Hashimoto's disease), atrophic autoimmune hypothyroidism, primary
myxoedema, phacogenic uveitis, primary vasculitis, vitiligo,
diseases of the central nervous system (e.g., depression,
schizophrenia, Alzheimers, Parkinsons, etc.), acute and chronic
pain, and lipid imbalance. The human antibodies, and antibody
portions of the invention can be used to treat humans suffering
from autoimmune diseases, in particular those associated with
inflammation, including, rheumatoid spondylitis, allergy,
autoimmune diabetes, autoimmune uveitis.
[0120] Preferably, the IL-1.alpha./IL-1.beta. dual specificity
antibodies of the invention or antigen-binding portions thereof,
are used to treat rheumatoid arthritis, Crohn's disease, multiple
sclerosis, insulin dependent diabetes, mellitus and psoriasis.
[0121] An IL-1.alpha./IL-1.beta. dual specificity antibody, or
antibody portion, of the invention also can be administered with
one or more additional therapeutic agents useful in the treatment
of autoimmune and inflammatory diseases.
[0122] Antibodies of the invention, or antigen binding portions
thereof can be used alone or in combination to treat such diseases.
It should be understood that the antibodies of the invention or
antigen binding portion thereof can be used alone or in combination
with an additional agent, e.g., a therapeutic agent, said
additional agent being selected by the skilled artisan for its
intended purpose. For example, the additional agent can be a
therapeutic agent art-recognized as being useful to treat the
disease or condition being treated by the antibody of the present
invention. The additional agent also can be an agent which imparts
a beneficial attribute to the therapeutic composition e.g., an
agent which effects the viscosity of the composition.
[0123] It should further be understood that the combinations which
are to be included within this invention are those combinations
useful for their intended purpose. The agents set forth below are
illustrative for purposes and not intended to be limited. The
combinations which are part of this invention can be the antibodies
of the present invention and at least one additional agent selected
from the lists below. The combination can also include more than
one additional agent, e.g., two or three additional agents if the
combination is such that the formed composition can perform its
intended function.
[0124] Preferred combinations are non-steroidal anti-inflammatory
drug(s) also referred to as NSAIDS which include drugs like
ibuprofen and COX-2 inhibitors. Other preferred combinations are
corticosteroids including prednisolone; the well known side-effects
of steroid use can be reduced or even eliminated by tapering the
steroid dose required when treating patients in combination with
the anti-IL-1 antibodies of this invention. Non-limiting examples
of therapeutic agents for rheumatoid arthritis with which an
antibody, or antibody portion, of the invention can be combined
include the following: cytokine suppressive anti-inflammatory
drug(s) (CSAIDs); antibodies to or antagonists of other human
cytokines or growth factors, for example, TNF, LT, IL-2, IL-6,
IL-7, IL-8, IL-12, IL-15, IL-16, IL-18, EMAP-II, GM-CSF, FGF, and
PDGF. Antibodies of the invention, or antigen binding portions
thereof, can be combined with antibodies to cell surface molecules
such as CD2, CD3, CD4, CD8, CD25, CD28, CD30, CD40, CD45, CD69,
CD80 (B7.1), CD86 (B7.2), CD90, or their ligands including CD154
(gp39 or CD40L).
[0125] Preferred combinations of therapeutic agents may interfere
at different points in the autoimmune and subsequent inflammatory
cascade; preferred examples include TNF antagonists like chimeric,
humanized or human TNF antibodies, D2E7, (PCT Publication No. WO
97/29131), CA2 (Remicade.TM.), CDP 571, CDP 870, Thalidamide and
soluble p55 or p75 TNF receptors, derivatives, thereof, (p75TNFR1gG
(Enbrel.TM.) or p55TNFR1gG (Lenercept), and also TNF.alpha.
converting enzyme (TACE) inhibitors; similarly IL-1 inhibitors
(Interleukin-1-converting enzyme inhibitors, IL-1RA etc.) may be
effective for the same reason. Other preferred combinations include
Interleukin 11. Yet another preferred combination are other key
players of the autoimmune response which may act parallel to,
dependent on or in concert with IL-1 function; especially preferred
are IL-12 and/or IL-18 antagonists including IL-12 and/or IL-18
antibodies or soluble IL-12 and/or IL-18 receptors, or IL-12 and/or
IL-18 binding proteins. It has been shown that IL-12 and IL-18 have
overlapping but distinct functions and a combination of antagonists
to both may be most effective. Yet another preferred combination
are non-depleting anti-CD4 inhibitors. Yet other preferred
combinations include antagonists of the co-stimulatory pathway CD80
(B7.1) or CD86 (B7.2) including antibodies, soluble receptors or
antagonistic ligands.
[0126] The antibodies of the invention, or antigen binding portions
thereof, may also be combined with agents, such as methotrexate,
6-MP, azathioprine sulphasalazine, mesalazine, olsalazine
chloroquinine/hydroxychloroquine, pencillamine, aurothiomalate
(intramuscular and oral), azathioprine, cochicine, corticosteroids
(oral, inhaled and local injection), beta-2 adrenoreceptor agonists
(salbutamol, terbutaline, salmeteral), xanthines (theophylline,
aminophylline), cromoglycate, nedocromil, ketotifen, ipratropium
and oxitropium, cyclosporin, FK506, rapamycin, mycophenolate
mofetil, leflunomide, NSAIDs, for example, ibuprofen,
corticosteroids such as prednisolone, phosphodiesterase inhibitors,
adensosine agonists, antithrombotic agents, complement inhibitors,
adrenergic agents, agents which interfere with signaling by
proinflammatory cytokines such as TNF.alpha. or IL-1 (e.g. IRAK,
NIK, IKK, p38 or MAP kinase inhibitors), IL-1.beta. converting
enzyme inhibitors, TNF.alpha. converting enzyme (TACE) inhibitors,
T-cell signalling inhibitors such as kinase inhibitors,
metalloproteinase inhibitors, sulfasalazine, azathioprine,
6-mercaptopurines, angiotensin converting enzyme inhibitors,
soluble cytokine receptors and derivatives thereof (e.g. soluble
p55 or p75 TNF receptors and the derivatives p75TNFRIgG (Enbrel.TM.
and p55TNFRIgG (Lenercept)), sIL-1RI, sIL-1RII, sIL-6R) and
antiinflammatory cytokines (e.g. IL-4, IL-10, IL-11, IL-13 and
TGF.beta.). Preferred combinations include methotrexate or
leflunomide and in moderate or severe rheumatoid arthritis cases,
cyclosporine.
[0127] Non-limiting examples of therapeutic agents for inflammatory
bowel disease with which an antibody, or antibody portion, of the
invention can be combined include the following: budenoside;
epidermal growth factor; corticosteroids; cyclosporin,
sulfasalazine; aminosalicylates; 6-mercaptopurine; azathioprine;
metronidazole; lipoxygenase inhibitors; mesalamine; olsalazine;
balsalazide; antioxidants; thromboxane inhibitors; IL-1 receptor
antagonists; anti-IL-1.beta. monoclonal antibodies; anti-IL-6
monoclonal antibodies; growth factors; elastase inhibitors;
pyridinyl-imidazole compounds; antibodies to or antagonists of
other human cytokines or growth factors, for example, TNF, LT,
IL-2, IL-6, IL-7, IL-8, IL-12, IL-15, IL-16, IL-18, EMAP-II,
GM-CSF, FGF, and PDGF. Antibodies of the invention can be combined
with antibodies to cell surface molecules such as CD2, CD3, CD4,
CD8, CD25, CD28, CD30, CD40, CD45, CD69, CD90 or their ligands. The
antibodies of the invention, or antigen binding portions thereof,
may also be combined with agents, such as methotrexate,
cyclosporin, FK506, rapamycin, mycophenolate mofetil, leflunomide,
NSAIDs, for example, ibuprofen, corticosteroids such as
prednisolone, phosphodiesterase inhibitors, adenosine agonists,
antithrombotic agents, complement inhibitors, adrenergic agents,
agents which interfere with signalling by proinflammatory cytokines
such as TNF.alpha. or IL-1 (e.g. IRAK, NIK, IKK, p38 or MAP kinase
inhibitors), IL-1.beta. converting enzyme inhibitors, TNF.alpha.
converting enzyme inhibitors, T-cell signalling inhibitors such as
kinase inhibitors, metalloproteinase inhibitors, sulfasalazine,
azathioprine, 6-mercaptopurines, angiotensin converting enzyme
inhibitors, soluble cytokine receptors and derivatives thereof
(e.g. soluble p55 or p75 TNF receptors, sIL-1RI, sIL-1RII, sIL-6R)
and antiinflammatory cytokines (e.g. IL-4, IL-10, IL-11, IL-13 and
TGF.beta.).
[0128] Preferred examples of therapeutic agents for Crohn's disease
in which an antibody or an antigen binding portion can be combined
include the following: TNF antagonists, for example, anti-TNF
antibodies, D2E7 (PCT Publication No. WO 97/29131), CA2
(Remicade.TM.), CDP 571, TNFR-Ig constructs, (p75TNFRIgG
(Enbrel.TM.) and p55TNFRIgG (Lenercept)) inhibitors and PDE4
inhibitors. Antibodies, or antigen binding portions thereof, of the
invention or antigen binding portions thereof, can be combined with
corticosteroids, for example, budenoside and dexamethasone.
Antibodies of the invention or antigen binding portions thereof,
may also be combined with agents such as sulfasalazine,
5-aminosalicylic acid and olsalazine, and agents which interfere
with synthesis or action of proinflammatory cytokines such as IL-1,
for example, IL-1.beta. converting enzyme inhibitors and IL-1ra.
Antibodies of the invention or antigen binding portion thereof may
also be used with T cell signaling inhibitors, for example,
tyrosine kinase inhibitors 6-mercaptopurines. Antibodies of the
invention or antigen binding portions thereof, can be combined with
IL-11.
[0129] Non-limiting examples of therapeutic agents for multiple
sclerosis with which an antibody, or antibody portion, of the
invention can be combined include the following: corticosteroids;
prednisolone; methylprednisolone; azathioprine; cyclophosphamide;
cyclosporine; methotrexate; 4-aminopyridine; tizanidine;
interferon-.beta.1a (Avonex; Biogen); interferon-.beta.1b
(Betaseron; Chiron/Berlex); Copolymer 1 (Cop-1; Copaxone; Teva
Pharmaceutical Industries, Inc.); hyperbaric oxygen; intravenous
immunoglobulin; clabribine; antibodies to or antagonists of other
human cytokines or growth factors, for example, TNF, LT, IL-2,
IL-6, IL-7, IL-8, IL-12, IL-15, IL-16, IL-18, EMAP-II, GM-CSF, FGF,
and PDGF. Antibodies of the invention, or antigen binding portions
thereof, can be combined with antibodies to cell surface molecules
such as CD2, CD3, CD4, CD8, CD25, CD28, CD30, CD40, CD45, CD69,
CD80, CD86, CD90 or their ligands. The antibodies of the invention,
or antigen binding portions thereof, may also be combined with
agents, such as methotrexate, cyclosporine, FK506, rapamycin,
mycophenolate mofetil, leflunomide, NSAIDs, for example, ibuprofen,
COX-2 inhibitors, corticosteroids such as prednisolone,
phosphodiesterase inhibitors, adensosine agonists, antithrombotic
agents, complement inhibitors, adrenergic agents, agents which
interfere with signalling by proinflammatory cytokines such as
TNF.alpha. or IL-1 (e.g. IRAK, NIK, IKK, p38 or MAP kinase
inhibitors), IL-1.beta. converting enzyme inhibitors, TACE
inhibitors, T-cell signalling inhibitors such as kinase inhibitors,
metalloproteinase inhibitors, sulfasalazine, azathioprine,
6-mercaptopurines, angiotensin converting enzyme inhibitors,
soluble cytokine receptors and derivatives thereof (e.g. soluble
p55 or p75 TNF receptors, sIL-1RI, sIL-1RII, sIL-6R) and
antiinflammatory cytokines (e.g. IL-4, IL-10, IL-13 and
TGF.beta.).
[0130] Preferred examples of therapeutic agents for multiple
sclerosis in which the antibody or antigen binding portion thereof
can be combined to include interferon-.beta., for example,
IFN.beta.1a and IFN.beta.1b; copaxone, corticosteroids, IL-1
inhibitors, TNF inhibitors, and antibodies to CD40 ligand and
CD80.
[0131] The pharmaceutical compositions of the invention may include
a "therapeutically effective amount" or a "prophylactically
effective amount" of an antibody or antibody portion of the
invention. A "therapeutically effective amount" refers to an amount
effective, at dosages and for periods of time necessary, to achieve
the desired therapeutic result. A therapeutically effective amount
of the antibody or antibody portion may vary according to factors
such as the disease state, age, sex, and weight of the individual,
and the ability of the antibody or antibody portion to elicit a
desired response in the individual. A therapeutically effective
amount is also one in which any toxic or detrimental effects of the
antibody or antibody portion are outweighed by the therapeutically
beneficial effects. A "prophylactically effective amount" refers to
an amount effective, at dosages and for periods of time necessary,
to achieve the desired prophylactic result. Typically, since a
prophylactic dose is used in subjects prior to or at an earlier
stage of disease, the prophylactically effective amount will be
less than the therapeutically effective amount.
[0132] Dosage regimens may be adjusted to provide the optimum
desired response (e.g., a therapeutic or prophylactic response).
For example, a single bolus may be administered, several divided
doses may be administered over time or the dose may be
proportionally reduced or increased as indicated by the exigencies
of the therapeutic situation. It is especially advantageous to
formulate parenteral compositions in dosage unit form for ease of
administration and uniformity of dosage. Dosage unit form as used
herein refers to physically discrete units suited as unitary
dosages for the mammalian subjects to be treated; each unit
containing a predetermined quantity of active compound calculated
to produce the desired therapeutic effect in association with the
required pharmaceutical carrier. The specification for the dosage
unit forms of the invention are dictated by and directly dependent
on (a) the unique characteristics of the active compound and the
particular therapeutic or prophylactic effect to be achieved, and
(b) the limitations inherent in the art of compounding such an
active compound for the treatment of sensitivity in
individuals.
[0133] An exemplary, non-limiting range for a therapeutically or
prophylactically effective amount of an antibody or antibody
portion of the invention is 0.1-20 mg/kg, more preferably 1-10
mg/kg. It is to be noted that dosage values may vary with the type
and severity of the condition to be alleviated. It is to be further
understood that for any particular subject, specific dosage
regimens should be adjusted over time according to the individual
need and the professional judgment of the person administering or
supervising the administration of the compositions, and that dosage
ranges set forth herein are exemplary only and are not intended to
limit the scope or practice of the claimed composition.
[0134] The present invention is further illustrated by the
following examples which should not be construed as limiting in any
way. The contents of all cited references, including literature
references, issued patents, and published patent applications, as
cited throughout this application are hereby expressly incorporated
by reference.
Example 1
Design of a Dual Specificity Antigen Based on a Contiguous
Topological Area of Identity
[0135] In this example, the largest contiguous topological area of
identity between two different but structurally related proteins,
IL-1.alpha. and IL-1.beta., was determined as a basis for designing
a dual specificity antigen for raising dual specificity antibodies
to IL-1.alpha. and IL-1.beta.. The BLAST algorithm was used to
compare the two proteins and allows one to measure the tendency of
one residue to replace another in similar structural or functional
regions. This analysis allowed for the identification of the
largest contiguous topological area of identity between IL-1.alpha.
and IL-1.beta. and to extend this area with any reasonable
stretches of similarity to create a linear peptide that serves as a
dual specificity antigen. The peptide that best fits these criteria
has an amino acid sequence as follows:
TABLE-US-00002 (SEQ ID NO: 1) NEAQNITDF * ****
The asterisk (*) indicates identical residues in both proteins and
the other residues are strongly similar according to the BLAST
algorithm. For example, lysine will often substitute for arginine
in homologous proteins, but not for phenylalanine. This peptide of
SEQ ID NO: 1 is a hybrid taken from two different sections of the
structure which are running in opposite directions, so another
reasonable representation of this epitope is:
TABLE-US-00003 dNdEdAdQNITDF
(wherein the "d" prefix indicates that the amino acid residue is a
D amino acid residue). Both the L amino acid version of the peptide
and the version partially substituted with D amino acid residues
are synthesized by standard chemical methods. The peptide is then
conjugated to a carrier protein (e.g., KLH or albumin) and the
conjugated peptide is used to select antibodies by in vitro or in
vivo methods.
Example 2
Design of a Dual Specificity Antigen Based on a Cyclic Peptide that
Mimics a Loop of a Common Fold
[0136] In this example, a cyclic peptide that structurally mimics a
key loop of a common fold between two different but structurally
related proteins, IL-1.alpha. and IL-1.beta., was constructed for
use as a dual specificity antigen for raising dual specificity
antibodies to IL-1.alpha. and IL-1.beta.. The chosen loop
represents residues 168-184 of IL-1.alpha. and residues 160-176 of
IL-1.beta.. The consensus sequence is:
TABLE-US-00004 (SEQ ID NO: 2) Cyclo-MAFLRANQNNGKISVAL(PG)
*cbcccccccc**c*b*
The asterisk (*) indicates identical residues between IL-1.alpha.
and IL-1.beta., c indicates consensus residues, i.e, residues
similar to IL-1.alpha. and IL-1.beta. but not actually present at
this location in either protein, and b indicates there was no clear
consensus residue so IL-1.beta. sequence identity was retained. The
linear peptide is synthesized by standard chemical synthesis
methods. To cyclize this peptide, a proline and a glycine residue
are added. The cyclic peptide may be synthesized using standard
coupling conditions at high dilution in N,N-dimethylformamide (1
mg/ml). Prototypical reactions are run at room temperature using
excess coupling reagent, such as
benzotriazole-1-yl-oxy-tris-pyrrolidino-phosphonium hexafluoro
phosphate (PyBOP; 2 eq) and sodium bicarbonate (10 eq). The peptide
is then conjugated to a carrier protein (e.g., KLH or albumin) and
the conjugated peptide is used to select antibodies by in vitro or
in vivo methods.
Example 3
Design of a Dual Specificity Antigen Based on a Hybrid Peptide
[0137] In this example, a hybrid peptide that includes alternating
or overlapping sequences of two different but structurally related
proteins, IL-1.alpha. and IL-1.beta., was constructed for use as a
dual specificity antigen for raising dual specificity antibodies to
IL-1.alpha. and IL-1.beta.. To create the hybrid peptide,
alternating and overlapping amino acid sequences of IL-1.alpha. and
IL-1.beta. were identified and spliced together to generate the
following peptide:
TABLE-US-00005 (SEQ ID NO: 3) TKGGQDITDFQILENQ bbbbbbbbbb
aaaaaaaaaa
The a and b indicate which protein is the source of the residues
(a=IL-1.alpha.; b=IL-1.beta.). The ITDF (SEQ ID NO: 4) motif common
to both proteins was included in the hybrid peptide. Moreover, this
hybrid peptide focuses on sequences from the carboxy termini of
both proteins, which is known to be antigenic for neutralizing
antibodies in both proteins as well. The hybrid peptide is
synthesized by standard chemical synthesis methods. The peptide is
then conjugated to a carrier protein (e.g., KLH or albumin) and the
conjugated peptide is used to select antibodies by in vitro or in
vivo methods.
Example 4
Generation of Dual Specific Antibodies to IL-1.alpha. and
IL-1.beta.
TABLE-US-00006 [0138] (SEQ ID NO: 1) NEAQNITDF (SEQ ID NO: 2)
Cyclo-MAFLRANQNNGKISVAL(PG) (SEQ ID NO: 3) TKGGQDITDFQILENQ
[0139] Peptides of SEQ ID NO; 1, 2 and 3 were conjugated with KLH
and individual rabbits were immunized. Antiserum from rabbits
immunized with each of the three peptides showed good antibody
response against the peptide used as antigen. However, only
antiserum from rabbit immunized with Peptide of SEQ ID NO: 3 was
able to bind both IL-1.alpha. protein and IL-1.beta. protein.
[0140] Five mice (BA119-BA123) were immunized subcutaneously with
peptide of SEQ ID NO: 3 conjugated with KLH plus Freund's
incomplete adjuvant (FIA) once every three weeks for a total of
three times, followed by two intravenous boosts with peptide of SEQ
ID NO: 3 conjugated with KLH. Each mouse was bled 10 days after
each immunization and antibody titer was determined by ELISA.
Spleen cells from mouse BA119 and BA123 respectively were fused
with myloma cell line P3X36Ag8.653 as described in section HA, and
the resulting fused cells were seeded one cell per well in several
96-well plates using limiting dilution. The hybridoma clones that
grew were first assayed for IgG and IgM production by standard
ELISA to identify antibody-producing clones. A total of 945 clones
from mouse #BA123 fusion were isolated. Supematants from 355 clones
tested in an ELISA showed antigen binding activity to IL-1.alpha.,
IL-1.beta. or both IL-1.alpha. and IL-1.beta..
TABLE-US-00007 Antigen Specificity (against full length # of clones
IL-1.alpha. and/or IL-1.beta.) Isotype 249 IL-1 .alpha. only IgG 19
IL-1 .alpha. only IgM 15 IL-1 .beta. only IgG 2 IL-1 .beta. only
IgM 57 IL-1 .alpha. and .beta. IgG 13 IL-1 .alpha. and .beta.
IgM
EQUIVALENTS
[0141] Those skilled in the art will recognize, or be able to
ascertain using no more than routine experimentation, many
equivalents to the specific embodiments of the invention described
herein. Such equivalents are intended to be encompassed by the
following claims.
SEQUENCE LISTING
TABLE-US-00008 [0142] (SEQ ID NO: 1) NEAQNITDF (SEQ ID NO: 2)
Cyclo-MAFLRANQNNGKISVAL(PG) (SEQ ID NO: 3) TKGGQDITDFQILENQ (SEQ ID
NO: 4) APVRSLNCTLRDSQQKSLVMSGPYELKALHLQGQDMEQQVVFSMGAYK
SSKDDAKITVILGLKEKNLYLSCVLKDDKPTLQLESVDPKNYPKKKME
KRFVFNKIEINNKLEFESAQFPNWYISTSQAENMPVFLGGTKGGQDIT DFTMQFVSS
Sequence CWU 1
1
419PRTArtificial Sequenceconsensus sequence fragment between human
IL-1alpha and IL-1beta 1Asn Glu Ala Gln Asn Ile Thr Asp Phe 1 5
219PRTArtificial Sequenceconsensus loop of a common structural
feature of IL-1alpha and IL-1beta 2Met Ala Phe Leu Arg Ala Asn Gln
Asn Asn Gly Lys Ile Ser Val Ala 1 5 10 15 Leu Xaa Xaa
316PRTArtificial Sequencehybrid peptide of alternating and
overlapping sequences of IL-1alpha and IL-1beta 3Thr Lys Gly Gly
Gln Asp Ile Thr Asp Phe Gln Ile Leu Glu Asn Gln 1 5 10 15
4153PRTArtificial Sequencehybrid peptide constructed for use as a
dual specificity antigen, having aa168-184 of huIL-1alpha embedded
in a corresponding segment of huIL-1beta 4Ala Pro Val Arg Ser Leu
Asn Cys Thr Leu Arg Asp Ser Gln Gln Lys 1 5 10 15 Ser Leu Val Met
Ser Gly Pro Tyr Glu Leu Lys Ala Leu His Leu Gln 20 25 30 Gly Gln
Asp Met Glu Gln Gln Val Val Phe Ser Met Gly Ala Tyr Lys 35 40 45
Ser Ser Lys Asp Asp Ala Lys Ile Thr Val Ile Leu Gly Leu Lys Glu 50
55 60 Lys Asn Leu Tyr Leu Ser Cys Val Leu Lys Asp Asp Lys Pro Thr
Leu 65 70 75 80 Gln Leu Glu Ser Val Asp Pro Lys Asn Tyr Pro Lys Lys
Lys Met Glu 85 90 95 Lys Arg Phe Val Phe Asn Lys Ile Glu Ile Asn
Asn Lys Leu Glu Phe 100 105 110 Glu Ser Ala Gln Phe Pro Asn Trp Tyr
Ile Ser Thr Ser Gln Ala Glu 115 120 125 Asn Met Pro Val Phe Leu Gly
Gly Thr Lys Gly Gly Gln Asp Ile Thr 130 135 140 Asp Phe Thr Met Gln
Phe Val Ser Ser 145 150
* * * * *
References