U.S. patent application number 11/527160 was filed with the patent office on 2008-02-28 for humanized immunoglobulin reactive with b7 molecules and methods of treatment therewith.
This patent application is currently assigned to Genetics Institute, LLC. Invention is credited to Abbie Cheryl Celniker, Gary S. Gray.
Application Number | 20080050368 11/527160 |
Document ID | / |
Family ID | 26939753 |
Filed Date | 2008-02-28 |
United States Patent
Application |
20080050368 |
Kind Code |
A1 |
Celniker; Abbie Cheryl ; et
al. |
February 28, 2008 |
Humanized immunoglobulin reactive with B7 molecules and methods of
treatment therewith
Abstract
The invention relates to humanized anti-B7-2 and anti-B7-1
antibodies, wherein each comprise a variable region of non-human
origin and at least a portion of an immunoglobulin of human origin.
The invention also pertains to methods of treatment for various
autoimmune diseases, transplant rejection, inflammatory disorders
and infectious diseases by administering humanized anti-B7-2 and/or
anti-B7-1 antibodies.
Inventors: |
Celniker; Abbie Cheryl;
(Newton, MA) ; Gray; Gary S.; (Brookline,
MA) |
Correspondence
Address: |
FOLEY HOAG, LLP/WYETH;PATENT GROUP, (w/WYS)
155 SEAPORT BLVD.
BOSTON
MA
02210-2600
US
|
Assignee: |
Genetics Institute, LLC
Cambridge
MA
|
Family ID: |
26939753 |
Appl. No.: |
11/527160 |
Filed: |
September 26, 2006 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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09501102 |
Feb 9, 2000 |
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11527160 |
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09339596 |
Jun 24, 1999 |
6913747 |
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09501102 |
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09249011 |
Feb 12, 1999 |
6972125 |
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09339596 |
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Current U.S.
Class: |
424/133.1 ;
424/130.1; 514/336 |
Current CPC
Class: |
A61P 3/00 20180101; A61P
29/00 20180101; A61P 1/00 20180101; A61P 17/00 20180101; A61P 19/02
20180101; A61P 31/00 20180101; C07K 2317/24 20130101; C07K 2319/30
20130101; A61K 2039/505 20130101; A61P 37/00 20180101; A61P 7/00
20180101; A61P 43/00 20180101; A61K 2300/00 20130101; A61P 35/02
20180101; A61P 37/06 20180101; A61P 1/04 20180101; A61P 7/06
20180101; A61P 1/18 20180101; A61K 39/395 20130101; A61P 3/10
20180101; A61P 25/00 20180101; A61P 35/00 20180101; A61P 37/02
20180101; C07K 2317/52 20130101; A61P 11/06 20180101; C07K 16/2827
20130101; C07K 2319/00 20130101; A61K 39/395 20130101 |
Class at
Publication: |
424/133.1 ;
424/130.1; 514/336 |
International
Class: |
A61K 31/44 20060101
A61K031/44; A61K 39/00 20060101 A61K039/00; A61K 39/395 20060101
A61K039/395; A61P 43/00 20060101 A61P043/00 |
Claims
1. A method for downmodulating the immune response in a subject
undergoing transplantation comprising preoperatively administering
to the subject at least on antibody that recognizes a B7 antigen
immediately prior to surgery and postoperatively administering to
the subject at least one antibody that recognizes a B7 antigen
immediately following surgery.
2. The method of claim 1, further comprising preoperatively
administering at least one antibody that recognizes a B7 antigen at
least about four days prior to surgery.
3. The method of claim 1, wherein two antibodies that recognize at
least two B7 antigens are administered to the subject.
4. The method of claim 1, wherein at least one antibody is a
humanized antibody.
5. The method of claim 1, wherein a higher dose of at least one
antibody is administered prior to surgery than after surgery.
6. The method of claim 1, further comprising postoperatively
administering at least one antibody that recognizes a B7 antigen at
weekly intervals for at least about 3 months.
7. A method for downmodulating the immune response in a subject
undergoing transplantation comprising preoperatively administering
to the subject at least one antibody that recognizes a B7 antigen
and postoperatively administering to the subject at least one
antibody that recognizes a B7 antigen in combination with an
immunosuppressive drug.
8. A method for downmodulating the immune response in a subject
undergoing transplantation comprising preoperatively administering
to the subject a combination of antibodies that recognize at least
two B7 antigens and postoperatively administering to the subject a
combination of antibodies that recognize at least two B7 antigens
in combination with an immunosuppressive drug.
9. The method of claim 9, wherein the immunosuppressive drug is a
rapamycin compound.
Description
RELATED APPLICATIONS
[0001] This application is a Continuation-In-Part of application
Ser. No. 09/339,596, filed Jun. 24, 1999, which is a
Continuation-In-Part of 09/249,011, filed Feb. 12, 1999, the entire
teachings of which are incorporated herein by reference.
BACKGROUND OF THE INVENTION
[0002] Antigen specific T-cell activation and the initiation of an
immune response depend initially on the interaction of the T-cell
receptor (TCR) complex with the peptide/major histocompatibility
complex (MHC) present on antigen presenting cells (APC). B7
molecules, B7-1 and B7-2, are molecules which are present on APCs.
A "co-stimulatory" signal, provided by the interaction of B7-1 and
B7-2 on the APC with their ligands CD28 and CTLA4 on T-cells, is
required to complete T-cell activation and the subsequent
regulation of an immune response. A need exists to regulate the
B7-1 and B7-2 pathway, referred to as the B7:CD28/CTLA4 pathway. A
further need exists to develop treatments for diseases that are
affected by this pathway.
SUMMARY OF THE INVENTION
[0003] The invention relates to humanized immunoglobulins having
binding specificity for B7 molecules. In particular, the invention
includes a humanized immunoglobulin having binding specificity for
B7-2 or B7-1, wherein the immunoglobulin comprises an antigen
binding region of non-human origin (e.g. rodent) and at least a
portion of human origin (e.g. a human constant region such as an
IgG constant region and/or a human framework region). In one
embodiment, the human constant region of either the humanized
anti-B7-2 or humanized anti-B7-1 immunoglobulin can also contain a
mutation that reduces the effector function of the humanized
immunoglobulin. The humanized B7-2 immunoglobulin described herein
can compete with murine 3D1 for binding to B7-2. Similarly, the
humanized B7-1 immunoglobulin described herein can compete with
murine 1F1 for binding to B7-1. In particular embodiments, the
antigen binding region of the humanized B7-2 immunoglobulin is
derived from the 3D1 monoclonal antibody, and the antigen binding
region of the humanized B7-1 immunoglobulin is derived from the 1F1
monoclonal antibody.
[0004] The humanized immunoglobulins of the present invention can
comprise a constant region of human origin and an antigen binding
region, wherein the antigen binding region of non-human origin
comprises one or more complementarity determining regions (CDRs) of
rodent origin (e.g., derived from 3D1 monoclonal antibody) that
binds to B7-2, or one or more CDRs of rodent origin (e.g., derived
from 1F1 monoclonal antibody) that binds to B7-1, and the portion
of an immunoglobulin of human origin is derived from a human
framework region (FR).
[0005] The antigen binding region of the humanized B7-2 antibody
can further comprise a light chain and a heavy chain, wherein the
light and heavy chain each have three CDRs derived from the 3D1
antibody. The FR of the light chain for the humanized B7-2 antibody
can be derived, for example, from the light chain of the human H2F
antibody and the heavy chain can be derived, for example, from the
heavy chain of the human I2R antibody. In a particular embodiment,
the invention is a humanized immunoglobulin having binding
specificity for B7-2 that is derived from the cell line deposited
with the American Type Culture Collection (A.T.C.C.), Accession No.
CRL-12524.
[0006] The antigen binding region of the humanized B7-1 antibody
can comprise a light chain and a heavy chain, wherein the light and
heavy chain each have three CDRs derived from the 1F1 antibody. The
FR of the humanized B7-1 light and heavy chain can be derived, for
example, from the light and heavy chains of the human III-2R
antibody. In a particular embodiment, the invention is a humanized
immunoglobulin having binding specificity for B7-1 that is derived
from the cell line deposited with the American Type Culture
Collection (A.T.C.C.), Accession No. PTA-263.
[0007] The invention also embodies a humanized immunoglobulin
having a binding specificity for B7-1 or B7-2 comprising a heavy
chain and/or a light chain. In one embodiment, the humanized
immunoglobulin has binding specificity for B7-2 and the light chain
comprises at least one CDR (e.g., CDR1, CDR2 and CDR3) derived from
an antibody of non-human origin which binds B7-2 and a FR derived
from a light chain of human origin (e.g., H2F antibody); and the
heavy chain comprises at least one CDR (e.g., CDR1, CDR2 and CDR3)
derived from an antibody of non-human origin which binds B7-2 and a
FR region derived from a heavy chain of human origin (e.g., the
human I2R antibody). In another embodiment, the humanized
immunoglobulin has binding specificity for the B7-1 and the light
and/or heavy chain comprise at least one CDR (e.g., CDR1, CDR2 and
CDR3) derived from an antibody of non-human origin which binds B7-1
and a FR derived from a light and/or heavy chain of human origin
(e.g., III-2R). The immunoglobulins can further comprise CDR1, CDR2
and CDR3 for the light or heavy chain having the amino acid
sequence set forth herein or an amino acid substantially the same
as the amino acid sequence such that the antibody specifically
binds to B7-2 or B7-1. The present invention also relates to
humanized immunoglobulin light chains and to humanized
immunoglobulin heavy chains. The invention further relates to
isolated nucleic acids comprising a sequence which encodes the
humanized immunoglobulins of the present invention (e.g., a single
chain antibody), and the invention also relates to isolated nucleic
acids that comprise a sequence which encodes the B7-2 or B7-1
humanized immunoglobulin light chain or heavy chain.
[0008] One embodiment of the invention is a humanized
immunoglobulin light chain having binding specificity for B7-2
comprising CDR1, CDR2 and/or CDR3 of the light chain of murine 3D1
antibody, and a human light chain FR (e.g., H2F antibody).
Similarly, the invention also comprises a humanized B7-1
immunoglobulin light chain having binding specificity for B7-1
comprising CDR1, CDR2 and/or CDR3 of the light chain of the murine
1F1 antibody, and a human light chain FR (e.g., III-2R antibody).
Another embodiment is a humanized B7-2 or B7-1 immunoglobulin light
chain that comprises a variable region shown in FIG. 2B (SEQ ID NO:
8) or the variable region shown in FIG. 7B (SEQ ID NO: 28). The
invention also relates to an isolated nucleic acid sequence that
encodes a humanized variable light chain specific for B7-2 or B7-1
that comprises a nucleic acid sequence shown in FIG. 2B (SEQ ID NO:
7) or FIG. 7B (SEQ ID NO.: 27), respectively, a nucleic acid
sequence that encodes the amino acid sequence shown in FIG. 2B (SEQ
ID NO: 8) or FIG. 7B (SEQ ID NO: 28), respectively, a nucleic acid
sequence which hybridizes thereto under stringent hybridization
conditions, or a nucleic acid sequence which is the complement
thereof.
[0009] Another embodiment of the invention is a humanized
immunoglobulin heavy chain that is specific for B7-2 and comprises
CDR1, CDR2 and/or CDR3 of the heavy chain of the 3D1 antibody, and
a human heavy chain FR (e.g., I2R antibody). Similarly, the
invention also relates to a B7-1 humanized immunoglobulin heavy
chain that is specific for B7-1 and comprises CDR1, CDR2, and/or
CDR3 of the heavy chain of the 1F1 antibody, and a human heavy
chain FR (e.g., III-2R antibody). The invention pertains to a
humanized immunoglobulin heavy chain that comprises a variable
region shown in FIG. 2A (SEQ ID NO: 6) or FIG. 7A (SEQ ID NO: 26).
The invention also pertains to an isolated nucleic acid sequence
that encodes a humanized variable heavy chain specific for B7-2
that comprises a nucleic acid sequence shown in FIG. 2A (SEQ ID NO:
5), an isolated nucleic acid sequence that encodes the amino acid
sequence shown in FIG. 2A (SEQ ID NO: 6), a nucleic acid sequence
which hybridizes thereto under stringent hybridization conditions,
or a nucleic acid sequence which is the complement thereof. The
invention relates to an isolated nucleic acid sequence that encodes
a humanized variable heavy chain specific for B7-1 that comprises a
nucleic acid sequence shown in FIG. 7A (SEQ ID NO: 25), an isolated
nucleic acid sequence that encodes the amino acid sequence shown in
FIG. 7A (SEQ ID NO: 26), a nucleic acid sequence which hybridizes
thereto under stringent hybridization conditions, or a nucleic acid
sequence which is the complement thereof.
[0010] In particular, an embodiment of the invention is a humanized
immunoglobulin which specifically binds to B7-2 and comprises a
humanized light chain comprising three light chain CDRs from the
mouse 3D1 antibody, and a light chain variable region framework
sequence from a human immunoglobulin light chain, and a humanized
heavy chain comprising three heavy chain CDRs from the mouse 3D1
antibody, and a heavy chain variable region framework sequence from
a human immunoglobulin heavy chain. The mouse 3D1 antibody can
further have a mature light chain variable domain, such as the
mature light chain variable domain shown in FIG. 1B (SEQ ID NO.: 4)
and a mature heavy chain variable domain such as the mature heavy
chain variable region shown in FIG. 1A (SEQ ID NO.: 2).
[0011] Another embodiment of the invention is a humanized
immunoglobulin which specifically binds to B7-1 and comprises a
humanized light chain comprising three light chain CDRs from the
mouse 1F1 antibody, and a light chain variable region framework
sequence from a human immunoglobulin light chain, and a humanized
heavy chain comprising three heavy chain CDRs from the mouse 1F1
antibody, and a heavy chain variable region framework sequence from
a human immunoglobulin heavy chain. Similarly, the mouse 1F1
antibody can further have a mature light chain variable domain,
such as the mature light chain variable domain shown in FIG. 6B
(SEQ ID NO.: 24) and a mature heavy chain variable domain such as
the mature heavy chain variable region shown in FIG. 6A (SEQ ID
NO.: 22).
[0012] The invention includes an expression vector that comprises a
fused gene which encodes humanized B7-1 and/or B7-2 immunoglobulin
light and/or heavy chains. The gene comprises a nucleotide sequence
encoding a CDR derived from a light and/or heavy chain of a
non-human antibody having binding specificity for B7-2 and/or B7-1
(e.g., murine 3D1 or 1F1 antibody, respectively) and a FR derived
from a light and/or heavy chain of human origin.
[0013] The present invention also relates to a host cell comprising
a nucleic acid of the present invention, including one or more
constructs comprising nucleic acid of the present invention. In one
embodiment, the invention encompasses a host cell comprising a
first B7-2 recombinant nucleic acid that encodes a humanized B7-2
immunoglobulin light chain and a second B7-2 recombinant nucleic
acid that encodes a humanized B7-2 immunoglobulin heavy chain. The
first B7-2 nucleic acid comprises a nucleotide sequence encoding at
least one CDR derived from the light chain of murine 3D1 antibody
and a FR derived from a light chain of human origin. The second
B7-2 nucleic acid comprises a nucleotide sequence encoding at least
one CDR derived from the heavy chain of murine 3D1 antibody and a
FR derived from a heavy chain of human origin. In another
embodiment, the invention encompasses a host cell comprising a
first B7-1 recombinant nucleic acid that encodes a humanized B7-1
immunoglobulin light chain and a second B7-1 recombinant nucleic
acid that encodes a humanized B7-1 immunoglobulin heavy chain. The
first B7-1 nucleic acid comprises a nucleotide sequence encoding at
least one CDR derived from the light chain of murine 1F1 antibody
and a FR derived from a light chain of human origin. The second
B7-1 nucleic acid comprises a nucleotide sequence encoding at least
one CDR derived from the heavy chain of murine 1F1 antibody and a
FR derived from a heavy chain of human origin. The invention
further relates to a host cell comprising a vector or a nucleic
acid that encodes the humanized B7-1 and/or B7-2 immunoglobulins,
as described herein.
[0014] The invention further pertains to methods of preparing
humanized immunoglobulins that comprise maintaining a host cell
that encodes a humanized immunoglobulin that is specific for B7-2
or B7-1, as described herein, under conditions appropriate for
expression of a humanized immunoglobulin, wherein a humanized
immunoglobulin chain (one or more) are expressed and a humanized
immunoglobulin is produced. The method further comprises the step
of isolating the humanized B7-1 or B7-2 immunoglobulin.
[0015] The invention encompasses methods of inhibiting the
interaction of a first cell bearing a B7-2 receptor with a second
cell bearing B7-2 comprising contacting the second cell with an
effective amount of a humanized B7-2 immunoglobulin, as described
herein. The invention also encompasses methods of inhibiting the
interaction of a first cell bearing a B7-1 receptor with a second
cell bearing B7-1 comprising contacting the second cell with an
effective amount of a humanized B7-1 immunoglobulin, as described
herein. Accordingly, the invention relates to inhibiting both a
B7-1 and B7-2 receptor with B7-1 and B7-2 ligands, comprising
contacting the cells having the B7-1 and B7-2 receptors with an
amount of humanized anti-B7-1 and B7-2 immunoglobulins. Thus, the
invention pertains to various methods of treatment. The invention
includes a method for modulating an immune response of an
individual (e.g., patient), or treating an individual having a
transplanted organ, tissue, cell or the like comprising
administering an effective amount of a humanized B7-1 and/or B7-2
immunoglobulin, as described herein, with or without a carrier
(e.g., pharmaceutical carrier), wherein the immune response is
modulated. The invention pertains to treating acute and/or chronic
transplant rejection, for example, for a prolonged periods of time
(e.g., days, months, or years). The invention also pertain to
methods of treating a disease associated with modulation of the
B7-2 and/or B7-1 molecules (e.g., autoimmune diseases, infectious
diseases, inflammatory disorders, systemic lupus erythematosus,
diabetes mellitus, asthma, insulitis, arthritis, inflammatory bowel
disease, inflammatory dermatitis, and multiple sclerosis),
comprising administering to an individual (e.g., patient) an
effective amount (e.g., a therapeutically effective amount) of the
B7-2 and/or B7-1 humanized immunoglobulins, as described herein,
with or without a carrier. Accordingly, the invention encompasses a
pharmaceutical composition comprising the B7-1 and/or B7-2
humanized immunoglobulins, as described herein.
[0016] The invention also embodies methods of making a humanized
immunoglobulin specific to B7-2 from a murine antibody specific to
B7-2, and/or methods of making a humanized immunoglobulin specific
to B7-1 from a murine antibody specific to B7-1. The methods
comprise determining or ascertaining the CDRs of an antibody of
non-human origin (e.g., murine origin) which has binding
specificity for B7-2 or B7-1; obtaining a human antibody having a
framework region amino acid sequence suitable for grafting of the
CDRs, and grafting the CDRs of an antibody of non-human origin into
the FR of the human antibody.
[0017] The invention also relates to methods for determining the
presence or absence of B7-2 and/or B7-1 in a sample. The methods
comprise obtaining the sample to be tested, contacting the sample
with the humanized antibody specific to B7-2 and/or B7-1, or a
fragment thereof, sufficiently to allow formation of a complex
between B7-2 and/or B7-1 and the anti-B7-2 and/or anti-B7-1
antibody, respectively, and detecting the presence or absence of
the complex formation. The presence of the complex indicates the
presence of B7-2 and/or B7-1 in the sample.
[0018] The invention relates to methods for treating an individual
having a disease comprising administering an amount (e.g.,
therapeutically effective amount) of a humanized immunoglobulin
specific to B7-1 and/or an amount (e.g., therapeutically effective
amount) of a humanized immunoglobulin specific to B7-2. The
diseases, as described herein, include, for example, autoimmune
diseases, infectious diseases, asthma, inflammatory disorders,
systemic lupus erythematosus, diabetes mellitus, insulitis,
arthritis, inflammatory bowel disease, inflammatory dermatitis, and
multiple sclerosis. This method also pertains to modulating the
immune response of an individual having a transplanted organ,
tissue, cell or the like comprising administering an effective
amount of a humanized immunoglobulin that binds to B7-1 and/or a
humanized immunoglobulin that binds to B7-2. This method further
includes administering a drug that is used to modulate the immune
response of an individual having a transplanted organ, tissue, cell
or the like. The drug can be, for example, methotrexate, rapamycin,
cyclosporin, steroids, anti-CD40 pathway inhibitors (e.g.,
anti-CD40 antibodies, anti-CD40 ligand antibodies and small
molecule inhibitors of the CD40 pathway), transplant salvage
pathway inhibitors (e.g., mycophenolate mofetil (MMF)), IL-2
receptor antagonists (e.g., Zeonpax.RTM. from Hoffmann-la Roche
Inc., and Simulet from Novartis, Inc.) and analogs thereof. These
drugs can be administered prior to, with or after administration of
the humanized immunoglobulins.
[0019] The invention also pertains to methods for transplanting
cells (e.g., bone marrow, blood cells, blood components and other
cells) to an individual in need thereof comprising obtaining cells
(e.g., bone marrow, or blood cells or components) from a donor, and
contacting the cells with an immunoglobulin specific to B7-1 and/or
an immunoglobulin specific to B7-2, and recipient cells, thereby
obtaining a mixture. The immunoglobulins and the recipient cells
are maintained for a period of time sufficient for tolerance
induction. The mixture (referred to as a bone marrow composition or
blood cell composition) is then introduced into the individual. The
recipient cells can be lymphocytes (e.g. lymphocytes that express
class I antigens (MHCI) or peripheral blood lymphocyte (PBL)).
Instead of using recipient cells, the method also comprise
utilizing tissue, organs or cells that express MHC Class I
antigens, B7-1 and/or B7-2 molecules. The cells can be engineered
to express recipient molecules. The cells from the donor can be
bone marrow cells, or cells/components from blood (e.g., stem cells
or immature cells). The B7 immunoglobulins are in contact with the
donor bone marrow and the recipient cells for a period of time that
is long enough to induce tolerance induction (e.g., about 1 to 96
hours, and, preferably about 36-48 hours). An individual in need of
such a transplant is one who has a disease that is benefited by or
treatable with a bone marrow transplant. Such diseases, for
example, are proliferative diseases (e.g. leukemia, lymphoma and
cancer), anemia (e.g. sickle-cell anemia, thalassemia, and aplastic
anemia), inborn errors of metabolism, congenital immunodeficiency
diseases, and myeloid dysplasia syndrome (MDS). The method further
includes administering to the individual a drug that is used to
modulate the immune response (e.g., methotrexate; rapamycin;
cyclosporin; steroids; anti-CD40 pathway inhibitors such as
anti-CD40 antibodies, anti-CD40 ligand antibodies and small
molecule inhibitors of the CD40 pathway; transplant salvage pathway
inhibitors such as mycophenolate mofetil (MMF), IL-2 receptor
antagonists such as Zeonpax.RTM. from Hoffmann-la Roche Inc., and
Simulet from Novartis, Inc.; or analogs thereof).
[0020] In particular, the invention includes methods for
transplanting bone marrow to an individual having a disease (e.g.,
proliferative diseases such as leukemia, lymphoma, cancer; anemia
such as sickle-cell anemia, thalassemia, and aplastic anemia;
inborn errors of metabolism; congenital immunodeficiency diseases;
and myeloid dysplasia syndrome) that is treated with a bone marrow
transplant comprising obtaining bone marrow from a donor, and
contacting the bone marrow with immunoglobulins specific to B7-1
and/or an immunoglobulin specific to B7-2, and recipient cells
(e.g., lymphocyte). The bone marrow, immunoglobulin(s) and
recipient cells are in contact for a period of time sufficient for
tolerance induction (e.g., about 1-96 hours, preferably about 36-48
hours). The method then comprises re-introducing the treated bone
marrow to the individual. The method further includes administering
to the individual a drug that is used to modulate the immune
response (e.g., methotrexate; rapamycin; cyclosporin; steroids;
anti-CD40 pathway inhibitors such as anti-CD40 antibodies,
anti-CD40 ligand antibodies and small molecule inhibitors of the
CD40 pathway; transplant salvage pathway inhibitors such as
mycophenolate mofetil (MMF), IL-2 receptor antagonists such as
Zeonpax.RTM. from Hoffmann-la Roche Inc., and Simulet from
Novartis, Inc.; or analogs thereof).
[0021] The invention also includes methods of treating a transplant
recipient or preventing transplant rejection in a transplant
recipient by administering to the recipient an effective amount of
an immunoglobulin specific to B7-1 and/or an effective amount of an
immunoglobulin specific to B7-2. The immunoglobulin specific to
B7-1 is administered in an amount between about 1 mg/kg and 100
mg/kg, and the immunoglobulin specific to B7-2 is administered in
an amount between about 1 mg/kg and about 100 mg/kg. The
immunoglobulins specific to B7-1 and B7-2 can be administered on
the day the recipient receives the transplantation (e.g., in an
amount between about 1 mg/kg and about 25 mg/kg), and can also be
administered periodically (e.g., daily, weekly or monthly) after
the recipient receives the transplantation (e.g., in an amount
between about 1 mg/kg and about 5 mg/kg). The method further
includes administering a composition that is used in transplant
rejection therapy, such as calcineurin inhibitors (e.g.,
cyclosporin A or FK506), steroids (e.g., methyl prednisone or
prednisone), or immunosuppressive agents that arrest the growth of
immune cells (e.g., rapamycin), anti-CD40 pathway inhibitors (e.g.,
anti-CD40 antibodies, anti-CD40 ligand antibodies and small
molecule inhibitors of the CD40 pathway) transplant salvage pathway
inhibitors (e.g., mycophenolate mofetil (MMF)), IL-2 receptor
antagonists (e.g., Zeonpax.RTM. from Hoffmann-la Roche Inc., and
Simulet from Novartis, Inc.), or analogs thereof. Also, the present
invention relates to a method of transplanting cells, tissue or
organs to an individual in need thereof, by transplanting the
cells, tissue or organs; and administering an effective amount of
an immunoglobulin specific to B7-1 and an effective amount of an
immunoglobulin specific to B7-2 to the individual, as described
above.
[0022] The invention also includes methods of decreasing an
antibody response to an antigen in a mammal by administering to the
individual an effective amount of a humanized immunoglobulin
specific to B7-1 and/or a humanized immunoglobulin specific to
B7-2, in the presence of the antigen. The method further includes
administering the antigen (e.g., tetanus toxoid, Factor VIII,
Factor IX, insulin, growth hormone, or a gene delivery vector) to
the individual. The antigen can be administered in a polypeptide
form or in a nucleic acid form (e.g., gene therapy with delivery
through adeno-associated viruses (AAV), retroviruses, naked DNA
vectors, etc.).
[0023] Advantages of the invention include the ability to regulate
or modulate the B7 co-stimulatory pathway. Manipulation of this
co-stimulatory pathway with humanized anti-B7-2 and/or anti-B7-1
antibodies provide methods of treatments for various diseases. The
humanized anti-B7-2 and anti-B7-1 antibodies maintain about the
same specificity for respective B7 molecule as the corresponding
murine antibody, but with a reduced immunogenicity in humans and an
extended half-life, as compared with the murine counterpart.
Accordingly, the invention can advantageously be used to treat
immune-related diseases/disorders, or diseases in which the B7-2
and/or B7-1 molecules play an important role. Particularly, the
invention relates to methods for treating autoimmune diseases, and
methods for modulating the immune response for individuals with
transplanted organs, tissue or cells.
BRIEF DESCRIPTION OF THE FIGURES
[0024] The foregoing and other embodiments, features and advantages
of the invention will be apparent from the following more
particular description of preferred embodiments of the invention,
as illustrated in the accompanying figures.
[0025] FIG. 1A is a sequence listing of the heavy chain variable
region nucleic acid and amino acid sequences (SEQ ID NOS: 1 and 2,
respectively) of the murine 3D1 antibody, wherein the amino acid
sequences of the CDRs (CDR1, CDR2 and CDR3) are underlined, and the
first amino acid of the mature, heavy chain is double
underlined.
[0026] FIG. 1B is a sequence listing of the light chain variable
region nucleic acid and amino acid sequences (SEQ ID NOS: 3 and 4,
respectively) of the murine 3D1 antibody wherein, the nucleic and
amino acid sequences of the CDRs (CDR1, CDR2 and CDR3) are
underlined, and the first amino acid of the mature light chain is
double underlined.
[0027] FIG. 2A is a sequence listing of the heavy chain variable
region nucleic acid and amino acid sequences (SEQ ID NOs: 5 and 6,
respectively) of the humanized 3D1 antibody, wherein the nucleic
and amino acid sequences of the CDRs (CDR1, CDR2 and CDR3), and
underlined and the first amino acid of the mature heavy chain is
double underlined.
[0028] FIG. 2B contains the light chain variable region nucleic
acid and amino acid sequences (SEQ ID NOs: 7 and 8, respectively)
of the humanized 3D1 antibody, wherein the nucleic and amino acid
sequences of CDR1, CDR2 and CDR3. The CDRs are underlined, and the
first amino acid of the mature light chain is double
underlined.
[0029] FIG. 3 is a graph depicting the results of a competitive
binding assay. The graph depicts the results of a competitive
binding assay of murine or humanized anti-human B7-2 mAbs to CHO
cells expressing rhB7-2 (CHO/hB7-2) on their surface. Increasing
concentrations of unlabeled competitor antibodies were incubated
with CHO/hB7-2 cells in the presence of radiolabeled tracer murine
anti-human B7-2 mAb and the ratio of bound/free antibody was
determined.
[0030] FIG. 4 is a graph depicting the results of a direct binding
assay of murine or humanized anti-human B7-2 mAbs to CHO/hB7-2
cells. Increasing concentrations of radiolabeled antibodies were
incubated with CHO or CHO/hB7-2 cells and the amount of specific
antibody bound to the CHO/hB7-2 cells was determined.
[0031] FIG. 5 is a graph depicting the results of a T cell
proliferation assay. Increasing concentrations of murine or
humanized anti-human B7-2 mAbs were added to CD28.sup.+ human T
cells stimulated with PMA and CHO/hB7-2 cells and the inhibition of
T cell proliferation by these mAbs was determined.
[0032] FIG. 6A is a sequence listing of the heavy chain variable
region nucleic acid and amino acid sequences (SEQ ID NOS: 21 and
22, respectively) of the murine 1F1 antibody, wherein the amino
acid sequences of the CDRs (CDR1, CDR2 and CDR3) are underlined,
and the first amino acid of the mature heavy chain is double
underlined.
[0033] FIG. 6B is a sequence listing of the light chain variable
region nucleic acid and amino acid sequences (SEQ ID NOS: 23 and
24, respectively) of the murine 1F1 antibody wherein, the nucleic
and amino acid sequences of the CDRs (CDR1, CDR2 and CDR3) are
underlined, and the first amino acid of the mature light chain is
double underlined.
[0034] FIG. 7A is a sequence listing of the heavy chain variable
region nucleic acid and amino acid sequences (SEQ ID NOs: 25 and
26, respectively) of the humanized 1F1 antibody (hu1F1), wherein
the nucleic and amino acid sequences of the CDRs (CDR1, CDR2 and
CDR3) are underlined, and the first amino acid of the mature heavy
chain is double underlined.
[0035] FIG. 7B contains the light chain variable region nucleic
acid and amino acid sequences (SEQ ID NOs: 27 and 28, respectively)
of the humanized 1F1 (hu1F1) antibody, wherein the nucleic and
amino acid sequences of CDR1, CDR2 and CDR3. The CDRs are
underlined, and the first amino acid of the mature light chain is
double underlined.
[0036] FIG. 8 is a graph depicting the results of a competitive
binding assay. The graph depicts the results of a competitive
binding assay of murine or humanized anti-human B7-1 mAbs to CHO
transfected with rhB7-1 (CHO/hB7-1). Increasing concentrations of
unlabeled competitor antibodies were incubated with CHO/hB7-1 cells
in the presence of radiolabeled tracer humanized 1F1, and the ratio
of bound/free antibody was determined.
[0037] FIG. 9A is a graph showing the Scatchard analysis of the
binding of mouse 1F1 antibodies to CHO cells transfected with
rhB7-1. Radiolabeled mouse 1F1 antibodies were incubated with CHO
cells transfected with rhB7-1, and the ratio of bound/free
radioactivity was determined.
[0038] FIG. 9B is a graph showing the Scatchard analysis of the
binding of humanized 1F1 antibodies to CHO cells transfected with
rhB7-1. Radiolabeled humanized 1F1 antibodies were incubated with
CHO cells transfected with rhB7-1, and the ratio of bound/free
radioactivity was determined.
[0039] FIG. 10 is a graph depicting the results of a competitive
binding assay of murine or humanized anti-human B7-1 mAbs to CHO
expressing rhB7-1 (CHO/hB7-1) on their surface. Increasing
concentrations of unlabeled competitor antibodies were incubated
with CHO/hB7-1 cells in the presence of radiolabeled tracer murine
anti-human B7-1 mAb and the ratio of bound/free antibody was
determined.
[0040] FIG. 11 is a graph depicting the results of a direct binding
assay of murine or humanized anti-human B7-1 mAbs to CHO/hB7-1
cells. Increasing concentrations of radiolabeled antibodies were
incubated with CHO or CHO/hB7-1 cells and the amount of specific
antibody bound to the CHO/hB7-1 cells was determined.
[0041] FIG. 12 is a graph depicting the results of a T cell
proliferation assay. Increasing concentrations of murine or
humanized anti-human B7-1 mAbs were added to CD28.sup.+ human T
cells stimulated with PMA and CHO/hB7-1 cells and the inhibition of
T cell proliferation by these mAbs was determined.
[0042] FIG. 13 is a graph depicting the results of a one way mixed
lymphocyte reaction (MLR) assay. Fixed concentrations of murine or
humanized anti-human B7-2 (IgG2.M3 isotype) or human CTLA4Ig were
added to a mixture of human responder and stimulator PBLs and the
proliferation of the responder PBLs was determined on days 3, 4,
and 5 by the addition of radiolabeled thymidine.
[0043] FIG. 14 is a graph depicting the results of a one way
secondary MLR assay using PBLs from a primary MLR as responders and
PBLs from the same or a different individual as in the primary MLR
as stimulators. The humanized anti-human B7-1 mAb was added to the
primary MLR only. Proliferation of the responder PBLs in the
secondary MLR was determined on days 3, 4, and 5 by the addition of
radiolabeled thymidine.
[0044] FIG. 15 is a graph depicting the results of a one way
secondary MLR assay using PBLs from a primary MLR as responders and
PBLs from the same or a different individual as in the primary MLR
as stimulators. The humanized anti-human B7-2 mAb (IgG2.M3 isotype)
was added to the primary MLR only. Proliferation of the responder
PBLs in the secondary MLR was determined on days 3, 4, and 5 by the
addition of radiolabeled thymidine.
[0045] FIG. 16 is a graph depicting the results of a one way
secondary MLR assay using PBLs from a primary MLR as responders and
PBLs from the same or a different individual as in the primary MLR
as stimulators. The humanized anti-human B7-1 and B7-2 mAbs
(IgG2.M3 isotype) were added to the primary MLR only. Proliferation
of the responder PBLs in the secondary MLR was determined on days
3, 4, and 5 by the addition of radiolabeled thymidine.
[0046] FIG. 17 is a graph showing the results from a one way
primary MRL assay using PBLs as responders and irradiated "B" PBL
stimulators. The responder and stimulator were treated with the
humanized anti-B7-2 antibody, humanized anti-B7-1 mAbs, combined
humanized anti-B7-1 and humanized anti-B7-2 mAbs, 10 .mu.g/ml CTLA4
Ig, 20 .mu.g/ml CTLA4 Ig, or control Ig. The culture proliferation
was measured on days 3, 4, and 5 by the addition of radiolabeled
thymidine.
[0047] FIG. 18 is a graph showing the results from a one way
primary MRL assay using PBLs as responders and irradiated "C" PBL
stimulators. The responder and stimulator were treated with the
humanized anti-B7-2 antibody, humanized anti-B7-1 mAbs, combined
humanized anti-B7-1 and humanized anti-B7-2 mAbs, 10 .mu.g/ml CTLA4
Ig, 20 .mu.g/ml CTLA4 Ig, or control Ig. The culture proliferation
was measured on days 3, 4, and 5 by the addition of radiolabeled
thymidine.
[0048] FIG. 19 is a graph showing the results from a one way
secondary MRL assay using responders from the "B" stimulator
primary MLR (See FIG. 17) and fresh "B" stimulators. The responders
and stimulators were treated with the humanized anti-B7-2 mAb,
humanized anti-B7-1 mAb, combined humanized anti-B7-1 and humanized
anti-B7-2 mAbs, 10 .mu.g/ml CTLA4 Ig, 20 .mu.g/ml CTLA4 Ig, or
control Ig in the primary MLR only. There were no additions to the
secondary MLR. The culture proliferation was measured on days 3, 4,
5 and 6 by the addition of radiolabeled thymidine.
[0049] FIG. 20 is a graph showing the results from a one way
secondary MRL assay using responders from the "B" stimulator
primary MLR (See FIG. 17) and fresh "C" stimulators. The responder
and stimulator were treated with the humanized anti-B7-2 mAb,
humanized anti-B7-1 mAb, combined humanized anti-B7-1 and humanized
anti-B7-2 mAbs, 10 .mu.g/ml CTLA4 Ig, 20 .mu.g/ml CTLA4 Ig, or
control Ig in the primary MLR only. There were no additions to the
secondary MLR. The culture proliferation was measured on days 3, 4,
5 and 6 by the addition of radiolabeled thymidine.
[0050] FIG. 21 is a graph showing the results from a one way
secondary MRL assay using responders from the "B" stimulator
primary MLR (See FIG. 17) and fresh "B" or "C" stimulators. The
responder and stimulator were treated with the humanized anti-B7-2
mAb, humanized anti-B7-1 mAb, combined humanized anti-B7-1 and
humanized anti-B7-2 mAbs, 10 .mu.g/ml CTLA4 Ig, 20 .mu.g/ml CTLA4
Ig, or control Ig in the primary MLR only. There were no additions
to the secondary MLR. The culture proliferation was measured on
days 3, 4, 5 and 6 by the addition of radiolabeled thymidine. FIG.
21 is a compilation of FIGS. 19 and 20.
[0051] FIG. 22 is a graph depicting the anti-tetanus response (log
titer) in non-human primates immunized with tetanus toxoid.
Cynomolgus monkeys were immunized with purified tetanus toxoid and
treated with humanized anti-B7-1 and humanized anti-B7-2
antibodies. Serum anti-tetanus antibody titers (IgM & IgG) were
measured weekly over a 26 week period.
[0052] FIG. 23 is a graph depicting the anti-tetanus response (log
titer) in non-human primates immunized with tetanus toxoid.
Cynomolgus monkeys were immunized with tetanus toxoid on Day 0 and
treated with a single IV dose of humanized anti-B7-1 and humanized
anti-B7-2 antibodies in combination or vehicle on Day 0. At Week 14
animals were immunized with tetanus toxoid a second time without
additional humanized anti-B7-1 or humanized anti-B7-2 antibody
treatment. Serum anti-tetanus antibody titers (IgM & IgG) were
measured weekly over an 18 week period.
[0053] FIG. 24 is a graph depicting the anti-tetanus response (log
titer) in non-human primates immunized with tetanus toxoid.
Cynomolgus monkeys were immunized with tetanus toxoid on Day 0 and
treated with a single IV dose of humanized anti-B7-1 antibody alone
or vehicle on Day 0. At Week 14 animals were immunized with tetanus
toxoid a second time without additional humanized anti-B7-1
antibody treatment. Serum anti-tetanus antibody titers (IgM &
IgG) were measured weekly over an 18 week period.
[0054] FIG. 25 is a graph depicting the anti-tetanus response (log
titer) in non-human primates immunized with tetanus toxoid.
Cynomolgus monkeys were immunized with tetanus toxoid on Day 0 and
treated with a single IV dose of humanized anti-B7-2 antibody alone
or vehicle on Day 0. At Week 14 animals were immunized with tetanus
toxoid a second time without additional humanized anti-B7-2
antibody treatment. Serum anti-tetanus antibody titers (IgM &
IgG) were measured weekly over an 18 week period.
[0055] FIG. 26 is a bar graph showing the area under the
anti-tetanus antibody titer curve (log titer) for each treatment
Group (Group designations: Group 1: vehicle control; Groups 2-4:
10, 1, or 0.1 mg/kg of h1F1 alone; Groups 5-7: 10, 1, or 0.1 mg/kg
of h3D1 alone; Groups 8-11: 10, 1, 0.1, or 0.01 mg/kg of h1F1 and
h3D1 in combination). Cynomolgus monkeys were immunized with
tetanus toxoid 0 and treated with a single IV dose of humanized
anti-B7-1 and humanized anti-B7-2 antibodies in combination,
humanized anti-B7-1 antibody or humanized anti-B7-2 antibody alone,
or vehicle (n=3 per group). Area Under the Curve (AUC) values were
calculated from time 0 to 14 weeks. All tetanus titers were
normalized to a baseline of zero before AUCs of the tetanus titer
curves were calculated. These AUC values were weighted by the
fraction of the number of animals in each group that produced
detectable antibody titers to account for the number of responding
animals in each group.
[0056] FIG. 27 is a graph showing the serum concentration of
anti-B7-1 and anti-B7-2 (IgG2.M3 isotype) mAbs at various times
after administration of an I.V. dose of 10 mg/kg.
[0057] FIG. 28 is a graph depicting the percent survival over about
1 year of Rhesus Monkeys that received a renal allotransplantation.
The monkeys were treated with humanized anti-B7-1 and humanized
anti-B7-2 antibodies in combination, humanized anti-B7-1 antibody
or humanized anti-B7-2 antibody alone, or a vehicle. The humanized
antibodies were given at an initial dose of 20 mg/kg followed by 5
mg/kg and then weekly doses of 5 mg/kg for between 60-80 days.
DETAILED DESCRIPTION OF THE INVENTION
[0058] The invention relates to humanized immunoglobulins having
binding specificity for B7-2 or B7-1, comprising an antigen binding
region of non-human origin and at least a portion of an
immunoglobulin of human origin. Preferably, the humanized
immunoglobulins can bind B7-2 or B7-1 with an affinity of at least
about 10.sup.7 M.sup.-1, preferably at least about 10.sup.8
M.sup.-1, and more preferably at least about 10.sup.9 M.sup.-1. In
one embodiment, the humanized immunoglobulins include an antigen
binding region of non-human origin which binds B7-2 or B7-1 and a
constant region derived from a human constant region. The human
constant region can have non-human residues in the framework region
(FR). In another embodiment, the humanized immunoglobulins which
binds B7-2 or B7-1 comprise a complementarity determining region
(one or more) of non-human origin and a variable framework region
(one or more) of human origin, and optionally, a constant region of
human origin. Optionally, the FR region of the immunoglobulins can
comprise residues of non-human origin. For example, the humanized
immunoglobulins can comprise a heavy chain and a light chain,
wherein the light chain comprises a complementarity determining
region derived from an antibody of non-human origin which binds
B7-2 and a framework region derived from a light chain of human
origin, and the heavy chain comprises a complementarity determining
region derived from an antibody of non-human origin which binds
B7-2 and a framework region derived from a heavy chain of human
origin. In another example, the humanized immunoglobulins can
comprise a heavy chain and a light chain, wherein the light chain
comprises a complementarity determining region derived from an
antibody of non-human origin which binds B7-1 and a framework
region derived from a light chain of human origin, and the heavy
chain comprises a complementarity determining region derived from
an antibody of non-human origin which binds B7-1 and a framework
region derived from a heavy chain of human origin. Also, the
invention, individually or in a functional combination, embodies
the light chain, the heavy chain, the variable region, the variable
light chain and the variable heavy chain.
[0059] The invention relates to a humanized B7-2 antibody that has
substantially the same binding specificity as the murine B7-2
antibody from which the humanized antibody is made, but with
reduced immunogenicity in primates (e.g., humans). Similarly, the
invention also relates to a humanized B7-1 antibody that has
substantially the same binding specificity as the murine B7-1
antibody, respectively, from which the humanized antibody is made,
but with reduced immunogenicity in primates (e.g., humans). The
humanized B7-2 or B7-1 antibody can have about a lesser,
substantially the same, or greater binding affinity as the murine
counterpart. See FIGS. 3, 4, 8, 9A and 9B.
[0060] Naturally occurring immunoglobulins have a common core
structure in which two identical light chains (about 24 kD) and two
identical heavy chains (about 55 or 70 kD) form a tetramer. The
amino-terminal portion of each chain is known as the variable (V)
region, also referred to as the "antigen binding" region, and can
be distinguished from the more conserved constant (C) regions of
the remainder of each chain. Within the variable region of the
light chain is a C-terminal portion known as the J region. Within
the variable region of the heavy chain, there is a D region in
addition to the J region. Most of the amino acid sequence variation
in immunoglobulins is confined to three separate locations in the V
regions known as hypervariable regions or complementarity
determining regions (CDRs) which are directly involved in antigen
binding. The variable region is the portion of the antibody that
binds to the antigen. The constant region allows for various
functions such as the ability to bind to Fc receptors on phagocytic
cells, placental cells, mast cells, etc. The light and heavy chains
each have a variable region and a constant region. Accordingly, the
invention relates to humanized immunoglobulins having binding
specificity to B7-2 or B7-1. The humanized B7-1 or B7-2
immunoglobulin comprises a light chain and a heavy chain in which
two light chains and two heavy chains form the tetramer.
[0061] The variable region further constitutes two types of
regions, a framework region (FR) and a complementarity determining
region (CDR). CDRs are hypervariable regions that contain most of
the amino acid sequence variation in between immunoglobulins.
Proceeding from the amino-terminus, these regions are designated
CDR1, CDR2 and CDR3, respectively. See FIGS. 1A-1B, 2A-2B, 6A-6B
and 7A-7B. The CDRs are connected by more conserved FRs. Proceeding
from the amino-terminus, these regions are designated FR1, FR2,
FR3, and FR4, respectively. The locations of CDR and FR regions and
a numbering system have been defined by Kabat et al. (Kabat, E. A.
et al., Sequences of Proteins of Immunological Interest, Fifth
Edition, U.S. Department of Health and Human Services, U.S.
Government Printing Office (1991); Kabat, E. A. Structural Concepts
in Immunology and Immunochemistry, Second Edition, Holt, Rinehart
and Winston, New York (1976); Kabat, E. A. Sequences of
Immunoglobulin Chains: Tabulation and Analysis of Amino Acid
Sequences of Precursors, V-regions, C-regions, J-Chain and
.beta.2-Microglobulins, U.S. Department of Health, Education and
Welfare, Public Health Service, (1979); Kabat, E. A. Structural
Concepts in Immunology and Immunochemistry, Holt, Rinehart and
Winston, New York (1968); Kabat, E. A. Experimental
Immunochemistry, Second Edition, Springfield, Thomas (1967). During
the process of humanizing an immunoglobulin, one or more of the
CDRs from an antibody having specificity for B7-2 or B7-1 from a
non-human species is grafted into the FRs of a human antibody. In
addition, certain non-human framework substitutes can be made
according to the methods described herein. The resulting humanized
antibody has CDRs from a non-human species such as a mouse and FRs
from a human antibody, whereby the humanized antibody maintains its
antigenic specificity and affinity to B7-1 or B7-2.
[0062] The invention also relates to a B7-1 or B7-2 humanized
immunoglobulin light chain, or a B7-1 or B7-2 humanized
immunoglobulin heavy chain. In one embodiment, the invention
relates to a humanized B7-2 light chain comprising one or more
light chain CDRs (e.g., CDR1 (SEQ ID NO: 16), CDR2 (SEQ ID NO: 18)
and/or CDR3 (SEQ ID NO: 20)) of non-human origin and a human light
chain framework region (See FIG. 2B). In another embodiment, the
invention relates to a humanized B7-2 immunoglobulin heavy chain
comprising one or more heavy chain CDRs (e.g., CDR1 (SEQ ID NO:
10), CDR2 (SEQ ID NO: 12), and/or CDR3 (SEQ ID NO: 14)) of
non-human origin and a human heavy chain framework region (See FIG.
2A). The CDRs can be derived from a non-human immunoglobulin such
as murine heavy (e.g., SEQ ID NO: 1, FIG. 1A) and light (e.g., SEQ
ID NO: 3, FIG. 1B) variable region chains of the 3D1 antibody which
are specific to B7-2.
[0063] In another embodiment, the invention relates to a humanized
B7-1 light chain comprising one or more light chain CDRs (e.g.,
CDR1 (SEQ ID NO: 36), CDR2 (SEQ ID NO: 38) and/or CDR3 (SEQ ID NO:
40)) of non-human origin and a human light chain framework region
(See FIG. 7B). The invention also pertains to a B7-1 humanized
immunoglobulin heavy chain comprising one or more heavy chain CDRs
(e.g., CDR1 (SEQ ID NO: 30), CDR2 (SEQ ID NO: 32), and/or CDR3 (SEQ
ID NO: 34)) of non-human origin and a human heavy chain framework
region (See FIG. 7A). The CDRs can be derived from a non-human
immunoglobulin such as murine heavy (e.g., SEQ ID NO: 21, FIG. 6A)
and light (e.g., SEQ ID NO: 23, FIG. 6B) variable region chains of
the 1F1 antibody which are specific to B7-1.
[0064] The invention also embodies the humanized anti-B7-2 antibody
expressed by a cell line deposited with the A.T.C.C., 10801
University Boulevard, Manassas, Va. 02110-2209, on May 5, 1998,
A.T.C.C. No: CRL-12524. The cell line which expresses the humanized
anti-B7-2 antibody, deposited with the A.T.C.C., is designated as a
recombinant CHO cell line (PA-CHO-DUKX-1538) expressing the
humanized anti-human B7-2 (CD86) monoclonal antibody (#HF2-3D1) of
the IgG2.M3 isotype.
[0065] The invention also embodies the humanized anti-B7-1 antibody
expressed by a cell line deposited with the A.T.C.C., 10801
University Boulevard, Manassas, Va. 02110-2209, on Jun. 22, 1999,
under A.T.C.C. No: PTA-263. The cell line which expresses the
humanized anti-B7-1 antibody, deposited with the A.T.C.C., is
designated as a recombinant CHO cell line (PA-CHO-DUKX-1538)
expressing the humanized anti-human B7-1 (CD80) monoclonal antibody
(#1F1).
[0066] Human immunoglobulins can be divided into classes and
subclasses, depending on the isotype of the heavy chain. The
classes include IgG, IgM, IgA, IgD and IgE, in which the heavy
chains are of the gamma (.gamma.), mu (.mu.), alpha (.alpha.),
delta (.delta.) or epsilon (.epsilon.) type, respectively.
Subclasses include IgG1, IgG2, IgG3, IgG4, IgA1 and IgA2, in which
the heavy chains are of the .gamma.1, .gamma.2, .gamma.3, .gamma.4,
.alpha.1 and .alpha.2 type, respectively. Human immunoglobulin
molecules of a selected class or subclass may contain either a
kappa (.kappa.) or lambda (.lamda.) light chain. See e.g., Cellular
and Molecular Immunology, Wonsiewicz, M. J., Ed., Chapter 45, pp.
41-50, W.B. Saunders Co, Philadelphia, Pa. (1991); Nisonoff, A.,
Introduction to Molecular Immunology, 2nd Ed., Chapter 4, pp.
45-65, Sinauer Associates, Inc., Sunderland, Mass. (1984).
[0067] The terms "HF2.3D1" and "3D1" refer to a murine
immunoglobulin specific to B7-2. The terms "humanized HF2.3D1,"
"humanized 3D1", "hu3D1," "h3D1," "B7-2 humanized immunoglobulin"
or "humanized B7-2 immunoglobulin" refer to a humanized
immunoglobulin specific to human B7-2 (e.g., mouse anti-human B7-2
antibody). The terms "1F1" or "mouse 1F1" refer to a murine
immunoglobulin that is specific to B7-1. The terms "humanized 1F1",
"hu1F1," "h1F1," "B7-1 humanized immunoglobulin" or "humanized B7-1
immunoglobulin" refer to a humanized immunoglobulin specific to
human B7-1 (e.g., mouse anti-human B7-1 antibody). The term "B7
molecules" refer to the B7-1 and B7-2 molecules. The term "B7
antibodies" refer to the anti-human B7-1 and anti-human B7-2
antibodies.
[0068] The terms "immunoglobulin" or "antibody" include whole
antibodies and biologically functional fragments thereof. Such
biologically functional fragments retain at least one antigen
binding function of a corresponding full-length antibody and
preferably, retain the ability to inhibit the interaction of B7-2
or B7-1 with one or more of its receptors (e.g., CD28, CTLA4). In a
preferred embodiment, biologically functional fragments can inhibit
binding of B7-2 and/or B7-1 for manipulation of the co-stimulatory
pathway. Examples of biologically functional antibody fragments
which can be used include fragments capable of binding to B7-2 or
B7-1, such as single chain antibodies, Fv, Fab, Fab' and
F(ab').sub.2 fragments. Such fragments can be produced by enzymatic
cleavage or by recombinant techniques. For instance, papain or
pepsin cleavage can be used to generate Fab or F(ab').sub.2
fragments, respectively. Antibodies can also be produced in a
variety of truncated forms using antibody genes in which one or
more stop codons have been introduced upstream of the natural stop
site. For example, a chimeric gene encoding the heavy chain of a
F(ab').sub.2 fragment can be designed to include DNA sequences
encoding the CH.sub.1 domain and hinge region of the heavy chain.
The invention includes single chain antibodies (e.g., a single
chain FV) that contain both portions of the heavy and light
chains.
[0069] The term "humanized immunoglobulin" as used herein refers to
an immunoglobulin comprising portions of immunoglobulins of
different origin, wherein at least one portion is of human origin.
For example, the humanized antibody can comprise portions derived
from an immunoglobulin of non-human origin with the requisite
specificity, such as a mouse, and from immunoglobulin sequences of
human origin (e.g., chimeric immunoglobulin). These portions can be
joined together chemically by conventional techniques (e.g.,
synthetic) or prepared as a contiguous polypeptide using genetic
engineering techniques (e.g., DNA encoding the protein portions of
the chimeric antibody can be expressed to produce a contiguous
polypeptide chain). Another example of a humanized immunoglobulin
of the invention is an immunoglobulin containing one or more
immunoglobulin chains comprising a CDR derived from an antibody of
non-human origin and a framework region derived from a light and/or
heavy chain of human origin (e.g., CDR-grafted antibodies with or
without framework changes). Chimeric or CDR-grafted single chain
antibodies are also encompassed by the term humanized
immunoglobulin. See, e.g., Cabilly et al., U.S. Pat. No. 4,816,567;
Cabilly et al., European Patent No. 0,125,023 B1; Boss et al., U.S.
Pat. No. 4,816,397; Boss et al., European Patent No. 0,120,694 B1;
Neuberger, M. S. et al., WO 86/01533; Neuberger, M. S. et al.,
European Patent No. 0,194,276 B1; Winter, U.S. Pat. No. 5,225,539;
Winter, European Patent No. 0,239,400 B1; Padlan, E. A. et al.,
European Patent Application No. 0,519,596 A1. See also, Ladner et
al., U.S. Pat. No. 4,946,778; Huston, U.S. Pat. No. 5,476,786; and
Bird, R. E. et al., Science, 242: 423-426 (1988), regarding single
chain antibodies.
[0070] As embodied in the exemplified antibody of the present
invention, the term "humanized immunoglobulin" also refers to an
immunoglobulin comprising a human framework, at least one CDR from
a non-human antibody, and in which any constant region present is
substantially identical to a human immunoglobulin constant region,
e.g., at least about 60-90%, preferably at least 95% identical.
Hence, all parts of a humanized immunoglobulin, except possibly the
CDR's, are substantially identical to corresponding parts of one or
more native human immunoglobulin sequences. In some instances, the
humanized immunoglobulin, in addition to CDRs from a non-human
antibody, would include additional non-human residues in the human
framework region.
[0071] The design of humanized immunoglobulins can be carried out
as follows. When an amino acid falls under the following
categories, the framework amino acid of a human immunoglobulin to
be used (acceptor immunoglobulin) is replaced by a framework amino
acid from a CDR-providing non-human immunoglobulin (donor
immunoglobulin): [0072] (a) the amino acid in the human framework
region of the acceptor immunoglobulin is unusual for human
immunoglobulin at that position, whereas the corresponding amino
acid in the donor immunoglobulin is typical for human
immunoglobulin in that position: [0073] (b) the position of the
amino acid is immediately adjacent to one of the CDR's; or [0074]
(c) the amino acid is capable of interacting with the CDR's in a
tertiary structure immunoglobulin model (see, Queen et al., op.
cit., and Co et al., Proc. Natl. Acad. Sci. USA 88, 2869
(1991)).
[0075] For a detailed description of the production of humanized
immunoglobulins, See Queen et al., op. cit. and Co et al, op. cit.
and U.S. Pat. Nos. 5,585,089; 5,693,762, 5,693,761, and
5,530,101.
[0076] Usually, the CDR regions in humanized antibodies are
substantially identical, and more usually, identical to the
corresponding CDR regions in the mouse antibody from which they
were derived. Although not usually desirable, it is sometimes
possible to make one or more conservative amino acid substitutions
of CDR residues without appreciably affecting the binding affinity
of the resulting humanized immunoglobulin. Occasionally,
substitutions of CDR regions can enhance binding affinity.
[0077] Other than for the specific amino acid substitutions
discussed above, the framework regions of humanized immunoglobulins
are usually substantially identical, and more usually, identical to
the framework regions of the human antibodies from which they were
derived. Of course, many of the amino acids in the framework region
make little or no direct contribution to the specificity or
affinity of an antibody. Thus, many individual conservative
substitutions of framework residues can be tolerated without
appreciable change of the specificity or affinity of the resulting
humanized immunoglobulin.
[0078] The antigen binding region of the humanized B7-2
immunoglobulin (the non-human portion) can be derived from an
immunoglobulin of non-human origin, referred to as a donor
immunoglobulin, having specificity for B7-2 (e.g., the 3D1
antibody) or B7-1 (e.g., the 1F1 antibody). For example, a suitable
antigen binding region for the humanized B7-2 antibody can be
derived from the HF2.3D1 monoclonal antibody, a murine anti-human
B7-2 antibody. U.S. Ser. No. 08/101,624, filed on Jul. 26, 1993,
Ser. No. 08/109,393, filed Aug. 19, 1993 and Ser. No. 08/147,773,
filed Nov. 3, 1993, entitled, "B7-2:CTLA4/CD28 Counter Receptor".
See also, Freeman, et al., WO 95/03408," B7-2: CTLA4/CD 28 Counter
Receptor, published on Feb. 2, 1995. A suitable antigen binding
region for the humanized B7-1 antibody can be derived from the
murine 1F1 monoclonal antibody, a murine-anti-human B7-1 antibody.
Other sources include B7-2 or B7-1 specific antibodies obtained
from non-human sources, such as rodent (e.g., mouse and rat),
rabbit, pig, goat or non-human primate (e.g., monkey) or camelid
animals (e.g., camels and llamas).
[0079] Additionally, other polyclonal or monoclonal antibodies,
such as antibodies which bind to the same or similar epitope as the
murine HF2.3D1 or 1F1 antibodies, can be made (e.g., Kohler et al.,
Nature, 256:495-497 (1975); Harlow et al., 1988, Antibodies: A
Laboratory Manual, (Cold Spring Harbor, N.Y.); and Current
Protocols in Molecular Biology, Vol. 2 (Supplement 27, Summer '94),
Ausubel et al., Eds. (John Wiley & Sons: New York, N.Y.),
Chapter 11 (1991)). For example, antibodies can be raised against
an appropriate immunogen in a suitable mammal such as a mouse, rat,
rabbit, sheep, or camelid. Cells bearing B7-2 or B7-1, membrane
fractions containing B7-2 or B7-1 immunogenic fragments of B7-2 or
B7-1, and a B7-2 or B7-1 peptide conjugated to a suitable carrier
are examples of suitable immunogens (e.g., DNA or peptide
immunogens). Antibody-producing cells (e.g., a lymphocyte) can be
isolated, for example, from the lymph nodes or spleen of an
immunized animal. The cells can then be fused to a suitable
immortalized cell (e.g., a myeloma cell line), thereby forming a
hybridoma. Fused cells can be isolated employing selective
culturing techniques. Cells which produce antibodies with the
desired specificity can be selected by a suitable assay, such as an
ELISA. Immunoglobulins of non-human origin having binding
specificity for B7-2 or B7-1 can also be obtained from antibody
libraries, such as a phage library comprising non-human Fab
molecules. Humanized immunoglobulins can be made using other
techniques.
[0080] In one embodiment, the antigen binding region of the
humanized immunoglobulins comprise a CDR of non-human origin. In
this embodiment, humanized immunoglobulins having binding
specificity for B7-2 or B7-1 comprise at least one CDR of non-human
origin. For example, CDRs can be derived from the light and heavy
chain variable regions of immunoglobulins of non-human origin, such
that a humanized B7-2 immunoglobulin includes substantially the
heavy chain CDR1 (e.g., SEQ ID NO: 10), CDR2 (e.g., SEQ ID NO: 12)
and/or CDR3 (e.g., SEQ ID NO: 14) amino acid sequences, and/or
light chain CDR1 (e.g., SEQ ID NO: 16), CDR2 (e.g., SEQ ID NO: 18)
and/or CDR3 (e.g., SEQ ID NO: 20) amino acid sequences, from one or
more immunoglobulins of non-human origin, and the resulting
humanized immunoglobulin has binding specificity for B7-2. CDRs can
also be derived from light and heavy chain variable regions of
immunoglobulins of non-human origin that are specific for B7-1. The
humanized B7-1 antibody comprises substantially the heavy chain
CDR1 (SEQ ID NO: 30), CDR2 (SEQ ID NO: 32) and/or CDR3 (e.g., SEQ
ID NO: 34) amino acid sequences, and/or light chain CDR1 (SEQ ID
NO: 36), CDR2 (SEQ ID NO: 38) and/or CDR3 (SEQ ID NO: 40) amino
acid sequences, from one or more immunoglobulins of non-human
origin, and the resulting humanized immunoglobulin has binding
specificity for B7-1. All three CDRs of a selected chain can be
substantially the same as the CDRs of the corresponding chain of a
donor, and preferably, all three CDRs of the light and heavy chains
are substantially the same as the CDRs of the corresponding donor
chain. The nucleic acid sequences of the B7-2 heavy chain CDR1
(e.g., SEQ ID NO: 9), CDR2 (e.g., SEQ ID NO: 11) and CDR3 (e.g.,
SEQ ID NO: 13) and/or B7-2 light chain CDR1 (e.g., SEQ ID NO: 15),
CDR2 (e.g., SEQ ID NO: 17), and CDR3 (e.g., SEQ ID NO: 19) can also
be used in grafting the CDRs into the human framework.
Additionally, the nucleic acid sequences of the B7-1 heavy chain
CDR1 (SEQ ID NO: 29), CDR2 (SEQ ID NO: 31) and CDR3 (SEQ ID NO: 33)
and/or B7-1 light chain CDR1 (SEQ ID NO: 35), CDR2 (SEQ ID NO: 37)
and CDR3 (SEQ ID NO:39) can be used in grafting the CDRs into the
human framework.
[0081] In another embodiment, the invention pertains to humanized
immunoglobulins having a binding specificity for either B7-2 or
B7-1 comprising a heavy chain and a light chain. The light chain
can comprise a CDR derived from an antibody of non-human origin
which binds B7-2 or B7-1 and a FR derived from a light chain of
human origin. For example, the light chain can comprise CDR1, CDR2
and/or CDR3 which have the amino acid sequence set forth below or
an amino acid sequence substantially the same as the amino acid
sequence such that the antibody specifically binds to B7-2: CDR1
KSSQSLLNSRTRENYLA (SEQ ID NO: 16), CDR2 WASTRES (SEQ ID NO: 18),
and CDR3 TQSYNLYT (SEQ ID NO: 20). The heavy chain can comprise a
CDR derived from an antibody of non-human origin which binds B7-2
and a FR derived from a heavy chain of human origin. For example,
the B7-2 heavy chain can comprise CDR1, CDR2 and CDR3 which have
the amino acid sequence set forth below or an amino acid sequence
substantially the same as said amino acid sequence such that the
antibody specifically binds to the B7-2: heavy chain: CDR1 DYAIQ
(SEQ ID NO: 10), CDR2 VINIYYDNTNYNQKFKG (SEQ ID NO: 12), CDR3
AAWYMDY (SEQ ID NO: 14).
[0082] The light chain that is specific to B7-1 can comprise CDR1,
CDR2 and/or CDR3 that have the amino acid sequence set forth below
or an amino acid sequence substantially the same as the amino acid
sequence such that the antibody specifically binds to B7-1: CDR1
SVSSSISSSNLH (SEQ ID NO: 30), CDR2 GTSNLAS (SEQ ID NO: 32) and CDR3
QQWSSYPLT (SEQ ID NO: 34). The heavy chain can comprise a CDR
derived from an antibody of non-human origin which binds B7-1 and a
FR derived from a heavy chain of human origin. The heavy chain that
is specific to B7-1 can comprise CDR1, CDR2 and/or CDR3 that have
the amino acid sequence set forth below or an amino acid sequence
substantially the same as the amino acid sequence such that the
antibody specifically binds to B7-1: CDR1 DYYMH (SEQ ID NO: 36),
CDR2 WIDPENGNTLYDPKFQG (SEQ ID NO: 38), and CDR3 EGLFFAY (SEQ ID
NO: 40).
[0083] An embodiment of the invention is a humanized immunoglobulin
which specifically binds to B7-2 and comprises a humanized light
chain comprising three light chain CDRs from the mouse 3D1 antibody
and a light chain variable region framework sequence from a human
immunoglobulin light chain. The invention further comprises a B7-2
humanized heavy chain that comprises three heavy chain CDRs from
the mouse 3D1 antibody and a heavy chain variable region framework
sequence from a human immunoglobulin heavy chain. The mouse 3D1
antibody can further have a mature light chain variable domain as
shown in FIG. 1B (SEQ ID NO.: 4) and a mature heavy chain variable
domain as shown in FIG. 1A (SEQ ID NO.: 2).
[0084] In another embodiment of the invention is a humanized
immunoglobulin which specifically binds to B7-1 and comprises a
humanized light chain comprising three light chain CDRs from the
mouse 1F1 antibody, and a light chain variable region framework
sequence from a human immunoglobulin light chain. The invention
further comprises a B7-1 humanized heavy chain that comprises three
heavy chain CDRs from the mouse 1F1 antibody, respectively, and a
heavy chain variable region framework sequence from a human
immunoglobulin heavy chain. The mouse 1F1 antibody can have a
mature light chain variable domain as shown in FIG. 6B (SEQ ID NO:
24) and a mature heavy chain variable domain, as shown in FIG. 6A
(SEQ ID NO: 22).
[0085] The portion of the humanized immunoglobulin or
immunoglobulin chain which is of human origin (the human portion)
can be derived from any suitable human immunoglobulin or
immunoglobulin chain. For example, a human constant region or
portion thereof, if present, can be derived from the .kappa. or
.lamda. light chains, and/or the .gamma. (e.g., .gamma.1, .gamma.2,
.gamma.3, .gamma.4), .mu., .alpha. (e.g., .alpha.1, .alpha.2),
.delta. or .epsilon. heavy chains of human antibodies, including
allelic variants. A particular constant region, such as IgG2 or
IgG4, variants or portions thereof can be selected to tailor
effector function. For example, a mutated constant region, also
referred to as a "variant," can be incorporated into a fusion
protein to minimize binding to Fc receptors and/or ability to fix
complement (see e.g., Winter et al., U.S. Pat. Nos. 5,648,260 and
5,624,821; GB 2,209,757 B; Morrison et al., WO 89/07142; Morgan et
al., WO 94/29351, Dec. 22, 1994). In addition, a mutated IgG2 Fc
domain can be created that reduces the mitogenic response, as
compared to natural Fc regions (see e.g., Tso et al., U.S. Pat. No.
5,834,597, the teachings of which are incorporated by reference
herein in their entirety). See Example 3 for mutations performed to
the humanized anti-B7-2 antibody and Example 10 for mutations
performed to the humanized anti-B7-1 antibody.
[0086] If present, human FRs are preferably derived from a human
antibody variable region having sequence similarity to the
analogous or equivalent region of the antigen binding region donor.
Other sources of FRs for portions of human origin of a humanized
immunoglobulin include human variable consensus sequences (See,
Kettleborough, C. A. et al., Protein Engineering 4:773-783 (1991);
Queen et al., U.S. Pat. Nos. 5,585,089, 5,693,762 and 5,693,761).
For example, the sequence of the antibody or variable region used
to obtain the non-human portion can be compared to human sequences
as described in Kabat, E. A., et al., Sequences of Proteins of
Immunological Interest, Fifth Edition, U.S. Department of Health
and Human Services, U.S. Government Printing Office (1991). In a
preferred embodiment, the FRs of the humanized immunoglobulin
chains are derived from a human variable region having at least
about 60% overall sequence identity, and preferably at least about
80% overall sequence identity, with the variable region of the
non-human donor (e.g., murine HF2.3D1 or 1F1 antibody). For
example, the overall sequence identity between the mouse HF2.3D1
and human H2F light chain variable framework regions is 82.5%, and
the overall sequence identity between the mouse HF2.3D1 and human
I2R heavy chain variable framework regions is 62.5%. For the B7-1
antibody, the overall sequence identity between the murine 1F1 and
humanized III-2R light chain variable frame-work region is 69%, and
the overall sequence identity between the murine III-2R heavy chain
variable frame-work region is 79%.
[0087] The phrase "substantially identical," in context of two
nucleic acids or polypeptides (e.g., DNAs encoding a humanized
immunoglobulin or the amino acid sequence of the humanized
immunoglobulin) refers to two or more sequences or subsequences
that have at least about 80%, most preferably 90-95% or higher
nucleotide or amino acid residue identity, when compared and
aligned for maximum correspondence, as measured using the following
sequence comparison method and/or by visual inspection. Such
"substantially identical" sequences are typically considered to be
homologous. Preferably, the "substantial identity" exists over a
region of the sequences that is at least about 50 residues in
length, more preferably over a region of at least about 100
residues, and most preferably the sequences are substantially
identical over at least about 150 residues, or over the full length
of the two sequences to be compared. As described below, any two
antibody sequences can only be aligned in one way, by using the
numbering scheme in Kabat. Therefore, for antibodies, percent
identity has a unique and well-defined meaning.
[0088] Amino acids from the variable regions of the mature heavy
and light chains of immunoglobulins are designated Hx and Lx
respectively, where x is a number designating the position of an
amino acid according to the scheme of Kabat, Sequences of Proteins
of Immunological Interest (National Institutes of Health, Bethesda,
Md., 1987 and 1991). Kabat lists many amino acid sequences for
antibodies for each subgroup, and lists the most commonly occurring
amino acid for each residue position in that subgroup. Kabat uses a
method for assigning a residue number to each amino acid in a
listed sequence. Kabat's scheme is extendible to other antibodies
not included in the compendium by aligning the antibody in question
with one of the consensus sequences in Kabat. The use of the Kabat
numbering system readily identifies amino acids at equivalent
positions in different antibodies. For example, an amino acid at
the L50 position of a human antibody occupies the equivalent
position to an amino acid position L50 of a mouse antibody.
[0089] The basic antibody structural unit is known to comprise a
tetramer. Each tetramer is composed of two identical pairs of
polypeptide chains, each pair having one "light" (about 25 kDa) and
one "heavy" chain (about 50-70 kDa). The amino-terminal portion of
each chain includes a variable region of about 100 to 110 or more
amino acids primarily responsible for antigen recognition. The
carboxy-terminal portion of each chain defines a constant region
primarily responsible for effector function. The variable regions
of each light/heavy chain pair form the antibody binding site.
Thus, an intact antibody has two binding sites.
[0090] Light chains are classified as either kappa or lambda. Heavy
chains are classified as gamma, mu, alpha, delta, or epsilon, and
define the antibody's isotype as IgG, IgM, IgA, IgD, and IgE,
respectively. Within light and heavy chains, the variable and
constant regions are joined by a "J" region of about 12 or more
amino acids, with the heavy chain also including a "D" region of
about 10 more amino acids. See generally, Fundamental Immunology,
Paul, W., ed., 3rd ed. Raven Press, N.Y., 1993, Ch. 9).
[0091] From N-terminal to C-terminal, both light and heavy chain
variable regions comprise alternating framework and (CDRs)'' FR1,
CDR1, FR2, CDR2, FR3, CDR3 and FR4. The assignment of amino acids
to each region is in accordance with the definitions of Kabat
(1987) and (1991), supra and/or Chothia & Lesk, J. Mol. Biol.
196:901-917 (1987); Chothia et al., Nature 342:878-883 (1989).
[0092] In one embodiment, the humanized immunoglobulins comprise at
least one of the FRs derived from one or more chains of an antibody
of human origin. Thus, the FR can include a FR1, FR2, FR3 and/or
FR4 derived from one or more antibodies of human origin.
Preferably, the human portion of a selected humanized chain
includes FR1, FR2, FR3 and/or FR4 derived from a variable region of
human origin (e.g., from a human immunoglobulin chain, from a human
consensus sequence). In a preferred embodiment, the FRs for the
B7-2 light chain variable region are from the H2F human antibody
and the FRs for the B7-2 heavy chain variable region are from the
I2R human antibody. The FRs for B7-1 heavy and light chain variable
regions are from the III-2R antibody.
[0093] The immunoglobulin portions of non-human and human origin
for use in the invention have sequences that are identical to
immunoglobulins or immunoglobulin portions from which they are
derived, or to variants thereof. Such variants include mutants
differing by the addition, deletion, or substitution of one or more
residues. As indicated above, the CDRs which are of non-human
origin are substantially the same as in the non-human donor, and
preferably are identical to the CDRs of the non-human donor. As
described herein, changes in the FR, such as those which substitute
a residue of the FR of human origin with a residue from the
corresponding position of the donor can be made. One or more
mutations in the FR can be made, including deletions, insertions
and substitutions of one or more amino acids. Several such
substitutions are described in the design of a humanized HF2.3D1
antibody in Example 2 and for the humanized 1F1 in Example 9. For a
selected humanized antibody or chain, framework mutations can be
designed as described herein. Preferably, the B7-2 and B7-1
humanized immunoglobulins can bind B7-2 and B7-1, respectively,
with an affinity similar to or better than that of the non-human
donor. Variants can be produced by a variety of suitable methods,
including mutagenesis of non-human donor or acceptor human
chains.
[0094] The humanized immunoglobulins of the invention have binding
specificity for human B7-2 or B7-1, and include humanized
immunoglobulins (including fragments) which can bind determinants
of B7-2 or B7-1. In a preferred embodiment, the humanized
immunoglobulin of the present invention has at least one functional
characteristic of murine HF2.3D1 or 1F1 antibody, such as binding
function (e.g., having specificity for B7-2 or B7-1, having the
same or similar epitopic specificity), and/or inhibitory function
(e.g., the ability to inhibit the binding of a cell bearing CD28 or
CTLA4 to the B7-2 or B7-1 ligand). Thus, preferred humanized
immunoglobulins can have the binding specificity of the murine
HF2.3D1 or 1F1 antibody, the epitopic specificity of the murine
HF2.3D1 or 1F1 antibody (e.g., can compete with murine HF2.3D1 or
1F1, a chimeric HF2.3D1 or 1F1 antibody, or humanized HF2.3D1 or
1F1 for binding to B7-2 or B7-1, respectively) and/or inhibitory
function.
[0095] The binding function of a humanized immunoglobulin having
binding specificity for B7-2 or B7-1 can be detected by standard
immunological methods, for example, using assays which monitor
formation of a complex between humanized immunoglobulin and B7-2 or
B7-1 (e.g., a membrane fraction comprising B7-2 or B7-1, or human
lymphocyte cell line or recombinant host cell comprising nucleic
acid which expresses B7-2 or B7-1).
[0096] Binding and/or adhesion assays or other suitable methods can
also be used in procedures for the identification and/or isolation
of humanized immunoglobulins (e.g., from a library) with the
requisite specificity (e.g., an assay which monitors adhesion
between a cell bearing a B7-2 or B7-1 receptor and B7 molecule, or
other suitable methods).
[0097] The immunoglobulin portions of non-human and human origin
for use in the invention include light chains, heavy chains and
portions of light and heavy chains. These immunoglobulin portions
can be obtained or derived from immunoglobulins (e.g., by de novo
synthesis of a portion), or nucleic acids encoding an
immunoglobulin or chain thereof having the desired property (e.g.,
binds B7-2 or B7-1, sequence similarity) can be produced and
expressed. Humanized immunoglobulins comprising the desired
portions (e.g., antigen binding region, CDR, FR, C region) of human
and non-human origin can be produced using synthetic and/or
recombinant nucleic acids to prepare genes (e.g., cDNA) encoding
the desired humanized chain. To prepare a portion of a chain, one
or more stop codons can be introduced at the desired position. For
example, nucleic acid sequences coding for newly designed humanized
variable regions can be constructed using PCR mutagenesis methods
to alter existing DNA sequences (see e.g., Kamman, M., et al.,
Nucl. Acids Res. 17:5404 (1989)). PCR primers coding for the new
CDRs can be hybridized to a DNA template of a previously humanized
variable region which is based on the same, or a very similar,
human variable region (Sato, K., et al., Cancer Research 53:851-856
(1993)). If a similar DNA sequence is not available for use as a
template, a nucleic acid comprising a sequence encoding a variable
region sequence can be constructed from synthetic oligonucleotides
(see e.g., Kolbinger, F., Protein Engineering 8:971-980 (1993)). A
sequence encoding a signal peptide can also be incorporated into
the nucleic acid (e.g., on synthesis, upon insertion into a
vector). If the natural signal peptide sequence is unavailable, a
signal peptide sequence from another antibody can be used (see,
e.g., Kettleborough, C. A., Protein Engineering 4:773-783 (1991)).
Using these methods, methods described herein or other suitable
methods, variants can be readily produced. In one embodiment,
cloned variable regions can be mutagenized, and sequences encoding
variants with the desired specificity can be selected (e.g., from a
phage library; see e.g., Krebber et al., U.S. Pat. No. 5,514,548;
Hoogengoom et al., WO 93/06213, published Apr. 1, 1993)).
Nucleic Acids and Constructs Comprising Same:
[0098] The invention also relates to isolated and/or recombinant
(including, e.g., essentially pure) nucleic acids comprising
sequences which encode a humanized B7-1 or B7-2 immunoglobulin, or
humanized B7-1 or B7-2 immunoglobulin light or heavy chain of the
present invention.
[0099] Nucleic acids referred to herein as "isolated" are nucleic
acids which have been separated away from the nucleic acids of the
genomic DNA or cellular RNA of their source of origin (e.g., as it
exists in cells or in a mixture of nucleic acids such as a
library), and include nucleic acids obtained by methods described
herein or other suitable methods, including essentially pure
nucleic acids, nucleic acids produced by chemical synthesis, by
combinations of biological and chemical methods, and recombinant
nucleic acids which are isolated (see e.g., Daugherty, B. L. et
al., Nucleic Acids Res., 19(9): 2471-2476 (1991); Lewis, A. P. and
J. S. Crowe, Gene, 101: 297-302 (1991)).
[0100] Nucleic acids referred to herein as "recombinant" are
nucleic acids which have been produced by recombinant DNA
methodology, including those nucleic acids that are generated by
procedures which rely upon a method of artificial recombination,
such as the polymerase chain reaction (PCR) and/or cloning into a
vector (e.g., plasmid) using restriction enzymes. "Recombinant"
nucleic acids are also those that result from recombination events
that occur through the natural mechanisms of cells, but are
selected for after the introduction to the cells of nucleic acids
designed to allow and make probable a desired recombination
event.
[0101] The invention also relates, more specifically, to isolated
and/or recombinant nucleic acids comprising a nucleotide sequence
which encodes a humanized HF2.3D1 or 1F1 immunoglobulin, also
referred to as "humanized 3D1" or "humanized 1F1," respectively,
(e.g., a humanized immunoglobulin of the invention in which the
non-human portion is derived from the murine HF2.3D1 or 1F1
monoclonal antibody), or chain thereof. In one embodiment, the
light chain comprises three complementarity determining regions
derived from the light chain of the HF2.3D1 or 1F1 antibody, and
the heavy chain comprises three complementarity determining regions
derived from the heavy chain of the HF2.3D1 or 1F1 antibody. Such
nucleic acids include, for example, (a) a nucleic acid comprising a
sequence which encodes a polypeptide comprising the amino acid
sequence of the heavy chain variable region of a humanized HF2.3D1
or 1F1 immunoglobulin (e.g., SEQ ID NO: 5, See FIG. 2A or SEQ ID
NO: 25, See FIG. 7A), (b) a nucleic acid comprising a sequence
which encodes a polypeptide comprising the amino acid sequence of
the light chain variable region of a humanized HF2.3D1 or 1F1
immunoglobulin (e.g., SEQ ID NO: 7, See FIG. 2B or SEQ ID NO: 27,
See FIG. 7B), (c) a nucleic acid comprising a sequence which
encodes at least a functional portion of the light or heavy chain
variable region of a humanized HF2.3D1 or 1F1 immunoglobulin (e.g.,
a portion sufficient for antigen binding of a humanized
immunoglobulin which comprises the chain). Due to the degeneracy of
the genetic code, a variety of nucleic acids can be made which
encode a selected polypeptide. In one embodiment, the nucleic acid
comprises the nucleotide sequence of the variable region as set
forth or substantially as set forth in FIG. 2A and/or FIG. 2B, or
FIG. 7A and/or FIG. 7B, including double or single-stranded
polynucleotides. Isolated and/or recombinant nucleic acids meeting
these criteria can comprise nucleic acids encoding sequences
identical to sequences of humanized HF2.3D1 or humanized 1F1
antibody or variants thereof, as discussed above.
[0102] Nucleic acids of the invention can be used in the production
of humanized immunoglobulins having binding specificity for B7-2 or
B7-1. For example, a nucleic acid (e.g., DNA) encoding a humanized
immunoglobulin of the invention can be incorporated into a suitable
construct (e.g., a vector) for further manipulation of sequences or
for production of the encoded polypeptide in suitable host
cells.
Method of Producing Humanized Immunoglobulins Having Specificity
for B7-2 and/or B7-1:
[0103] Another aspect of the invention relates to a method of
preparing a humanized immunoglobulin which has binding specificity
for B7-2 or B7-1. The humanized immunoglobulin can be obtained, for
example, by the expression of one or more recombinant nucleic acids
encoding a humanized immunoglobulin having binding specificity for
B7-2 or B7-1 in a suitable host cell.
[0104] Constructs or expression vectors suitable for the expression
of a humanized immunoglobulin having binding specificity for B7-2
and/or B7-1 are also provided. The constructs can be introduced
into a suitable host cell, and cells which express a humanized
immunoglobulin of the invention, can be produced and maintained in
culture. Suitable host cells can be procaryotic, including
bacterial cells such as E. coli, B. subtilis and or other suitable
bacteria, or eucaryotic, such as fungal or yeast cells (e.g.,
Pichia pastoris, Aspergillus species, Saccharomyces cerevisiae,
Schizosaccharomyces pombe, Neurospora crassa), or other lower
eucaryotic cells, and cells of higher eucaryotes such as those from
insects (e.g., Sf9 insect cells (WO 94/26087, O'Connor, published
Nov. 24, 1994)). Suitable host cells can also come from plants,
transgenic animals, or mammals (e.g., COS cells, NSO cells, SP2/0,
Chinese hamster ovary cells (CHO), HuT 78 cells, 293 cells). (See,
e.g., Ausubel, F. M. et al., eds. Current Protocols in Molecular
Biology, Greene Publishing Associates and John Wiley & Sons
Inc., (1993)).
[0105] Host cells which produce a humanized immunoglobulin having
binding specificity for B7-2 and/or B7-1 can be produced as
follows. For example, a nucleic acid encoding all or part of the
coding sequence for the desired humanized immunoglobulin can be
inserted into a nucleic acid vector, e.g., a DNA vector, such as a
plasmid, virus or other suitable expression unit. A variety of
vectors are available, including vectors which are maintained in
single copy or multiple copy, or which become integrated into the
host cell chromosome.
[0106] Suitable expression vectors can contain a number of
components, including, but not limited to one or more of the
following: an origin of replication; a selectable marker gene; one
or more expression control elements, such as a transcriptional
control element (e.g., a promoter, an enhancer, terminator), and/or
one or more translation signals; a signal sequence or leader
sequence for membrane targeting or secretion. In a construct, a
signal sequence can be provided by the vector or other source. For
example, the transcriptional and/or translational signals of an
immunoglobulin can be used to direct expression.
[0107] A promoter can be provided for expression in a suitable host
cell. Promoters can be constitutive or inducible. For example, a
promoter can be operably linked to a nucleic acid encoding a
humanized immunoglobulin or immunoglobulin chain, such that it
directs expression of the encoded polypeptide. A variety of
suitable promoters for procaryotic (e.g., lac, tac, T3, and T7
promoters for E. coli) and eucaryotic (e.g., yeast alcohol
dehydrogenase (ADH) and SV40, CMV) hosts are available.
[0108] In addition, the expression vectors typically comprise a
selectable marker for selection of host cells carrying the vector,
and, in the case of replicable expression vector, an origin of
replication. Genes encoding products which confer antibiotic or
drug resistance are common selectable markers and can be used in
procaryotic (e.g., .beta.-lactamase gene (ampicillin resistance)
and Tet gene (tetracycline resistance)) and eucaryotic cells (e.g.,
neomycin (G418 or geneticin), gpt (mycophenolic acid), ampicillin,
and hygromycin resistance genes). Dihydrofolate reductase marker
genes permit selection with methotrexate in a variety of hosts.
Genes encoding the gene product of auxotrophic markers of the host
(e.g., LEU2, URA3 and HIS3) are often used as selectable markers in
yeast. Use of viral (e.g., baculovirus) or phage vectors, and
vectors which are capable of integrating into the genome of the
host cell, such as retroviral vectors, are also contemplated. The
invention also relates to cells carrying these expression
vectors.
[0109] For example, a nucleic acid (e.g., one or more nucleic
acids) encoding the heavy and light chains of a humanized
immunoglobulin having binding specificity for B7-2 or B7-1, or a
construct (e.g., one or more constructs) comprising such nucleic
acid(s), can be introduced into a suitable host cell by a method
appropriate to the host cell selected (e.g., transformation,
transfection, electroporation, infection), such that the nucleic
acid(s) are operably linked to one or more expression control
elements (e.g., in a vector, in a construct created by processes in
the cell, integrated into the host cell genome). Host cells can be
maintained under conditions suitable for expression (e.g., in the
presence of inducer, suitable media supplemented with appropriate
salts, growth factors, antibiotic, nutritional supplements, etc.),
whereby the encoded polypeptide(s) are produced. If desired, the
encoded protein (e.g., humanized HF2.3D1 or 1F1 antibody) can be
isolated from, for example, host cells, medium or milk. This
process encompasses expression in a host cell of a transgenic
animal (see e.g., WO 92/03918, GenPharm International, published
Mar. 19, 1992).
[0110] Fusion proteins can be produced in which a humanized
immunoglobulin or immunoglobulin chain is linked to a
non-immunoglobulin moiety (e.g., a moiety which does not occur in
immunoglobulins as found in nature) in an N-terminal location,
C-terminal location or internal to the fusion protein. For example,
some embodiments can be produced by the insertion of a nucleic acid
encoding immunoglobulin sequences into a suitable expression
vector, such as a pET vector (e.g., pET-15b, Novagen), a phage
vector (e.g., pCANTAB 5 E, Pharmacia), or other vector (e.g.,
pRIT2T Protein A fusion vector, Pharmacia). The resulting construct
can be introduced into a suitable host cell for expression. Upon
expression, some fusion proteins can be isolated or purified from a
cell lysate by means of a suitable affinity matrix (see e.g.,
Current Protocols in Molecular Biology (Ausubel, F. M. et al.,
eds., Vol. 2, Suppl. 26, pp. 16.4.1-16.7.8 (1991)).
Therapeutic Methods and Compositions:
[0111] Two types of T-cells exist: helper T cells and cytotoxic T
cells. The helper T cells can recognize an antigen that is coupled
with a major histocompatibility complex (MHC). Antigen presenting
cells internalize an antigen and re-express the antigen with the
MHC molecule. Upon recognition of the antigen, secretion of
cytokines occur. Cytokine secretion activates B-lymphocytes,
cytotoxic T cells, phagocytes and other cells. However, cytokine
secretion and cellular proliferation require more than recognition
of the antigen. Complete T-cell activation requires a second signal
referred to as the "co-stimulatory signal." These co-stimulatory
signals serve to initiate, maintain, and regulate the activation
cascade. An important co-stimulatory pathway is called the
B7:CD28/CTLA4 pathway.
[0112] The B7:CD28/CTLA4 pathway involves two co-stimulatory
ligands, B7-1 (CD80) and B7-2 (CD86). The B7-1 and B7-2 ligands
which are present on the antigen presenting cell each bind to two
receptors on T-cells called CD28 and CTLA4.
[0113] The expression of B7 polypeptides, B7-1 (CD80) and B7-2
(CD86), is tightly regulated. (Linsley, P S et al., Immunity
1:793-801 (1994). Unstimulated antigen-presenting cells generally
do not express B7-1 and B7-2, except in dendritic cells. After
activation, dendritic cells, epidermal Langerhans' cells, B cells,
and macrophages up-regulate the expression of B7-2 and B7-1.
Additionally, B7-2 can be expressed on granulocytes and on T-cell
molecules, and B7-1 is expressed in fibroblasts and T-cell
molecules. (Reiser, et al., New England J. of Med., 335:18,
1369-1377, 1371 (1996).
[0114] In most immune responses, B7-2 is induced earlier than B7-1
and rises to higher levels. B7-2 also affects the production of
interleukin-4 (IL-4) and the generation of type 2 helper cells. B7
molecules (B7-1 and B7-2) are also responsible for costimulating
CD8 T cells in the absence of CD4 T cells which can be helpful in
generating vaccines against melanoma. B7 molecules can costimulate
natural killer cells and .gamma./.delta. T cells. Hence, modulation
of B7 molecules is helpful in anti-tumor and anti-microbial
immunity.
[0115] The B7:CD28/CTLA4 pathway participates in various disease
states including the pathogenesis of infectious diseases, asthma,
autoimmune diseases, inflammatory disorders, the rejection of
grafted organs and graft versus host disease. This pathway also
participates in prophylaxis and mechanisms that stimulate the
immune system. Transfection with genes encoding costimulators, such
as B7, are applicable for anti-tumor and anti-viral vaccines. Also,
the B7 molecules participate in autoimmune diseases such as
systemic lupus erythematosus, diabetes mellitus, asthma, insulitis,
arthritis, inflammatory bowel disease, inflammatory dermatitis
(psoriasis vulgaris and atopic dermatitis), and multiple sclerosis.
(Reiser, et al., New England J. of Med., 335 (18): 1369 (1996).
Accordingly, the invention encompasses methods for treating the
disease, as described herein, comprising administering
immunoglobulin(s) that binds to B7-1 and/or B7-2. The
immunoglobulin should be administered in therapeutically effective
amounts and, optionally, in a carrier.
[0116] Treating an individual having a disease refers to minimizing
or alleviating one or more symptoms associated with the disease.
Treating an individual with a transplant rejection means minimizing
or alleviating one or more symptoms associated with transplant
rejection (e.g., fever, loss of kidney function, distended kidneys,
T-cell/APC cell attack the rejection). Preventing a disease in an
individual refers to preventing the occurrence of one or more
symptoms of the disease. Preventing a transplant rejection means
reducing one or more of the immune responses associated with such a
transplant rejection.
[0117] Therefore, modulating or influencing the B7 molecules role
can be useful in treating individuals with the diseases, described
herein. B7 modulation is also useful in treating individuals with
immune-related or autoimmune diseases and disorders in which B7-2
and/or B7-1 participates. The modulation of B7-2 or B7-1 can also
be used for diseases related to or affected by IL-4 and/or the
generation of type 2 helper cells. These disorders/diseases can be
treated using an antibody specific to B7-2 and/or B7-1. Preferably,
the antibody is a humanized antibody specific to B7-2 or B7-1.
Treatment of these diseases can be facilitated with
co-administration of an anti-B7-2 antibody, an anti-B7-1 antibody,
including chimeric and humanized versions thereof, and/or
antibodies to the corresponding receptors, CD28 and CTLA4.
[0118] In addition to the diseases described herein, the
immunoglobulins that bind B7-1 and/or B7-2 can be administered to a
person having transplanted tissue, organ or cells. Inhibiting the
B7 pathway prevents or reduces the rejection of the transplanted
tissue, organ or cell. The invention pertains to treating acute
and/or chronic transplant rejection for a prolonged period of time
(e.g., days, months, years). Acute transplant rejection generally
occurs within the first few weeks of the transplant, whereas
chronic rejection occurs after the first few weeks. The amounts of
the anti-B7-1 and anti-B7-2 antibodies administered are described
herein.
[0119] In particular, the invention pertains to methods of treating
a transplant recipient or preventing transplant rejection. This
method involves administering the humanized anti-B7-1 and humanized
anti-B7-2 antibodies in sufficient amounts to prevent rejection of
the transplant. The transplant can be cells, tissue, or an organ.
The antibodies can be administered before and/or after the
transplant, or at the time of the transplant. The antibodies are
administered in doses (e.g., between about 1 mg/kg and about 100
mg/kg). In particular, the antibodies are administered in higher
doses (e.g., between about 1 mg/kg and about 25 mg/kg) on the day
of the transplant, and then at lower doses periodically after the
transplant (e.g., weekly at a dose between about 1 mg/kg and about
5 mg/kg). Remarkably, such administration results in a significant
decrease and even complete prevention of an immune response
directed toward the transplant rejection. See Examples 22 and
23.
[0120] Methods of treatment also involve co-administration of a
humanized anti-B7-2 antibody and/or humanized anti-B7-1 antibody
with other known standard of care drugs (e.g., drugs that are used
to modulate the immune response of an individual having a
transplanted organ, tissue, cell or the like). Such drugs include,
for example, methotrexate, immunosuppressive agents that arrest the
growth of immune cells or inhibit cell cycle progression (e.g.,
rapamycin), steroids (e.g., prednisone or derivative thereof),
calcineurin inhibitors (e.g., cyclosporin or FK506), anti-CD40
pathway inhibitors (e.g., anti-CD40 antibodies, anti-CD40 ligand
antibodies and small molecule inhibitors of the CD40 pathway),
transplant salvage pathway inhibitors (e.g., mycophenolate mofetil
(MMF)), IL-2 receptor antagonists (e.g., Zeonpax.RTM. from
Hoffmann-la Roche Inc., and Simulet from Novartis, Inc.) and
analogs thereof, or transplant rejection drugs developed in the
future. The data described herein show that Cyclosporin A,
prednisone and rapamycin work particularly well in preventing
transplant rejection when any one of these compounds are
administered with humanized anti-B7-1 and humanized anti-B7-2
antibodies. The amounts administered of these compounds vary. The
amounts administered depend their serum concentrations in the
individual. A higher serum concentration warrants a lower dosage,
and lower serum concentration warrants a higher dosage. For
example, Cyclosporin A can be administered in an amount between
about 150 ng/ml and about 100 mg/ml (e.g., 200-300 ng/ml),
prednisone can be administered between about 0.2 mg/kg and about
2.0 mg/ml, methylprednisone can be administered between about 0.2
mg/kg and 2.0 mg/kg, and rapamycin can be administered between
about 0.5 mg/kg and about 2.0 mg/kg, when administered with the
humanized anti-B7-1 and humanized anti-B7-2 antibodies.
[0121] The invention includes ex vivo methods for transplanting
cells (e.g., blood cells or components, or bone marrow) to an
individual in need thereof. An individual in need thereof is one,
for example, having a disease that is treated with such a
transplant (e.g., proliferative diseases such as leukemia,
lymphoma, cancer; anemia such as sickle-cell anemia, thalassemia,
and aplastic anemia; inborn errors of metabolism; congenital
immunodeficiency diseases; and myeloid dysplasia syndrome). The
method comprises obtaining cells from a donor. Generally, donor
bone marrow contains both immature and mature lymphocytes. The
blood cells from a donor can be stem cells or immature blood cells
in addition to bone marrow cells. The cells of the donor preferably
comes from, but is not limited to a person who has similar
characteristics as the patient/recipient (e.g., the donor's bone
marrow is a match to the patient's bone marrow). The
characteristics that are analyzed to determine whether a donor is a
match to the patient are MHC class 1 and 2 (e.g., HLA-A, HLA-B,
and/or HLA-DR). The method involves contacting the cells (e.g.,
bone marrow or other blood components) with an immunoglobulin
specific to B7-1 and/or an immunoglobulin specific to B7-2 and
recipient cells (e.g., lymphocyte from the patient) to obtain a
mixture, referred to as "treated cells". The amount of antibody
utilized depends on the number of cells present. A greater amount
of cells requires more antibody, and a lesser amount of cells
requires less antibody. The experiments described herein utilized
10 mg/ml of each antibody which is in excess of 10-100 fold. The
amount of the anti-B7-1 and/or anti-B7-2 antibodies used in this
embodiment should be sufficient to induce anergy, e.g., about 0.01
to about 10 mg/ml. The donor cells, immunoglobulin(s) and recipient
cells are in contact for a period of time sufficient for tolerance
induction (e.g., about 1-96 hours, preferably about 36-48 hours).
Tolerance induction (e.g., anergy) refers to the lack of
responsiveness to an antigen that has been induced with a treatment
with B7-1 and/or B7-2 antibodies, such that the T-cell can no
longer adequately or fully respond to that antigen. Example 18. The
recipient cells (e.g., Peripheral Blood Lymphocytes (PBL), or
lymphocytes that express class I antigens (MHC-I)) are irradiated
to prevent cells from dividing. A substitute for recipient cells
can be tissue, organs or engineered cells that express MHC class I
antigens, and B7-1 and/or B7-2 molecules. The method then includes
introducing the mixture (e.g., the treated cells) or treated bone
marrow to the patient. This method of treatment is aimed at
preventing graft vs. host disease. For example, cells in the
treated bone marrow become tolerant to recipient alloantigen
thereby reducing or eliminating graft vs. host disease, and
improving engraphment of donor marrow (e.g., stem cells).
Accordingly, the methods of the present invention include treating,
preventing or aiding in the prevention of graft vs. host disease.
The anti-B7-1 and anti-B7-2 antibodies reduce rejection by donor
bone marrow or donor cells of the recipient. However, the methods
are able to reduce rejection without significantly compromising the
patient's ability to detect and develop an immune response to other
foreign cells and antigens. Hence, these methods allow the
transplantation to be recipient specific, and allow for the
rejection of foreign antigens without compromising the transplant.
See Exemplification Section.
[0122] The invention also relates to methods for decreasing an
antibody response to an antigen in an individual by administering
the humanized anti-B7-1 and/or humanized anti-B7-2 antibodies to
the individual. The antibodies can be administered in the presence
of the antigen. The antigen can be growth factors, clotting
factors, cytokines, chemokines, gene therapy vehicles or hormones.
In particular, the antigen can be, for example, tetanus toxoid,
Factor VIII, Factor IX, insulin, growth hormone, or a gene delivery
vector. The antigens can be administered in a polypeptide form, or
in a nucleic acid form (e.g., gene delivery through
adeno-associated viruses, retroviruses, naked DNA vectors, etc.).
Suppression of the antibody response to these antigens is helpful
in the treatment of a number of diseases, such as in hemophiliacs
who develop antibody responses to their administered Factor VIII or
Factor IX, thereby leading to blood clotting problems. The data
described herein shows that administering effective amounts of the
humanized anti-B7-1 and/or humanized anti-B7-2 antibodies in
conjunction with the model antigen, tetanus toxoid, inhibit an
antibody response to the tetanus toxoid. The antibodies can be
administered together with the antigen, or close enough in time
(e.g., shortly before or shortly thereafter) to confer the desired
effect, e.g., suppression of the antibody response. The humanized
anti-B7-1 and humanized anti-B7-2 antibodies are administered
within about 3 weeks (the 1/2 life of the antibodies) of the
antigen, e.g., between about 14 days before and about 2 days after
administration of the antigen. The humanized anti-B7-1 and
humanized anti-B7-2 antibodies are administered between about 0.01
mg/kg and about 10 mg/kg.
[0123] The invention pertains to a pharmaceutical composition
comprising a humanized anti-B7-1 and/or humanized anti-B7-2
antibody, with or without a carrier. A preferred embodiment is to
administer the B7-1 and/or B7-2 immunoglobulins in injectable or
capsule form. In particular, the injectable form can be intravenous
or subcutaneous injections. The terms "pharmaceutically acceptable
carrier" or a "carrier" refer to any generally acceptable excipient
or drug delivery composition that is relatively inert and
non-toxic. Exemplary carriers include calcium carbonate, sucrose,
dextrose, mannose, albumin, starch, cellulose, silica gel,
polyethylene glycol (PEG), dried skim milk, rice flour, magnesium
stearate, and the like. Suitable formulations and additional
carriers are described in Remington's Pharmaceutical Sciences,
(17th Ed., Mack Pub. Co., Easton, Pa.).
[0124] Suitable carriers (e.g., pharmaceutical carriers) also
include, but are not limited to sterile water, salt solutions (such
as Ringer's solution), alcohols, polyethylene glycols, gelatin,
carbohydrates such as lactose, amylose or starch, magnesium
stearate, talc, silicic acid, viscous paraffin, fatty acid esters,
hydroxymethylcellulose, polyvinyl pyrolidone, etc. Such
preparations can be sterilized and, if desired, mixed with
auxiliary agents, e.g., lubricants, preservatives, stabilizers,
wetting agents, emulsifiers, salts for influencing osmotic
pressure, buffers, coloring, and/or aromatic substances and the
like which do not deleteriously react with the immunoglobulin. They
can also be combined where desired with other active substances,
e.g., enzyme inhibitors, to reduce metabolic degradation. A carrier
(e.g., a pharmaceutically acceptable carrier) is preferred, but not
necessary to administer the immunoglobulins.
[0125] For parenteral application, particularly suitable are
injectable, sterile solutions, preferably oily or aqueous
solutions, as well as suspensions, emulsions, or implants,
including suppositories. In particular, carriers for parenteral
administration include aqueous solutions of dextrose, saline, pure
water, ethanol, glycerol, propylene glycol, peanut oil, sesame oil,
polyoxyethylene-polyoxypropylene block polymers, and the like.
Ampules, vials and syringes are convenient unit dosages.
[0126] Immunoglobulins of the invention can be administered
intravenously, parenterally, intramuscular, subcutaneously, orally,
nasally, by inhalation, by implant, by injection, or by
suppository. The composition can be administered in a single dose
or in more than one dose over a period of time to confer the
desired effect.
[0127] The actual effective amounts of immunoglobulin can vary
according to the specific immunoglobulin being utilized, the
particular composition formulated, the mode of administration, and
the age, weight, condition of the patient, and severity of the
disorder or disease, for example. As used herein, an effective
amount of the B7-2 and/or B7-2 immunoglobulins is an amount which
modulates or inhibits the B7/CD28/CTLA4 pathway. Dosages for a
particular patient are described herein and can be determined by
one of ordinary skill in the art using conventional considerations,
(e.g. by means of an appropriate, conventional pharmacological
protocol).
[0128] The administration of the humanized B7-1 antibody, humanized
B7-2 antibody and/or other drugs can occur simultaneously or
sequentially in time. These compounds or compositions can be
administered before, after or at the same time. Thus, the term
"co-administration" is used herein to mean that the humanized B7-1
and/or B7-2 antibodies and/or other compositions are administered
at times to treat the diseases described herein or induce
tolerization (e.g., methotrexate; rapamycin; cyclosporin; steroids;
anti-CD40 pathway inhibitors such as anti-CD40 antibodies,
anti-CD40 ligand antibodies and small molecule inhibitors of the
CD40 pathway; transplant salvage pathway inhibitors such as
mycophenolate mofetil (MMF); IL-2 receptor antagonists such as
Zeonpax.RTM. from Hoffmann-la Roche Inc. and Simulet from Novartis,
Inc. and analogs thereof). The methods of the present invention are
not limited to the sequence in which the antibodies or compositions
are administered, so long as they are administered close enough in
time to produce the desired effect.
[0129] The invention also pertains to methods for determining the
presence, absence or level of B7-2 or B7-1 using a humanized
anti-B7-2 or B7-1 antibody, respectively. The presence or absence
of B7-2 or B7-1 can be detected in an assay (e.g., ELISA,
radioimmunoassay (RIA), FACS or Immunohistochemistry). The assay
can be a direct detection or an indirect detection (e.g. a
competitive assay).
[0130] For example, to determine the presence or absence of B7-2 or
B7-1 using an ELISA assay in a suitable sample, the method
comprises combining a suitable sample with a composition comprising
a humanized or murine anti-B7-2 or B7-1 antibody as detector (e.g.,
biotinylated anti-B7-2 or B7-1 mAb and HRP-streptavidin, or
HRP-conjugated anti-B7-2 or B7-1 mAb) and a solid support (e.g., a
microtiter plate), having an anti-B7-2 or B7-1 capture antibody
bound (directly or indirectly) thereto. The detector antibody can
bind to a different B7-2 or B7-1 epitope from that recognized by
the capture antibody, under conditions suitable for the formation
of a complex between the anti-B7-2 or B7-1 antibodies and B7-2 or
B7-1, respectively. The method further comprises determining the
formation of complex in the sample.
[0131] The presence of B7-2 or B7-1 can also be determined in a
radioimmunoassay or a fluorescent assay. For example, the presence
of B7-2 or B7-1 can be assessed by an immunobinding assay
comprising obtaining a sample, contacting the sample with a
composition comprising an anti-B7-2 or B7-1 antibody (e.g., a
humanized or murine anti-B7-2 or B7-1 antibody comprising a
radioactive or fluorescent label; or a humanized or murine
anti-B7-2 or B7-1 antibody comprising a binding site for a second
antibody which comprises a radioactive or fluorescent label),
preferably in an amount in excess of that required to bind the B7-2
or B7-1, under conditions suitable for the formation of labeled
complexes. The method further comprises determining (detecting or
measuring) the formation of complex in the samples. Similarly, the
present, absence or level of B7-1 or B7-2 can be determined using
Fluorescent-Activated Cell Sorting (FACS) analysis, or
histochemical analysis of tissues, using the humanized anti-B7-1 or
B7-2 antibody of the present invention.
EXEMPLIFICATION
[0132] The present invention will now be illustrated by the
following Examples, which are not intended to be limiting in any
way.
Example 1
Cloning and Sequencing of Mouse 3D1 Variable Region cDNAs
[0133] Mouse 3D1 (also referred to as HF2.3D1) heavy and light
chain variable region cDNAs were cloned from mRNA isolated from
hybridoma cells using anchored PCR (Co et al., J. Immunol. 148:
1149 (1992)). The 5' primers used annealed to the poly-dG tails
added to the cDNA, and the 3' primers annealed to the constant
regions. The amplified gene fragments were then inserted into the
plasmid pUC18. Nucleotide sequences were determined from several
independent clones for both V.sub.L and V.sub.H cDNA. For the heavy
chain, a single, unique sequence was identified, typical of a mouse
heavy chain variable region. For the light chain, two unique
sequences, both homologous to murine light chain variable region
sequences, were identified. However, one sequence was not
functional because of a missing nucleotide that caused a frame
shift at the V-J junction, and was identified as the non-productive
allele. The other sequence was typical of a functional mouse kappa
chain variable region. The variable region cDNA sequences of the
heavy chain and the functional light chain and the translated amino
acid sequences are shown in FIGS. 1A-1B. The mouse V.sub.L sequence
belongs to Kabat's mouse kappa chain subgroup I. The mouse V.sub.H
belongs to Kabat's heavy chain subgroup II(A).
Example 2
Design of Humanized 3D1 Variable Regions
[0134] To retain the binding affinity of the mouse antibody in the
humanized antibody, the general procedures of Queen et al. were
followed (Queen et al., Proc. Natl. Acad. Sci. USA 86: 10029
(1989), U.S. Pat. Nos. 5,585,089 and 5,693,762, the teachings of
which are incorporated herein in their entirety). The choice of
framework residues can be critical in retaining high binding
affinity. In principle, a framework sequence from any human
antibody can serve as the template for CDR grafting; however, it
has been demonstrated that straight CDR replacement into such a
framework can lead to significant loss of binding affinity to the
antigen (Tempest et al., Biotechnology 9: 266 (1992); Shalaby et
al., J. Exp. Med. 17: 217 (1992)). The more homologous a human
antibody is to the original murine antibody, the less likely the
human framework will introduce distortions into the mouse CDRs that
could reduce affinity. Based on a sequence homology, I2R was
selected to provide the framework for the humanized 3D1 heavy chain
and H2F for the humanized 3D1 light chain variable region.
Manheimer-Lory, A. et al., J. Exp. Med. 174(6):1639-52 (1991).
Other highly homologous human antibody chains would also be
suitable to provide the humanized antibody framework, especially
kappa light chains from human subgroup 4 and heavy chains from
human subgroup 1 as defined by Kabat.
[0135] Normally the heavy chain and light chain from the same human
antibody are chosen to provide the framework sequences, so as to
reduce the possibility of incompatibility in the assembling of the
two chains. The I2R antibody shows a high homology to the 3D1 heavy
and light chains and thus, was chosen to provide the framework for
the initial humanized antibody model. The 3D1 light chain variable
region, however, shows a significantly higher homology to the H2F
framework compared to any others, including I2R. Therefore, H2F was
chosen instead to provide the framework for the humanized 3D1 light
chain variable region, while I2R was selected to provide the
framework for the heavy chain variable region.
[0136] The computer programs ABMOD and ENCODE (Levitt et al., J.
Mol. Biol. 168: 595 (1983)) were used to construct a molecular
model of the 3D1 variable domain, which was used to locate the
amino acids in the 3D1 framework that are close enough to the CDRs
to potentially interact with them. To design the humanized 3D1
heavy and light chain variable regions, the CDRs from the mouse 3D1
heavy chain were grafted into the framework regions of the human
I2R heavy chain and the CDRs from the mouse 3D1 light chain grafted
into the framework regions of the human H2F light chain. At
framework positions where the computer model suggested significant
contact with the CDRs, the amino acids from the mouse antibody were
substituted for the original human framework amino acids. For
humanized 3D1, this was done at residues 27, 30, 48, 67, 68, 70 and
72 of the heavy chain and at residue 22 of the light chain.
Furthermore, framework residues that occurred only rarely at their
positions in the database of human antibodies were replaced by a
human consensus amino acid at those positions. For humanized 3D1
this was done at residue 113 of the heavy chain and at residue 3 of
the light chain.
[0137] The sequence of the humanized 3D1 antibody heavy chain and
light chain variable regions is shown in FIGS. 2A-2B. However, many
of the potential CDR-contact residues are amenable to substitutions
of other amino acids that may still allow the antibody to retain
substantial affinity to the antigen. Table 1 lists a number of
positions in the framework where alternative amino acids may be
suitable (LC=light chain, HC=heavy chain). The position specified
in the table is the number of amino acids from the first amino acid
of the mature chain, which is indicated by a double underline
(FIGS. 2A-2B). For example, position LC-22 is the twenty second
amino acid beginning from the doubled underlined Aspartic Acid, D,
(or the forty second amino acid from the start codon).
TABLE-US-00001 TABLE 1 Amino Acids Substitutes and/or Alternatives
Position Humanized 3D1 Alternatives LC-22 S N HC-27 Y G HC-30 T S
HC-48 I M HC-67 K R HC-68 A V HC-70 M I HC-72 V A
[0138] Likewise, many of the framework residues not in contact with
the CDRs in the humanized 3D1 heavy and light chains can
accommodate substitutions of amino acids from the corresponding
positions of I2R and H2F frameworks, from other human antibodies,
from the mouse 3D1 antibody, or from other mouse antibodies,
without significant loss of the affinity or non-immunogenicity of
the humanized antibody. Table 2 lists a number of additional
positions in the framework where alternative amino acids may be
suitable.
TABLE-US-00002 TABLE 2 Framework Region Amino Acid Substitutes
and/or Alternatives Position Humanized 3D1 Alternatives LC-3 V Q
HC-113 T I
[0139] Selection of various alternative amino acids may be used to
produce versions of humanized 3D1 that have varying combinations of
affinity, specificity, non-immunogenicity, ease of manufacture, and
other desirable properties. Thus, the examples in the above tables
are offered by way of illustration, not of limitation.
Example 3
Construction of Humanized 3D1
[0140] Once the humanized variable region amino acid sequences had
been designed, as described above, genes were constructed to encode
them, including signal peptides, splice donor signals and
appropriate restriction sites (FIG. 2A-2B). The light and heavy
chain variable region genes were constructed and amplified using
eight overlapping synthetic oligonucleotides ranging in length from
approximately 65 to 80 bases (see He et al., J. Immunol. 160: 1029
(1998)). The oligos were annealed pairwise and extended with the
Klenow fragment of DNA polymerase I, yielding four double-stranded
fragments. The resulting fragments were denatured, annealed, and
extended with Klenow, yielding two fragments. These fragments were
denatured, annealed pairwise, and extended once again, yielding a
full-length gene. The resulting product was amplified by polymerase
chain reaction (PCR) using Taq polymerase, gel-purified, digested
with XbaI, gel-purified again, and subcloned into the XbaI site of
the pVk for the expression of light chain and pVg4 or pVg2.M3 for
the expression of heavy chains. The pVk vector for kappa light
chain expression has been previously described (See Co et al., J.
Immunol. 148:1149 (1992)). The pVg4 vector for the .gamma.4 heavy
chain expression was constructed by replacing the XbaI-BamHI
fragment of pVg1 containing the .gamma.1 constant region gene (See
Co et al., J. Immunol. 148: 1149 (1992)) with an approximately 2000
bp fragment of the human g4 constant region gene (Ellison and Hood,
Proc. Natl. Acad. Sci. USA 79: 1984 (1982)) that extended from the
HindIII site preceding the C.sub.H1 exon of the .gamma.4 gene to
270 bp after the NsiI site following the C.sub.H4 exon of the gene.
The pVg2.M3 vector for the .gamma.2 heavy chain expression was
described in Cole, et al., J. Immunol. 159: 3613 (1997). The
pVg2.M3 is mutated from the human wildtype IgG2 by replacing the
amino acids Val and Gly at positions 234 and 237 with Ala. This
variant has a reduced interaction with its Fc receptors and thus
has minimal antibody effector activity.
[0141] The structure of the final plasmids was verified by
nucleotide sequencing and restriction mapping. All DNA
manipulations were performed by standard methods well-known to
those skilled in the art.
[0142] Two humanized 3D1, an IgG4 and an IgG2.M3, were generated
for comparative studies. To construct a cell line producing
humanized 3D1 (IgG4 or IgG2.M3), a light chain and the respective
heavy chain plasmids were transfected into the mouse myeloma cell
line Sp2/0-Ag14 (ATCC CRL 1581). Plasmids were also transfected
into CHO cells using known methods in the art. Before transfection,
the heavy and light chain-containing plasmids were linearized using
restriction endonucleases. The kappa chain and the .gamma.2 chain
were linearized using FspI; the .gamma.4 chain was linearized using
BstZ17I. Approximately 20 .mu.g of the light chain and a heavy
chain plasmid was transfected into 1.times.10.sup.7 cells in PBS.
Transfection was by electroporation using a Gene Pulser apparatus
(BioRad) at 360 V and 25 .mu.FD capacitance according to the
manufacturer's instructions. The cells from each transfection were
plated in four 96-well tissue culture plates, and after two days,
selection medium (DMEM, 10% FCS, 1.times.HT supplement (Sigma),
0.25 mg/mL xanthine, 1 .mu.g/mL mycophenolic acid) was applied.
[0143] After approximately two weeks, the clones that appeared were
screened for antibody production by ELISA. Antibody from a
high-producing clone was prepared by growing the cells to
confluency in regular medium (DMEM with 10% FCS), then replacing
the medium with a serum-free medium (Hybridoma SMF; Gibco) and
culturing until maximum antibody titers were achieved in the
culture. The culture supernatant was run through a protein
A-Sepharose column (Pharmacia); antibody was eluted with 0.1 M
Glycine, 100 mM NaCl, pH 3, neutralized and subsequently exchanged
into phosphate-buffered saline (PBS). The purity of the antibody
was verified by analyzing it on an acrylamide gel, and its
concentration was determined by an OD.sub.280 reading, assuming 1.0
mg of antibody protein has an OD.sub.280 reading of 1.4.
Example 4
Affinity of Humanized Anti-B7-2 Antibody
Competitive Binding Assay:
[0144] The relative affinities, of the murine and humanized 3D1
antibodies for the B7-2 antigen were determined by competitive
binding assays. Three-fold serial dilutions of unlabeled humanized
or murine 3D1 antibodies were mixed with a fixed amount of
radio-iodinated murine 3D1 antibody (40,000-50,000 cpm per test in
PBS containing 2% fetal calf serum).
[0145] 1.times.10.sup.5 CHO cells expressing cell surface rhB7-2
(CHO/hB7-2) were added subsequently and the mixture (in a total
volume of 200 .mu.L) was incubated for 2 hr at 4.degree. C. with
gentle shaking. The cell-antibody suspension was then transferred
to Sarstedt Micro Tubes (part #72.702) containing 100 .mu.L of 80%
dibutyl phthalate-20% olive oil. After centrifugation in a
microfuge, the Sarstedt tubes were plunged into dry ice for several
minutes. Cell-bound .sup.125I was determined by clipping tips of
each tube (containing cell pellets) into scintillation vials and
counting in a gamma counter. Bound and free counts were determined
and the ratio plotted against the concentrations of the cold
competitor antibodies according to the method of Berzofsky and
Berkower (J. A. Berzofsky and I. J. Berkower, in Fundamental
Immunology 9ed. W. E. Paul), Raven Press (New York), 595
(1984)).
Cell Line:
[0146] Recombinant Chinese Hamster Ovary (CHO) cell lines
expressing hB7-2 on their membrane surfaces were cloned from cells
transfected with B7-2 cDNA sequence and G418 resistance. Expression
of hB7-2 on the CHO cell surface over many passages under selective
pressure has been confirmed using murine anti-B7 antibodies and
FACS analysis.
Preparation of .sup.125I Labeled Anti-hB7 mAb and
Characterization:
[0147] Anti-hB7 antibodies were labeled with .sup.125I by reaction
with .sup.125I-Bolton-Hunter reagent according to manufacturers
instructions (Amersham Corp., Arlington Hts, Ill.). Protein was
separated from free reagent with a NAP-25 column. An HPLC
size-exclusion column was used to confirm that the antibodies
remained intact and were not aggregated, and to measure protein
concentration against standards prepared from non-labeled antibody.
Labeling typically resulted in 4 to 8 microcuries per microgram of
protein, or approximately 30 to 60% of the antibody molecules
labeled.
Results:
[0148] The competitive binding graph is shown in FIG. 3. Each data
point represents the average of triplicate determinations. Results
showed that both humanized IgG4 and humanized IgG2.M3 anti-human
B7-2 antibodies have a similar high binding affinity as the murine
anti-human B7-2 antibody (approximately 1.times.10.sup.9 M.sup.-1),
indicating no loss of affinity for B7-2 in the humanization of 3D1.
Both murine and humanized anti-B7-2 recognize cell surface
expressed hB7-2 with high affinity.
Example 5
Direct Binding of Humanized Anti-B7 mAbs to CHO/hB7 Cells
Cell Binding Assay:
[0149] Binding assays were begun by plating cells onto 96-well
tissue culture plates at 10,000 CHO/hB7-2 cells per well. Two days
later, adherent cells were gently washed with assay buffer
containing nonfat dry milk protein (for blocking nonspecific
binding) and sodium azide (to prevent internalization of antibodies
by cells). For direct binding assays, .sup.125I-labeled anti-B7
antibodies (.sup.125I-murine anti-human B7-2; 826 cpm/fmol;
humanized anti-human B7-2, 883 cpm/fmol) were serially diluted in
assay buffer and incubated on cells overnight, allowing antibodies
to bind to cell-surface B7 and come to equilibrium. Unbound
antibody was gently washed from cells, and bound .sup.125I-labeled
antibody was detected using an .sup.125I scintillant and
photodetector system. Non-specific binding to CHO cells was
determined for each dilution in the same manner, but on cells
expressing the B7-1 molecule that is not recognized by the antibody
being tested.
Results:
[0150] The direct binding graph is shown in FIG. 4. The data, means
of triplicate wells with nonspecific binding subtracted, were fit
to a hyperbolic saturation curve using Graphpad PrismJ software.
K.sub.D of the antibodies determined as the concentration
corresponding to half-maximal binding indicated that the murine and
humanized anti-B7-2 mAbs had similar and high binding affinities
(.about.10.sup.-9 m) for B7-2. Both murine and humanized anti-B7-2
antibodies recognize cell surface expressed hB7-2 with high
affinity.
Example 6
Binding of Humanized Anti-B7 mAbs to Protein Ligands
Affinity Determination by BIACORE.RTM.:
[0151] The BIACORE.RTM. biosensor (BIACORE.RTM.; Uppsalla, Sweden)
was used to determine binding kinetics of murine and humanized
anti-B7-2 human antibodies to human B7-2Ig. Human B7-2Ig (hB7-2Ig)
was immobilized onto the dextran matrix of a BIACORE.RTM. sensor
chip. Humanized and murine anti-human B7-2 were tested at 200, 100,
50, and 20 nM. Each dilution was tested 4 times per run and a total
of three separate runs performed. Anti-human B7-2 antibody binding
was measured in real time by Surface Plasmon Resonance (SPR) and
global analysis was performed using the bivalent binding model in
BIA evaluation software (version 3.1). For each sample, the
association (k.sub.a), dissociation (k.sub.d), and equilibrium
dissociation constant (K.sub.D) were determined.
Preparation of hB7-2 Ig:
[0152] A soluble form of hB7-2 Ig was recovered from culture medium
of CHO cells engineered to secrete this protein. Recombinant hB7-2
Ig was derived by fusing the DNA coding sequences corresponding to
the extracellular domain of B7-2 gene to the hinge-CH2-CH3 domains
of the human IgG1 heavy chain. Recombinant hB7-2 Ig was purified
from the culture medium by protein A.
Results:
[0153] Table 3 reports the mean values obtained for both murine and
humanized anti-human B7-2 mAbs. The binding constants for the
murine and humanized anti-B7-2 mAbs determined by SPR shows that
the murine and humanized forms of the anti-B7-2 mAbs are similar
and that the murine anti-B7-2 mAb has a slightly higher binding
constant for the immobilized hB7-2 Ig than does the humanized
anti-B7-2. The approximately 2.8 fold higher affinity calculated
for the murine anti-B7-2 mAb may represent a real, but slight
difference between the murine and humanized anti-B7-2 mAbs
introduced during the humanization process. Another possibility may
be due to technical variation in the preparation, processing and
analysis of these antibodies. As shown in Examples 4, 5, and 7, a
difference was not observed in humanized hB7-2 binding affinity in
cell based assays.
TABLE-US-00003 TABLE 3 Affinity of anti-B7-2 mAbs as determined by
BIACORE .RTM. mAb Mean K.sub.D murine Anti-B7-2 1.8 .times.
10.sup.-9 M humanized 5.1 .times. 10.sup.-9 M Anti-B7-2
Example 7
Inhibition of T Cell Costimulation by Humanized Anti-B7-2
CD28.sup.+ T Cell/CHO-B7 Proliferation Assay
[0154] CD28.sup.+ T cells, isolated as described herein, were
washed once and resuspended in RPMI complete medium, supplemented
with 2 ng/mL PMA (Calbiochem), to a cell density of
5.times.10.sup.5 cells/mL. The CD28.sup.+ T cells (100 .mu.L,
5.times.10.sup.4 cells) were added to the antibody/CHO/hB7-2
mixture (see below), incubated for 3 days at 37.degree. C., 5%
CO.sub.2, and T cell proliferation was measured by pulsing for the
last 6 hours of culture with 1 uCi of [.sup.3H]-thymidine (NEN,
Boston, Mass.). The cells were harvested on a filter and the
incorporated radioactivity was measured in a scintillation
counter.
Materials:
[0155] CD28.sup.+ human T cells were isolated by negative selection
with immunoabsorption from human peripheral blood lymphocytes, as
described (June et al., Mol. Cell. Biol. 7:4472-4481 (1987)). Buffy
coats were obtained by leukophoresis of healthy human donors and
peripheral blood lymphocytes (PBL) were isolated by density
gradient centrifugation. Monocytes were depleted from the PBL by
plastic absorption. CD28.sup.+ T cells were isolated from the
non-adherent cells by negative selection using antibodies to CD11,
CD20, CD16 and CD14, (this set of antibodies will coat all B cells,
monocytes, large granular lymphocytes, and CD28 T cells) and
magnetic bead separation using goat anti-mouse
immunoglobulin-coated magnetic particles.
[0156] CHO/hB7-2 cells were detached from the tissue culture plates
by incubation in phosphate-buffered saline without Ca.sup.2+ and
Mg.sup.2+ (PBS) with 0.5 mM EDTA, washed, and fixed with freshly
prepared paraformaldehyde.
[0157] Various concentrations of anti-B7-2 antibody (in duplicate)
were preincubated for 1 hour at 37.degree. C., 5% CO.sub.2 with
1.times.10.sup.4 CHO/hB7-2 cells in 100 .mu.L RPMI complete medium
(RPMI 1640 medium, 10% fetal bovine serum (FBS),100 U/mL
penicillin, 100 .mu.g/mL streptomycin) in a microtiter plate
(flat-bottom, 96-well, Costar, Cambridge, Mass.).
Results:
[0158] FIG. 5 shows the results of the inhibition of human
CD28.sup.+ T cell proliferation by the murine and humanized
anti-hB7-2 mAbs. Both antibodies exhibit dose dependent inhibition
of B7-2 driven T cell proliferation with similar IC.sub.50
(Inhibitory concentration 50%; amount of antibody required to
inhibit the maximal T cell proliferation by 50%) values of 72 pm
(murine anti-hB7-2) and 50 pm (humanized anti-hB7-2) indicating
that both antibodies were similar and very effective in inhibiting
the B7-2 T cell stimulatory signal. This demonstrates that the high
affinity anti-B7-2 mAbs can block B7-2 functionality by inhibiting
(e.g., preventing) the activation and/or proliferation of human T
cells. These mAbs are expected to exhibit similar capability in in
vivo use to inhibit T cell response.
Example 8
Cloning and Sequencing of Mouse 1F1 Variable Region cDNAs
[0159] Mouse 1F1 heavy and light chain variable region cDNAs were
cloned from mRNA isolated from hybridoma cells using anchored PCR
(Co et al., J. Immunol. 148: 1149 (1992)). The 5' primers used
annealed to the poly-dG tails added to the cDNA, and the 3' primers
annealed to the constant regions. The amplified gene fragments were
then inserted into the plasmid pUC19. Nucleotide sequences were
determined from several independent clones for both V.sub.H and
V.sub.L cDNA. For the heavy chain, a single, unique sequence was
identified, typical of a mouse heavy chain variable region. For the
light chain, two unique sequences, both homologous to mouse light
chain variable regions, were identified. However, one sequence was
not functional because of a missing nucleotide that caused a frame
shift at the V-J junction, and was identified as the non-productive
allele. The other sequence was typical of a functional mouse kappa
chain variable region. The variable region cDNA sequences of the
heavy chain and the functional light chain, and the translated
amino acid sequences are shown in FIGS. 6A and 6B, respectively.
The mouse V.sub.H belongs to Kabat's heavy chain subgroup II(C).
The mouse V.sub.K sequence belongs to Kabat's mouse kappa chain
subgroup IV.
Example 9
Design of Humanized 1F1 Variable Regions
[0160] To retain the binding affinity of the mouse antibody in the
humanized antibody, the general procedures of Queen et al., were
followed (Queen et al., Proc. Natl. Acad. Sci. USA 86: 10029 (1989)
and U.S. Pat. Nos. 5,585,089 and 5,693,762). The choice of
framework residues can be critical in retaining high binding
affinity. In principle, a framework sequence from any human
antibody can serve as the template for CDR grafting; however, it
has been demonstrated that straight CDR replacement into such a
framework can lead to significant loss of binding affinity to the
antigen (Tempest et al., Biotechnology 9: 266 (1992); Shalaby et
al., J. Exp. Med. 17: 217 (1992)). The more homologous a human
antibody is to the original mouse antibody, the less likely that
the human framework will introduce distortions into the mouse CDRs
that could reduce affinity. Based on a sequence homology search
against the Kabat antibody sequence database (Kabat et al.,
Sequences of Proteins of Immunological Interest, 5th ed., U.S.
Department of Health and Human Services (1991)), III-2R
(Manheimer-Lory et al., J. Exp. Med. 176: 309 (1992)) was selected
to provide the framework for both the humanized 1F1 heavy chain
variable region and for the humanized 1F1 light chain variable
region. Other highly homologous human antibody chains would also be
suitable to provide the humanized antibody framework, especially
heavy chains from human subgroup 1 and kappa light chains from
human subgroup 1 as defined by Kabat.
[0161] Normally the heavy chain and light chain from the same human
antibody are chosen to provide the framework sequences, so as to
reduce the possibility of incompatibility in the assembly of the
two chains. The III-2R antibody shows a high homology to the 1F1
heavy and light chains and thus was chosen to provide the framework
for the humanized antibody. The humanized 1F1 heavy chain variable
domain has 69 residues out of 87 framework residues that are
identical to those of the mouse 1F1 heavy chain framework, or 79%
sequence identity. The humanized 1F1 light chain variable domain
has 55 residues out of 80 framework residues that are identical to
those of the mouse 1F1 light chain framework, or 69% sequence
identity.
[0162] The computer programs ABMOD and ENCAD (Levitt et al., J.
Mol. Biol. 168: 595 (1983)) were used to construct a molecular
model of the 1F1 variable domain, which was used to locate the
amino acids in the 1F1 framework that are close enough to the CDRs
to potentially interact with them. To design the humanized 1F1
heavy and light chain variable regions, the CDRs from the mouse 1F1
heavy chain were grafted into the framework regions of the human
III-2R heavy chain and the CDRs from the mouse 1F1 light chain were
grafted into the framework regions of the human III-2R light chain.
At framework positions where the computer model suggested
significant contact with the CDRs, the amino acids from the mouse
antibody were substituted for the original human framework amino
acids. For humanized 1F1, this was done at residues 1, 24, 27, 28,
29, 30, 48, 67, and 68 of the heavy chain and at residues 47 and 72
of the light chain. Furthermore, framework residues that occurred
only rarely at their positions in the database of human antibodies
were replaced by human consensus amino acids at those positions.
For humanized 1F1 this was done at residues 16, 74, and 113 of the
heavy chain and at residue 44 of the light chain. Overall, the
humanized 1F1 heavy chain variable domain has 88 residues that are
identical to the human III-2R heavy chain variable domain, and the
humanized 1F1 light chain variable domain has 88 residues that are
identical to the III-2R light chain variable domain.
[0163] The sequences of the humanized 1F1 antibody heavy chain and
light chain variable regions are shown in FIGS. 7A and 7B. However,
many of the potential CDR-contact residues are amenable to
substitutions of other amino acids that still allow the antibody to
retain substantial affinity to the antigen. Table 4 lists a number
of positions in the framework where alternative amino acids may be
suitable (LC=light chain, HC=heavy chain).
TABLE-US-00004 TABLE 4 Position Humanized 1F1 Alternatives HC-1 E Q
HC-24 P A HC-27 F G, Y HC-28 N T HC-29 I F HC-30 K S, T HC-48 I M
HC-67 K R HC-68 A V LC-72 Y F
[0164] Likewise, many of the framework residues not in contact with
the CDRs in the humanized 1F1 heavy and light chains can
accommodate substitutions of amino acids from the corresponding
positions of the III-2R framework, from other human antibodies,
from the mouse 1F1 antibody, or from other mouse antibodies,
without significant loss of the affinity or non-immunogenicity of
the humanized antibody. Table 5 lists a number of additional
positions in the framework where alternative amino acids may be
suitable.
TABLE-US-00005 TABLE 5 Position Humanized 1F1 Alternatives HC-16 A
S HC-74 T K HC-113 T I LC-44 A S, V
[0165] Selection of various alternative amino acids may be used to
produce versions of humanized 1F1 that have varying combinations of
affinity, specificity, non-immunogenicity, ease of manufacture, and
other desirable properties. Thus, the examples in the above tables
are offered by way of illustration, not of limitation.
Example 10
Construction of Humanized 1F1
[0166] Once the humanized variable region amino acid sequences had
been designed as described above, genes were constructed to encode
them, including signal peptides, splice donor signals and
appropriate restriction sites (FIGS. 7A and 7B). The heavy and
light chain variable region genes were constructed and amplified
using eight overlapping synthetic oligonucleotides ranging in
length from approximately 65 to 80 bases (see He et al., J.
Immunol. 160: 1029 (1998)). The oligos were annealed pairwise and
extended with the Klenow fragment of DNA polymerase I, yielding
four double-stranded fragments. The resulting fragments were
denatured, annealed pairwise, and extended with Klenow, yielding
two fragments. These fragments were denatured, annealed pairwise,
and extended once again, yielding a full-length gene. The resulting
product was amplified by polymerase chain reaction (PCR) using Taq
polymerase, gel-purified, digested with XbaI, gel-purified again,
and subcloned into the XbaI site of pVg2.M3 for the expression of
heavy chain, and pVk for the expression of light chain. The pVg2.M3
vector for human .gamma.2 heavy chain expression has been
previously described (Cole et al., J. Immunol. 159: 3613 (1997)).
The human .gamma.2 constant region in the pVg2.M3 plasmid is
mutated from the wildtype human .gamma.2 constant region by
replacing the amino acids Val and Gly at positions 234 and 237 with
Ala. This variant has a reduced interaction with its Fc receptors
and thus has minimal antibody effector activity. The pVk vector for
human kappa light chain expression has been previously described
(see Co et al., J. Immunol. 148: 1149 (1992)).
[0167] The structures of the final plasmids were verified by
nucleotide sequencing and restriction mapping. All DNA
manipulations were performed by standard methods well-known to
those skilled in the art.
[0168] An IgG2.M3 form of the humanized 1F1 antibody was generated
for binding studies. To construct a cell line producing humanized
1F1, the heavy and light chain plasmids were transfected into mouse
myeloma cell line Sp2/0-Ag14 (ATCC CRL 1581). Before transfection,
the heavy and light chain plasmids were linearized using
restriction endonucleases. The .gamma.2 heavy chain plasmid and the
kappa light chain plasmid were linearized using FspI. Approximately
40 .mu.g of the heavy chain plasmid and 20 .mu.g of the light chain
plasmid were transfected into 1.times.10.sup.7 cells in PBS.
Transfection was by electroporation using a Gene Pulser apparatus
(BioRad) at 360 V and 25 .mu.FD capacitance according to the
manufacturer's instructions. The cells from each transfection were
plated in four 96-well tissue culture plates, and after two days
selection medium (DMEM, 10% FCS, 1.times.HT supplement (Sigma),
0.25 mg/mL xanthine, 1 .mu.g/mL mycophenolic acid) was applied.
[0169] After approximately two weeks, the clones that appeared were
screened for antibody production by ELISA. Antibody from a
high-producing clone was prepared by growing the cells to
confluency in regular medium (DMEM with 10% FCS), then replacing
the medium with a serum-free medium (Hybridoma SFM; GIBCO) and
culturing until maximum antibody titers were achieved in the
culture. The culture supernatant was run through a protein
A-Sepharose column (Pharmacia); antibody was eluted with 0.1 M
Glycine, 100 mM NaCl, pH 3, neutralized, and subsequently exchanged
into phosphate-buffered saline (PBS). The purity of the antibody
was verified by analyzing it on an acrylamide gel, and its
concentration was determined by an OD.sub.280 reading, assuming 1.0
mg of antibody protein has an OD.sub.280 reading of 1.4.
[0170] An IgG4 form of the humanized 1F1 antibody was also
generated and purified following the methods described above.
[0171] In order to permit high level expression in CHO cells, the
complete humanized human 1F1 (h1F1) and human 3D1 (h3D1) light
chain and heavy chain genes were each independently subcloned into
the selectable, amplifiable expression vector pED (Kaufman R. J.,
et al., Nucl Acids Res., 19:4485-4490 (1991)). The pED-derived
expression plasmids were sequenced to confirm that they encoded the
appropriate h1F1 and h3D1 light and heavy chains. The penultimate
amino acid of the IgG2 m3 CH3 domain was found to be serine in
contrast to the glycine residue reported at this position for all
published IgG2 and IgG1 sequences. This serine was replaced with
the more common glycine for the pED expression constructs. The h1F1
light chain and heavy chain expression plasmids (pED. 1F1v2KA and
pED.1F1v2G2m3gly) were linearized and cotransfected into the CHO
PA-DUKX.153.8 cell line, which has been pre-adapted for growth in
serum-free suspension culture (Sinacore M. S. et al., Biotechnol.
Bioeng. 52:518-528 (1996)). Cell lines expressing h3D1 were
generated in the same manner by cotransfection with linearized
plasmids pED.3D1KA and pED.3D1G2 m3gly. In each case, stable
integration of light chain and heavy chain genes into the CHO cell
genome, followed by methotrexate selection and amplification,
resulted in recombinant h1F1 or h3D1 cell lines. The CHO cell lines
expressing anti-B7-1 or anti-B7-2 were cultured in serum-free
growth medium and the secreted antibodies purified from the
conditioned culture supernatant by chromatography on protein A
sepharose. Bound antibody was eluted in acid buffer followed by
neutralization to pH 7.0 with. The purified antibody was buffer
exchanged into PBS and sterile filtered.
Example 11
Properties of Humanized 1F1
[0172] The affinity of the mouse and humanized 1F1 antibodies for
the B7-1 antigen was determined by competitive binding with
radio-iodinated humanized 1F1 antibody. Three-fold serial dilutions
of unlabeled mouse or humanized 1F1 antibodies were mixed with a
fixed amount of radio-iodinated humanized 1F1 antibody
(40,000-50,000 cpm per test) in PBS containing 2% fetal calf serum,
0.1% sodium azide. 3.times.10.sup.4 CHO cells expressing cell
surface rhB7-1 were added subsequently and the mixture (in a total
volume of 200 .mu.L) was incubated for 2 hr at 4.degree. C. with
gentle shaking. The cell-antibody suspension was then transferred
to Sarstedt Micro Tubes (part #72.702) containing 100 .mu.L of 80%
dibutyl phthalate-20% olive oil. After centrifugation in a
microfuge, the Sarstedt tubes were plunged into dry ice for several
minutes. Cell-bound .sup.125I was determined by clipping the tips
of each tube (containing cell pellets) into scintillation vials and
counting in a gamma counter. Bound and free counts were determined
and the ratio plotted against the concentrations of the cold
competitor antibodies according to the method of Berzofsky and
Berkower (J. A. Berzofsky and I. J. Berkower, in Fundamental
Immunology, W. E. Paul, Raven Press, New York, pp. 595-644
(1984)).
[0173] The competitive binding graphs are shown in FIG. 8. Each
data point represents the average of triplicate determinations. The
results showed that the IgG2.M3 antibody has a similar binding
affinity to that of the mouse antibody (approximately
1.times.10.sup.9 M.sup.-1), indicating no loss of affinity in the
humanization of 1F1.
[0174] The affinity of the mouse and humanized 1F1 antibodies for
the B7-1 antigen was confirmed by Scatchard analysis of binding of
radiolabeled antibodies. Two-fold serial dilutions of radiolabeled
mouse or humanized 1F1 antibodies were incubated with
5.times.10.sup.4 CHO cells expressing cell surface rhB7-1 in PBS
containing 2% FCS, 0.1% sodium azide in a total volume of 200
.mu.L. The mixture was incubated for 6 hr at 4.degree. C. with
gentle shaking. The cell-antibody suspension was then transferred
to Sarstedt Micro Tubes (part #72.702) containing 100 .mu.L of 80%
dibutyl phthalate-20% olive oil. After centrifugation in a
microfuge, the Sarstedt tubes were plunged into dry ice for several
minutes. Cell-bound .sup.125I was determined by clipping the tips
of each tube (containing cell pellets) into scintillation vials and
counting in a gamma counter. Bound and free counts were determined
and the ratio plotted against the concentration of bound antibody
following the method of Scatchard (Scatchard, Ann. N.Y. Acad. Sci.
51: 660 (1949)). A least squares method was used to fit a line to
the data, and the apparent Ka was determined from the slope of the
line.
[0175] The Scatchard plots are shown in FIGS. 9A and 9B. Each data
point represents the average of duplicate determinations. The
results showed that the IgG2.M3 antibody has a similar binding
affinity to that of the mouse antibody (approximately
1.times.10.sup.9 M.sup.-1), confirming no loss of affinity in the
humanization of 1F1.
Example 12
Affinity of Humanized Anti-B7-1 Monoclonal Antibody
Competitive Binding Assay:
[0176] The relative affinities of the murine and humanized
anti-B7-1 (1F1) antibodies for the B7-1 antigen were determined by
competitive binding assays. Three-fold serial dilutions of the
unlabeled murine or humanized anti-B7-1 mAbs were mixed with a
fixed amount of radio-labeled murine anti-B7-1 mAb
(.sup.125I-anti-B7-1,2800 cpm/fmol; 40,000-50,000 cpm/test in PBS
containing 2% fetal calf serum). 1.times.10.sup.5 CHO/hB7-1 cells
expressing hB7-1 on their surface were added subsequently and the
mixture (total volume=200 .mu.L) incubated for 2 hrs at 4.degree.
C. with gentle shaking. The cell antibody mixture was then
transferred to Sarstedt Micro tubes (Part # 72.702) containing 100
.mu.L of 80% dibutyl phthlate-20% olive oil. After centrifugation
in a microfuge, the Sarstedt tubes were plunged into dry ice for
several minutes. Cell bound .sup.125I-labeled mAb was determined by
clipping the cell pellet containing tips of each tube into
scintillation vials and counting in a gamma counter. Bound and free
counts were determined. The bound counts were plotted against the
concentration of the cold competitor mAbs.
CHO/hB7-1 Cell Line:
[0177] A recombinant Chinese Hamster Ovary (CHO) cell line
expressing hB7-1 on its surface was cloned form CHO cells
transfected with the hB7-1 cDNA sequence and G418 resistance
marker. Stable expression of the hB7-1 on the CHO cell surface over
many passages under selective pressure has been confirmed using the
murine anti-B7-1 mAb and FACS analysis.
Preparation of .sup.125I-labeled anti-B7-1 mAbs:
[0178] Murine and Humanized anti-B7-1 mAbs were labeled with
.sup.125Iodine by reaction with .sup.125I-Bolton Hunter reagent
according to the manufacturers' instructions (Amersham Corp,
Arlington Heights, Ill.). Protein was separated from free reagent
with a NAP-25 column. HPLC size-exclusion chromatography was used
to verify antibody integrity and aggregation state post labeling
and to determine protein concentration. Labeling typically resulted
in 4 to 8 microcuries .sup.125I/.mu.g of mAb with an estimated 30
to 60% of the antibody molecules labeled. The murine and humanized
anti-B7-1 mAbs had a specific activity of 2800 cpm/fmol and 950
cpm/fmol, respectively.
Results:
[0179] The graphical representation of the competitive binding data
is shown in FIG. 10. Each data point represents the average of
triplicate determinations. Results show that both the humanized
anti-B7-1 mAb and the murine anti-B7-1 mAb from which it was
derived have a similar high affinity for B7-1 (approximately
1.times.10.sup.-9 M) indicating no loss of affinity for the
humanized anti-B7-1 mAb. Both the murine and humanized anti-B7-1
compete similarly and effectively with labeled murine anti-B7-1 mAb
for binding to cell surface expressed B7-1
Example 13
Direct Binding of Humanized Anti-B7-1 mAb to B7-1
Cell Binding Assay:
[0180] Binding assays were begun by plating CHO/hB7-1 cells onto 96
well tissue culture plates at 10,000 cells/well in complete medium
and the plates incubated at 37.degree. C. for two days. Adherent
cells were washed gently with assay buffer (PBS containing nonfat
dry milk and sodium azide). Murine and humanized anti-B7-1 mAbs
labeled with .sup.125I were serially diluted in assay buffer and
incubated with the cells overnight at 4.degree. C. to allow biding
to reach equilibrium. Unbound labeled antibody was removed from the
cells by a series of gentle washings with assay buffer and the
bound .sup.125I-labeled antibody was detected using a .sup.125I
scintillant and detector system. Non-specific binding was
determined for each dilution of labeled antibody in an identical
manner as described above except that the target CHO cells
expressed hB7-2 which is not bound by either the murine or
humanized anti-B7-1 mAbs.
Results:
[0181] The graphical depiction of the results of the direct binding
experiment is shown in FIG. 11. The mean value of triplicate
determinations minus the non-specific binding was calculated and
fit to a hyperbolic saturation binding curve using Graphpad Prism
software. Binding constants (K.sub.D), determined as the
concentration of antibody corresponding to half-maximal saturation
of binding sites, for both the murine and humanized antibodies
indicated that both antibodies had similar and high affinities for
B7-1 (approximately 10.sup.-9M). Both murine and humanized
anti-B7-1 mAbs recognize cell surface expressed human B7-1
Example 14
Binding of Murine and Humanized Anti-B7-1 mAbs to Protein
Ligands
Affinity Determination by BIACORE.RTM.:
[0182] The BIACORE.RTM. biosensor (BIACORE.RTM., Uppsalla, Sweden)
was used to determine binding kinetics of murine and humanized
anti-B7-1 monoclonal antibodies to human B7-1 Ig (hB7-1 Ig)
protein. Human B7-1 Ig was immobilized onto the dextran matrix of a
BIACORE.RTM. sensor chip. Humanized and murine anti-B7-1 mAbs were
tested at 200, 100, 50, and 20 nM on the immobilized hB7-1 Ig. Each
mAb dilution was tested 4 times per run and a total of three
separate runs were performed. Anti-human B7-1 Ig binding was
measured in real time by Surface Plasmon Resonance (SPR) and global
analysis was performed using the bivalent binding model in BIA
evaluation software (Version 3.1). For each sample, the association
(k.sub.a), dissociation (k.sub.d) and equilibrium dissociation
constant (K.sub.D) were determined.
[0183] Table 6 reports the mean values determined for both the
murine and humanized anti-B7-1 mAbs. The binding constants for the
murine and humanized anti-B7-1 mAbs determined by SPR shows that
the murine and humanized forms of the anti-B7-1 mAbs are similar
and that the murine anti-B7-1 mAb had a slightly higher binding
affinity for the hB7-1 Ig protein than did the humanized anti-B7-1.
The 5 fold higher binding affinity found for the murine anti-B7-1
mAb may represent a real but slight difference between the murine
and humanized anti-B7-1 mAbs introduced during the humanization
process. Alternatively, technical variations in the preparation,
processing, and analysis of the individual mAbs may explain these
minor differences. As shown in Examples 12, 14, and 19, no
difference was observed between the murine and humanized anti-B7-1
mAbs in cell binding or functional assays.
TABLE-US-00006 TABLE 6 Affinity of anti-B7-1 mAbs as determined by
BIACORE .RTM. mAb Mean K.sub.D Std deviation Murine anti-B7-1 5.6
.times. 10.sup.-10 M 1.9 .times. 10.sup.-10 M Humanized anti-B7-1
.sup. 2.8 .times. 10.sup.-9 M .sup. 1.2 .times. 10.sup.-9 M
Preparation of hB7-1 Ig:
[0184] A soluble form of hB7-1 Ig was recovered from culture medium
of CHO cells engineered to secrete this protein. Recombinant hB7-1
Ig was derived by fusing the DNA sequences encoding the
extracellular domain of hB7-1 gene to the hinge-CH2-CH3 domains of
human IgG1 heavy chain. Recombinant protein was purified from
culture medium by protein A chromatography.
Example 15
Inhibition of T Cell Costimulation by Humanized B7-1 mAb
CD28.sup.+ T Cell/CHO-B7Proliferation Assay
[0185] CD28.sup.+ T cells, isolated as described herein, were
washed once and resuspended in RPMU complete medium supplemented
with 2 ng/mL PMA (Calbiochem) to a cell density of 5.times.10.sup.5
cells/mL. The CD28.sup.+ T cells (100 .mu.L; 5.times.10.sup.4
cells) were added to the antibody/CHO/hB7-1 cell mixture (see
below), incubated for 3 days at 37.degree. C., 5% CO.sub.2, and the
T cell proliferation measured by pulsing for the last 6 hours of
culture with 1 uCi of [.sup.3H]-thymidine (NEN, Boston, Mass.). The
cells were harvested on a filter and the incorporated radioactivity
was measured by scintillation counting.
Materials:
[0186] CD28.sup.+ human T cells were isolated by negative selection
with immunoabsorption from human peripheral blood lymphocytes as
described (June et al., Mol. Cell. Biol. 7:4472-4481 (1987)).
Briefly, buffy coats were obtained by leukophoresis of healthy
human donors and the peripheral blood lymphocytes (PBL) were
isolated by density gradient centrifugation. Monocytes were
depleted from the PBLs by adsorption onto plastic. CD28.sup.+ T
cells were isolated from the non-adherent cells be negative
selection using antibodies to CD11, CD20, CD16, and CD14 (this set
of antibodies will coat B cells, monocytes, large granular
lymphocytes, and CD28.sup.- T cells) and magnetic bead separation
using goat anti-mouse immunoglobulin-coated magnetic beads.
[0187] CHO/hB7-1 cells were detached from the tissue culture plates
by incubation with phosphate-buffered saline lacking Ca.sup.2+ or
Mg.sup.2+ with 0.5 mM EDTA. The cells were washed and then fixed
with freshly prepared paraformaldehyde.
[0188] Various concentrations of the anti-B7-1 antibody (in
duplicate) were preincubated with 1.times.10.sup.4 CHO/hB7-1 cells
for 1 hour at 37.degree. C. in 100 .mu.L RPMI complete medium (RPMI
1640, 10% FBS, 100 U/mL penicillin, 100 .mu.g/mL streptomycin) in a
microtiter plate (flat bottomed, 96 well, Costar, Cambridge
Mass.)
Results:
[0189] FIG. 12 shows the results of the inhibition of human
CD28.sup.+ T cell proliferation by the murine and humanized forms
of the anti-B7-1 mAbs. Both monoclonal antibodies exhibit dose
dependent inhibition of B7-1 driven proliferation of human T cells
with similar IC50 (Inhibitory concentration 50%, amount of antibody
required to inhibit maximal T cell proliferation by 50%) values of
110 pM (humanized anti-B7-1) and 220 pM (murine anti-B7-1)
indicating that both antibodies were similar and very effective in
inhibiting the B7-1 T cell co-stimulatory signal. This demonstrates
that the high affinity anti-B7-1 mAbs can block B7-1 functionally
by inhibiting or preventing the activation and/or proliferation of
human T cells. These mAbs are expected to exhibit similar
capability in in vivo use to inhibit T cell responses.
Example 16
Inhibition of Mixed Lymphocyte Reactions by Anti-B7-1 and Anti-B7-2
mAbs
[0190] Mixed lymphocyte reactions (MLR): Human normal peripheral
blood lymphocytes (PBL) (responders) were cultured with irradiated
(2,500 cGy) normal donor PBL (stimulators) in RPMI 1640 containing
5% heat-inactivated human AB serum at 37.degree. C. in 5% CO2 at a
final concentration of 10.sup.6 cells/mL. Where indicated, murine
anti-hB7-1 or murine anti-hB7-2 antibodies were added alone (10
.mu.g/mL), in combination (10 .mu.g/mL each), and in comparison
with CTLA4Ig (10 or 20 .mu.g/mL). Cells were cultured in triplicate
in microtiter plates in a final volume of 200 .mu.L and
proliferation was assessed by [.sup.3H]-thymidine incorporation for
the last 16 hours of culture. Secondary MLR was performed using the
cells derived from the primary MLRs as responders. These cells were
washed, cultured overnight, and restimulated as above using the
same or different, third party stimulator PBLs. No inhibitors were
added to the secondary MLRs.
Results:
[0191] The determinations shown in FIG. 13 were made by performing
primary one-way MLRs in the absence or presence of B7 inhibitors
(anti-B7, CTLA4Ig). The proliferation was measured after 3, 4, or 5
days of culture.
[0192] In the primary MLR, the additional anti-B7-1 mAb alone had
no inhibitory effect indicating a minor role for B7-1 alone in
driving proliferation of responder T cells. Anti-B7-2 alone
inhibited T cell proliferation on all days tested at a level
comparable to human CTL4Ig (hCTL4Ig), a recombinant protein known
to bind to both B7-1 and B7-2. The combination of anti-B7-1 and
anti-B7-2 was the most effective inhibitor of T cell proliferation
that completely inhibited this response on all days tested. The
superior ability of the combined anti-B7-1 and anti-B7-2 to inhibit
T cell proliferation, as compared to hCTL4Ig, reflects the higher
affinity of the anti-B7 mAbs for B7-1 and B7-2 as compared to
hCTL4Ig. The combined anti-B7-1 and anti-B7-2 mAbs were better
inhibitors of T cell proliferation than anti-B7-2 alone,
demonstrating the need to block both stimulatory receptors to
completely inhibit T cell responses. These results show that
complete blockade of the B7-1 and B7-2 costimulators more
completely abrogates alloresponsiveness in the MLR. Accordingly,
these results indicate that methods of treatment including both
anti-B7-1 and anti-B7-2 antibodies will be even more effective than
either of the antibodies alone, especially where both
co-stimulatory molecules are functional. While the
responder/stimulator pair, described herein, was not sensitive to
inhibition by anti-B7-1 alone, some responder/stimulator pairs do
exhibit moderate (0-50%) anti-B7-1 sensitivity.
[0193] To determine whether treatment with anti-B7 mAbs in the
primary MLR had resulted in the development of T cell
hyporesponsiveness or anergy, the responder T cells from the
primary MLRs were tested in secondary MLRs where the stimulators
were either from the same donor as the primary MLR or from a third
party. FIG. 14 shows that the responder T cells obtained from the
primary MLR that was treated with anti-B7-1 alone show full
proliferative responses to both the original sensitizing cells and
to third party cells when tested in a secondary MLR with no
immunosuppression indicating that blocking the B7-1 receptor alone
by treatment with the anti-B7-1 mAb had no tolerizing effect on
these responding T cells. This is in contrast to the lack of
response to the primary stimulators seen in the secondary MLR when
the primary MLR was treated with anti-B7-2 alone. The results in
FIG. 15 show that the responder T cells from the primary MLR
treated with anti-B7-2 alone failed to respond to the same
stimulators as used in the primary MLR but retained normal
proliferative response to third party, unrelated stimulators
indicating that these responder T cells were rendered tolerant to
the original stimulator PBLs by treatment with anti-B7-2 and that
the tolerization was specific for the stimulator antigens present
in the primary MLR. With this responder/stimulator pair, treatment
with anti-B7-2 alone resulted in tolerance to the stimulator cells;
however, with other responder/stimulator pairs, the induction of
tolerance may not be complete.
[0194] FIG. 16 shows that the responder T cells from the primary
MLR treated with anti-B7-1 and anti-B7-2 failed to respond to the
same stimulators as used in the primary MLR, but retained normal
proliferative response to third party, unrelated stimulators. This
indicates that these responder T cells were rendered tolerant to
the original stimulator PBLs by treatment with the combined
anti-B7-1 and anti-B7-2. The results obtained with this
responder/stimulator pair are typical for other
responder/stimulator pairs in that tolerance induction is the
rule.
Example 17
Inhibition of Primary and Secondary MLRs by Treatment with
Humanized Anti-B7-1 and Anti-B7-2
[0195] The ability of the anti-B7 mAbs to inhibit primary MLRs and
to induce specific durable hyper-responsiveness or "tolerance" and
secondary MLR were investigated. Primary MLRs were treated with the
individual or combined anti-B7 mAbs or with CTLA4Ig and
proliferation measured on days 3, 4, and 5. The combined
anti-B7-1+anti-B7-2 mAbs and CTLA4Ig inhibited proliferation
whereas the individual anti-B7 mAbs were less effective. Cells from
the primary MLRs (after 48 hours of treatment) were then put into
secondary MLRs with no inhibitors. Cells from primary MLRs treated
with either the combined anti-B7 mAbs or CTLA4Ig showed minimal
response to the original stimulators in the secondary MLR whereas
medium or individual anti-B7 mAbs treated cells responded well.
Cells form all conditions gave good responses against third party
stimulators thereby demonstrating that the hyporesponsiveness seen
in primary MLRs for cells treated with the combined anti-B7 mAbs or
CTLA4Ig was both specific and durable.
Materials and Methods:
Experimental Design:
Cells:
[0196] Responders "A" Cells were PBLs prepared from freshly drawn
blood. PBLs were purified by ficoll gradient centrifugation, the
cells washed twice with culture medium and suspended at
1.times.10.sup.6/mL in culture medium. Original Stimulator "B"
Cells were PBLs prepared from leukophoresis. Cells purified by
ficoll separation and washed twice with culture medium. Cells
suspended at 2.times.10.sup.6/mL in culture medium for primary MLR
and at 1.times.10.sup.6/mL for secondary MLR. Third Party
Stimulator "C" Cells were PBLs prepared from a leukophoresis. Cells
purified by ficoll separation and washed twice with culture medium.
Cells suspended at 1.times.10.sup.6/mL in culture medium. "B" and
"C" stimulators are PBLs obtained from two genetically different
human donors.
Culture Medium:
[0197] RPMI 1640 containing 2 mM glutamine, 10 mM HEPES, 10% heat
inactivated human AB serum, 100 U/mL penicillin, 100 ug/mL
streptomycin sulfate, and 5 ug/mL gentamicin sulfate.
Test Articles:
[0198] Control Ig is a chimeric mAb with the variable domains
derived form a murine anti-HIV envelope protein mAb and the
constant domains derived from human IgG1. All antibody, CTLA4Ig,
and control Ig solutions were prepared in culture medium at the
following concentration: anti-B7-1 (40 ug/mL), anti-B7-2 (40
ug/mL), anti-B7-1+anti-B7-2 (40 ug/mL each), CTLA4Ig (40 ug/mL),
CTLA4Ig (80 ug/mL), and control Ig (40 ug/mL).
Methods for Primary MLR:
[0199] The following was performed: [0200] Irradiate "B" cells
(stimulators) at 3.5 Gy [0201] In a 96 well "U" bottom microtiter
plate, mix 50 uL of irradiated "B" cells+50 uL of antibody,
CTLA4Ig, Control Ig, or medium. Total volume=100 uL containing
1.times.10.sup.5 B cells (stimulators) and 2.times. final
concentration of inhibitory reagents. Incubate for 30 minutes on
ice. [0202] Add 100 uL of "A" cells (responders) [0203] Incubate at
37 C, 5% CO.sub.2 [0204] On day 2, 3, and 4, add 1 uCi
[3H]-thymidine and continue incubation overnight [0205] Harvest
cells and determine incorporated radioactivity by scintillation
counting. Report results as day 3, 4, and 5.
Methods for Coupled Primary/Secondary MLR:
[0206] The following was performed: [0207] Irradiate B cells
(stimulators) at 3.5 Gy [0208] Bulk primary MLR. In a T-25 flask,
mix 2.5 mL of irradiated "B" cells (2.times.10.sup.6/mL)+2.5 mL of
antibody, CTLA4Ig, Control Ig, or medium. Total volume=5 mL
containing 1.times.10.sup.6/mL "B" cells (stimulators) and 2.times.
final concentration of inhibitory reagents. Incubate for 30 minutes
on ice. [0209] Add 5 mL of "A" cells (responders). Total volume in
T-25=10 mL [0210] Incubate at 37 C, 5% CO.sub.2 for .about.48 hours
[0211] Collect cells by ficoll gradient centrifugation, wash twice
with ice cold medium and suspend at 1.times.10.sup.6/mL in culture
medium. Hold for 8 hours on ice. Wash cells twice with ice cold
medium and suspend at 1.times.10.sup.6/mL in culture medium. [0212]
Irradiate "C" cells (third party stimulators) at 3.5 Gy. Irradiate
"B" cells (original stimulators) at 3.5 Gy. Both "B" cells and "C"
cells at 1.times.10.sup.6/mL. [0213] In a 96 well "U" bottom
microtiter plate, mix 100 uL of irradiated "B" cells or irradiated
"C" cells and 100 uL cells from bulk MLR. [0214] Incubate at 37 C,
5% CO.sub.2 [0215] On day 2, 3, 4, and 5, add 1 uCi [3H]-thymidine
and continue incubation overnight. [0216] Harvest cells and
determine incorporated radioactivity by scintillation counting.
Report results as day 3, 4, 5, and 6
Results:
[0217] Primary MLRs performed using one responder and two different
stimulators were treated with the individual anti-B7 mAbs, the
combined anti-B7 mAbs, CTLA4Ig, or control Ig and the culture
proliferation measured on days 3, 4, and 5. Anti-B7-1 alone had
minimal inhibitory effect on proliferation (1-35%). Anti-B7-2 alone
had moderate inhibitory effect on proliferation (30-50%). The
combined anti-B7 mAbs or CTLA4Ig had maximal inhibition of
proliferation (86-92%, anti-B7s; 82-91%, CTLA4Ig; FIGS. 17 and
18).
[0218] After 48 hours of incubation in primary MLRs using one of
the stimulators and containing inhibitors, the cells were washed,
rested, and placed in secondary MLR using the original and third
party stimulators in cultures lacking inhibitors. Culture
proliferation was measured on days 3, 4, 5, & 6 (FIGS. 19 and
20). A compilation of the data from the secondary MLR experiments
is presented in FIG. 21.
[0219] Using data obtained form the peak proliferative response,
regardless of the day of peak proliferative response, the response
against the original and third party stimulators for cells from
primary MLR treated with anti-B7-1 alone were as follows:
TABLE-US-00007 TABLE 7 Response Against Original Response Against
Third Treatment in Primary MLR Stimulators (%) Party Stimulators
(%) Medium 100 100 anti-B7-1 70 89 anti-B7-2 47 89 anti-B7-1 +
anti-B7-2 15 89 CTLA4Ig 10 19 90 CTLA4Ig 20 17 95 Control Ig 99
105
[0220] Primary MLRs treated with either the combined anti-B7 mAbs
or CTLA4Ig resulted in cells that were refractory to proliferation
against the same stimulators in a secondary MLR. Treatment with
either of the anti-B7 mAbs alone was less effective. Regardless of
the treatment in the primary MLR, cells form all treatment
conditions responded normally to third party cells demonstrating
that treatment with the combined anti-B7 mAbs or CTLA4Ig resulted
in specific, durable hyporesponsiveness or anergy.
Conclusion:
[0221] Humanized anti-B7 mAbs h1F1 and 3D1 used in combination
inhibit primary MLRs. This inhibition is specific and durable as
responders from a primary MLR treated with the combined anti-B7
mAbs respond poorly to the same stimulators in a secondary MLR
performed without inhibitors. Responders from a primary MLR treated
with the combined anti-B7 mAbs respond normally to third party
stimulators in the secondary MLR. The combined anti-B7 mAbs are as
effective as CTLA4Ig in inhibiting a primary and secondary
responses. The individual anti-B7 mAbs are less effective in
inhibiting a primary MLR and treatment with the individual anti-B7
mAbs did not lead to the development of anergy in the secondary
MLR.
Example 18
Inhibition of Immune Responses in Non-Human Primates by Anti-B7
mAbs; Inhibition of Anti-Tetanus Responses
Abstract:
[0222] The ability of the anti-B7 mAbs to inhibit primary and
secondary (recall) antibody responses was determined in a non-human
primate tetanus immunization model. Four cohorts of cynomolgus
monkeys (n=3) were immunized with tetanus toxoid on Day 0 and six
weeks later on Day 42. Anti-tetanus titers were evaluated weekly.
In groups administered a single 10 mg/kg dose of a combination of
both human anti-B7-1 antibody (h1F1) and human anti-B7-2 antibody
(h3D1) on Day 0, anti-tetanus antibody response was dramatically
suppressed, with 0 of 6 treated animals developing a significant
titer within six weeks of immunization. In addition, the animals
that had been treated with the combination of antibodies on Day 0
did not respond to challenge with tetanus antigen on Day 42
regardless of whether they received saline or another dose of h1F1
and h3D1 on Day 42. In the cohort of animals that received saline
on Day 0 and h1F1 and h3D1 on Day 42 only, the mean titer of the
secondary antibody response to tetanus was lower than the mean
titer observed in the saline control cohort. All cohorts of animals
treated with h1F1 and h3D1 were re-immunized with a third dose of
tetanus antigen 112 days after the last dose, when serum levels of
h1F1 and h3D1 were below detectable limits. At this timepoint, the
animals responded by making anti-tetanus antibodies with kinetics
that appeared similar to a primary or secondary antibody response.
These data show that inhibition of costimulation by blocking B7-1
and B7-2 with anti-B7-1 and anti-B7-2 antibodies is capable of
dramatically suppressing the primary antibody response to tetanus
as well as reducing the secondary antibody response. In addition,
these results show recovery from immunosuppression, indicating that
long-term tolerance against tetanus was not achieved in this
study.
Materials and Methods:
Test and Control Articles:
Anti-B7-1 (h1F1):
[0223] Anti-B7-1 antibody was administered to non-human primates by
slow IV infusion. Each animal in the treated groups received 10
mg/kg on Day 0 and/or Day 42 of the study.
Anti-B7 (h3D1):
[0224] Anti-B7-2 antibody was administered to non-human primates by
slow IV infusion. Each animal in the treated groups received 10
mg/kg on Day 0 and/or Day 42 of the study.
Saline Control:
[0225] Saline (9% Sodium Chloride for injection, was administered
to non-human primates by slow IV infusion. Each animal in the
Saline control group and groups B and C received 15 ml of saline on
Day 0 and/or Day 42 of the study.
Purified Tetanus Toxoid Antigen:
[0226] Tetanus toxoid antigen (University of Massachusetts Medical
Center, Biologic Laboratories) was administered on Day 0. All
animals were immunized with 10 limit of flocculation units (LfU) by
intramuscular (IM) injection and 1 Lf unit by intradermal (ID)
injection ninety minutes after saline or antibody administration.
On Day 42, all animals were immunized with 10 Lf units by IM
injection, 90 minutes after saline or antibody administration. On
Day 84, all animals were injected with 1 Lf unit by ID injection to
test for tetanus specific delayed type hypersensitivity (DTH).
Animals in groups B, C, and D were immunized a third time with 10
Lf units by IM injection 112 days after the last dose (second
immunization).
Experimental Design:
[0227] Twelve tetanus naive, 4-6 kg male Cynomolgus macaques
(Macaca fasicularis) were divided into four experimental groups of
three animals per group: [0228] Group A; received 2 immunizations
with 10 Lf Units (Flocculation Units) i.m. tetanus toxoid on day 0
and 42 (controls). [0229] Group B; received 10 mg/kg of each
humanized anti-B7-1 (1F1) and anti-B7-2 (3D1) i.v., at least 90
minutes before 10 Lf units i.m. tetanus toxoid on day 0; tetanus
toxoid immunization only (without mAb pretreatment) on day 42 and
Day 112 (Costimulation blockade with primary immunization). [0230]
Group C; received tetanus toxoid immunization only (without mAb
pretreatment) on day 0; 10 mg/kg of each humanized anti-B7-1 and
anti-B7-2 i.v., at least 90 minutes before 10 Lf units i.m. tetanus
toxoid on day 42 and day 154 (Costimulation blockade with secondary
immunization). [0231] Group D; received 10 mg/kg of each humanized
anti-B7-1 (1F1) and anti-B7-2 (3D1) i.v., at least 90 minutes
before 10 Lf units i.m. tetanus toxoid on day 0; received 10 mg/kg
of each humanized anti-B7-1 and anti-B7-2 i.v., at least 90 minutes
before 10 Lf units i.m. tetanus toxoid on day 42 and a tetanus
toxoid immunization only (without mAb pretreatment) on Day 154
(Costimulation blockade with primary and secondary
immunization).
[0232] All groups received tetanus immunization on Days 0 and 42.
Groups B, C, D received a third tetanus immunization 112 days post
anti-B7 dosing (Table 8).
TABLE-US-00008 TABLE 8 Treatment Groups 1.degree. Tetanus 2.degree.
Tetanus Group Immunization Immunization 3.degree. Tetanus N =
3/group Day 0 Day 42 Immunization A Saline Saline None Saline
Control B Anti B7-1/B7-2 Saline Day 112 anti-B7 after 1.degree. 10
mg/kg IV bolus immunization C Saline Anti B7-1/B7-2 Day 154 anti-B7
after 2.degree. 10 mg/kg immunization IV bolus D Anti B7-1/B7-2
Anti B7-1/B7-2 Day 154 anti-B7 after 1.degree. 10 mg/kg IV bolus 10
mg/kg and 2.degree. IV bolus immunization All groups received
tetanus immunization on Days 0 and 42. Groups B, C, and D received
a third tetanus immunization 112 days post anti-B7 dosing.
Anti-Tetanus Antibody ELISA:
[0233] 96-well ELISA plates were coated with tetanus toxoid at 4
.mu.g/mL. A four-log titration of serum samples was performed
starting at 1:100. Ab binding to tetanus was detected with a
combination of monoclonal anti-human IgG and polyclonal goat
anti-rhesus IgM HRP-conjugated antibodies, and developed with TNB
substrate.
Results and Discussion:
[0234] FIG. 22 shows the anti-tetanus IgM+IgG responses in monkeys
immunized with tetanus toxoid and treated with the combined
anti-B7-1 and anti-B7-2 mAbs.
[0235] Cynomolgus monkeys were administered either saline or a
combination of 10 mg/kg anti-B7.1 antibody (h1F1) and 10 mg/kg
anti-B7.2 antibody (h3D1) by IV infusion. Ninety minutes post
antibody or saline infusion, each animal was immunized with 10 Lf
units of purified tetanus toxoid by IM injection and 1 Lf unit by
ID injection. Anti-tetanus antibody titers were measured weekly. In
the saline control group, mean log titers of anti-tetanus antibody
were increased over baseline by Day 14, peaked at Day 49 and
remained elevated over baseline throughout the study (FIG. 22). In
the groups receiving the combination of h1F1 and h3D1 on Day 0
(Groups B and D), 0 of 6 treated animals had a significant
anti-tetanus antibody titer by day 42. Upon rechallange with
tetanus toxoid on Day 42, animals in Group B, despite receiving no
additional anti-B7 antibody did not mount a significant antibody
response to tetanus (FIG. 22). Analysis of serum showed significant
levels of h1F1 and h3D1 remained in the serum (mean serum
concentration >10 mg/mL) on Day 42. Animals in Group D, received
another infusion of both h1F1 and h3D1 on Day 42 prior to
re-immunization with tetanus toxoid. All 3 animals in this group
had anti-tetanus titers that remained below detectable limits
throughout the study. In Group C, animals that received saline on
Day 0 and h1F1 and h3D1 on Day 42 only, had a primary anti-tetanus
antibody responses similar to the Saline control group (Group A).
The mean antibody titer observed in the secondary response of Group
C, however, was lower than the mean antibody titer observed in the
secondary response of Group A, (FIG. 22).
[0236] Serum concentrations of h1F1 and h3D1 were monitored
throughout the study and when they were below detectable limits,
Groups B, C, and D were immunized a third time with tetanus toxoid.
Each Group was re-immunized 112 days after their last anti-B7
antibody infusion and received no additional anti-B7 antibody
treatment. At this time point, the animals in all groups responded
by generating anti-tetanus antibodies with kinetics that appeared
similar to a primary antibody response (FIG. 22).
Conclusion:
[0237] These data show that inhibition of costimulation by blocking
B7-1 and B7-2 with anti-B7-1 and anti-B7-2 antibodies is capable of
dramatically suppressing the primary antibody response to tetanus
as well as reducing the secondary antibody response. In addition,
these results show recovery from immunosuppression, indicating that
long-term tolerance against tetanus was not achieved in this
study.
[0238] Therefore, the administration of anti-B7 antibodies
concurrent with exposure to a new antigen (tetanus immunization)
can prevent the development of a new antibody response and can
lessen the strength of a secondary response to the same antigen.
Since many disease states are exacerbated by the development of
such antibodies, treatment with the combined anti-B7 mAbs will
prevent the development of such antibodies. One such disease state
is the development of inhibitor antibodies to administered Factor
VIII or Factor IX in hemophiliacs, thereby reducing the
effectiveness of these life saving compounds. Treatment of
hemophiliacs with anti-B7 mAbs prevents or reduces the formation of
the inhibitor antibodies.
Example 19
Pharmacodynamics of Anti-B7-1 (h1F1) and Anti-B7-2 (h3D1) in
Combination or Individually at Doses of 0.01, 0.1, 1, or 10 mg/kg
in a Tetanus Toxoid Challenge Cynomolgus Monkey Model
Abstract:
[0239] The ability of varying doses of individual anti-B7-1 or
anti-B7-2 antibodies, or the combined anti-B7-1 and anti-B7-2
antibodies to inhibit a primary and secondary (recall) response to
tetanus was determined in a Cynomolgus monkey immunization model.
Cynomolgus monkeys (total n=33) were administered a single
intravenous dose of 10, 1, 0.1, or 0.01 mg/kg of h1F1 and h3D1 in
combination, 10, 1, or 0.1 mg/kg of h1F1 or h3D1 alone, or vehicle
control. All animals were immunized with purified tetanus toxoid
one hour after administration of h1F1 and h3D1. Animals were
immunized with tetanus toxoid again at 14 weeks to determine if
normal immune function returned when h1F1 and h3D1 levels fell
below detectable levels.
[0240] A complete suppression of a primary anti-tetanus antibody
response was observed following a single IV dose of 10 or 1 mg/kg
of h1F1 and h3D1 in combination, while a dose of 0.1 mg/kg resulted
in only partial suppression. Treatment with h1F1 or h3D1 alone was
not as effective as equal doses of h1F1 and h3D1 in combination in
suppressing the anti-tetanus antibody response. All animals in all
groups produced a high anti-tetanus antibody titer following a
second immunization with tetanus toxoid, after h1F1 and h3D1
concentrations fell below detection (<50 ng/mL). This indicated
that normal immune function returned following cessation of
treatment with h1F1 and h3D1, and that long-term tolerance against
tetanus was not achieved in this study.
Materials and Methods:
[0241] Animals were administered a single IV dose of test or
control (vehicle) article on Day 0 according to Table 9. The test
or control article was infused via a peripheral vein using a
syringe pump set at 0.1 .mu.L/min/kg (maximum dose rate of 1
mg/min/kg). At t=1 hour on Day 0 all animals received purified
tetanus toxoid (Massachusetts Biologic Laboratories, UMass, Jamaica
Plain, Mass.) at a dose of 10 Lf units (limit of flocculation) by
IM injection and 10 Lf units by ID injection. At t=14 weeks all
animals received a second administration of purified tetanus toxoid
at a dose of 10 Lf units by IM injection and 10 Lf units by ID
injection. Blood samples were collected via venipuncture at
specified timepoints up to 3024 hours (126 days) after dosing.
Serum levels of anti-tetanus antibody (IgM and IgG) were determined
by ELISA.
TABLE-US-00009 TABLE 9 Group Designations and Dose Levels Number of
Dose Level of Antibody Test Group Animals (mg/kg) Agent(s) 1 3 0
Administered Vehicle 2 3 10 h1F1 3 3 1 h1F1 4 3 0.1 h1F1 5 3 10
h3D1 6 3 1 h3D1 7 3 0.1 h3D1 8 3 10.sup.a h1F1 and h3D1 9 3 .sup.
1.sup.a h1F1 and h3D1 10 3 .sup. 0.1.sup.a h1F1 and h3D1 11 3 .sup.
0.01.sup.a h1F1 and h3D1
Anti-Tetanus Antibody Formation:
[0242] All animals treated with vehicle produced a detectable
anti-tetanus antibody titer beginning 14 days after immunization
(Table 10, FIGS. 23, 24, and 25). Treatment with the two highest
doses (10 or 1 mg/kg) of h1F1 and h3D1 in combination completely
suppressed the formation of a detectable antibody titer within the
14-week observation period in all animals. A partial suppression of
the antibody response was observed in animals treated with 0.1
mg/kg, one out of three animals produced a detectable antibody
titer. All animals treated with 0.01 mg/kg of h1F1 and h3D1 in
combination produced a detectable antibody titer; however, this
titer was lower in magnitude when compared to the titers observed
in the vehicle control group, indicating that some suppression of
the antibody response was achieved.
TABLE-US-00010 TABLE 10 Number of Cynomolgus Monkeys that Produced
a Detectable Anti-Tetanus Antibody Titer Following a Primary
Challenge with Tetanus Toxoid During Treatment with h1F1 and h3D1
Number of Animals that Group Treatment Produced a Detectable 1
Vehicle Control 3 2 10 mg/kg h1F1 1 3 1 mg/kg h1F1 3 4 0.1 mg/kg
h1F1 2 5 10 mg/kg h3D1 1 6 1 mg/kg h3D1 1 7 0.1 mg/kg h3D1 3 8 10
mg/kg h1F1 and h3D1 0 9 1 mg/kg h1F1 and h3D1 0 10 0.1 mg/kg h1F1
and h3D1 1 11 0.01 mg/kg h1F1 and h3D1 3
[0243] When the 0.1, 1, and 10 mg/kg dose groups are taken
together, treatment with either h1F1 (FIG. 24) or h3D1 (FIG. 25)
alone is not as effective as treatment with h1F1 and h3D1 in
combination (FIG. 23). Treatment with h1F1 alone (Groups 2, 3, and
4) resulted in 6/9 (66%) animals with a detectable anti-tetanus
antibody response. Treatment with h3D1 alone (Groups 5, 6, and 7)
resulted in 5/9 (55%) animals with detectable antibodies, whereas
only 1/9 (11%) animals had detectable antibodies following
treatment with the combination of h1F1 and h3D1 (Groups 8, 9, and
10). Group 11 was not included in these comparisons since h1F1 and
h3D1 were not administered individually at 0.01 mg/kg.
[0244] At 1 and 10 mg/kg, there was no detectable anti-tetanus
antibody response in any of the animals treated with h1F1 and h3D1
in combination; however, at both of these doses, there were
detectable antibodies in animals treated with h1F1 or h3D1 alone.
When animals were treated with 0.1 mg/kg, there were detectable
anti-tetanus antibodies in animals from all three treatment groups;
however, there was a lower incidence (1/3) in the combination
treatment group than in the h1F1 (2/3) or h3D1 ( 3/3) groups.
[0245] FIG. 26 shows the area under the anti-tetanus antibody titer
curve (AUC) for each treatment group. These AUC values were
calculated using the antibody titer curves shown in FIGS. 23, 24,
and 25. The AUC values were calculated from Weeks 0 to 14. To
account for the number of responding animals in each group, the AUC
values were weighted by the fraction of the number of animals in
each group that produced detectable antibody titers. This plot
gives an indication of the cumulative magnitude of the antibody
titer or strength of the antibody response throughout the 14 week
observation period. Animals treated with either 10 or 1 mg/kg of
h1F1 and h3D1 in combination (Groups 8 or 9) showed 100%
suppression (AUC=0) of the antibody response relative to the
control group. Treatment with 0.1 or 0.01 mg/kg of h1F1 and h3D1 in
combination (Groups 10 or 11) suppressed the antibody response by
78% or 86% (AUC=402 or 253 Log titerhr), respectively, relative to
the control group. The antibody response was suppressed between
8.6% and 99% (AUC=1640 to 5.04 Log titerhr) relative to the control
group in the animals treated with h1F1 or h3D1 alone (Groups 2-7).
These data indicate that treatment with h1F1 or h3D1 alone was not
as effective as equal doses of h1F1 and h3D1 in combination in
suppressing the anti-tetanus antibody response.
[0246] Following the re-immunization with tetanus toxoid 14 weeks
after administration of h1F1 and h3D1, all animals in all groups
produced a high anti-tetanus antibody titer (>3 log titer),
indicating that normal immune function returned following cessation
of treatment with h1F1 and h3D1, and that long-term tolerance
against tetanus was not achieved in this study. Animals treated
with 10 mg/kg of h1F1 and h3D1 in combination showed a delayed
antibody response with detectable antibody titers observed 14 days
after immunization similar to a primary antibody response,
indicating that a response to the first immunization was completely
blocked. All other animals showed increased antibody titers 7 days
after immunization, which resembles a memory response rather than a
primary response.
Conclusions:
[0247] A complete suppression of a primary anti-tetanus antibody
response was observed following a single IV dose of 10 or 1 mg/kg
of h1F1 and h3D1 in combination, while a dose of 0.1 mg/kg resulted
in only partial suppression. Treatment with h1F1 or h3D1 alone was
not as effective as equal doses of h1F1 and h3D1 in combination in
suppressing the anti-tetanus antibody response. All animals in all
groups produced a high anti-tetanus antibody titer following a
second immunization with tetanus toxoid after h1F1 and h3D1
concentrations fell below detection. This indicates that normal
immune function returns following cessation of treatment with h1F1
and h3D1.
Example 20
Serum Half-Life of Anti-B7 Antibodies in Non-Human Primates
[0248] The murine-anti-hB7-1 and murine-anti-hB7-2 mAbs were tested
in non-human primates for serum half-life and target cell
saturation. Three Cynomolgus monkeys were dosed with one dose each
of a combination of the anti-hB7-1 and anti-hB7-2 mAbs at 2, 8, or
20 mg each mAb/kg body weight. The monkeys were analyzed for mAb
binding to PBMC (Proliferative Blood Mononuclear Cells), serum mAb
concentration, and primate anti-mouse antibody (PAMA) response
(Table 11). PBMC saturation was determined by flow cytometry (FACS)
where PBMCs isolated from the blood of mAb dosed primates were
stained with goat-anti-murine Ig-PE (% in vivo) or the PBMC were
first reacted with the anti-hB7-1 and anti-hB7-2 mAbs followed by
detection with the goat-anti-murine Ig-PE (% ex vivo). The level of
PBMC saturation at the various time points was calculated by (% in
vivo/% ex vivo).times.100. This study shows that PBMC saturation
for the anti-hB7-1 and anti-B7-2 mAbs falls below 80% between days
4 to 6 (mAbs @ 2 mg/ks), days 6 to 8 (mAbs @ 8 mg/kg), and days 13
to 20 (mAbs @ 20 mg/kg) depending upon mAb dose. Although not
measured directly, there was no apparent dramatic decrease in the
numbers of circulating B7.sup.+ cells.
[0249] Serum half-lives of the anti-hB7-1 and anti-hB7-2 mAbs were
measured with a specific ELISA for each mAb using hB7-1Ig or
hB7-2Ig as target and goat-anti-murine Ig HRP/ABTS for detection.
These assays were sensitive to 400 ng/mL and 200 ng/mL for
anti-hB7-2 and anti-hB7-1, respectively. PAMA responses were
measured using a commercially available kit. The serum
concentrations of the two anti-hB7 mAbs and the PAMA responses are
shown at the individual dosage levels for each mAb. Both mAbs
exhibit similar serum half lines of 48 hours as determined at all
three dosage levels. Increasing mAb dosage increased serum mAb
concentrations by a comparable factor at all dosages and times
tested. When dosed at 20 mg/kg, circulating mAb levels of >30
.mu.g/mL were found for each mAb at 6 days post dosing.
[0250] PAMA responses to the anti-hB7-1 and anti-hB7-2 mAbs were
low and were first measurable beginning 10 days after serum mAb
levels had fallen below 10 .mu.g/mL.
[0251] The serum half-life of humanized anti-human B7-2 and B7-1
antibodies were also determined in Cynomolgus monkeys (n=6) dosed
once with 10 mg/kg of humanized anti-B7-2 antibody. Serum
concentration was monitored by specific ELISA assay for each
antibody using HRP-anti human IgG2 and ABTS.
[0252] FIG. 27 shows the serum concentration of the humanized B7-2
and humanized B7-1 mAbs in Cynomolgus monkeys through 42 days after
dosing.
[0253] The humanized anti-human B7-2 and anti-human B7-1 mAbs
exhibited an extended serum half-life in Cynomolgus monkeys, as
compared to a value of approximately 2 days for the murine
anti-human B7-2 and anti-human B7-1 mAbs when dosed at the same
level, demonstrating that the humanized anti-human B7 mAbs were
retained in circulation much longer than the murine anti-B7
mAbs.
TABLE-US-00011 TABLE 11 Results from the Preclinical primate
studies Dose @ 2 mg each mAb/kg Dose @ 8 mg each mAb/kg Dose @ 20
mg each mAb/kg Time PBL PBL PBL Hours Anti-hB7-2 PAMA Saturation
Anti-hB7-2 PAMA Saturation Anti-hB7-2 PAMA Saturation (Days)
.mu.g/mL ng/mL % .mu.g/mL ng/mL % .mu.g/mL ng/mL % 0 BQL Neg. 0 BQL
Neg. 0 BQL Neg. 0 .167 61 NT 206 NT 580 NT .5 59 NT 100 229 NT 25
570 NT 65 1 52 NT 227 NT 527 NT 3 52 NT 100 230 NT 100 548 NT 100 5
50 NT 139 NT 464 NT 8 44 NT 169 NT 412 NT 24 (1 D) 26 NT 70 103 NT
100 286 NT 80 48 (2 D) 15 NT 100 59 NT 100 196 NT 100 96 (4 D) 2.4
NT 75 18 NT 100 83 NT 100 144 (6 D) BQL NT 95 3.9 NT 100 32 NT 100
192 (8 D) BQL NT 65 BQL NT 100 13 NT 100 240 (10 D) BQL NT BQL NT
3.9 NT 312 (13 D) BQL Neg. 5 BQL Neg. 55 BQL Neg. 80 480 (20 D)
2908 10 4080 10 517 20 684 (27 D) 1260 1460 1094 816 (34 D) BQL =
Below Quantifiable Limit; NT = Not Tested
Example 21
Inhibition of Specific T-Cell Responses to Superantigens (Toxic
Shock Syndrome Toxin-1; TSST-1)
[0254] NODscid mice were populated with human lymphocytes by the
administration of 10.sup.8 human PBLs. After 28 days, the mice were
treated with TSST-1 (10 mg, I.P.) with or without the treatment
with the combined antibodies to human B7-1 and B7-2 (500 mg, I.V.).
After 14 additional days, the presence of human lymphocytes,
T-cells, and TSST-1 specific T-cells (V.beta.2-TCR-cells) in the
peritoneal cavity was measured by FACS using antibodies specific
for human CD45, CD4, and human V.beta.2-TCR.
TABLE-US-00012 TABLE 12 Human Addition T-cells (%) TSST-1 Anti-B7-1
+ Anti-B7-2 Total V.beta.2.sup.+ - - 10.2 3.9 + - 27.4 12.0 + +
23.4 3.8
Results:
[0255] Table 12 shows the proportion of total human T cells and
V.sub..beta.2.sup.+-TCR human T cells (TSST-1 specific) found in
the peritoneal cavity of hu-NODscid mice. Treatment with TSST-1
greatly increased the percentage of human T cells and of TSST-1
specific human T cells (V.sub..beta.2.sup.+) in the huNOD-scid
mice. Treatment with the anti-human B7-1 and B7-2 mAbs moderately
diminished the total human T cell response and completely inhibited
the expansion of the TSST-1 specific human T cells indicating that
the anti-B-7 mAbs could effectively inhibit human T cell
superantigen mediated responses.
Example 22
Evaluation of Anti-B7 Antibodies h1F1 (Anti-B7-1) and h3D1
(Anti-B7-2) in a Life-Supporting Renal Transplant Model in
Cynomolgus Monkeys
Abstract:
[0256] This study was performed to evaluate the efficacy and
compatibility of the monoclonal antibodies h1F1 (anti-B7-1) and
h3D1 (anti-B7-2) in a life-supporting renal transplant model in
Cynomolgus monkeys. The efficacy and compatibility of these
antibodies were evaluated when they were given as a monotherapy as
well as in combination with conventional immunosuppressive agents
such as cyclosporin A (CsA), rapamycin, and steroids.
[0257] Twenty four male cynomolgus monkeys received blood group
compatible, mixed lymphocyte reaction (MLR) mismatched, renal
allografts. The recipients were studied consecutively in 6
treatment groups, which included the following combinations of
immunosuppressive agents for the first 56 postoperative days
(poDay) and then followed for an additional 65-66 days (total
maximum follow-up period 119-120 days) without additional
treatment: [0258] Group 1 received combined anti-B7 antibodies
according to Schedule A: h1F1 20 mg/kg+h3D1 20 mg/kg
preoperatively, then h1F1 5 mg/kg and h3D1 5 mg/kg postoperatively
and h1F1 5 mg/kg and h3D1 5 mg/kg every 7 days repeated until poDay
56; 9 dosing time points. [0259] Group 2 received combined anti-B7
antibodies according to Schedule B: h1F1 5 mg/kg+h3D1 5 mg/kg
preoperatively, then h1F1 10 mg/kg and h3D1 10 mg/kg immediately
postoperatively, and again on poDay 3, then 5 mg/kg of each
antibody for 8 consecutive doses given in weekly intervals starting
poDay 7 and ending poDay 56. [0260] Group 3 received the combined
anti-B7 antibodies according to Schedule A plus microemulsion CsA
at a dose designed to reach 24-hour trough level concentrations of
200 to 300 ng/mL from poDay 0 to poDay 14, then at a reduced dose
to reach 24-hour CsA trough levels of 150 to 250 ng/mL from poDay
15 to the last dose on poDay 56. [0261] Group 4 received the
combined anti-B7 antibodies (h1F1 and h3D1) according to Schedule A
in combination with a tapering dose of steroids: methylprednisolone
2 mg/kg IV postoperatively and then daily on poDay 2, then
prednisone 0.5 mg/kg reduced by 0.05 mg/kg every three days until
0.2 mg/kg was reached then 0.2 mg/kg was continued until poDay 56
[0262] Group 5 was given combined anti-B7 antibodies (h1F1 and
h3D1) according to Schedule A in combination with rapamycin:
rapamycin 1 mg/kg postoperatively and then daily through poDay 13,
then 0.5 mg/kg daily was continued until poDay 56 [0263] Group 6
was given microemulsion CsA at a dose designed to reach 24-hour
trough level concentrations of 200 to 300 ng/mL from poDay 0 to
poDay 14, then at a reduced dose to reach 24-hour CsA trough levels
of 150 to 250 ng/mL from poDay 15 to the last dose on poDay 56.
[0264] Group 7 was given rapamycin at 1 mg/kg from poDay 0 to poDay
13, and then at 0.5 mg/kg from poDay 14 through poDay 56.
[0265] Animals were euthanized before poDay 120 if the creatinine
levels rose above 8.0 mg/dL, which was interpreted as evidence of
terminal renal rejection.
[0266] The survival of transplanted, treated cynomolgus monkeys is
depicted in Table 13.
TABLE-US-00013 TABLE 13 Survival and Diagnosis of Transplanted,
Treated Cynomolgus Monkeys Treatment Schedule anti-B7-1 + anti-B7-
Treatment Group 2/Other Therapies Survival (poDay)* historical
controls none 10 1 schedule A/none 9, 48, 119, 119 2 schedule
B/none 12, 14, 18, 120 3 schedule A/CsA 96, 119, 119, 119 4
schedule A/steroids 6, 77, 111, 119 5 schedule A/rapamycin 69, 73,
81, 114 6 no mAbs/CsA 22, 25, 38, 71 7 no mAbs/rapamycin 11, 18,
27, 35 *kidney recipients surviving until day 119/120 were
sacrificed as per study protocol
[0267] The results of this study show that treatment of non-human
primate, renal allograft recipients with the combination of the
B7-1 and B7-2 monoclonal antibodies can lead to long-term graft
survival. Graft survival was observed for up to 66 days post
termination of treatment in 50% of the Group 1 animals. In
contrast, antibodies dosed according to Schedule B, used in Group 2
were not sufficient to overcome the early acute rejection episode
in 3 of the 4 animals treated, and only one animal of this group
survived 120 days. Combining Schedule A with microemulsion CsA
resulted in long-term survival in all animals with no evidence of
an antagonistic immunosuppressive effect of CsA on the previously
demonstrated efficacy of the antibodies. In Group 4,
coadministration of high dose steroids with the antibodies did not
antagonize the immunosuppressive effect of the antibodies in this
non-human primate model. In Group 5, coadministration of rapamycin
with the antibodies did not antagonize the immunosuppressive effect
of the antibodies in this non-human primate model. In Group 6,
treatment with CsA alone resulted in early rejection in most kidney
recipients. The results with this treatment group were inferior to
those obtained with the combined treatment with the anti-B7
antibodies alone (Group 1) or with the combined antibodies+CsA
(Group 4). In group 7, treatment with rapamycin alone resulted in
early rejection in all kidney recipients. The result in this
treatment group was inferior to that obtained with the combined
treatment with the anti-B7 antibodies alone (Group 1) or with the
combined antibodies and rapamycin (Group 5).
[0268] Thus, the monoclonal antibodies h1F1 and h3D1 are
efficacious in avoiding early terminal rejection in a
life-supporting renal transplant model in non-human primates. The
antibodies are compatible with other immunosuppressants and their
efficacy appears to be increased by combining them with
microemulsion CsA rapamycin, or steroids.
[0269] The following study was divided into two phases. The purpose
of the first phase was to define the efficacy of the novel
immunosuppressive antibodies h1F1 and h3D1 when given as a
monotherapy to cynomolgus monkeys that had received a renal
transplant in a life supporting model. Animals transplanted without
concurrent immunosuppressant treatment reject their grafts within
10 days of transplant. The study was designed to test two different
schedules of antibody therapy. The antibody treatment was
discontinued after poDay 56 in both schedules and animals were then
followed until graft rejection or until Day 119-120.
[0270] The second phase of this study was designed to test if: a)
h1F1 and h3D1, when given in combination with either CsA,
rapamycin, or steroids, would antagonize the immunosuppressive
effect of CsA, rapamycin, or steroids, thus resulting in shortened
graft survival, or b) CsA, rapamycin, or steroids would antagonize
the immunosuppressive efficacy of h1F1 and h3D1 either during the
time when treatment was given simultaneously (poDay 0 to poDay 56)
or following termination of all immunosuppressive therapy
(>poDay 56).
Materials and Methods:
Animals:
[0271] Male cynomolgus monkeys, Macaca fascicularis, with a weight
between 5 and 8 kg were used in this study. The animals underwent
blood group typing. Donor and recipient monkeys were paired based
on an ABO blood group match, a negative cross-match, and a
stimulation index of at least 2.5 in a two-way MLR.
Test and Control Articles:
[0272] h1F1 and h3D1:
[0273] The anti-B7 antibodies were administered in a syringe
connected to a syringe pump. h1F1 and h3D1 were always given in
combination and administered at a maximum infusion rate of 1
mg/kg/min through a peripheral venous catheter.
[0274] The antibodies were administered according to different
protocols as defined for each group.
Microemulsion CsA (Neoral):
[0275] The microemulsion CsA (Neoral; 100 mg/mL (Novartis)) was
kept at room temperature and protected from exposure to light. The
calculated volume of Neoral based on the required dose (in mg/kg)
was drawn directly into the smallest appropriate syringe and
administered without any dilution through a nasogastric tube
directly into the stomach.
Methylprednisolone and Prednisone:
[0276] Methylprednisolone (Solu-Medrol, Pharmacia & Upjohn Co.,
Kalamazoo, Mich.) was supplied in vials with self-contained sterile
water for reconstitution and was reconstituted following
manufacturer's instructions. The drug was kept refrigerated after
reconstitution and discarded after 48 hours.
[0277] Prednisone (Mutual Pharmaceutical Co., Inc., Philadelphia,
Pa.) was supplied in tablets (5 mg). Tablets were dissolved in
sterile water, one tablet per 5 mL water, with a resulting solution
concentration of 1 mg/mL. The remaining drug was discarded at the
end of dosing each day.
Experimental Design:
[0278] A total of 24 monkeys received unilateral renal transplants
in a life-supporting model.
Group 1: h1F1 and h3D1 Monotherapy (Schedule A):
[0279] Group 1 received combination anti-B7-1+anti-B7-2 antibody
monotherapy according to Schedule A: h1F1 20 mg/kg+h3D1 20 mg/kg
preoperatively, then h1F1 5 mg/kg and h3D1 5 mg/kg postoperatively
and h1F1 5 mg/kg and h3D1 5 mg/kg every 7 days repeated until poDay
56.
Group 2: h1F1 and h3D1 Monotherapy (Schedule B):
[0280] The main differences between Schedules A and B were that in
Schedule B, the first preoperative dose was reduced from 20 mg/kg
to 5 mg/kg, the immediate postoperative dose was increased from 5
mg/kg to 10 mg/kg, and an additional dose of 10 mg/kg was given on
poDay 3. The remainder of the postoperative dosing schedule was the
same as Schedule A. Therefore, the total amount of antibody given
over the postoperative course for each animal was the same in
Schedules A and B.
Group 3: h1F1 and h3D1 (Schedule A) Plus CsA:
[0281] In Group 3, antibody treatment (Schedule A) was combined
with daily oral administration of microemulsion CsA. Group 3
received the antibodies according to Schedule A plus microemulsion
CsA at a dose designed to reach 24-hour trough level concentrations
of 200 to 300 ng/mL from poDay 0 to poDay 14, then at a reduced
dose to reach 24-hour CsA trough levels of 150 to 250 ng/mL from
poDay 15 to the last dose on poDay 56.
[0282] Microemulsion CsA was administered daily by gavage following
short Ketamine sedation by gavage. Twenty-four hour trough levels
of CsA were measured three times per week and the daily dose was
adjusted to meet the target CsA trough levels. Additional
modifications of the CsA dose were performed to prevent excessive
weight loss or when renal function impairment was thought to be
related to CsA trough levels.
Group 4: h1F1 and h3D1 (Schedule A) Plus Steroids:
[0283] In Group 4, h1F1 and h3D1 were administered according to
Schedule A in combination with a tapering dose of steroids:
methylprednisolone 2 mg/kg IV postoperatively and then daily on
poDay 2, then prednisone 0.5 mg/kg reduced by 0.05 mg/kg every
three days until 0.2 mg/kg was reached then 0.2 mg/kg was continued
until poDay 56
[0284] For the first 3 postoperative days (poDay 0 through 2), the
animals received methylprednisolone at a dose of 2 mg/kg given
intravenously as a bolus. From poDay 3 to poDay 56, prednisone was
given to the ketamine sedated animals by gavage in a tapering
schedule (see FIG. 4) starting at 0.5 mg/kg and ending at a dose of
0.2 mg/kg.
Group 5: h1F1 and h3D1 (Schedule A) Plus Rapamycin:
[0285] In Group 5, h1F1 and h3D1 were administered according to
Schedule A in combination with rapamycin: rapamycin 1 mg/kg
postoperatively and then daily through poDay 13, then 0.5 mg/kg
daily was continued until poDay 56
Group 6: CsA Alone:
[0286] In Group 6, microemulsion CsA was administered at a dose
designed to reach 24-hour trough level concentrations of 200 to 300
ng/mL from poDay 0 to poDay 14, then at a reduced dose to reach
24-hour CsA trough levels of 150 to 250 ng/mL from poDay 15 to the
last dose on poDay 56.
[0287] Microemulsion CsA was administered daily by gavage following
short Ketamine sedation by gavage. Twenty-four hour trough levels
of CsA were measured three times per week and the daily dose was
adjusted to meet the target CsA trough levels. Additional
modifications of the CsA dose were performed to prevent excessive
weight loss or when renal function impairment was thought to be
related to CsA trough levels.
Group 7: Rapamycin
[0288] Rapamycin was administered daily by gavage following short
Ketamine sedation by gavage. Group 7 was given rapamycin at 1 mg/kg
from poDay 0 to poDay 13, and then at 0.5 mg/kg from poDay 14
through poDay 56.
Results and Discussion:
Historical Control Group: Renal Transplantation with No
Treatment:
[0289] Historical data shows that cynomolgus monkeys receiving
renal transplants and no treatment uniformly reject the
transplanted kidney by day 10 post transplant.
Group 1: Monotherapy with h1F1 and h3D1 According to Schedule
A:
[0290] Two of four animals treated with the combined
anti-B7-1+anti-B7-2 mAbs according to schedule A retained a
functional allograft until the end of the study period (120 days)
whereas the other two rejected the transplanted kidney during the
treatment period (day 9 and day 48). Therefore, three out of four
kidney recipients treated according to schedule A retained the
transplanted kidney for much longer than historical controls
demonstrating the efficacy of anti-B7 therapy in preventing the
rejection of transplanted kidneys.
Group 2: Monotherapy with h1F1 and h3D1 According to Schedule
B:
[0291] In contrast to Group 1, only 1 of the 4 animals treated with
the anti-B7 antibodies according to schedule B retained a
functional renal allograft beyond poDay 18. However, this animal
retained the transplanted kidney until the end of the study period
(poDay 120). This data suggests that treatment with anti-B7 mAbs
according to schedule A results in better immunosuppression of
kidney rejection than animals treated according to schedule B. This
result may be due to the higher concentrations of anti-B7 mAbs
present early after transplantation in animals treated according to
schedule A.
Group 3: Therapy with h1F1 and h3D1 According to Schedule A Plus
Microemulsion CsA:
[0292] All animals treated with the anti-B7 mAbs according to
schedule A in combination with CsA exhibited greatly delayed
rejection of the transplanted kidneys with three out of four
animals retaining the functional allograft until the study end
(poDay 119). These results are superior to those obtained after
treatment with the anti-B7 mAbs alone (Group 1) or with CsA alone
(Group 6) demonstrating that there is no antagonism between
treatment with the anti-B7 mAbs+CsA and that cotreatment with these
agents provides and additional benefit of delayed organ
rejection.
Group 4: Therapy with h1F1 and h3D1 According to Schedule A Plus
Methylprednisolone/Prednisone:
[0293] One of four animals treated with the combined anti-B7
mAbs+steroids retained the transplanted kidney until the end of the
study period (poDay 119). Of the remaining three kidney recipients,
two exhibited delayed rejection of the transplanted kidneys. The
third monkey rejected the transplanted kidney early (poDay 6) due
to ureter necrosis believed to be associated with the
administration of the high dose steroids. Taken together, these
data suggest that the combination of the anti-B7 mAbs+steroids are
effective in preventing the rejection of the transplanted kidney
and the use of these agents in combination is not
contraindicated.
Group 5: Therapy with h1F1 and h3D1 According to Schedule A Plus
Rapamycin:
[0294] All four animals treated with the combined anti-B7 mAbs
according to schedule A+rapamycin retained functional renal
allografts beyond the treatment period for a duration ranging from
69 to 114 poDay. This demonstrated that the treatment with the
combined anti-B7 mAbs+rapamycin is beneficial in delaying organ
rejection and does not antagonize the treatment with the anti-B7
mabs alone.
Group 6: Therapy with CsA Alone:
[0295] All four animals treated with CsA alone rejected their
transplanted kidneys with three out of four animals rejecting
during the CsA treatment period. These results are inferior to
those obtained with in animals treated with the combined anti-B7
mAbs+CsA.
Group 7: Therapy with Rapamycin Alone:
[0296] All four animals treated with rapamycin alone rejected their
transplanted kidneys during the treatment period.
Conclusion:
[0297] The purpose of the study was to evaluate the efficacy and
compatibility of the novel monoclonal antibodies, h1F1 and h3D1, in
a life-supporting non-human primate kidney transplant model.
Efficacy was assessed by evaluating the incidence of terminal acute
rejection and ultimately allograft (animal) survival. Terminal
rejection in untreated animals would be expected to occur within 10
days post-transplantation in the model used. Compatibility and
efficacy were tested in a total of 7 different treatment groups.
The treatment regimens used in later groups evolved as the study
progressed and were based on the results of previous groups.
Evaluation of Efficacy:
Group 1: h1F1 and h3D1 Monotherapy (Schedule A):
[0298] Animals in the first therapeutic group were treated with
only h1F1 and h3D1, given weekly for 56 days without any additional
immunosuppressive drug therapy. The results showed that monotherapy
with h1F1 and h3D1 was able to prevent terminal acute rejection in
2 of the 4 animals of this group and delay the occurrence of
terminal rejection in an additional monkey until poDay 48. The
administration of the antibodies did not prolong graft survival in
one monkey beyond that seen in the historical control group.
Group 2: h1F1 and h3D1 Monotherapy (Schedule B):
[0299] The regimen and schedule of antibody administration was
changed in treatment Group 2 in attempt to prevent the early acute
rejection episodes seen in Group 1 between poDay 5 and 7. The
preoperative dose in this group was reduced from 20 mg/kg of each
mAb to 5 mg/kg of each mAb and the first postoperative dose was
increased from 5 to 10 mg/kg of each mAb. The total amount of
antibody administered perioperatively was therefore reduced from 25
mg/kg to 15 mg/kg. An additional dose of 10 mg/kg was given on
poDay 3. Thereafter, the administration schedule in Group 1 and 2
were identical.
[0300] Three of the 4 animals did not recover from this first
rejection episode. Only one animal survived for the entire
follow-up period. This demonstrates the critical aspect of timing
and dosing of the antibodies during the early peri- and
postoperative periods. The reduction in the perioperative dose most
likely resulted in a more vigorous rejection response, and the
administration of the additional dose of antibodies on poDay 3 did
not affect this outcome.
[0301] In both Groups 1 and 2, there were animals with long-term
survival without any additional immunosuppressive therapy.
[0302] A direct comparison of graft outcome following Schedule A
(Group 1) and Schedule B (Group 2) favored Schedule A. The
combination of antibodies were administered according to Schedule A
for the next two groups.
Group 3: h1F1 and h3D1 (Schedule A) Plus CsA:
[0303] A dose of microemulsion CsA, that aimed at delivering
24-hour trough levels in the 200 to 300 ng/mL range was
administered in Group 3. All animals in this group survived
long-term (>56 days) and 3 of the 4 animals showing no evidence
of terminal acute rejection until the end of the follow-up period.
Therefore, neither the anti-B7 antibodies nor the CsA negatively
affected the efficacy of the other. The median survival was 119
days, and the mean survival was 113 days in this group, which was
at least as good as, if not better than, the length of survival in
Group 1 (median: 84 days; mean: 74 days). An additional treatment
group in which the recipients are treated with only microemulsion
CsA (Group 6) dosed to achieve similar trough levels as in Group 3
(for 56 days) showed that all four transplant recipients rejected
their transplanted kidneys and that three out of four rejected
during the treatment period.
[0304] The combination of the monoclonal antibodies with
microemulsion CsA was sufficient to avoid an early acute rejection
episode.
Group 4: h1F1 and h3D1 (Schedule A) Plus Steroids:
[0305] The combination of h1F1 and h3D1 was administered with a
tapered dose of steroids in Group 4. Monotherapy with steroids
alone is generally not sufficient to prevent terminal allograft
rejection in this model. A high dose of steroids was chosen for
this group to determine if the steroids and antibodies affected the
efficacy of each other. One of four animals treated with the
combined anti-B7 mAbs+steroids retained the transplanted kidney
until the end of the study period (poDay 119). Of the remaining
three kidney recipients, two exhibited delayed rejection of the
transplanted kidneys. The third monkey rejected the transplanted
kidney early (poDay 6) due to ureter necrosis believed to be
associated with the administration of the high dose steroids.
[0306] There did not appear to be any negative effect of
co-administration of high dose steroids on the efficacy of the
antibodies in this group. It is unclear if the antibody
administration had an effect on the immunosuppressive efficacy of
steroids as there are no data available on the efficacy of a
similar steroid regimen used as monotherapy in a non-human primate
renal transplant model.
Group 5: Therapy with h1F1 and h3D1 According to Schedule A Plus
Rapamycin:
[0307] All four animals treated with the combined anti-B7 mAbs
according to schedule A+rapamycin retained functional renal
allografts beyond the treatment period for a duration ranging from
69 to 114 poDay. This demonstrated that the treatment with the
combined anti-B7 mAbs+rapamycin is beneficial in delaying organ
rejection and does not antagonize the treatment with the anti-B7
mabs alone. An additional treatment group in which the recipients
were treated only with rapamycin (Group 7) dosed to achieve similar
levels as in Group 5 showed that all four transplant recipients
rejected their transplanted kidneys during the treatment
period.
Group 6: Therapy with CsA Alone:
[0308] All four animals treated with CsA alone rejected their
transplanted kidneys with three out of four animals rejecting
during the CsA treatment period. These results are inferior to
those obtained in animals treated with the combined anti-B7
mAbs+CsA.
Group 7: Rapamycin Alone:
[0309] All four animals treated with rapamycin alone rejected their
transplanted kidneys during the treatment period. These results are
inferior to those obtained in animals treated with the combined
anti-B7 mAbs and rapamycin (Group 5).
Example 23
Induction Therapy with Monoclonal Antibodies Against B7-1 and B7-2
Delays the Onset of Renal Allograft Rejection in Non-Human
Primates
[0310] In this study, the administration of monoclonal antibodies
anti-B7-1 (h1F1) and anti-B7-2 (h3D1) alone and in combination were
tested for their ability to delay the onset of acute renal
allograft rejection in rhesus monkeys. The most durable effect
resulted from simultaneous blockade of both B7 ligands. The
mechanism of action did not involve global depletion of T or B
cells.
Materials and Methods:
Experimental Design:
MHC Typing and Donor-Recipient Selection:
[0311] Donor-recipient combinations were selected based on genetic
non-identity at major histocompatibility complex (MHC) class II.
This was established by denaturing gradient gel electrophoresis and
direct sequencing of the second exon of the major
histocompatibility antigen, HLA-DRB. T cell responsiveness of the
recipient towards the donor was confirmed in vitro for all
donor-recipient pairs using the mixed lymphocyte reaction (MLR)
assay. Each animal was tested against all potential donors to
establish the highest responder pairs for transplantation.
Renal Allografts:
[0312] Renal allotransplantation was performed as previously
described. Knechtle S J, et al., Transplantation, 63:1-6 (1997);
Kirk A D, et al., Proc Natl Acad Sci USA. 94:8789-8794 (1997); Kirk
A D, et al., Nature Medicine, 5:686-693 (1999). Briefly, outbred
juvenile rhesus monkeys (age 18 to 36 months, male), seronegative
for simian immunodeficiency virus, and herpes B virus, were
obtained from LABS of Virginia, Inc. (Yemassee, S.C.).
[0313] All procedures were performed under general anesthesia.
Renal transplantation was performed between genetically distinct
donor-recipient pairs as determined by MHC analysis. The animals
were heparinized (100 units/kg) during organ harvest and
implantation. The allograft was implanted using standard
microvascular techniques to create an end to side anastomosis
between the donor renal artery and recipient distal aorta as well
as the donor renal vein and recipient vena cava. A primary
ureteroneocystostomy was then created. Bilateral native nephrectomy
was completed prior to closure. Skin sutures were removed after 7
to 10 days.
[0314] Anti-B7-1 and/or anti-B7-2 antibodies were administered
intravenously at doses detailed below. Animals were euthanized at
the time of renal failure as determined by a rising serum
creatinine level or if a weight loss of 15% of pre-transplant body
weight occurred in accordance with AAALAC standards. Complete gross
and histopathological analysis was performed at necropsy on all
sacrificed animals.
Results and Discussion:
Group I: Control Animals:
[0315] Five animals received renal allografts without any treatment
to prevent rejection. All five lost their grafts to acute rejection
within 8 days (Table 14, FIG. 28).
Group II: Monotherapy with h1F1 Alone:
[0316] Two animals were treated solely with h1F1 (Table 14, FIG.
28). The antibody was given at a dose of 20 mg/kg beginning prior
to graft reperfusion. Subsequent doses of 5 mg/kg were given every
seven days until rejection. These two animals had trivial
prolongation of their graft survival with rejection occurring in 8
and 9 days.
Group III: Monotherapy with h3D1 Alone:
[0317] Two animals were treated solely with h3D1 (Table 14, FIG.
28). The antibody was given at a dose of 20 mg/kg beginning prior
to graft reperfusion. Subsequent doses of 5 mg/kg were given every
seven days until rejection. These two animals also had minimal
prolongation of their graft survival with rejection occurring in 8
and 28 days.
Group IV: Combination Therapy with h1F1 Together with h3D1:
[0318] Four animals were treated with both h1F1 and h3D1 (Table 14,
FIG. 28). The antibodies were given at a dose of 20 mg/kg beginning
prior to graft reperfusion. In one animal (AT48) a dose of 5 mg/kg
was given immediately post transplant. In all animals doses of 5
mg/kg were given every seven days for a fixed period of time or
until rejection. Antibody administration was scheduled to be halted
after 60 days in three of the four animals in Group IV. One animal
was dosed out to 80 days (AC2B). All animals had prolonged graft
survivals of 47, 67, 227, and >365 days. One animal remained
alive and well without rejection at a time point of one year
post-transplant. This animal was not sacrificed; however, for the
purposes of this study, monitoring was discontinued. Creatinine
began to rise from baseline approximately one week prior to
sacrifice in those animals who rejected.
[0319] The animals receiving combination therapy with both h1F1 and
h3D1 had significantly prolonged allograft function (p=0.016)
compared to Group I. The prolongation of survival was also markedly
increased in the combination group when compared to both of the
monotherapy groups.
TABLE-US-00014 TABLE 14 Survival and Diagnosis of Rhesus Monkeys
Date of Survival Transplant Group Treatment Recipient (poDay) Dec.
1, 1996 I none X9X 5 Nov. 30, 1996 I none 1FE 7 Nov. 15, 1996 I
none T4T 7 Apr. 2, 1997 I none 95052 8 May 3, 1999 I none AT5H 8
Nov. 2, 1998 II h1F1 AC74 9 Feb. 10, 1999 II h1F1 2WN 8 Feb. 3,
1999 III h3D1 AT5J 8 Feb. 16, 1999 III h3D1 2WF 28 Oct. 26, 1998 IV
h1F1 + h3D1 AC2B >365.sup.a Oct. 28, 1998 IV h1F1 + h3D1 AC8V 47
Mar. 1, 1999 IV h1F1 + h3D1 AT5P 67.sup.b Mar. 2, 1999 IV h1F1 +
h3D1 AT48 227.sup.b .sup.a80 days of post-operative treatment
.sup.b60 days of post-operative treatment The treatment regimen and
outcomes of all animals transplanted are shown. Humanized
antibodies were given at an initial dose of 20 mg/kg followed by 5
mg/kg and then weekly doses of 5 mg/kg for between 60-80 days.
Animal AT48 also received a 5 mg/kg dose immediately post
transplant.
[0320] The teachings of all the references, patents and/or patent
applications cited herein are incorporated herein by reference in
their entirety.
[0321] While this invention has been particularly shown and
described with references to preferred embodiments thereof, it will
be understood by those skilled in the art that various changes in
form and details may be made therein without departing from the
scope of the invention encompassed by the appended claims.
Sequence CWU 1
1
401405DNAMus sp.CDS(1)..(405) 1atg ggt tgg aac tgt atc atc ttc ttt
ctg gtt aca aca gct aca ggt 48Met Gly Trp Asn Cys Ile Ile Phe Phe
Leu Val Thr Thr Ala Thr Gly1 5 10 15gtg cac tcc cag gtc cag ctg cag
cag tct ggg cct gag ctg gtg agg 96Val His Ser Gln Val Gln Leu Gln
Gln Ser Gly Pro Glu Leu Val Arg 20 25 30cct ggg gaa tca gtg aag att
tcc tgc aag ggt tcc ggc tac aca ttc 144Pro Gly Glu Ser Val Lys Ile
Ser Cys Lys Gly Ser Gly Tyr Thr Phe 35 40 45act gat tat gct ata cag
tgg gtg aag cag agt cat gca aag agt cta 192Thr Asp Tyr Ala Ile Gln
Trp Val Lys Gln Ser His Ala Lys Ser Leu 50 55 60gag tgg att gga gtt
att aat att tac tat gat aat aca aac tac aac 240Glu Trp Ile Gly Val
Ile Asn Ile Tyr Tyr Asp Asn Thr Asn Tyr Asn65 70 75 80cag aag ttt
aag ggc aag gcc aca atg act gta gac aaa tcc tcc agc 288Gln Lys Phe
Lys Gly Lys Ala Thr Met Thr Val Asp Lys Ser Ser Ser 85 90 95aca gcc
tat atg gaa ctt gcc aga ttg aca tct gag gat tct gcc atc 336Thr Ala
Tyr Met Glu Leu Ala Arg Leu Thr Ser Glu Asp Ser Ala Ile 100 105
110tat tac tgt gca aga gcg gcc tgg tat atg gac tac tgg ggt caa gga
384Tyr Tyr Cys Ala Arg Ala Ala Trp Tyr Met Asp Tyr Trp Gly Gln Gly
115 120 125acc tca gtc acc gtc tcc tca 405Thr Ser Val Thr Val Ser
Ser 130 1352135PRTMus sp. 2Met Gly Trp Asn Cys Ile Ile Phe Phe Leu
Val Thr Thr Ala Thr Gly1 5 10 15Val His Ser Gln Val Gln Leu Gln Gln
Ser Gly Pro Glu Leu Val Arg 20 25 30Pro Gly Glu Ser Val Lys Ile Ser
Cys Lys Gly Ser Gly Tyr Thr Phe 35 40 45Thr Asp Tyr Ala Ile Gln Trp
Val Lys Gln Ser His Ala Lys Ser Leu 50 55 60Glu Trp Ile Gly Val Ile
Asn Ile Tyr Tyr Asp Asn Thr Asn Tyr Asn65 70 75 80Gln Lys Phe Lys
Gly Lys Ala Thr Met Thr Val Asp Lys Ser Ser Ser 85 90 95Thr Ala Tyr
Met Glu Leu Ala Arg Leu Thr Ser Glu Asp Ser Ala Ile 100 105 110Tyr
Tyr Cys Ala Arg Ala Ala Trp Tyr Met Asp Tyr Trp Gly Gln Gly 115 120
125Thr Ser Val Thr Val Ser Ser 130 1353396DNAMus sp.CDS(1)..(396)
3atg gat tca cag gcc cag gtt ctt ata ttg ctg ctg cta tgg gta tct
48Met Asp Ser Gln Ala Gln Val Leu Ile Leu Leu Leu Leu Trp Val Ser1
5 10 15ggt acc tgt ggg gac att gtg ctg tca cag tct cca tcc tcc ctg
gct 96Gly Thr Cys Gly Asp Ile Val Leu Ser Gln Ser Pro Ser Ser Leu
Ala 20 25 30gtg tca gca gga gag aag gtc act atg agc tgc aaa tcc agt
cag agt 144Val Ser Ala Gly Glu Lys Val Thr Met Ser Cys Lys Ser Ser
Gln Ser 35 40 45ctg ctc aac agt aga acc cga gag aac tac ttg gct tgg
tac cag cag 192Leu Leu Asn Ser Arg Thr Arg Glu Asn Tyr Leu Ala Trp
Tyr Gln Gln 50 55 60aaa cca ggg cag tct cct aaa ctg ctg atc tac tgg
gca tcc act agg 240Lys Pro Gly Gln Ser Pro Lys Leu Leu Ile Tyr Trp
Ala Ser Thr Arg65 70 75 80gaa tct ggg gtc cct gat cgc ttc aca ggc
agt gga tct ggg aca gat 288Glu Ser Gly Val Pro Asp Arg Phe Thr Gly
Ser Gly Ser Gly Thr Asp 85 90 95ttc act ctc acc atc agc agt gtg cag
gct gaa gac ctg gca gtt tat 336Phe Thr Leu Thr Ile Ser Ser Val Gln
Ala Glu Asp Leu Ala Val Tyr 100 105 110tac tgc acg caa tct tat aat
ctt tac acg ttc gga ggg ggg acc aag 384Tyr Cys Thr Gln Ser Tyr Asn
Leu Tyr Thr Phe Gly Gly Gly Thr Lys 115 120 125ctg gaa ata aaa
396Leu Glu Ile Lys 1304132PRTMus sp. 4Met Asp Ser Gln Ala Gln Val
Leu Ile Leu Leu Leu Leu Trp Val Ser1 5 10 15Gly Thr Cys Gly Asp Ile
Val Leu Ser Gln Ser Pro Ser Ser Leu Ala 20 25 30Val Ser Ala Gly Glu
Lys Val Thr Met Ser Cys Lys Ser Ser Gln Ser 35 40 45Leu Leu Asn Ser
Arg Thr Arg Glu Asn Tyr Leu Ala Trp Tyr Gln Gln 50 55 60Lys Pro Gly
Gln Ser Pro Lys Leu Leu Ile Tyr Trp Ala Ser Thr Arg65 70 75 80Glu
Ser Gly Val Pro Asp Arg Phe Thr Gly Ser Gly Ser Gly Thr Asp 85 90
95Phe Thr Leu Thr Ile Ser Ser Val Gln Ala Glu Asp Leu Ala Val Tyr
100 105 110Tyr Cys Thr Gln Ser Tyr Asn Leu Tyr Thr Phe Gly Gly Gly
Thr Lys 115 120 125Leu Glu Ile Lys 1305405DNAArtificial
SequenceDescription of Artificial Sequence Synthetic nucleotide
sequence 5atg ggt tgg aac tgt atc atc ttc ttt ctg gtt acc aca gct
aca ggt 48Met Gly Trp Asn Cys Ile Ile Phe Phe Leu Val Thr Thr Ala
Thr Gly1 5 10 15gtg cac tcc cag gtc cag ctg gtg cag tct ggg gct gag
gtg aag aag 96Val His Ser Gln Val Gln Leu Val Gln Ser Gly Ala Glu
Val Lys Lys 20 25 30cct ggg agc tca gtg aag gtg tcc tgc aaa gct tcc
ggc tac aca ttc 144Pro Gly Ser Ser Val Lys Val Ser Cys Lys Ala Ser
Gly Tyr Thr Phe 35 40 45act gat tat gct ata cag tgg gtg aga cag gct
cct gga cag ggc ctc 192Thr Asp Tyr Ala Ile Gln Trp Val Arg Gln Ala
Pro Gly Gln Gly Leu 50 55 60gag tgg att gga gtt att aat att tac tat
gat aat aca aac tac aac 240Glu Trp Ile Gly Val Ile Asn Ile Tyr Tyr
Asp Asn Thr Asn Tyr Asn65 70 75 80cag aag ttt aag ggc aag gcc aca
atg act gta gac aag tcg acg agc 288Gln Lys Phe Lys Gly Lys Ala Thr
Met Thr Val Asp Lys Ser Thr Ser 85 90 95aca gcc tat atg gaa ctt agt
tct ttg aga tct gag gat acg gcc gtt 336Thr Ala Tyr Met Glu Leu Ser
Ser Leu Arg Ser Glu Asp Thr Ala Val 100 105 110tat tac tgt gca aga
gcg gcc tgg tat atg gac tac tgg ggt caa ggt 384Tyr Tyr Cys Ala Arg
Ala Ala Trp Tyr Met Asp Tyr Trp Gly Gln Gly 115 120 125acc ctt gtc
acc gtc tcc tca 405Thr Leu Val Thr Val Ser Ser 130
1356135PRTArtificial SequenceDescription of Artificial Sequence
Synthetic peptide sequence 6Met Gly Trp Asn Cys Ile Ile Phe Phe Leu
Val Thr Thr Ala Thr Gly1 5 10 15Val His Ser Gln Val Gln Leu Val Gln
Ser Gly Ala Glu Val Lys Lys 20 25 30Pro Gly Ser Ser Val Lys Val Ser
Cys Lys Ala Ser Gly Tyr Thr Phe 35 40 45Thr Asp Tyr Ala Ile Gln Trp
Val Arg Gln Ala Pro Gly Gln Gly Leu 50 55 60Glu Trp Ile Gly Val Ile
Asn Ile Tyr Tyr Asp Asn Thr Asn Tyr Asn65 70 75 80Gln Lys Phe Lys
Gly Lys Ala Thr Met Thr Val Asp Lys Ser Thr Ser 85 90 95Thr Ala Tyr
Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val 100 105 110Tyr
Tyr Cys Ala Arg Ala Ala Trp Tyr Met Asp Tyr Trp Gly Gln Gly 115 120
125Thr Leu Val Thr Val Ser Ser 130 1357396DNAArtificial
SequenceDescription of Artificial Sequence Synthetic nucleotide
sequence 7atg gat tca cag gcc cag gtt ctt ata ttg ctg ctg cta tgg
gta tct 48Met Asp Ser Gln Ala Gln Val Leu Ile Leu Leu Leu Leu Trp
Val Ser1 5 10 15ggc acc tgt ggg gac att gtg ctg aca cag tct cca gat
tcc ctg gct 96Gly Thr Cys Gly Asp Ile Val Leu Thr Gln Ser Pro Asp
Ser Leu Ala 20 25 30gta agc tta gga gag agg gcc act att agc tgc aaa
tcc agt cag agt 144Val Ser Leu Gly Glu Arg Ala Thr Ile Ser Cys Lys
Ser Ser Gln Ser 35 40 45ctg ctc aac agt aga acc cga gag aac tac ttg
gct tgg tac cag cag 192Leu Leu Asn Ser Arg Thr Arg Glu Asn Tyr Leu
Ala Trp Tyr Gln Gln 50 55 60aaa cca ggg cag cct cct aaa ctg ctg atc
tac tgg gca tcc act agg 240Lys Pro Gly Gln Pro Pro Lys Leu Leu Ile
Tyr Trp Ala Ser Thr Arg65 70 75 80gaa tct ggg gtc cct gat cgc ttc
agt ggc agt gga tct ggg aca gat 288Glu Ser Gly Val Pro Asp Arg Phe
Ser Gly Ser Gly Ser Gly Thr Asp 85 90 95ttc act ctc acc atc agc agt
ctg cag gct gaa gac gtg gca gtt tat 336Phe Thr Leu Thr Ile Ser Ser
Leu Gln Ala Glu Asp Val Ala Val Tyr 100 105 110tac tgc acg caa tct
tat aat ctt tac acg ttc gga cag ggg acc aag 384Tyr Cys Thr Gln Ser
Tyr Asn Leu Tyr Thr Phe Gly Gln Gly Thr Lys 115 120 125gtg gaa ata
aaa 396Val Glu Ile Lys 1308132PRTArtificial SequenceDescription of
Artificial Sequence Synthetic peptide sequence 8Met Asp Ser Gln Ala
Gln Val Leu Ile Leu Leu Leu Leu Trp Val Ser1 5 10 15Gly Thr Cys Gly
Asp Ile Val Leu Thr Gln Ser Pro Asp Ser Leu Ala 20 25 30Val Ser Leu
Gly Glu Arg Ala Thr Ile Ser Cys Lys Ser Ser Gln Ser 35 40 45Leu Leu
Asn Ser Arg Thr Arg Glu Asn Tyr Leu Ala Trp Tyr Gln Gln 50 55 60Lys
Pro Gly Gln Pro Pro Lys Leu Leu Ile Tyr Trp Ala Ser Thr Arg65 70 75
80Glu Ser Gly Val Pro Asp Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp
85 90 95Phe Thr Leu Thr Ile Ser Ser Leu Gln Ala Glu Asp Val Ala Val
Tyr 100 105 110Tyr Cys Thr Gln Ser Tyr Asn Leu Tyr Thr Phe Gly Gln
Gly Thr Lys 115 120 125Val Glu Ile Lys 130915DNAArtificial
SequenceDescription of Artificial Sequence Synthetic nucleotide
sequence 9gat tat gct ata cag 15Asp Tyr Ala Ile Gln1
5105PRTArtificial SequenceDescription of Artificial Sequence
Synthetic peptide sequence 10Asp Tyr Ala Ile Gln1 51151DNAMus
sp.CDS(1)..(51) 11gtt att aat att tac tat gat aat aca aac tac aac
cag aag ttt aag 48Val Ile Asn Ile Tyr Tyr Asp Asn Thr Asn Tyr Asn
Gln Lys Phe Lys1 5 10 15ggc 51Gly1217PRTMus sp. 12Val Ile Asn Ile
Tyr Tyr Asp Asn Thr Asn Tyr Asn Gln Lys Phe Lys1 5 10
15Gly1321DNAMus sp.CDS(1)..(21) 13gcg gcc tgg tat atg gac tac 21Ala
Ala Trp Tyr Met Asp Tyr1 5147PRTMus sp. 14Ala Ala Trp Tyr Met Asp
Tyr1 51551DNAMus sp.CDS(1)..(51) 15aaa tcc agt cag agt ctg ctc aac
agt aga acc cga gag aac tac ttg 48Lys Ser Ser Gln Ser Leu Leu Asn
Ser Arg Thr Arg Glu Asn Tyr Leu1 5 10 15gct 51Ala1617PRTMus sp.
16Lys Ser Ser Gln Ser Leu Leu Asn Ser Arg Thr Arg Glu Asn Tyr Leu1
5 10 15Ala1721DNAMus sp.CDS(1)..(21) 17tgg gca tcc act agg gaa tct
21Trp Ala Ser Thr Arg Glu Ser1 5187PRTMus sp. 18Trp Ala Ser Thr Arg
Glu Ser1 51924DNAMus sp.CDS(1)..(24) 19acg caa tct tat aat ctt tac
acg 24Thr Gln Ser Tyr Asn Leu Tyr Thr1 5208PRTMus sp. 20Thr Gln Ser
Tyr Asn Leu Tyr Thr1 521405DNAMus sp.CDS(1)..(405) 21atg aaa tgc
agc tgg gtc atc ttc ttc ctg atg gca gtg gtt aca ggg 48Met Lys Cys
Ser Trp Val Ile Phe Phe Leu Met Ala Val Val Thr Gly1 5 10 15gtc aat
tca gag gtt cac ctg cag cag tct ggg gct gag ctt gtg agg 96Val Asn
Ser Glu Val His Leu Gln Gln Ser Gly Ala Glu Leu Val Arg 20 25 30cca
ggg gcc tta gtc aag ttg tcc tgc aaa cct tct ggc ttc aac att 144Pro
Gly Ala Leu Val Lys Leu Ser Cys Lys Pro Ser Gly Phe Asn Ile 35 40
45aaa gac tac tat atg cac tgg gtg aag cag agg cct gaa cag ggc ctg
192Lys Asp Tyr Tyr Met His Trp Val Lys Gln Arg Pro Glu Gln Gly Leu
50 55 60gag tgg att gga tgg att gat cct gag aat ggt aat act cta tat
gac 240Glu Trp Ile Gly Trp Ile Asp Pro Glu Asn Gly Asn Thr Leu Tyr
Asp65 70 75 80ccg aag ttc cag ggc aag gcc agt ata aca gca gac aca
tcc tcc aac 288Pro Lys Phe Gln Gly Lys Ala Ser Ile Thr Ala Asp Thr
Ser Ser Asn 85 90 95aca gcc tac ctg cag ctc agc agc ctg aca tct gag
gac act gcc gtc 336Thr Ala Tyr Leu Gln Leu Ser Ser Leu Thr Ser Glu
Asp Thr Ala Val 100 105 110tat tac tgt gct aga gag ggg ctt ttt ttt
gct tac tgg ggc caa ggg 384Tyr Tyr Cys Ala Arg Glu Gly Leu Phe Phe
Ala Tyr Trp Gly Gln Gly 115 120 125act ccg gtc act gtc tct gca
405Thr Pro Val Thr Val Ser Ala 130 13522135PRTMus sp. 22Met Lys Cys
Ser Trp Val Ile Phe Phe Leu Met Ala Val Val Thr Gly1 5 10 15Val Asn
Ser Glu Val His Leu Gln Gln Ser Gly Ala Glu Leu Val Arg 20 25 30Pro
Gly Ala Leu Val Lys Leu Ser Cys Lys Pro Ser Gly Phe Asn Ile 35 40
45Lys Asp Tyr Tyr Met His Trp Val Lys Gln Arg Pro Glu Gln Gly Leu
50 55 60Glu Trp Ile Gly Trp Ile Asp Pro Glu Asn Gly Asn Thr Leu Tyr
Asp65 70 75 80Pro Lys Phe Gln Gly Lys Ala Ser Ile Thr Ala Asp Thr
Ser Ser Asn 85 90 95Thr Ala Tyr Leu Gln Leu Ser Ser Leu Thr Ser Glu
Asp Thr Ala Val 100 105 110Tyr Tyr Cys Ala Arg Glu Gly Leu Phe Phe
Ala Tyr Trp Gly Gln Gly 115 120 125Thr Pro Val Thr Val Ser Ala 130
13523390DNAMus sp.CDS(1)..(390) 23atg gat ttt cat gtg cag att ttc
agc ttc atg cta atc agt gtc aca 48Met Asp Phe His Val Gln Ile Phe
Ser Phe Met Leu Ile Ser Val Thr1 5 10 15gtc ata ttg tcc agt gga gaa
att gtg ctc acc cag tct cca gca ctc 96Val Ile Leu Ser Ser Gly Glu
Ile Val Leu Thr Gln Ser Pro Ala Leu 20 25 30atg gct gca tct cca ggg
gag aag gtc acc atc acc tgc agt gtc agc 144Met Ala Ala Ser Pro Gly
Glu Lys Val Thr Ile Thr Cys Ser Val Ser 35 40 45tca agt ata agt tcc
agc aac ttg cac tgg tac cag cag aag tca gaa 192Ser Ser Ile Ser Ser
Ser Asn Leu His Trp Tyr Gln Gln Lys Ser Glu 50 55 60acc tcc ccc aaa
ccc tgg att tat ggc aca tcc aac ctg gct tct gga 240Thr Ser Pro Lys
Pro Trp Ile Tyr Gly Thr Ser Asn Leu Ala Ser Gly65 70 75 80gtc cct
gtt cgc ttc agt ggc agt gga tct ggg acc tct tat tct ctc 288Val Pro
Val Arg Phe Ser Gly Ser Gly Ser Gly Thr Ser Tyr Ser Leu 85 90 95aca
atc agc agc atg gag gct gaa gat gct gcc act tat tac tgt caa 336Thr
Ile Ser Ser Met Glu Ala Glu Asp Ala Ala Thr Tyr Tyr Cys Gln 100 105
110cag tgg agt agt tac cca ctc acg ttc ggt gct ggg acc aag ctg gag
384Gln Trp Ser Ser Tyr Pro Leu Thr Phe Gly Ala Gly Thr Lys Leu Glu
115 120 125ctg aaa 390Leu Lys 13024130PRTMus sp. 24Met Asp Phe His
Val Gln Ile Phe Ser Phe Met Leu Ile Ser Val Thr1 5 10 15Val Ile Leu
Ser Ser Gly Glu Ile Val Leu Thr Gln Ser Pro Ala Leu 20 25 30Met Ala
Ala Ser Pro Gly Glu Lys Val Thr Ile Thr Cys Ser Val Ser 35 40 45Ser
Ser Ile Ser Ser Ser Asn Leu His Trp Tyr Gln Gln Lys Ser Glu 50 55
60Thr Ser Pro Lys Pro Trp Ile Tyr Gly Thr Ser Asn Leu Ala Ser Gly65
70 75 80Val Pro Val Arg Phe Ser Gly Ser Gly Ser Gly Thr Ser Tyr Ser
Leu 85 90 95Thr Ile Ser Ser Met Glu Ala Glu Asp Ala Ala Thr Tyr Tyr
Cys Gln 100 105 110Gln Trp Ser Ser Tyr Pro Leu Thr Phe Gly Ala Gly
Thr Lys Leu Glu 115 120 125Leu Lys 13025405DNAArtificial
SequenceDescription of Artificial Sequence Synthetic nucleotide
sequence 25atg aaa tgc agc tgg gtc atc ttc ttc ctg atg gca gtg gtt
aca ggg 48Met Lys Cys Ser Trp Val Ile Phe Phe Leu Met Ala Val Val
Thr Gly1 5 10 15gtc aat tca gag gtt cag ctg gtg cag tct ggg gct gag
gtt aag aag 96Val Asn Ser
Glu Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys 20 25 30cca ggg
gcc tca gtc aag gtg tcc tgc aaa cct tct ggc ttc aac att 144Pro Gly
Ala Ser Val Lys Val Ser Cys Lys Pro Ser Gly Phe Asn Ile 35 40 45aaa
gac tac tat atg cac tgg gtg agg cag gcg cct gga cag ggc ctc 192Lys
Asp Tyr Tyr Met His Trp Val Arg Gln Ala Pro Gly Gln Gly Leu 50 55
60gag tgg att gga tgg att gat cct gag aat ggt aat act cta tat gac
240Glu Trp Ile Gly Trp Ile Asp Pro Glu Asn Gly Asn Thr Leu Tyr
Asp65 70 75 80ccg aag ttc cag ggc aag gcc act ata act gca gac aca
tcc acc agc 288Pro Lys Phe Gln Gly Lys Ala Thr Ile Thr Ala Asp Thr
Ser Thr Ser 85 90 95aca gcc tac atg gag ctg agc agc ctg aga tct gag
gac act gcc gtc 336Thr Ala Tyr Met Glu Leu Ser Ser Leu Arg Ser Glu
Asp Thr Ala Val 100 105 110tat tac tgt gct aga gag ggg ctt ttt ttt
gct tac tgg ggc caa ggt 384Tyr Tyr Cys Ala Arg Glu Gly Leu Phe Phe
Ala Tyr Trp Gly Gln Gly 115 120 125acc ctg gtc act gtc tct tca
405Thr Leu Val Thr Val Ser Ser 130 13526135PRTArtificial
SequenceDescription of Artificial Sequence Synthetic peptide
sequence 26Met Lys Cys Ser Trp Val Ile Phe Phe Leu Met Ala Val Val
Thr Gly1 5 10 15Val Asn Ser Glu Val Gln Leu Val Gln Ser Gly Ala Glu
Val Lys Lys 20 25 30Pro Gly Ala Ser Val Lys Val Ser Cys Lys Pro Ser
Gly Phe Asn Ile 35 40 45Lys Asp Tyr Tyr Met His Trp Val Arg Gln Ala
Pro Gly Gln Gly Leu 50 55 60Glu Trp Ile Gly Trp Ile Asp Pro Glu Asn
Gly Asn Thr Leu Tyr Asp65 70 75 80Pro Lys Phe Gln Gly Lys Ala Thr
Ile Thr Ala Asp Thr Ser Thr Ser 85 90 95Thr Ala Tyr Met Glu Leu Ser
Ser Leu Arg Ser Glu Asp Thr Ala Val 100 105 110Tyr Tyr Cys Ala Arg
Glu Gly Leu Phe Phe Ala Tyr Trp Gly Gln Gly 115 120 125Thr Leu Val
Thr Val Ser Ser 130 13527390DNAArtificial SequenceDescription of
Artificial Sequence Synthetic nucleotide sequence 27atg gat ttt cat
gtg cag att ttc agc ttc atg cta atc agt gtc aca 48Met Asp Phe His
Val Gln Ile Phe Ser Phe Met Leu Ile Ser Val Thr1 5 10 15gtc ata ttg
tcc agt gga gat att cag atg acc cag tct cca tca tcc 96Val Ile Leu
Ser Ser Gly Asp Ile Gln Met Thr Gln Ser Pro Ser Ser 20 25 30ctg tct
gca tct gta ggg gat agg gtc acc atc acc tgc agt gtc agc 144Leu Ser
Ala Ser Val Gly Asp Arg Val Thr Ile Thr Cys Ser Val Ser 35 40 45tca
agt ata agt tcc agc aac ttg cac tgg tac cag cag aag cca ggc 192Ser
Ser Ile Ser Ser Ser Asn Leu His Trp Tyr Gln Gln Lys Pro Gly 50 55
60aag gcc ccc aaa ccc ttg att tat ggc aca tcc aac ctg gct tct gga
240Lys Ala Pro Lys Pro Leu Ile Tyr Gly Thr Ser Asn Leu Ala Ser
Gly65 70 75 80gtc cct agt cgc ttc agt ggc agt gga tct ggg acc gat
tat act ctc 288Val Pro Ser Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp
Tyr Thr Leu 85 90 95aca atc agc agc ttg cag cct gaa gat gtt gcc act
tat tac tgt caa 336Thr Ile Ser Ser Leu Gln Pro Glu Asp Val Ala Thr
Tyr Tyr Cys Gln 100 105 110cag tgg agt agt tac cca ctc acg ttc ggt
caa ggg acc aag gtg gag 384Gln Trp Ser Ser Tyr Pro Leu Thr Phe Gly
Gln Gly Thr Lys Val Glu 115 120 125atc aaa 390Ile Lys
13028130PRTArtificial SequenceDescription of Artificial Sequence
Synthetic peptide sequence 28Met Asp Phe His Val Gln Ile Phe Ser
Phe Met Leu Ile Ser Val Thr1 5 10 15Val Ile Leu Ser Ser Gly Asp Ile
Gln Met Thr Gln Ser Pro Ser Ser 20 25 30Leu Ser Ala Ser Val Gly Asp
Arg Val Thr Ile Thr Cys Ser Val Ser 35 40 45Ser Ser Ile Ser Ser Ser
Asn Leu His Trp Tyr Gln Gln Lys Pro Gly 50 55 60Lys Ala Pro Lys Pro
Leu Ile Tyr Gly Thr Ser Asn Leu Ala Ser Gly65 70 75 80Val Pro Ser
Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Tyr Thr Leu 85 90 95Thr Ile
Ser Ser Leu Gln Pro Glu Asp Val Ala Thr Tyr Tyr Cys Gln 100 105
110Gln Trp Ser Ser Tyr Pro Leu Thr Phe Gly Gln Gly Thr Lys Val Glu
115 120 125Ile Lys 1302936DNAMus sp.CDS(1)..(36) 29agt gtc agc tca
agt ata agt tcc agc aac ttg cac 36Ser Val Ser Ser Ser Ile Ser Ser
Ser Asn Leu His1 5 103012PRTMus sp. 30Ser Val Ser Ser Ser Ile Ser
Ser Ser Asn Leu His1 5 103121DNAMus sp.CDS(1)..(21) 31ggc aca tcc
aac ctg gct tct 21Gly Thr Ser Asn Leu Ala Ser1 5327PRTMus sp. 32Gly
Thr Ser Asn Leu Ala Ser1 53327DNAMus sp.CDS(1)..(27) 33caa cag tgg
agt agt tac cca ctc acg 27Gln Gln Trp Ser Ser Tyr Pro Leu Thr1
5349PRTMus sp. 34Gln Gln Trp Ser Ser Tyr Pro Leu Thr1 53515DNAMus
sp.CDS(1)..(15) 35gac tac tat atg cac 15Asp Tyr Tyr Met His1
5365PRTMus sp. 36Asp Tyr Tyr Met His1 53751DNAMus sp.CDS(1)..(51)
37tgg att gat cct gag aat ggt aat act cta tat gac ccg aag ttc cag
48Trp Ile Asp Pro Glu Asn Gly Asn Thr Leu Tyr Asp Pro Lys Phe Gln1
5 10 15ggc 51Gly3817PRTMus sp. 38Trp Ile Asp Pro Glu Asn Gly Asn
Thr Leu Tyr Asp Pro Lys Phe Gln1 5 10 15Gly3921DNAMus
sp.CDS(1)..(21) 39gag ggg ctt ttt ttt gct tac 21Glu Gly Leu Phe Phe
Ala Tyr1 5407PRTMus sp. 40Glu Gly Leu Phe Phe Ala Tyr1 5
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