U.S. patent application number 15/515461 was filed with the patent office on 2017-08-24 for methods of treating systemic lupus erythematosus using a domain antibody directed against cd28.
The applicant listed for this patent is BRISTOL-MYERS SQUIBB COMPANY. Invention is credited to Dominique Duchesne, Marek Honczarenko, Xiaoni Liu, Steven G. Nadler, Diane E. Shevell, Rong Shi, Suzamme J. Suchard, John P. Throup, Xavier Valencia, Jenny H. Xie.
Application Number | 20170240636 15/515461 |
Document ID | / |
Family ID | 54291723 |
Filed Date | 2017-08-24 |
United States Patent
Application |
20170240636 |
Kind Code |
A1 |
Valencia; Xavier ; et
al. |
August 24, 2017 |
METHODS OF TREATING SYSTEMIC LUPUS ERYTHEMATOSUS USING A DOMAIN
ANTIBODY DIRECTED AGAINST CD28
Abstract
Methods of treating autoimmune diseases, such as systemic lupus
erythematosus using domain antibodies that specifically bind human
CD28 are provided. The methods may comprise at least one
administration cycle comprising one dose of the domain antibody.
The method reduces symptoms of systemic lupus erythematosus
compared to placebo.
Inventors: |
Valencia; Xavier;
(Princeton, NJ) ; Throup; John P.; (New Hope,
PA) ; Nadler; Steven G.; (Princeton, NJ) ;
Suchard; Suzamme J.; (Wilmington, DE) ; Duchesne;
Dominique; (Yardley, PA) ; Liu; Xiaoni;
(Yardley, PA) ; Shi; Rong; (Princeton, NJ)
; Shevell; Diane E.; (Westfield, NJ) ; Xie; Jenny
H.; (Princeton, NJ) ; Honczarenko; Marek;
(Princeton, NJ) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
BRISTOL-MYERS SQUIBB COMPANY |
Princeton |
NJ |
US |
|
|
Family ID: |
54291723 |
Appl. No.: |
15/515461 |
Filed: |
September 30, 2015 |
PCT Filed: |
September 30, 2015 |
PCT NO: |
PCT/US2015/053233 |
371 Date: |
March 29, 2017 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
|
|
62057981 |
Sep 30, 2014 |
|
|
|
Current U.S.
Class: |
1/1 |
Current CPC
Class: |
A61P 37/02 20180101;
C07K 2317/569 20130101; C07K 2317/24 20130101; A61K 39/3955
20130101; A61K 2039/54 20130101; C07K 2319/31 20130101; A61K
2039/505 20130101; A61P 37/06 20180101; A61K 2039/545 20130101;
C07K 2317/76 20130101; C07K 2317/94 20130101; A61K 45/06 20130101;
A61K 47/60 20170801; C07K 2317/565 20130101; C07K 16/2818 20130101;
C07K 2317/92 20130101 |
International
Class: |
C07K 16/28 20060101
C07K016/28; A61K 45/06 20060101 A61K045/06; A61K 39/395 20060101
A61K039/395 |
Claims
1. A method of treating an immune disease in a patient, comprising
administering to the patient a therapeutically effective amount of
an anti-CD28 domain antibody which comprises a variable domain,
wherein the variable domain comprises the amino acid sequence of
SEQ ID NO: 12 (1h-239-891(D70C)) or differs from SEQ ID NO: 12 by
up to 5 amino acids, wherein at least one dose of the anti-CD28
domain antibody is administered at a dose from about 1.25 mg to
about 12.5 mg.
2. The method of claim 1, wherein the variable domain of the
anti-CD28 domain antibody comprises: (1) a CDR1 region having the
amino acid sequence of SEQ ID NO: 1; (2) a CDR2 region having the
amino acid sequence of SEQ ID NO: 2; and (3) a CDR3 region having
the amino acid sequence of SEQ ID NO: 3.
3. The method of claim 1, wherein the anti-CD28 domain antibody
comprises the amino acid sequence of SEQ ID NO: 12.
4. The method of claim 1, wherein the anti-CD28 domain antibody
comprises a 40 kDa branched polyethylene glycol.
5. The method of claim 1, wherein the anti-CD28 domain antibody is
BMS-931699.
6. The method of claim 1, wherein the immune disease is systemic
lupus erythematosus (SLE).
7. The method of claim 1, wherein the dose is selected from about
1.25 mg, about 5 mg, and about 12.5 mg.
8. The method of claim 1, wherein the dose is at least 1.25 mg.
9. The method of claim 1, wherein the dose is at least 5 mg.
10. The method of claim 7, wherein the dose is about 12.5 mg.
11. The method of claim 1, wherein the dose is administered every
week.
12. The method of claim 1, wherein the dose is administered every
two weeks.
13. The method of claim 1, wherein at least 2 doses are
administered.
14. The method of claim 13, wherein the at least 2 doses are the
same or different.
15. The method of claims 13, wherein at least 12 doses are
administered.
16. The method of claims 13, wherein at least 24 doses are
administered.
17. The method of claim 1, wherein the anti-CD28 domain antibody is
administered subcutaneously.
18. The method of claim 1, further comprises administering to the
patient an immunosuppressive/immunomodulatory and/or
anti-inflammatory agent.
19. The method of claim 18, wherein the
immunosuppressive/immunomodulatory and/or anti-inflammatory agent
is administered before or after the anti-CD28 domain antibody.
20. The method of claims 18, wherein the
immunosuppressive/immunomodulatory and/or anti-inflammatory agent
is administered concurrently with the anti-CD28 domain antibody.
Description
CROSS-REFERENCE TO RELATED APPLICATION
[0001] This application claims priority to U.S. Provisional
Application Ser. No. 62/057,981, filed Sep. 30, 2014, the entire
content of which is incorporated herein by reference.
FIELD OF THE INVENTION
[0002] Methods of treating autoimmune diseases, such as systemic
lupus erythematosus using domain antibodies that specifically bind
human CD28 are provided
SEQUENCE LISTING
[0003] Reference to a Sequence Listing is submitted electronically
herewith and is incorporated herein by reference.
BACKGROUND
[0004] Systemic lupus erythematosus (SLE) is a chronic progressive
autoimmune disease characterized by pleiotropic organ/tissue
involvement and clinical manifestations. The progression of the
disease is punctuated by flares, which by definition require
therapy modification. Uncontrolled SLE will lead to end organ
damage with increased morbidity and mortality. The clinical
manifestations are variegated and often in a single patient many
organs and tissue are involved. Most commonly targeted tissues are
skin (with the typical malar or butterfly rush), joints and kidney,
but practically any organ and tissue can be targeted.
[0005] Autoantibodies, such as anti-double-stranded
deoxyribonucleic acid (anti-dsDNA) or anti-nuclear antibody (ANA),
and have helped to define the autoimmune nature of SLE. These
autoantibodies, however, are not pathognomonic. SLE, with its
pleiotropic clinical manifestations and lack of specific
autoantibodies is the archetype of the non-organ specific
autoimmune diseases. American College of Rheumatology (ACR) has
developed an 11 factor set of guidelines to diagnose SLE. This
intrinsic complexity for diagnosis and monitoring disease
progression has hampered the validation of new treatments.
[0006] The full pathogenic cascade leading to SLE, with all its
clinical facets, is complex and not yet fully defined. Despite
this, it is now well accepted that T cells have a pivotal role in
SLE. SLE is characterized by hyper-responsive T cells, excessive
autoantibody production, and antigen presenting cell (APC)
hyperactivation. The autoantibodies (in particular, anti-nuclear
antibodies, and anti-dsDNA antibodies) in SLE patients are
dependent on T-cell help that is provided by co-stimulatory
molecules and cytokines. In addition to providing B-cell help, T
cells can directly infiltrate the joints, skin, kidney, and brain
causing damage directly through cytotoxicity or indirectly through
the recruitment and activation of macrophages and neutrophils.
[0007] Lymphocytes from patients with SLE show signs of increased
activation; e.g., the percentage of CD4+ T cells expressing CD25
increase as does the expression of CD86 on CD19+ B cells. The
increased CD86 expression is thought to render (autoreactive) B
cells more susceptible to T-cell help and thus facilitate
autoantibody production. Consistent with this observation, the
number of activated B cells and levels of anti-dsDNA antibodies
increase with diseased activity. In addition, peripheral blood
dendritic cells (DCs) and DCs derived from peripheral blood
monocytes of SLE patients show increased expression of CD86 and the
ratio of CD86/CD80 is higher in SLE patients compared with healthy
donors. Unlike CD86, CD28 expression on CD4+ and CD8+ T cells in
lupus patients appears to be more variable. Regardless of the
levels of CD28 expression, T cells from SLE patients appear to be
more responsive to anti-CD28-mediated activation and patients with
active disease have increased gene expression of CD28 when compared
to normal controls. CTLA4 (a co-inhibitory molecule) is also
increased in T cells from SLE patients but this does not seem to
control aberrant T-cell activation. Taken together, these data
suggest that the CD28-CD86/CD80 pathway plays a central role in the
defective immune response observed in SLE patients.
[0008] Currently SLE patients are treated, depending on the
severity of the disease, with antimalarials, CS, such as oral
predinisone and immunosuppressive drugs such as MTX, AZA,
mycophenolate mofetil, and cyclophosphamide. Although
corticosteroids and immunosuppressive drugs are generally effective
in temporarily controlling flares and disease progression, their
reduced efficacy and serious adverse effects significantly limit
their prolonged use. This has led to the off-label use of many
medicines to treat Lupus patients. The paucity of satisfactory
therapeutic options is stressed by the approval of only one new
medicine (Belimumab) for SLE in the last fifty years. Despite
Belimumab approval SLE patients still have a very high unmet
medical need and novel therapies are needed to satisfactorily treat
SLE.
[0009] Inhibition of CD28 mediated T cell activation could inhibit
undesired T cell responses occurring during autoimmunity,
transplant rejection, or allergic responses. For example,
inhibiting CD28 mediated T cell activation could delay graft
rejection, prevent acute allograft rejection, induce donor specific
tolerance, and prevent development and interrupt the progression of
chronic allograft rejection, as well as prevent graft versus host
disease (GVH), i.e., when transplanted T cells mount a vigorous
immune response against host tissue alloantigens (Salama et al.
(2001) J. Clin. Invest. 108: 943-48). Not only would inhibiting
CD28 mediated T cell activation dampen the immune response through
negating activation signaling through CD28, it should not impact
the interaction of CD86 and CD80 to CTLA4, thereby preserving CTLA4
mediated inhibition of the T cell response. Thus, inhibiting CD28
mediated T cell activation could be used to prevent induction of
autoimmunity and moderate the progression and/or severity of lupus
as well as other autoimmune diseases. (Saloman et al. (2001) Ann.
Rev. Immunol. 19: 225-252).
[0010] Accordingly, it is an object of this invention to provide
improved methods for treating subjects with SLE without stimulation
of CD28 signaling pathways.
SUMMARY
[0011] In certain embodiments, the present invention provides a
method of treating an immune disease in a patient, comprising
administering to the patient a therapeutically effective amount of
an anti-CD28 domain antibody which comprises a variable domain,
wherein the variable domain comprises the amino acid sequence of
SEQ ID NO: 12 (1h-239-891(D70C)) or differs from SEQ ID NO: 12 by
up to 5 amino acids, wherein at least one dose of the anti-CD28
domain antibody is administered at a dose from about 1.25 mg to
about 12.5 mg. In a specific embodiments, the immune disease is
systemic lupus erythematosus (SLE). Preferably, the patient is a
human patient.
[0012] In certain aspects, the anti-CD28 domain antibody is
administered at a dose selected from about 1.25 mg, about 5 mg, and
about 12.5 mg. For example, the dose is at least 1.25 mg or at
least 5 mg. Optionally, the dose is about 1.25 mg, about 5 mg, or
about 12.5 mg. Optionally, the dose is administered every week or
every two weeks. Optionally, at least 2 doses are administered,
wherein the at least 2 doses are the same or different. For
example, at least 12 doses are administered. For example, at least
24 doses are administered.
[0013] In certain aspects, the variable domain of the anti-CD28
domain antibody comprises: (1) a CDR1 region having the amino acid
sequence of SEQ ID NO: 1; (2) a CDR2 region having the amino acid
sequence of SEQ ID NO: 2; and (3) a CDR3 region having the amino
acid sequence of SEQ ID NO: 3. To illustrate, the anti-CD28 domain
antibody comprises the amino acid sequence of SEQ ID NO: 12.
Optionally, the anti-CD28 domain antibody comprises a 40 kDa
branched polyethylene glycol. In certain specific embodiments, the
anti-CD28 domain antibody is BMS-931699.
[0014] In certain aspects, the anti-CD28 domain antibody is
administered subcutaneously. For example, the anti-CD28 domain
antibody is formulated in a pharmaceutical composition for
subcutaneous administration. Alternatively, the anti-CD28 domain
antibody is administered intraveneously. For example, the anti-CD28
domain antibody is formulated in a pharmaceutical composition for
intraveneous administration.
[0015] In certain aspects, the method of the invention further
comprises administering to the patient an
immunosuppressive/immunomodulatory and/or anti-inflammatory agent.
Optionally, the immunosuppressive/immunomodulatory and/or
anti-inflammatory agent is administered before the anti-CD28 domain
antibody. Optionally, the immunosuppressive/immunomodulatory and/or
anti-inflammatory agent is administered after the anti-CD28 domain
antibody. Optionally, the immunosuppressive/immunomodulatory and/or
anti-inflammatory agent is administered concurrently with the
anti-CD28 domain antibody.
[0016] Included is method of antagonizing CD28, comprising
administering an effective amount of an anti-CD28 dAb disclosed
herein to an individual. Also included is a method of antagonizing
the binding of CD28 comprising administering an effective amount of
the anti-CD28 dAb disclosed herein to an individual, wherein the
anti-CD28 dAb antagonizes the binding of CD28 to CD80 and/or CD86
in the individual.
[0017] Further included is a method of treating, alleviating, or
preventing a symptom of an immune disease, such as an autoimmune
disease or a graft-related disease, comprising administering an
effective amount of an anti-CD28 dAb disclosed herein to an
individual having or at risk of having an immune disease. Included
is a method of treating, alleviating, or preventing an immune
disease, comprising administering an effective amount of an
anti-CD28 dAb disclosed herein to an individual having or at risk
of having an immune disease.
[0018] Included is the use of an anti-CD28 dAb disclosed herein for
preparing a medicament for treating or preventing an immune disease
in a patient in need thereof. Also included is the use of an
anti-CD28 dAb disclosed herein for preparing a medicament for
treating or preventing a symptom of an immune disease in a patient
in need thereof. Further included herein is the use of an anti-CD28
dAb disclosed herein for preparing a medicament for alleviating at
least one symptom of an immune disease in a patient in need
thereof.
[0019] Further provided is an anti-CD28 domain antibody formulated
in a pharmaceutical composition for subcutaneous administration.
The pharmaceutical composition may further comprise a
pharmaceutically acceptable carrier. The domain antibody may be
administered subcutaneously.
[0020] A kit for treating an immune disease in a patient is also
provided, the kit comprising: (a) a dose of domain antibody
comprising an anti-CD28 domain antibody, and (b) instructions for
using the domain antibody in the disclosed methods.
BRIEF DESCRIPTION OF THE DRAWINGS
[0021] FIG. 1 shows Part 1 of an FDA Phase 2 parallel-arm,
randomized, double-blinded, multicenter, international study,
adaptive design schematic for BMS-931699.
[0022] FIG. 2 shows Part 2 of an FDA Phase 2 parallel-arm,
randomized, double-blinded, multicenter, international study,
adaptive design schematic for BMS-931699.
[0023] FIG. 3 shows the long term extension (LTE) design
schematic.
[0024] FIG. 4 demonstrates the expected receptor occupancy for
BMS-931699 at steady state for 12.5 mg Weekly (QW), 12.5 mg Every 2
Weeks (QOW), 5 mg Every 2 Weeks (QOW) and 1.25 mg Every 2 Weeks
(QOW).
DETAILED DESCRIPTION
[0025] The present disclosure provides anti-CD28 domain antibodies
to antagonize CD28 activity and methods of treating immune
diseases, such as lupus, using said domain antibodies. The domain
antibodies may be linked to polymers to improve pharmacokinietic
properties, such as stability and half-life. Included herein are
compositions and methods for the attachment of polymer molecules
(e.g., polyethylene glycol; PEG) to proteins to modulate the
pharmacokinetic properties of the modified proteins. For example,
PEG modification of proteins has been shown to alter the in vivo
circulating half-life, antigenicity, solubility, and resistance to
proteolysis of the protein (Abuchowski et al. (1977) J. Biol.
Chem., 252: 3578; Nucci et al. (1991) Adv. Drug Delivery Reviews 6:
133; Francis et al., Pharmaceutical Biotechnology Vol. 3
(Borchardt, R. T. ed.); and Stability of Protein Pharmaceuticals:
in vivo Pathways of Degradation and Strategies for Protein
Stabilization 1991 pp235-263, Plenum, NY).
[0026] The disclosed domain antibodies, including BMS-931699
(otherwise known as lulizumab or 1h-239-891(D70C) formatted with a
40 kDa branched polyethylene glycol), monovalently bind CD28, and
inhibit the interaction of CD80 and CD86 with CD28, the key
co-stimulatory receptor of T lymphocytes. Ultimately, targeting
CD28 with the domain antibodies can provide opportunity to inhibit
autoimmune processes leading to systemic lupus erythematosus among
other graft-related or autoimmune diseases. Such monovalent domain
antibodies can also avoid potential undesirable effects that can
occur with antibodies capable of divalent or multivalent binding of
CD28. Domain antibodies described herein also do not block the
interaction of CD80 and CD86 to CTLA4. The domain antibodies
described herein do not cross-react with CTLA4, and thus do not
bind the common motif on CTLA4 and CD28 that binds CD80/86. The
domain antibodies can thus provide improved therapeutic benefits
and reduced side-effects for autoimmune disease patients. The
domain antibody thus offers a novel therapeutic modality, currently
not available to patients. Given the lack of therapeutic options
for systemic lupus erythematosus and autoimmune disease subjects
who have failed all conventional therapies, the domain antibody
with its distinct mechanism of action and known safety profile
demonstrates a favorable Risk/Benefit profile.
1. Definitions
[0027] In accordance with this detailed description, the following
abbreviations and definitions apply. It must be noted that as used
herein, the singular forms "a", "an", and "the" include plural
referents unless the context clearly dictates otherwise. Thus, for
example, reference to "an antibody" includes a plurality of such
antibodies and reference to "the dosage" includes reference to one
or more dosages and equivalents thereof known to those skilled in
the art, and so forth.
[0028] Unless defined otherwise, all technical and scientific terms
used herein have the same meaning as commonly understood by one of
ordinary skill in the art. Unless otherwise stated, all ranges
described herein are inclusive of the specific endpoints. The
following terms are provided below.
[0029] As used herein, a "fixed dose" is a dose administered
regardless of the subjects' body weight.
[0030] As used herein, the term "human" when applied to a domain
antibody or to an immunoglobulin variable domain means that the
polypeptide has a sequence derived from a human immunoglobulin. A
sequence is "derived from" a human immunoglobulin coding sequence
when the sequence is either: a) isolated from a human individual or
from cells or a cell line from a human individual; b) isolated from
a library of cloned human antibody gene sequences (or a library of
human antibody V domain sequences); or c) when a cloned human
antibody gene sequence (or a cloned human V region sequence
(including, e.g., a germline V gene segment)) was used to generate
one or more diversified sequences that were then selected for
binding to a desired target antigen.
[0031] At a minimum, a human domain antibody has at least 70%
identical, at least 75% identical, at least 80% identical, at least
85% amino acid identity (including, for example, 87%, 90%, 93%,
95%, 97%, 99%, or higher identity) to a naturally-occurring human
immunoglobulin variable domain sequence, e.g., a
naturally-occurring human immunoglobulin variable domain sequence
disclosed in Kabat ("Sequences of Proteins of Immunological
Interest", US Department of Health and Human Services 1991).
[0032] As used herein, the term "domain" refers to a folded protein
structure which retains its tertiary structure independently of the
rest of the protein. Generally, domains are responsible for
discrete functional properties of proteins, and in many cases may
be added, removed, or transferred to other proteins without loss of
function of the remainder of the protein and/or of the domain.
[0033] By "domain antibody" is meant a folded polypeptide domain
which comprises a sequence characteristic of immunoglobulin
variable domains and which specifically binds an antigen (e.g.,
dissociation constant of 500 nM or less). A "domain antibody"
therefore includes complete antibody variable domains as well as
modified variable domains, for example in which one or more loops
have been replaced by sequences which are not characteristic of
antibody variable domains, or antibody variable domains which have
been truncated or comprise N- or C-terminal extensions, as well as
folded fragments of variable domains which retain a dissociation
constant of 500 nM or less (e.g., 450 nM or less, 400 nM or less,
350 nM or less, 300 nM or less, 250 nM or less, 200 nM or less, 150
nM or less, 100 nM or less) and the target antigen specificity of
the full-length domain. Where necessary or in case of any doubt,
the numbering convention and boundaries set forth by Kabat et al.
(Kabat et al. (1991) Sequences of Immunological Interest, 5.sup.th
ed. U.S. Dept. Health & Human Services, Washington, D.C.) are
applicable to immunoglobulin variable and constant domains referred
to herein.
[0034] A "dAb" is used interchangeably with "domain antibody"
herein. A "domain antibody" or "dAb" used in the present invention
refers to an "anti-CD28 domain antibody".
[0035] As used herein, the phrase "sequence characteristic of
immunoglobulin variable domains" refers to an amino acid sequence
that is identical, over 20 or more, 25 or more, 30 or more, 35 or
more, 40 or more, 45 or more, or even 50 or more contiguous amino
acids, to a sequence comprised by an immunoglobulin variable domain
sequence. Sequences similar or identical (e.g., at least about 70%
sequence identity) to the sequences disclosed herein are also
included herein.
[0036] As used herein, the term "identity" refer to the degree with
which two nucleotide or amino acid sequences structurally resemble
each other. As used herein, sequence "similarity" is a measure of
the degree to which amino acid sequences share similar amino acid
residues at corresponding positions in an alignment of the
sequences. Amino acids are similar to each other where their side
chains are similar. Specifically, "similarity" encompasses amino
acids that are conservative substitutes for each other. A
"conservative" substitution is any substitution that has a positive
score in the blosum62 substitution matrix (Hentikoff and Hentikoff
(1992) Proc. Natl. Acad. Sci. USA 89: 10915-10919). By the
statement "sequence A is n % similar to sequence B" is meant that n
% of the positions of an optimal global alignment between sequences
A and B consists of identical amino acids or conservative
substitutions. Typical conservative substitutions are exchanges
among Met, Val, Leu, and Ile; among Ser and Thr; among the residues
Asp, Glu, and Asn; among the residues Gln, Lys, and Arg; or
aromatic residues Phe and Tyr.
[0037] As used herein, the term "epitope" refers to a unit of
structure conventionally bound by an immunoglobulin V.sub.H/V.sub.L
pair. Epitopes define the minimum binding site for an antibody, and
thus represent the target of specificity of an antibody. In the
case of a domain antibody, an epitope represents the unit of
structure bound by a domain antibody in isolation. That is, the
binding site is provided by one, single immunoglobulin variable
domain. Epitopes can be linear or conformational, and can be as
small as three amino acids.
[0038] As used herein, "CD28 activity" is an activity involving or
resulting from the binding of CD80, CD86 and/or another ligand to
CD28, and includes, but is not limited to, activation of
CD28-mediated cell signaling. CD28 activity also includes the
induction of T cell proliferation and the induction of cytokine
secretion, e.g., interleukin 2 (IL-2), by T cells.
[0039] As used herein, the term "does not substantially agonize"
means that a given agent, e.g., a domain antibody, does not
substantially activate one or more of the CD28 activities as the
term "activate" is defined herein. Specifically, an agent that
"does not substantially agonize" means that the agent does not
activate more than 20% of the activity which is activated by CD80
and/or CD86 binding to CD28, and in an aspect, the agent does not
activate more than about 10%, 8%, 5%, 3%, or 2% or less, including
zero activation, of the activity which is activated by CD80 and/or
CD86 binding to CD28. By way of a non-limiting example, a domain
antibody set forth herein that does not substantially agonize CD28
activity does not agonize CD28 activity more than 5% of the
activity obtained upon agonism of CD28 activity by anti-CD28 mAb
9.3 (Gibson, et al. (1996) JBC, 271: 7079-7083) under otherwise
identical assay conditions.
[0040] As used herein, the terms "inhibit," "inhibits" and
"inhibited" refer to a decrease in a given measurable activity
(e.g., binding activity) by at least 10% relative to a reference.
Where inhibition is desired, such inhibition is at least about 20%,
30%, 40%, 50%, 60%, 70%, 80%, 90%, or more, up to and including
100%, i.e., complete inhibition or absence of the given activity.
Inhibition of CD28 binding to CD80 or CD86 can be measured as
described in the working examples herein. As used herein, the term
"substantially inhibits" refers to a decrease in a given measurable
activity (e.g., the binding of CD28 to CD80 or CD86) by at least
50% relative to a reference. For example, "substantially inhibits"
refers to a decrease in a given measurable activity of at least
about 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, and up to
and including 100% relative to a reference. As used herein,
"inhibits the binding", with reference to the binding of a domain
antibody binding to CD28, or CD80 binding to CD28, or CD86 binding
to CD28, refers to a decrease in binding by at least 10% relative
to a reference. "Inhibits the binding" refers to a decrease in
binding of at least about 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%,
or more, up to and including 100%.
[0041] As used herein, the terms "activate," "activates" and
"activated" refer to an increase in a given measurable activity by
at least 5% relative to a reference, for example, at least 10%,
25%, 50%, 75%, or even 100%, or more.
[0042] As used herein, the term "monovalent" means that a given
domain antibody can bind only a single molecule of its target.
Naturally-occurring antibodies are generally divalent, in that they
have two functional antigen-binding loops, each comprising a VH and
a VL domain. Where steric hindrance is not an issue, a divalent
antibody can bind two separate molecules of the same antigen. In
contrast, a "monovalent" antibody has the capacity to bind only one
such antigen molecule. As the term is used herein, a "monovalent"
antibody can also comprise more than one antigen binding site,
e.g., two antigen binding sites, but the binding sites must be for
different antigens, such that the antibody can only bind one
molecule of CD28 at a time. The antigen-binding domain of a
monovalent antibody can comprise a V.sub.H and a V.sub.L domain,
but in an aspect, comprises only a single immunoglobulin variable
domain, i.e., a V.sub.H or a V.sub.L domain, that has the capacity
to bind CD28 without the need for a corresponding V.sub.L or
V.sub.H domain, respectively. A monovalent antibody lacks the
capacity to cross link molecules of a single antigen.
[0043] As used herein, the terms "V.sub.H domain" and "V.sub.L
domain" refer to immunoglobulin variable regions as defined by
Kabat et al. (Kabat et al. (1991) Sequences of Immunological
Interest, 5th ed. U.S. Dept. Health & Human Services,
Washington, D.C.), which is incorporated herein by reference.
[0044] As used herein, "linked" refers to the attachment of a
polymer moiety, such as PEG to an amino acid residue of a domain
antibody. Attachment of a PEG polymer to an amino acid residue of a
domain antibody, e.g., a domain antibody, is referred to as
"PEGylation" and may be achieved using several PEG attachment
moieties including, but not limited to N-hydroxylsuccinimide (NHS)
active ester, succinimidyl propionate (SPA), maleimide (MAL), vinyl
sulfone (VS), or thiol. A PEG polymer, or other polymer, can be
linked to a domain antibody at either a predetermined position, or
may be randomly linked to the domain antibody molecule. The PEG
polymer may be linked to a domain antibody at a predetermined
position. A PEG polymer may be linked to any residue in a domain
antibody, however, it is preferable that the polymer is linked to
either a lysine or cysteine, which is either naturally occurring in
the domain antibody or which has been engineered into the domain
antibody, for example, by mutagenesis of a naturally occurring
residue in the domain antibody to either a cysteine or lysine.
PEG-linkage can also be mediated through a peptide linker attached
to a domain antibody. That is, the PEG moiety can be attached to a
peptide linker fused to a domain antibody, where the linker
provides the site, e.g., a free cysteine or lysine, for PEG
attachment. As used herein, "linked" can also refer to the
association of two or more domain antibodies, e.g., dAb monomers,
to form a dimer, trimer, tetramer, or other multimer. Domain
antibody monomers can be linked to form a multimer by several
methods known in the art, including, but not limited to, expression
of the domain antibody monomers as a fusion protein, linkage of two
or more monomers via a peptide linker between monomers, or by
chemically joining monomers after translation, either to each other
directly, or through a linker by disulfide bonds, or by linkage to
a di-, tri- or multivalent linking moiety (e.g., a multi-arm PEG).
While dAb multimers are specifically contemplated herein, e.g., in
the context of dual- or multi-specific domain antibody constructs,
it is emphasized that for any given domain antibody construct, the
construct should only be able to bind one molecule of CD28, i.e.,
the constructs should have only one CD28-binding element, and
should not cross link CD28.
[0045] As used herein, "polymer" refers to a macromolecule made up
of repeating monomeric units, and can refer to a synthetic or
naturally occurring polymer such as an optionally substituted
straight or branched chain polyalkylene, polyalkenylene, or
polyoxyalkylene polymer or a branched or unbranched polysaccharide.
A "polymer" as used herein, specifically refers to an optionally
substituted or branched chain poly(ethylene glycol), poly(propylene
glycol), or poly(vinyl alcohol) and derivatives thereof.
[0046] As used herein, "PEG" or "PEG polymer" refers to
polyethylene glycol, and more specifically can refer to a
derivatized form of PEG, including, but not limited to
N-hydroxylsuccinimide (NHS) active esters of PEG such as
succinimidyl propionate, benzotriazole active esters, PEG
derivatized with maleimide, vinyl sulfones, or thiol groups. For
example, PEG formulations can include
PEG-O--CH.sub.2CH.sub.2CH.sub.2--CO.sub.2--NHS;
PEG-O--CH.sub.2--NHS; PEG-O--CH.sub.2CH.sub.2--CO.sub.2--NHS;
PEG-S--CH.sub.2CH.sub.2--CO--NHS;
PEG-O.sub.2CNH--CH(R)--CO.sub.2--NHS;
PEG-NHCO--CH.sub.2CH.sub.2--CO--NHS; and
PEG-O--CH.sub.2--CO.sub.2--NHS; where R is
(CH.sub.2).sub.4)NHCO.sub.2(mPEG). PEG polymers set forth herein
may be linear molecules, or may be branched wherein multiple PEG
moieties are present in a single polymer.
[0047] The attachment of PEG or another agent, e.g., HSA, to a
domain antibody as described herein in an exemplary embodiment,
will not impair the ability of the polypeptide to specifically bind
CD28. That is, the PEG-linked domain antibody will retain its
binding activity relative to a non-PEG-linked counterpart. As used
herein, "retains activity" refers to a level of activity of a
PEG-linked domain antibody which is at least 10% of the level of
activity of a non-PEG-linked domain antibody, including at least
about 20%, 30%, 40%, 50%, 60%, 70%, 80%, and up to 90%, including
up to about 95%, 98%, and up to 100% of the activity of a
non-PEG-linked domain antibody comprising the same antigen-binding
domain or domains. More specifically, the activity of a PEG-linked
domain antibody compared to a non-PEG linked domain antibody should
be determined on a molar basis; that is equivalent numbers of moles
of each of the PEG-linked and non-PEG-linked domain antibody should
be used in each trial. In determining whether a particular
PEG-linked domain antibody "retains activity", the activity of a
PEG-linked domain antibody may be compared with the activity of the
same domain antibody in the absence of PEG.
[0048] As used herein, the term "IC.sub.50" refers to the
concentration of an inhibitor necessary to inhibit a given activity
by about 50%. IC.sub.50 is determined by assaying a given activity,
e.g., binding of CD28 to CD80 or CD86, in the presence of varying
amounts of the inhibitor (e.g., domain antibody), and plotting the
inhibitor concentration versus the activity being targeted. Binding
of CD28 to CD80 or CD86 is measured herein by the method described
the working examples. Alternatively, surface plasmon resonance
(SPR) can be used.
[0049] As used herein, the term "EC.sub.50" refers to the
concentration of compound or domain antibody that provokes a
response in a subject, wherein the response is halfway between the
baseline and the maximum response. The baseline and maximum
responses of a subject, with respect to a compound or domain
antibody, can be determined by any technique known in the art.
[0050] As used herein, the term "fused to a domain antibody"
generally means that a polypeptide is fused to a given antibody
through use of recombinant DNA techniques, though fusion may occur
chemically at the protein level. Thus, an antibody "fused to"
another polypeptide, e.g., to another antibody of different binding
specificity, does not exist in nature and is generated through
recombinant means. The term "fused to a domain antibody" also
encompasses the linkage of a polypeptide to a given domain antibody
through, for example, disulfide or other chemical linkages, where
the fused polypeptide is not naturally found fused to the domain
antibody. Recombinant and chemical methods of fusing a polypeptide
to another polypeptide, e.g., to an antibody, are well known in the
art.
[0051] As used herein, the term "Fc domain" refers to the constant
region antibody sequences comprising CH2 and CH3 constant domains
as delimited according to Kabat et al., supra. The Fc portion of
the heavy chain polypeptide has the ability to self-associate, a
function which facilitates the formation of divalent antibodies.
The term "lacks an Fc domain" means that a given domain antibody
lacks at least the portion of an immunoglobulin Fc domain (as such
domains are defined according to Kabat et al., 1991, Sequences of
Immunological Interest, 5.sup.th ed. U.S. Dept. Health & Human
Services, Washington, D.C.) sufficient to mediate the dimerization
of Fc-containing domain antibodies. Dimerization of Fc-containing
domain antibodies is measured, for example, by chromatographic
methods or by surface plasmon resonance. A domain antibody lacking
an Fc domain avoids Fc-platelet interactions and therefore avoids
induction of platelet aggregation.
[0052] As used herein, the term "universal framework" refers to a
single antibody framework sequence corresponding to the regions of
an antibody conserved in sequence as defined by Kabat (Kabat et al.
(1991) Sequences of Immunological Interest, 5.sup.th ed. U.S. Dept.
Health & Human Services, Washington, D.C.) or corresponding to
the human germline immunoglobulin repertoire or structure as
defined by Chothia and Lesk, (1987) J. Mol. Biol. 196: 910-917. The
use of a single framework, or a set of such frameworks, which has
been found to permit the derivation of virtually any binding
specificity though variation in the hypervariable regions alone, is
included herein.
[0053] The term "about" will be understood by persons of ordinary
skill in the art and will vary to some extent on the context in
which it is used. Generally, about encompasses a range of values
that are plus/minus 10% of a referenced value.
2. Anti-CD28 Domain Antibodies
[0054] The present invention relates to domain antibodies that
specifically bind and inhibit human CD28 ("anti-CD28 domain
antibodies") and that are useful in the treatment of diseases
involving the CD28 pathway. Accordingly, a method of treating an
immune disease in a patient in need of such treatment is provided
comprising administering to the patient a therapeutically effective
amount of the anti-CD28 domain antibody.
[0055] Domain antibodies are provided that are monovalent for
binding to CD28. While not wishing to be bound by any particular
theory, it is believed that monovalency for CD28 binding removes
the possibility for cross-linking cell surface receptors that
occurs with prior art antibodies. Thus, in one aspect, the domain
antibodies disclosed herein not only inhibit or antagonize the
binding of CD80 or CD86 to CD28, they do not substantially agonize
CD28 activity.
[0056] In one aspect, the antibodies monovalent for CD28 binding
are human domain antibodies. Human domain antibodies can be
administered to human patients while largely avoiding the
anti-antibody immune response often provoked by the administration
of antibodies from other species, e.g., mouse. While murine
antibodies can be "humanized" by grafting human constant domains
onto the murine antigen-binding domains, human antibodies as
disclosed herein are produced without the need for laborious and
time-consuming genetic manipulation of a murine antibody
sequence.
[0057] In certain embodiments, domain antibodies may include one or
more of the following CDRs:
TABLE-US-00001 CDR1: (SEQ ID NO: 1) RASRPIWPFLE CDR2: (SEQ ID NO:
2) FTSRLRH CDR3: (SEQ ID NO: 3) LQNVANPAT
[0058] In one embodiment, the anti-CD28 domain antibody has a CDR1
sequence that is at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%,
98%, or 99% identical to SEQ ID NO: 1. In another embodiment, the
CDR1 differs from SEQ ID NO: 1 by up to 5 amino acids (e.g., by 5,
4, 3, 2, 1, or 0 amino acids).
[0059] In one embodiment, the anti-CD28 domain antibody has a CDR2
sequence that is at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%,
98%, or 99% identical to SEQ ID NO: 2. In another embodiment, the
CDR2 differs from SEQ ID NO: 2 by up to 5 amino acids (e.g., by 5,
4, 3, 2, 1, or 0 amino acids).
[0060] In one embodiment, the anti-CD28 domain antibody has a CDR3
sequence that is at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%,
98%, or 99% identical to SEQ ID NO: 3. In another embodiment, the
CDR3 differs from SEQ ID NO: 3 by up to 5 amino acids (e.g., by 5,
4, 3, 2, 1, or 0 amino acids).
[0061] In certain embodiments, the anti-CD28 domain antibody
comprises an amino acid sequence at least 70%, 75%, 80%, 85%, 90%,
95%, 96%, 97%, 98%, or 99% identical to an amino acid sequence
selected from SEQ ID NOs: 4-15. In another embodiment, the
anti-CD28 domain antibody differs from the selected amino acid
sequence by up to 5 amino acids. For example, the domain antibody
differs from SEQ ID NO: 12 by up to 5 amino acids (e.g., by 5, 4,
3, 2, 1, or 0 amino acids).
[0062] In certain embodiments, the anti-CD28 domain antibody
comprises an amino acid sequence selected from SEQ ID NOs: 4-15. In
a specific embodiment, the anti-CD28 domain antibody comprises the
amino acid sequence of SEQ ID NO: 12.
[0063] Certain exemplary sequences of the anti-CD28 domain
antibodies are provided below.
TABLE-US-00002 1h-239-891 (SEQ ID NO: 4):
DIQMTQSPSSLSASVGDRVTITCRASRPIWPFLEWYQQKPGKAPKLLIYF
TSRLRHGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCLQNVANPATFSQ GTKVEIKR
1h-239-891(Q3C) (SEQ ID NO: 5):
DICMTQSPSSLSASVGDRVTITCRASRPIWPFLEWYQQKPGKAPKLLIYF
TSRLRHGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCLQNVANPATFSQ GTKVEIKR
1h-239-891(S9C) (SEQ ID NO: 6):
DIQMTQSPCSLSASVGDRVTITCRASRPIWPFLEWYQQKPGKAPKLLIYF
TSRLRHGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCLQNVANPATFSQ GTKVEIKR
1h-239-891(R18C) (SEQ ID NO: 7):
DIQMTQSPSSLSASVGDCVTITCRASRPIWPFLEWYQQKPGKAPKLLIYF
TSRLRHGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCLQNVANPATFSQ GTKVEIKR
1h-239-891(G41C) (SEQ ID NO: 8):
DIQMTQSPSSLSASVGDRVTITCRASRPIWPFLEWYQQKPCKAPKLLIYF
TSRLRHGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCLQNVANPATFSQ GTKVEIKR
1h-239-891(K42C) (SEQ ID NO: 9):
DIQMTQSPSSLSASVGDRVTITCRASRPIWPFLEWYQQKPGCAPKLLIYF
TSRLRHGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCLQNVANPATFSQ GTKVEIKR
1h-239-891(K45C) (SEQ ID NO: 10):
DIQMTQSPSSLSASVGDRVTITCRASRPIWPFLEWYQQKPGKAPCLLIYF
TSRLRHGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCLQNVANPATFSQ GTKVEIKR
1h-239-891(S60C) (SEQ ID NO: 11):
DIQMTQSPSSLSASVGDRVTITCRASRPIWPFLEWYQQKPGKAPKLLIYF
TSRLRHGVPCRFSGSGSGTDFTLTISSLQPEDFATYYCLQNVANPATFSQ GTKVEIKR
1h-239-891(D70C) (SEQ ID NO: 12):
DIQMTQSPSSLSASVGDRVTITCRASRPIWPFLEWYQQKPGKAPKLLIYF
TSRLRHGVPSRFSGSGSGTCFTLTISSLQPEDFATYYCLQNVANPATFSQ GTKVEIKR
1h-239-891(T74C) (SEQ ID NO: 13):
DIQMTQSPSSLSASVGDRVTITCRASRPIWPFLEWYQQKPGKAPKLLIYF
TSRLRHGVPSRFSGSGSGTDFTLCISSLQPEDFATYYCLQNVANPATFSQ GTKVEIKR
1h-239-891(Q79C) (SEQ ID NO: 14):
DIQMTQSPSSLSASVGDRVTITCRASRPIWPFLEWYQQKPGKAPKLLIYF
TSRLRHGVPSRFSGSGSGTDFTLTISSLCPEDFATYYCLQNVANPATFSQ GTKVEIKR
1h-239-891(K103C) (SEQ ID NO: 15):
DIQMTQSPSSLSASVGDRVTITCRASRPIWPFLEWYQQKPGKAPKLLIYF
TSRLRHGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCLQNVANPATFSQ GTCVEIKR
[0064] In certain specific embodiments, the anti-CD28 domain
antibody may comprise 1h-239-891(D70C) (SEQ ID NO: 543). The
anti-CD28 domain antibody may comprise a 40 kDa branched
polyethylene glycol. The anti-CD28 domain antibody can be
BMS-931699. For example, BMS-931699 is a potent inhibitor of T cell
proliferation and cytokine production, with an EC.sub.50 of
35.+-.14 ng/mL and 25.+-.6 ng/mL, respectively. BMS-908613-P40B (a
macaque surrogate for BMS-931699) is equipotent at inhibiting both
CD80- and CD86-driven T cell proliferation. Importantly, no agonist
or co-agonist activity, as measured by T cell proliferation or
cytokine release, was observed with BMS-931699.
[0065] In one embodiment, the binding of the domain antibody to
CD28 does not substantially agonize CD28 activity. In particular,
the dAb does not agonize CD28 signaling in combination with T cell
receptor signaling. In another embodiment, the domain antibody
inhibits the binding of CD28 to CD80. In another embodiment, the
domain antibody inhibits the binding of CD28 to CD80, and does not
substantially agonize signaling by CD28. In yet another embodiment,
the domain antibody inhibits the binding of CD28 to CD86. In
another embodiment, the domain antibody inhibits the binding of
CD28 to CD86, and does not substantially agonize signaling by
CD28.
[0066] In an aspect, the dAb does not substantially induce T cell
proliferation in combination with T cell receptor signaling. In
another aspect, the dAb does not substantially induce cytokine
secretion by T cells in combination with T cell receptor signaling.
In an embodiment, a cytokine is at least one cytokine selected from
the group consisting of GM-CSF, IL-2, IL-3, IL-4, IL-5, IL-6,
IL-10, IL-12 IL-13, IL-15, IL-17, IL-21, IL-22, IL-24, TGF.beta.,
TNF-.alpha., TNF-.beta., IFN-.alpha., IFN-.beta., IFN-.gamma..
[0067] In one aspect, because human antibodies will avoid the
generation of an immune response to the antibodies when
administered to human subjects for the treatment or prevention of
disease, the domain antibody is a human domain antibody that
monovalently binds CD28, and in an exemplary embodiment, without
substantially agonizing CD28 activity.
[0068] In one embodiment, the domain antibody interacts with human
CD28 with a K.sub.d in the range of 50 nM to 1 pM, inclusive, as
measured by surface plasmon resonance. For example, the K.sub.d for
human CD28 can be 25 nM to 20 pM, 10 nM to 20 pM, 5 nm to 20 pM, 1
nM to 20 pM, 0.5 nM to 20 pM, 0.1 nM to 20 pM, 0.1 nM to 50 pM, 75
pM to 20 pM, or even 50 pM to 20 pM. In an embodiment, the K.sub.d
for human CD28 is about 50 pM.
[0069] In one embodiment, the domain antibody inhibits binding of
CD80 to CD28 with an EC.sub.50 of 50 nM or less. In one embodiment,
the domain antibody inhibits binding of CD86 to CD28 with an
EC.sub.50 of 50 nM or less. In a further embodiment, the domain
antibody has binding specificity to CD28 with a K.sub.off rate
constant of 1.times.10.sup.-3 s.sup.-1 or less, 1.times.10.sup.-4
s.sup.-1 or less, 1.times.10.sup.-5 s.sup.-1 or less, or
1.times.10.sup.-6 s.sup.-1 or less, as determined by surface
plasmon resonance. In one embodiment, the domain antibody
neutralizes CD28 in a standard assay with a EC.sub.50 of 50 nM or
less.
[0070] In another embodiment, the domain antibody comprises a
single immunoglobulin variable domain that binds CD28. In one
embodiment, the single immunoglobulin variable domain is a V.sub.H
or a V.sub.L domain. In another embodiment, the domain antibody
comprises a homomultimer or heteromultimer of two variable domains,
e.g., a V.sub.H and V.sub.L domain, but one of the variable domains
has the capacity to bind CD28 without the need for a corresponding
V.sub.L or V.sub.H domain. That is, the dAb binds an antigen
independently of the additional V.sub.H or V.sub.L domains. The
variable domains in these embodiments may comprise three
complementarity determining regions (CDRs). In another embodiment,
the domain antibody is free of an Fc domain. The limits of an Fc
domain are set out in Kabat et al. (1991, Sequences of
Immunological Interest, 5.sup.th ed. U.S. Dept. Health & Human
Services, Washington, D.C.; incorporated herein by reference). In
the alternative, an Fc domain consists of the CH2--CH3 regions,
optionally including a hinge region linked to the CH2.
[0071] In one aspect, the domain antibody comprises a universal
framework. In this aspect, a domain antibody may comprise one or
more framework regions comprising an amino acid sequence that is
the same as the amino acid sequence of a corresponding framework
(FW) region encoded by a human germline antibody gene segment, or
the amino acid sequence of one or more of said framework regions
collectively comprising up to 5, e.g., 1, 2, 3, 4 or 5, amino acid
differences relative to the amino acid sequence of said
corresponding framework region encoded by a human germline antibody
gene segment.
[0072] In one embodiment, the dAb comprises amino acid sequences of
FW1, FW2, FW3, and FW4 that correspond to the FW1, FW2, FW3, and
FW4 of a human antibody, e.g., a human germline antibody. In a
further embodiment, some or all of the amino acid sequences of FW1,
FW2, FW3, and FW4 of the domain antibody are the same as the amino
acid sequences of corresponding framework regions encoded by human
germline antibody gene segments. For example, FW2 may be identical
to the FW2 of a human antibody. In another embodiment, the amino
acid sequences of FW1, FW2, FW3, and FW4 collectively contain up to
10 amino acid differences relative to the amino acid sequences of
corresponding framework regions encoded by said human germline
antibody gene segment. In a further embodiment of the foregoing,
the human germline antibody gene segment can be selected from the
group consisting of DP47, DP45, DP48, and DPK9. In one embodiment,
the universal framework comprises a V.sub.H framework selected from
the group consisting of DP47, DP45, and DP38, and/or the V.sub.L
framework is DPK9.
[0073] In one aspect, a domain antibody is formatted to increase
its in vivo half-life. In particular, the domain antibody has an
increased in vivo t-.alpha. or t-.beta. half-life relative to the
same unformatted domain antibody.
[0074] In one embodiment, the t.alpha.-half-life of the domain
antibody composition is increased by 10% or more when compared to
an unmodified protein assayed under otherwise identical conditions.
In another embodiment, the t.alpha.-half-life of the domain
antibody composition is increased by 50% or more. In another
embodiment, the t.alpha.-half-life of the domain antibody
composition is increased by 2.times. or more. In another
embodiment, the t.alpha.-half-life of the domain antibody
composition is increased by 5.times. or more, e.g., 10.times.,
15.times., 20.times., 25.times., 30.times., 40.times., 50.times.,
or more. In another embodiment, the t.alpha.-half-life of the
domain antibody composition is increased by 100.times., 200.times.,
300.times., 400.times., 500.times., or more.
[0075] In another embodiment, the domain antibody has a t.alpha.
half-life of 0.25 to 6 hours, inclusive. In another embodiment, the
t.alpha. half-life is in the range of 30 minutes to 12 hours,
inclusive. In another embodiment, the t.alpha.-half-life of the
domain antibody is in the range of 1 to 6 hours.
[0076] In another embodiment, the t.beta.-half-life of the domain
antibody is increased by 10% or more when compared to an unmodified
protein assayed under otherwise identical conditions. In another
embodiment, the t.beta.-half-life of the domain antibody is
increased by 50% or more. In another embodiment, the
t.beta.-half-life of the antibody domain antibody is increased by
2.times. or more. In another embodiment, the t.beta.-half-life of
the domain antibody is increased by 5.times. or more, e.g.,
10.times., 15.times., 20.times., 25.times., 30.times., 40.times.,
or more. In another embodiment, the t.beta.-half-life of the domain
antibody is increased by 50.times. or more.
[0077] In another embodiment, the domain antibody has a t.beta.
half-life of 1 hour to 744 hours, inclusive. In another embodiment,
the t.beta.-half-life is in the range of 12 to 48 hours, inclusive.
In another embodiment, the t.beta. half-life is in the range of 12
to 26 hours, inclusive. In yet another embodiment, the t.beta.
half-life is about 336 hours.
[0078] In addition to, or alternative to the above criteria, a
domain antibody-containing composition is provided comprising a
ligand having an AUC value (area under the curve) in the range of 1
mgmin/ml or more. In one embodiment, the lower end of the range is
5, 10, 15, 20, 30, 100, 200, or 300 mgmin/ml. In addition, or
alternatively, a ligand or composition has an AUC in the range of
up to 600 mgmin/ml. In one embodiment, the upper end of the range
is 500, 400, 300, 200, 150, 100, 75, or 50 mgmin/ml. Advantageously
a ligand will have an AUC in the range selected from the group
consisting of the following: 15 to 150 mgmin/ml, 15 to 100
mgmin/ml, 15 to 75 mgmin/ml, and 15 to 50 mgmin/ml.
[0079] In another embodiment, the formatting comprises PEGylation
of the dAb. In one embodiment, the PEG is covalently linked. In
another embodiment, the PEG is linked to the domain antibody at a
cysteine or lysine residue. In yet another embodiment, the
PEG-linked domain antibody has a hydrodynamic size of at least 24
kD. In yet another embodiment, the total PEG size is from 20 to 60
kD, inclusive. In yet another embodiment, the PEG-linked domain
antibody has a hydrodynamic size of at least 200 kD.
[0080] In another embodiment, the PEG-linked domain antibody has an
increased in vivo half-life relative to the same polypeptide
composition lacking linked polyethylene glycol. In another
embodiment, the t.alpha.-half-life of the domain antibody
composition is increased by 10% or more. In another embodiment, the
t.alpha.-half-life of the domain antibody composition is increased
by 50% or more. In another embodiment, the t.alpha.-half-life of
the domain antibody composition is increased by 2.times. or more.
In another embodiment, the t.alpha.-half-life of the domain
antibody composition is increased by 5.times. or more, e.g.,
10.times., 15.times., 20.times., 25.times., 30.times., 40.times.,
50.times., or more. In another embodiment, the t.alpha.-half-life
of the domain antibody composition is increased by 100.times.,
200.times., 300.times., 400.times., 500.times., or more.
[0081] In another embodiment, the PEG-linked domain antibody has a
t.alpha. half-life of 0.25 to 6 hours, inclusive. In another
embodiment, the t.alpha. half-life is in the range of 30 minutes to
12 hours, inclusive. In another embodiment, the t.alpha.-half-life
of the domain antibody is in the range of 1 to 6 hours.
[0082] In another embodiment, the t.beta.-half-life of the
PEG-linked domain antibody is increased by 10% or more. In another
embodiment, the t.beta.-half-life of the PEG-linked domain antibody
is increased by 50% or more. In another embodiment, the
t.beta.-half-life of the PEG-linked domain antibody is increased by
2.times. or more. In another embodiment, the t.beta.-half-life of
the PEG-linked domain antibody is increased by 5.times. or more,
e.g., 10.times., 15.times., 20.times., 25.times., 30.times.,
40.times., or more. In another embodiment, the t.beta.-half-life of
the PEG-linked domain antibody is increased by 50.times. or
more.
[0083] In another embodiment, the PEG-linked domain antibody has a
t.beta. half-life of 1 to 170 hours, inclusive. In another
embodiment, the t.beta.-half-life is in the range of 12 to 48
hours, inclusive. In another embodiment, the t.beta.-half-life is
in the range of 12 to 26 hours, inclusive.
[0084] In another embodiment, the PEG-linked domain antibody has an
AUC value (area under the curve) in the range of 1 mgmin/ml or
more. In one embodiment, the lower end of the range is about 5, 10,
15, 20, 30, 100, 200, or 300 mgmin/ml. In addition, or
alternatively, a ligand or composition has an AUC in the range of
up to about 600 mgmin/ml. In one embodiment, the upper end of the
range is about 500, 400, 300, 200, 150, 100, 75, or 50 mgmin/ml.
Advantageously a ligand will have an AUC in the range selected from
the group consisting of the following: about 15 to 150 mgmin/ml,
about 15 to 100 mgmin/ml, about 15 to 75 mgmin/ml, and about 15 to
50 mgmin/ml.
[0085] The dAb may inhibit binding of CD28 to CD80 and/or CD86 with
an EC.sub.50 of about 100 nM, about 50 nM, about 1 nM, about 500
pM, about 100 pM, about 50 pM, about 10 pM, about 5 pM, or about 1
pM. For example, the domain antibody inhibits binding of CD28 to
CD80 with an EC.sub.50 in the range of 1 pM to 1.5 .mu.M,
inclusive; EC.sub.50 for inhibition of CD28 binding to CD80. The
EC.sub.50 can be in the range of 1 pM to 1 .mu.M, 1 pM to 900 nM, 1
pM to 800 nM, 1 pM to 700 nM, 1 pM to 600 nM, 1 pM to 500 nM, 1 pM
to 400 nM, 1 pM to 300 nM, 1 pM to 200 nM, 1 pM to 100 nM, 1 pM to
50 nM, 1 pM to 10 nM, 1 pM to 1 nM, 1 pM to 500 pM, 1 pM to 100 pM,
1 pM to 50 pM, 1 pM to 10 pM, or 1 pM to 5 pM. Further acceptable
ranges include, for example, 50 pM to 1 .mu.M, 100 pM to 500 nM,
125 pM to 250 nM, 150 pM to 200 nM, 150 pM to 100 nM, and 200 pM to
50 nM.
[0086] In another embodiment, the domain antibody inhibits binding
of CD28 to CD86 with an EC.sub.50 in the range of 1 pM to 1.5
.mu.M, inclusive; EC.sub.50 for inhibition of CD28 binding to CD86.
The EC.sub.50 can be in the range of 1 pM to 1 .mu.M, 1 pM to 900
nM, 1 pM to 800 nM, 1 pM to 700 nM, 1 pM to 600 nM, 1 pM to 500 nM,
1 pM to 400 nM, 1 pM to 300 nM, 1 pM to 200 nM, 1 pM to 100 nM, 1
pM to 50 nM, 1 pM to 10 nM, 1 pM to 1 nM, 1 pM to 500 pM, 1 pM to
100 pM, 1 pM to 50 pM, 1 pM to 10 pM, or 1 pM to 5 pM. Further
acceptable ranges include, for example, 50 pM to 1 .mu.M, 100 pM to
500 nM, 125 pM to 250 nM, 150 pM to 200 nM, 150 pM to 100 nM, and
200 pM to 50 nM.
[0087] The domain antibody may comprise one or more framework
regions comprising an amino acid sequence that is the same as the
amino acid sequence of a corresponding framework region encoded by
a human germline antibody gene segment, or the amino acid sequence
of one or more of said framework regions collectively comprises up
to 5 amino acid differences relative to the amino acid sequence of
said corresponding framework region encoded by a human germline
antibody gene segment.
[0088] In one embodiment, the amino acid sequences of FW1, FW2,
FW3, and FW4 of the domain antibody are the same as the amino acid
sequences of corresponding framework regions encoded by a human
germline antibody gene segment, or the amino acid sequences of FW1,
FW2, FW3, and FW4 collectively contain up to 10 amino acid
differences relative to the amino acid sequences of corresponding
framework regions encoded by said human germline antibody gene
segment.
[0089] In one embodiment, the amino acid sequences of said FW1,
FW2, and FW3 of the domain antibody are the same as the amino acid
sequences of corresponding framework regions encoded by human
germline antibody gene segments. The human germline antibody gene
segments may be selected from the group consisting of DP47, DP45,
DP48, and DPK9.
3. Uses of Domain Antibodies
[0090] Domain antibodies as described herein are useful for
antagonizing the activity of CD28. Therefore, domain antibodies as
described herein can be used to treat a patient having a condition,
disease or disorder mediated in whole or in part by CD28 activity.
For example, domain antibodies as described herein are useful for
the treatment or prevention of diseases or disorders in which
inappropriate activation of a CD28-mediated pathway is involved,
such as systemic lupus erythematosus (SLE).
[0091] As used herein "treat", "reduce", "prevent", or "alleviate"
as it relates to a symptom of disease refer to a decrease of a
symptom by at least 10% based on a clinically measurable parameter,
or by at least one point on a clinically-accepted scale of disease
or symptom severity. As used herein, the term "symptom(s) of
systemic lupus erythematosus" refers to any of the clinically
relevant symptoms of SLE known to those of skill in the art,
including, but not limited to, BICLA (BILAG-Based Composite Lupus
Assessment), SRI (Systemic Lupus Erythematosus Responder Index).
Non-limiting examples include the accumulation of IgG
autoantibodies (e.g., against nuclear antigens such as chromatin,
snRNPs (especially U1, Sm, Ro/SSA and La/SSB), phospholipids and
cell surface molecules), hemolytic anemia, thrombocytopenia,
leukopenia, glomerulonephritis, vasculitis, arthritis, and
serositis). A reduction in such a symptom of a patient is a
reduction by at least 10% in a clinically measurable parameter, or
by at least one point on a clinically-accepted scale of disease
severity, compared to a patient treated with a placebo.
[0092] In an aspect, autoimmune diseases frequently involve
inappropriate regulation or activity of CD28 pathways.
Administration of a domain antibody as described herein to an
individual suffering from such a disease, including an autoimmune
disease, can reduce one or more symptoms of the disease.
Non-limiting examples of diseases for which the domain antibodies
described herein can be therapeutically useful include, but are not
limited to, Addison's disease, allergy, ankylosing spondylitis,
asthma, atherosclerosis, autoimmune diseases of the ear, autoimmune
diseases of the eye, autoimmune atrophic gastritis, autoimmune
hepatitis, autoimmune hymolytic anemia, autoimmune parotitis,
primary biliary cirrhosis, benign lymphocytic aniitis, colitis,
coronary heart disease, Crohn's disease, diabetes (Type I),
diabetes, including Type 1 and/or Type 2 diabetes, epididymitis,
glomerulonephritis, Goodpasture's syndrome, Graves' disease,
Guillain-Barre syndrome, Hashimoto's disease, hemolytic anemia,
idiopathic thrombocytopenic purpura, inflammatory bowel disease
(IBD), immune response to recombinant drug products, e.g., factor
VII in hemophilia, systemic lupus erythematosus, lupus nephritis,
male infertility, mixed connective tissue disease, multiple
sclerosis, myasthenia gravis, primary myxedema, pemphigus,
pernicious anemia, polymyositis, psoriasis, psoriatic arthritis,
rheumatic fever, rheumatoid arthritis, sarcoidosis, scleroderma,
Sjogren's syndrome, spondyloarthropathies, sympathetic ophthalmia,
T-cell lymphoma, T-cell acute lymphoblastic leukemia, testicular
antiocentric T-cell lymphoma, thyroiditis, transplant rejection,
ulcerative colitis, autoimmune uveitis, and vasculitis.
Autoimmune-mediated conditions include, but are not limited to,
conditions in which the tissue affected is the primary target, and
in some cases, the secondary target. Such conditions include, but
are not limited to, AIDS, atopic allergy, bronchial asthma, eczema,
leprosy, schizophrenia, inherited depression, transplantation of
tissues and organs, chronic fatigue syndrome, Alzheimer's disease,
Parkinson's disease, myocardial infarction, stroke, autism,
epilepsy, Arthus's phenomenon, anaphylaxis, and alcohol and drug
addiction.
[0093] The domain antibodies described herein also can be
therapeutically useful in graft-related diseases, such as graft
versus host disease (GVHD), acute transplantation rejection, and
chronic transplantation rejection.
[0094] In certain embodiments, the present invention provides a
method of treating an immune disease in a patient, comprising
administering to the patient a therapeutically effective amount of
an anti-CD28 domain antibody. To illustrate, the immune disease may
be systemic lupus erythematosus. The patient receiving an anti-CD28
domain antibody may have decreased SLE symptoms compared to a
patient receiving placebo. For example, the patient receiving an
anti-CD28 domain antibody may have lower levels of C3, C4,
anti-dsDNA, and/or anti-ANA compared to a patient receiving
placebo. For example, the patient receiving an anti-CD28 domain
antibody may have decreased arthritis symptoms compared to a
patient receiving placebo. For example, the patient receiving an
anti-CD28 domain antibody may have decreased inflammatory skin
disease symptoms compared to a patient receiving placebo.
[0095] The treatment may further comprise administering an
immunosuppressive/immunomodulatory and/or anti-inflammatory agent.
The treatment may be administered in combination with an
immunosuppressive/immunomodulatory and/or anti-inflammatory agent.
Such additional immunosuppressive/immunomodulatory and/or
anti-inflammatory agents or therapies may comprise calcineuirin
inhibitor, cyclosporine, cytoxan, prednisone, azathioprine,
methotrexate, corticosteroids, nonsteroidal antiinflammatory
drugs/Cox-2 inhibitors, hydroxychloroquine, sulphasalazopryine,
gold salts, etanercept, infliximab, anakinra, mizoribine,
mycophenolic acid, mycophenolate mofetil, interferon beta-1a,
interferon beta-1b, glatiramer acetate, mitoxantrone hydrochloride,
and/or other biologics like anti-TNF. The domain antibodies also
may be administered in combination with one or more of the
following agents to regulate an immune response: CTLA4, soluble
gp39 (also known as CD40 ligand (CD40L), CD154, T-BAM, TRAP),
soluble CD29, soluble CD40, soluble CD80, soluble CD86, soluble
CD56, soluble Thy-1, soluble CD3, soluble TCR, soluble VLA-4,
soluble VCAM-1, soluble LECAM-1, soluble ELAM-1, soluble CD44,
antibodies reactive with gp39, antibodies reactive with CD40,
antibodies reactive with B7, antibodies reactive with CD28,
antibodies reactive with LFA-1, antibodies reactive with LFA-2,
antibodies reactive with IL-2, antibodies reactive with IL-12,
antibodies reactive with IFN-gamma, antibodies reactive with CD2,
antibodies reactive with CD48, antibodies reactive with any ICAM
(e.g., ICAM-2), antibodies reactive with CTLA4, antibodies reactive
with Thy-1, antibodies reactive with CD56, antibodies reactive with
CD3, antibodies reactive with CD29, antibodies reactive with TCR,
antibodies reactive with VLA-4, antibodies reactive with VCAM-1,
antibodies reactive with LECAM-1, antibodies reactive with ELAM-1,
antibodies reactive with CD44, monoclonal antibodies to leukocyte
receptors, e.g., MHC, CD2, CD3, CD4, CD11a/CD18, CD7, CD25, CD 27,
B7, CD40, CD45, CD58, CD 137, ICOS, CD150 (SLAM), OX40, 4-1BB or
their ligands.
[0096] Where domain antibodies of the invention are administered in
combination with another immunosuppressive/immunomodulatory or
anti-inflammatory agent or therapy, e.g., as specified above, the
administration may be made concomitantly or in sequence. When the
dAbs are administered concomitantly with another agent, such as an
agent specified above, the dAb and agent may administered in the
same pharmaceutical composition.
[0097] The treatment may produce at least one therapeutic effect
measurable by a biomarker selected from the group consisting of:
CD28 receptor occupancy on T cells, C3, C4, anti-dsDNA, anti-ANA,
anti-Ro autoantibodies, anti-La autoantibodies, anti-RNP
autoantibodies, anti-Sm autoantibodies, anti-APL autoantibodies,
CRP, total IgG, total IgM, RNA transcripts in whole blood, NGAL in
urine, TWEAK in urine, MCP-1 in urine, IL-18 in urine, IL-1 in
urine, total soluble CD28, T cell activation, leukocyte surface
CD4, leukocyte surface CD8, leukocyte surface CD28, leukocyte
surface CD57 and leukocyte intracellular granzyme B, serum IL-6,
serum IL-18, serum TNF-.alpha., serum .alpha.-interferon, serum
BLyS(BAFF), CD154, sCD28 and microvessicles.
4. Pharmaceutical Compositions, Dosage, and Administration
[0098] The anti-CD28 domain antibodies set forth herein can be
incorporated into pharmaceutical compositions suitable for
administration to a subject. Typically, the pharmaceutical
composition comprises the domain antibody and a pharmaceutically
acceptable carrier. As used herein, "pharmaceutically acceptable
carrier" includes any and all solvents, dispersion media, coatings,
antibacterial, and antifungal agents, isotonic and absorption
delaying agents, and the like that are physiologically compatible.
The term "pharmaceutically acceptable carrier" excludes tissue
culture medium comprising bovine or horse serum. Examples of
pharmaceutically acceptable carriers include one or more of water,
saline, phosphate buffered saline, dextrose, glycerol, ethanol and
the like, as well as combinations thereof. In many cases, it will
be preferable to include isotonic agents, for example, sugars,
polyalcohols such as mannitol, sorbitol, or sodium chloride in the
composition. Pharmaceutically acceptable substances include minor
amounts of auxiliary substances such as wetting or emulsifying
agents, preservatives or buffers, which enhance the shelf life or
effectiveness of the domain antibody.
[0099] The compositions as described herein may be in a variety of
forms. These include, for example, liquid, semi-solid, and solid
dosage forms, such as liquid solutions (e.g., injectable and
infusible solutions), dispersions or suspensions, powders,
liposomes, and suppositories. The preferred form depends on the
intended mode of administration and therapeutic application.
Typical preferred compositions are in the form of injectable or
infusible solutions, such as compositions similar to those used for
passive immunization of humans with other antibodies. One mode of
administration is parenteral (e.g., intravenous, subcutaneous,
intraperitoneal, intramuscular).
[0100] Therapeutic compositions typically must be sterile and
stable under the conditions of manufacture and storage. The
composition can be formulated as a solution, microemulsion,
dispersion, liposome, or other ordered structure suitable to high
drug concentration. Sterile injectable solutions can be prepared by
incorporating the active compound in the required amount in an
appropriate solvent with one or a combination of ingredients
enumerated above, as required, followed by filter sterilization.
Generally, dispersions are prepared by incorporating the active
compound into a sterile vehicle that contains a basic dispersion
medium and the required other ingredients from those enumerated
above. In the case of sterile powders for the preparation of
sterile injectable solutions, methods of preparation include vacuum
drying and freeze-drying that yields a powder of the active
ingredient plus any additional desired ingredient from a previously
sterile-filtered solution thereof. The proper fluidity of a
solution can be maintained, for example, by the use of a coating
such as lecithin, by the maintenance of the required particle size
in the case of dispersion and by the use of surfactants.
[0101] The domain antibodies described herein can be administered
by a variety of methods known in the art, although for many
therapeutic applications. The polypeptide can be administered by
instravenous (IV), intramuscular (IM) or subcutaneous (SC)
injection.
[0102] As will be appreciated by the skilled artisan, the route
and/or mode of administration will vary depending upon the desired
results. In certain embodiments, the active compound may be
prepared with a carrier that will protect the compound against
rapid release, such as a controlled release formulation, including
implants, and microencapsulated delivery systems. Domain antibodies
are well suited for formulation as extended release preparations
due, in part, to their small size, the number of moles per dose can
be significantly higher than the dosage of, e.g., full sized
antibodies. Biodegradable, biocompatible polymers can be used, such
as ethylene vinyl acetate, polyanhydrides, polyglycolic acid,
collagen, polyorthoesters, and polylactic acid. Prolonged
absorption of injectable compositions can be brought about by
including in the composition an agent that delays absorption, for
example, monostearate salts and gelatin. Many methods for the
preparation of such formulations are patented or generally known to
those skilled in the art. See, e.g., Sustained and Controlled
Release Drug Delivery Systems, J. R. Robinson, ed., Marcel Dekker,
Inc., New York, 1978. Additional methods applicable to the
controlled or extended release of polypeptide agents such as the
monovalent domain antibodies disclosed herein are described, for
example, in U.S. Pat. Nos. 6,306,406 and 6,346,274, as well as, for
example, in U.S. Patent Publication Nos. US20020182254 and
US20020051808, all of which are incorporated herein by reference
for all purposes. For example, the domain antibody can be
formulated in a pharmaceutical composition for subcutaneous
administration, which can include a pharmaceutically acceptable
carrier. The pharmaceutical composition can further comprise 12.5
mg/mL BMS-931699 in 20 mM phosphate, pH 5.9, with 5% (w/v)
sorbitol.
[0103] Additional active compounds can also be incorporated into
the compositions. In certain embodiments, a domain antibody is
co-formulated with and/or co-administered with one or more
additional therapeutic agents. For example, a domain antibody can
be co-formulated and/or co-administered with one or more additional
antibodies that bind other targets (e.g., antibodies that bind
other cytokines or that bind cell surface molecules), or, for
example, one or more cytokines. Such combination therapies may
utilize lower dosages of the administered therapeutic agents, thus
avoiding possible toxicities or complications associated with the
various monotherapies.
[0104] The pharmaceutical compositions disclosed herein can include
a "therapeutically effective amount" or a "prophylactically
effective amount" of a domain antibody. A "therapeutically
effective amount" refers to an amount effective, at dosages and for
periods of time necessary, to achieve the desired therapeutic
result. A therapeutically effective amount of the domain antibody
can vary according to factors such as the disease state, age, sex,
and weight of the individual, and the ability of domain antibody to
elicit a desired response in the individual. A therapeutically
effective amount is also one in which any toxic or detrimental
effects of the antibody or antibody portion are outweighed by the
therapeutically beneficial effects. A "prophylactically effective
amount" refers to an amount effective, at dosages and for periods
of time necessary, to achieve the desired prophylactic result.
Typically, because a prophylactic dose is used in subjects prior to
or at an earlier stage of disease, the prophylactically effective
amount will be less than the therapeutically effective amount.
[0105] Dosage regimens may be adjusted to provide the optimum
desired response (e.g., a therapeutic or prophylactic response).
For example, a single bolus may be administered, several divided
doses may be administered over time or the dose may be
proportionally reduced or increased as indicated by the exigencies
of the therapeutic situation. It is advantageous to formulate
parenteral compositions in dosage unit form for ease of
administration and uniformity of dosage. Dosage unit form as used
herein refers to physically discrete units suited as unitary
dosages for the mammalian subjects to be treated; each unit
containing a predetermined quantity of active compound calculated
to produce the desired therapeutic effect in association with the
required pharmaceutical carrier.
[0106] A non-limiting range for a therapeutically or
prophylactically effective amount of a domain antibody is 0.1-20
mg/kg, and in an aspect, 1-10 mg/kg. For example, the domain
antibody can be administered at a dose of about 1.25 mg to about
12.5 mg; i.e. the dose can be at least 1.25 mg, at least 5 mg, at
least 12.5 mg, about 1.25 mg, about 5 mg, or about 12.5 mg. 1.25 mg
to about 12.5 mg mg of domain antibody can be administered
subcutaneously. The dose may be administered on a fixed or varied
schedule. For example, the dose may be administered on a weekly or
bi-weekly basis. The dose can be administered over a set treatment
regimen, such as once every two weeks for 24 weeks (12 doses total)
or once a week for 24 weeks (24 doses total). It is to be noted
that dosage values can vary with the type and severity of the
condition to be alleviated. It is to be further understood that for
any particular subject, specific dosage regimens should be adjusted
over time according to the individual need and the professional
judgment of the administering clinician.
[0107] The efficacy of treatment with a domain antibody as
described herein is judged by the skilled clinician on the basis of
improvement in one or more symptoms or indicators of the disease
state or disorder being treated. An improvement of at least 10%
(increase or decrease, depending upon the indicator being measured)
in one or more clinical indicators is considered "effective
treatment," although greater improvements are included, such as
about 20%, 30%, 40%, 50%, 75%, 90%, or even 100%, or, depending
upon the indicator being measured, more than 100% (e.g., two-fold,
three-fold, ten-fold, etc., up to and including attainment of a
disease-free state. Indicators can be physical measurements, e.g.,
enzyme, cytokine, growth factor or metabolite levels, rate of cell
growth or cell death, or the presence or amount of abnormal cells.
One can also measure, for example, differences in the amount of
time between flare-ups of symptoms of the disease or disorder
(e.g., for remitting/relapsing diseases, such as multiple
sclerosis). Alternatively, non-physical measurements, such as a
reported reduction in pain or discomfort or other indicator of
disease status can be relied upon to gauge the effectiveness of
treatment. Where non-physical measurements are made, various
clinically acceptable scales or indices can be used, for example,
the Crohn's Disease Activity Index, or CDAI (Best et al. (1976)
Gastroenterology 70: 439), which combines both physical indicators,
such as hematocrit and the number of liquid or very soft stools,
among others, with patient-reported factors such as the severity of
abdominal pain or cramping and general well-being, to assign a
disease score.
[0108] As the term is used herein, "prophylaxis" performed using a
composition as described herein is "effective" if the onset or
severity of one or more symptoms is delayed or reduced by at least
10%, or abolished, relative to such symptoms in a similar
individual (human or animal model) not treated with the
composition.
[0109] Whereas the domain antibodies described herein bind human
CD28, where one is to evaluate its effect in an animal model
system, the polypeptide must cross-react with one or more antigens
in the animal model system, in an aspect, at high affinity. One of
skill in the art can readily determine if this condition is
satisfied for a given animal model system and a given domain
antibody. If this condition is satisfied, the efficacy of the
domain antibody can be examined by administering it to an animal
model under conditions which mimic a disease state and monitoring
one or more indicators of that disease state for at least a 10%
improvement.
EXAMPLES
Example 1
Pharmacokinetics of PEGylated 1h-239-891 (D70C) in Cynomolgus
Monkeys
[0110] Studies were performed in the cynomolgus monkeys to evaluate
the pharmacokinetics of 1h-239-891(D70C) PEGylated with a 30 kDa
linear (P30L) or a 40 kDa branched PEG (P40B),following single
intravenous (IV) doses of 0.05 and 5 mg/kg, or subcutaneous (SC)
doses of 0.05, 0.5 and 5 mg/kg (mpk).
[0111] Tables 1 and 2 summarize the IV and SC PK parameters of
1h-239-891(D70C)-P30L and -P40B, respectively. Nonlinear
pharmacokinetics were observed for both P30L and P40B PEG formats.
The terminal half-lives (T1/2) of P30L and P40B were 1.6 and 2.5
days, respectively. The absorption of 1h-239-891(D70C) was nearly
complete after SC dosing for P30L at 0.05 mg/kg and for P40B at
0.05 and 5 mg/kg (bioavailability F>90%), but reduced to 70% for
P30L at 5 mg/kg. The steady-state volume of distribution (Vss) was
comparable between the two PEG formats. However, the systemic
clearance (CL) of P40B was 3-fold lower than that of 30L, largely
accounting for differences in the AUC and T1/2 observed between the
two formats. At 0.05 mg/kg, for example, T1/2 could not be
accurately determined because the drug concentration at the
terminal phase was below the level of quantitation (LLQ).
TABLE-US-00003 TABLE 1 PK Parameters of 1h-239-891(D70C)-P30L iv sc
0.05 5 0.05 0.5 5 Dose mpk (n = 1) (n = 1) Dose mpk (n = 2) (n = 3)
(n = 2) AUC.sub.tot nM * h 996 182161 C.sub.max nMolar 21 225 1980
T.sub.1/2 h nd* 39 T.sub.max h 8 8 16 MRT h 15.3 20.1 AUC.sub.tot
nM * h 905 12774 125742 CL mL/min/kg 0.069 0.038 T.sub.1/2 h 33 34
36 V.sub.ss L/kg 0.063 0.045 MRT h 48 56 56 F % 91 69 *Cannot be
accurately determined because drug concentrations at the terminal
phase were below LLQ.
TABLE-US-00004 TABLE 2 PK Parameters of 1h-239-891(D70C)-P40B iv sc
0.05 5 0.05 0.5 5 Dose mpk (n = 1) (n = 1) Dose mpk (n = 2) (n = 3)
(n = 2) AUC.sub.tot nM * h 3704 499857 C.sub.max nMolar 27 410 5075
T.sub.1/2 h 49 70 T.sub.max h 36 27 36 MRT h 61 65 AUC.sub.tot nM *
h 3561 52646 556918 CL mL/min/kg 0.018 0.014 T.sub.1/2 h 56 65 61
V.sub.ss L/kg 0.067 0.054 MRT h 96 107 94 F % 96 100 *Cannot be
accurately determined because drug concentrations at the terminal
phase were below LLQ.
Example 2
BMS-931699 Surrogate Pharmacology
[0112] Surrogate CD28 dAbs that recognize mouse CD28, with
potencies similar to BMS-931699, were used to evaluate the impact
of direct inhibition of CD28 in murine efficacy models. In a mouse
KLH antibody response model, BMS1m-74-14982-P40B (a murine
surrogate for BMS-931699) completely suppressed IgG titers at 0.1
mg/kg in mice, with an in vivo EC.sub.50 of 18 ng/mL. CD28 receptor
occupancy (RO) of .about.100% 24 hours post dose was required for
maximal efficacy. In the same model, mCTLA4Ig completely suppressed
the IgG response at 3 mg/kg, with an in vivo EC.sub.50 of 1656
ng/mL.
[0113] Treatment with BMS1m-74-14982-P40B after disease onset
(therapeutic mode) in the NZB/NZW F1 lupus model, was effective in
reducing proteinuria, serum autoantibody titers, local cytokine
gene expression, and glomerulonephritis/immune complex deposition.
Most endpoints were fully impacted at the 0.5 mg/kg dose, with
survival and anti-dsDNA requiring higher concentrations of 2 and 8
mg/kg, respectively. Taken as a whole, the in vitro and in vivo
studies provide proof of concept for the dAb technology and
confidence that a CD28 dAb should be efficacious in autoimmune
diseases in the clinic.
[0114] The nonclinical safety assessment that supports the clinical
development of BMS-931699 includes: 1) single-dose PK/PD studies
conducted with BMS-908613-P40B in monkeys to support the minimal
anticipated biological effect level (MABEL) dose rationale; 2) a
single-dose exploratory toxicity study in mice with
BMS1m-74-14982-S60C-P40B to assess potential toxicity of CD28
antagonism in a rodent model; 3) pivotal GLP 1- and 6-month
repeat-dose toxicity studies of BMS-931699 in cynomolgus monkeys;
4) an exploratory in vitro study of potential BMS-931699-related
effects (cytokine release, T-cell activation/proliferation) on
human T cells; and 5) a Good Laboratory Practice (GLP) human tissue
cross-reactivity study to demonstrate target distribution and
inform of any potential unexpected epitope binding.
[0115] The cynomolgus monkey was selected as the toxicology species
because BMS-931699 binds comparably to macaque CD28, is
pharmacologically active in monkeys, and does not cross-react with
rodent CD28.
[0116] Intended PD effects in cynomolgus monkeys have been
demonstrated in vivo with BMS-908613-P40B (inhibition of TDARs) and
with BMS-931699 (decreases in cortical lymphocytes in various lymph
nodes which is reflective of decreased germinal center activity).
In vitro, BMS-931699 and BMS-908613-P40B showed similar binding
affinities for human CD28 and similar potency in in vitro mixed
lymphocyte reaction (MLR) assays. The rodent surrogate of
BMS-931699, BMS1m-74-14982-S60C-P40B, was used to assess the
potential for toxicity in mice.
[0117] In the single-dose exploratory toxicity study in mice,
BMS1m-74-14982-S60C-P40B at SC doses up to 18 mg/kg
(AUC.ltoreq.5184 .mu.gh/mL) was not associated with any adverse
drug-related findings. In the single-dose exploratory PK/PD studies
in monkeys, BMS-908613-P40B at SC doses up to 5 mg/kg
(AUC.ltoreq.6793 .mu.gh/mL) was not associated with any adverse
drug-related findings including any effects on plasma cytokines.
BMS-908613-P40B-related effects at doses .gtoreq.0.5 mg/kg were
limited to reversible dose-dependent suppression of primary TDARs
to KLH (IgG), when KLH was administered 24-hours postdose, an
expected pharmacologic effect.
[0118] In a 5-week intermittent dose toxicity study in cynomolgus
monkeys with a 8-week recovery, IV doses of BMS-931699 up to 15
mg/kg (combined sex mean AUC from time zero to 168 hours [AUC(0-168
h)].ltoreq.16,700 .mu.ghr/mL) or a SC dose of 3.5 mg/kg (mean
AUC.ltoreq.3520 .mu.ghr/mL) administered once weekly for 5 weeks
were clinically well tolerated. Notably, BMS-931699-related effects
at all doses (mean [AUC(0-168 h)].gtoreq.1860 .mu.gh/mL on Day 22)
included non-adverse reductions in peripheral blood regulatory T
lymphocytes (Tregs), minimal to mild decreases in cortical
lymphocytes in lymph nodes, and minimal to slight macrophage and/or
epithelial cell vacuolation in various tissues that was not
associated with inflammation or necrotic changes or altered organ
function. Reductions in Tregs and cortical lymphocytes in lymph
nodes were expected pharmacologic effects, while vacuolation in
various tissues was attributed to the PEG moiety of BMS-931699. All
BMS-931699-related effects showed partial to complete resolution
following an 8-week recovery period with the exception of
vacuolation in the choroid plexus epithelium and pituitary gland.
Based on the low severity and lack of associated inflammatory or
degenerative changes, the no-observed-adverse-effect level (NOAEL)
was considered to be 15 mg/kg/week IV mean AUC[0-168 h] of 15,200
.mu.gh/mL on Day 22) and 3.5 mg/kg/week SC (mean AUC[0-168 h] of
3330 .mu.gh/mL on Day 22).
[0119] In a 6-month intermittent dose toxicity study with a 6-month
recovery, BMS-931699 was clinically well tolerated by cynomolgus
monkeys for 6 months when administered as weekly SC doses of
.ltoreq.10 mg/kg (AUC[[0-168 h] 12,100 .mu.gh/mL). The primary
BMS-931699-related findings at all doses were
pharmacologically-mediated decreases in peripheral blood Tregs, B
lymphocytes, serum IgG, and cortical lymphocytes in lymph nodes or
spleen. Given the changes in Tregs were minimal, reversible, and
not associated with any other correlative adverse findings, they
were not considered adverse. Other nonadverse microscopic findings
at .gtoreq.1 mg/kg/week (AUC[0-168 h] 1,350 .mu.gh/mL) included
PEG-related vacuolation of macrophages and/or epithelial cells in
several tissues (choroid plexus of the brain, kidney, axillary
lymph node, and injection sites), and increased thickness of the
kidney interstitium. At .gtoreq.3.5 mg/kg/week (AUC[0-168 h] of
4,450 .mu.gh/mL), PEG-related vacuolation of macrophages in the
pituitary gland and increased incidence and severity of
inflammation and hemorrhage at the subcutaneous injection sites
were noted. Additional findings at 10 mg/kg/week included
PEG-related vacuolation of macrophages in the mandibular and
mesenteric lymph nodes, circumventricular organs of the brain,
urinary bladder, ovaries, uterus, and spleen. All of the
aforementioned findings showed evidence of reversibility (partial
or complete) with the exception of increased thickness of the
kidney interstitium and vacuolation of macrophages in the pituitary
gland, circumventricular organs of the brain, urinary bladder, and
uterus. These findings were not accompanied by degenerative or
inflammatory changes and were considered not adverse. Lymphoma was
noted in 1 female at 1 mg/kg/week that was considered secondary to
BMS-931699-induced immunosuppression in cynomolgus monkeys latently
infected with LCV; based on this finding a NOAEL was not determined
in this study.
[0120] In both the single-dose exploratory PK and PD studies, and
repeat-dose monkey toxicity studies, immunogenicity occurred with
low incidence. Also, the presence of the anti-drug antibodies
(ADAs) did not affect the PK, PD or toxicokinetics of BMS-931669 in
monkeys. No adverse irritation or local intolerance was observed at
the BMS-931699 IV or SC injection sites in either the 5-week or the
6-month studies in monkeys using BMS-931699 concentrations and
injection rates greater than or equal to those recommended for
human use. In an in vitro assay system, purified human T cells were
incubated with BMS-931699 or the superagonist anti-CD28 monoclonal
antibody (mAb) TGN 5.11A1 to monitor for potential effects on
T-lymphocyte activation, proliferation, and cytokine release. There
were no BMS-931699-related effects, while TGN 5.11A1 induced both
cytokine release and cellular activation. In the GLP
tissue-cross-reactivity study using a comprehensive panel of 23
human tissues, binding of BMS-931699 was limited to mononuclear
cells in most human tissues. As CD28 is expressed primarily by T
cells, the staining of blood lymphocytes and mononuclear cells
throughout the human tissue panel was expected reactivity. Overall,
BMS-931699 has demonstrated acceptable pharmacologic, nonclinical
PK, PD, and toxicologic properties that support continued clinical
development.
[0121] Evaluations of the potential effects of IV and/or SC
administration of BMS-931699 on the cardiovascular,
central/peripheral nervous, and/or respiratory systems were
included as part of the pivotal GLP repeat-dose toxicity study in
monkeys. Following 5-weekly IV/SC doses or 6-months of weekly IV
doses of BMS-931699 in monkeys, clinical assessments yielded no
findings for physical, neurologic, and ophthalmologic examinations;
body temperature; heart rates; qualitative and quantitative
electrocardiographic evaluations; respiratory rates; evaluations of
lung sounds and mucous membrane color; and arterial oxygen
saturation determinations, that were considered to be related to
BMS-931699.
Example 3
Preclinical Metabolism and Pharmacokinetics
[0122] The PK of BMS1m-74-14982-S60C-P40Br and BMS-908613-P40Br,
were evaluated in mice and cynomolgus monkeys, respectively. After
intravenous (IV) administration (0.2 mg/kg in mice; 0.05, and 5
mg/kg in monkeys), circulating BMS-1m74-14982-S60C-P40B and
BMS-908613-P40B concentrations exhibited bi-exponential declines.
The steady-state volumes of distribution (Vss) for
BMS1m-74-14982-S60C-P40B and BMS-908613-P40B in mice were (0.13
L/kg) which is greater than the reported plasma volume in mice
indicating that the drug largely resides in the extracellular
space. However, in monkeys, the Vss (0.053 L/kg) for
BMS-908613-P40B was similar to the reported plasma volume,
indicating very limited extravascular distribution. The serum
clearance of BMS-1m74-14982-S60C-P40B in mice and plasma clearance
of BMS-908613-P40B in monkeys were 3.9 mL/h/kg and 0.82 to 1.1
mL/h/kg, respectively. The apparent elimination half-life (T-HALF)
after IV administration was 27 hours in mice and 50 to 71 hours in
monkeys.
[0123] Following single SC administration (0.2 mg/kg in mice; 0.05,
0.5, and 5 mg/kg in monkeys), BMS-1m74-14982-S60C-P40B and
BMS-908613-P40B were well absorbed, with bioavailability of 78% in
mice and 96 to 111% in monkeys, respectively. The time of peak
plasma or serum concentration (Tmax) was generally around 24 hours
(range=8 to 36 hours). In a SC PK study (DS09012), BMS-908613-P40B
exhibited a more than dose-proportional increase in exposure in
female monkeys (at 0.05, 0.5, and 5 mg/kg, peak plasma
concentrations [Cmax] were 0.335, 5.0, and 61.9 .mu.g/mL,
respectively, and the area under the concentration-time curve from
time zero to infinity [AUC(INF)] values were 43.4, 642, and 6790
.mu.g*h/mL, respectively). In a subsequent monkey SC PK/PD study
(DS09013), in which monkeys were immunized with ovalbumin and KLH,
linear PK was observed between 0.05 and 5 mg/kg in males, but, as
in Study DS09012, there was a trend for a greater-than-proportional
increase in exposure in females, but not in males.
[0124] Following intraperitoneal administration (0.08, 0.4, and 2
mg/kg) to mice, BMS 1m74-14982-S60C-P40Br exhibited an
approximately dose-proportional increase in exposure. The Tmax was
4 to 9 hours.
Example 4
First-In-Humans Study: Clinical Pharmacology and Safety
[0125] The early clinical program for BMS-931699 consists of a
First-in-Humans (FIH) single ascending dose (SAD) study (Part 1)
along with a neo-antigen immunization SAD study (Part 2), followed
by a multiple ascending dose (MAD) study in healthy subjects. A
total of 156 subjects were enrolled in the FIH SAD study IM128001,
including 108 subjects in Part 1 (single-ascending dose study) and
48 subjects in SAD Part 2 (KLH immunization). A total of 108
subjects received active study drug and 48 received placebo. In
summary, BMS-931699 was safe and well tolerated after a single dose
of up to 100 mg IV. There were no deaths. No clinically relevant
changes in vital signs, physical findings, or ECGs were reported.
No clinically meaningful changes in pro-inflammatory cytokines were
observed following a single dose of BMS-931699 confirming the lack
of CD28 receptor agonistic activity in humans. Two serious adverse
events (SAEs) were reported, but both were considered to be
unrelated to study drug. These included an event of acute pre-renal
failure and an event of appendicitis. Acute infusion reactions
occurred in 7 subjects; 3 of these events led to discontinuation of
study drug prior to completion of infusion. All acute infusion
reactions were moderate in intensity. Isolated asymptomatic alanine
aminotransferase (ALT) increases were reported in both study
drug-treated and placebo groups and no subject met Hy's criteria.
The most frequent adverse effects (AEs) included headache, feeling
hot, oropharyngeal pain, back pain, pruritus, and upper respiratory
tract infection.
[0126] The MAD study IM128003 was performed in 24 subjects (3
cohorts of 8 subjects) treated for 5 weeks with either SC doses of
BMS-931699 (6.25 mg every other week [EOW], 12.5 mg weekly [QW], or
37.5 mg QW) or placebo (3:1 randomization). As reported in the SAD
study, preliminary results from the MAD study show no evidence of
CD28 agonistic activity, as defined by clinically significant
cytokine release and/or lymphocyte changes. Infections were
observed in the BMS-931699-treated healthy subject cohorts (5/18;
27.8%) but no correlation was observed between exposure and
infection rate.
[0127] In total, 6 infections were reported in 5 subjects, with 5
infections in 4 subjects considered related events. One subject,
dosed at the 12.5 mg weekly regimen, experienced 2 infective
episodes: oral herpes on study Day 40 followed after 7 days by
upper respiratory infection, in both cases the severity was defined
as mild. One subject, dosed at the 12.5 mg weekly schedule,
presented with furuncle (mild severity) on study Day 69. One
subject, dosed at 37.5 mg weekly, on Day 89 presented with a
peritonsillar abscess of moderate severity, which required
antibiotic treatment (500 mg amoxicillin three times a day [TID]
for 10 days. One subject had a mild viral infection on Day 81
following administration of 37.5 mg BMS-931699 weekly, which was
considered unrelated.
[0128] One subject had a SAE of severe cellulitis following
administration of 6.25 mg every 2 weeks. The subject required
hospitalization on Day 49 for cellulitis that developed in his
right hand after damage of the skin at the base of his 3rd finger.
When hospitalized, the patient was treated with IV antibiotics and
the lesion was surgically drained. The SAE followed traumatic skin
damage to his right hand, providing a breach of skin integrity for
the development of cellulitis. However it could not be excluded
that BMS-931699 might have made the subject more susceptible to the
subsequent infection. Therefore the SAE was considered possibly
related to the study drug.
Pharmacokinetics of BMS-931699
Pharmacokinetics Summary of IM128001: Single Ascending Dose
Study
[0129] The SAD study IM128001 evaluated BMS-931699 in the dose
range of 0.01 mg to 100 mg (0.01, 0.05, 0.25, 3, 9, 25, 50, and 100
mg) following 30-minute IV infusion, and doses of 9, 25, and 50 mg
following SC administration. BMS-931699 exhibited linear PK after
single IV and SC administration. The Cmax and AUC(INF) of
BMS-931699 administered IV and SC increased approximately in
proportion to dose over the range of 3 mg to 100 mg. AUC and Cmax
values of BMS-931699 increased in a dose-proportional manner
following single doses of 3 mg to 100 mg IV and 9 mg to 50 mg SC in
healthy subjects.
[0130] The mean total body clearance (CLT), V.sub.Z and V.sub.SS
were in the range of 0.42-0.59 L/min, 3.4-5.1 L, and 3.2-4.5 L
respectively, and relatively consistent among all the dose groups
following single IV administration. The mean apparent total body
clearance (CLT)/F and V.sub.Z/F were in the range of 0.59-0.70
L/min and 6.0-7.3 L, respectively, and relatively consistent among
all the dose groups following single SC administration. The T-HALF
of BMS-931699 was similar following a single dose or multiple doses
in healthy subjects (4 to 5.5 days for single dose and 6 to 7 days
for multiple doses). Bioavailability of BMS-931699 following SC
administration on AUC(INF) was 68.2%.
Pharmacokinetics Summary of IM128003: Multiple Ascending Dose
Study
[0131] The MAD study IM128003 evaluated BMS-931699 in three
treatment groups: 6.25 mg every other week (3 doses), 12.5 mg
weekly (5 doses), and 37.5 mg weekly (5 doses). Following every
other week and weekly SC administration, the pharmacokinetics of
BMS-931699 is linear over the range of 6.25 mg every other week to
37.5 mg weekly. The geometric mean of accumulation index of AUC
were 1.3, 2.4 and 3 for 6.25 mg EOW, 12.5 mg QW and 37.5 mg QW,
respectively.
[0132] Following multiple doses of BMS-931699, and a median T-HALF
of 6 to 7 days were observed. The mean CL/F (0.345 to 0.46 L/min)
is consistent with what was observed in SAD study. The reason of a
slightly longer T-HALF reported in the MAD compared to the SAD
study is likely due to the fact that more measurable concentration
data at the terminal phase was available to estimate T-HALF of the
BMS-931699 in the MAD study.
Example 5
Phase 2 Clinical Trials Dose Selection Rationale
[0133] Four treatments of BMS-931699 are selected for initial
evaluation in humans in a Phase 2 dose ranging study, including
1.25 mg every other week, 5 mg every other week, 12.5 mg every
other week, and 12.5 mg weekly. These dosing regimens were selected
based on the PK/PD relationship using receptor occupancy (RO) and
IgG suppression following Keyhole Limpet Hemocyacin (KLH)-antigen
challenge as predictive markers for immunosuppressive activity and
clinical efficacy in SLE patients.
[0134] Using the PK/PD model for receptor occupancy based on MAD
data in humans, simulations were performed to identify dosing
regimens that would provide a wide range of receptor occupancy to
span the PK-receptor occupancy curve. These simulations predict
that the 1.25 mg every other week, 5 mg every other week, 12.5 mg
every other week, and 12.5 mg every week regimens provide
approximately .gtoreq.40%, .gtoreq.70%, .gtoreq.80%, .gtoreq.90%
receptor occupancy, respectively, throughout the dosing interval
for majority of the patients (FIG. 4). The distribution of the
receptor occupancy associated with the 1.25 mg every other week and
5 mg every other week regimen, fall on the steep portion of the
PK-receptor occupancy curve, while the 12.5 mg every other week and
every week regimens fall on the maximal plateau portion.
[0135] The preclinical monkey data suggested that >80% receptor
occupancy for 2 weeks is needed for maximum immunosuppression as
measured by IgG suppression following KLH-antigen challenge, and as
levels of receptor fall below 80%, the immunosuppressive activity
lessens and anti-KLH antibody formation rises. Results from the
single ascending dose (SAD) study substantiate this premise. Near
maximal IgG suppression was observed across treatment groups when
approximately 80% receptor occupancy was achieved. Notably, in the
lowest KLH treated group (9 mg), when the % RO declined below 80%,
there was a subsequent rebound in IgG formation. Therefore, it is
believed that high levels of target engagement, for example 80%
receptor occupancy or greater, are needed to elicit
immunosuppressive activity.
[0136] The exposures associated with the lowest proposed dose of
1.25 mg every other week dosing regimen are expected to be very low
(5 times lower than the lowest dose tested in the MAD study in
normal healthy volunteers) with a large projected safety margin
(50.times.) from the lowest dose tested in 6-month monkey study.
The predicted receptor occupancy distribution for the 1.25 mg every
other week is expected to fall below 80% for the entire treatment
population, while this dose is expected to maintain receptor
occupancy above 40% for majority of the human subjects. Based on
the above findings of the correlation between 80% receptor
occupancy and KLH suppression, the 5 mg every other week regimen is
expected to elicit >80% receptor occupancy in approximately 50%
of the patients, thereby providing some immunosuppressive activity,
which is expected to induce some clinical response, albeit
suboptimal. Furthermore the exposure (AUC(TAU)) associated with
this dosing regimen are fairly low with a large projected safety
margin (12.7.times.) from the lowest tested dose in 6-month monkey
toxicology study. The higher doses of 12.5 mg every other week and
every week are expected to provide >80% and >90% receptor
occupancy for the entire treatment population, and expected to
elicit near maximal immunosuppressive activity potentially leading
to near maximal efficacious response. The exposures associated with
these 2 regimens are considerably lower (5.1.times. and
2.5.times.), than that of the lowest tested dose in 6-month monkey
study. The projected exposures of the four doses are within the
range of exposures tested in the MAD study in healthy human
subjects. The highest dose in this POC study will still be one
third of the highest dose in the MAD study.
Example 6
Study Design and Duration
[0137] A Phase 2, parallel-arm, randomized, double-blinded,
multicenter, international study, with an adaptive design is
conducted. The study comprises a short-term period which has two
parts: Part 1 (FIG. 1) and Part 2 (FIG. 2), and a long-term
extension (LTE) period (FIG. 3).
[0138] Part 1 focuses on assessing safety and receptor occupancy.
Part 1 is limited to 30 to 50 patients (approximately 6-10
patients/arm) are included in the Safety/RO interim analysis (IA).
The 6 to 10 patients per arm complete 28 days (4 weeks) of
treatment. These patients continue study treatment beyond the IA
for Safety/RO and be treated for up to 24 weeks and be followed for
6 additional weeks after treatment is completed. A total of up to
50 randomized subjects are treated for up to 6 months. Treatment
could be shorter if IA for Safety/RO analysis indicates that one or
more arms should be dropped.
[0139] Part 2 is opened once the cumulative safety profile is
considered acceptable and receptor occupancy data for 6-10 subjects
per cohort dosed for >28 days is available. Approximately 300
subjects are randomized into this part of the study (number of
patient to be randomized into Part 2 may increase based on the
results of the futility interim analysis). All subjects undergo
screening evaluations to determine eligibility and allow down
titration of prednisone (or prednisone equivalent) within 28 days
prior to administration of their first dose of study medication.
The dose may be adapted as follows: [0140] Safety and receptor
occupancy (RO) interim analysis: a safety and receptor occupancy
analysis is performed when approximately 6 to 10 subjects per arm
have reached Study Day 29 (4 weeks of treatment). Based on the
results of the analysis, the arm dose may be adjusted or dropped
completely. [0141] Interim analysis for futility and dose adaption
on BICLA response, SLE Responder Index response, ACR28 and some SLE
biomarkers (such as auto-antibodies, complement levels, etc.) with
possible exploratory exposure response analysis, is performed when
approximately 30 subjects per arm have reached Study Day 85 (12
weeks of treatment or discontinued). Analysis is performed by an
unblinded Sponsor team, while maintaining blind at the site and
subject level. Based on the results, the dose levels and sample
size may be modified. [0142] Ongoing assessment of safety is
performed by an independent Data Monitoring Committee (DMC) and an
internal unblinded safety monitoring team. Both entities may make
recommendations to the Sponsor regarding conduct of study and dose
adjustment based on safety observations.
[0143] The long-term extension (LTE) period is optional and
includes eligible subjects who have completed Day 169 (week 24) of
treatment and consent to participate. This period of the study
remains blinded but no longer has a placebo arm. Eligible subjects
remain on their originally-assigned dose arm, unless they were on
the placebo arm during the short-term period. Placebo-arm subjects
are re-randomized into one of the existing active arms at Day 169
(24 weeks). Re-randomization of the placebo subjects is done by
IVRS and only the unblinded pharmacist/drug preparer knows the new
randomization arm. The LTE remains blinded to the study team and
study personnel.
[0144] The approximate duration of the study is 28 days (4 weeks)
of screening, 168 days (24 weeks) of treatment, and 42 days (6
weeks) of safety follow-up, for a total of approximately 238 days
(34 weeks) in the short-term period. If the subject is eligible and
opts to continue into the LTE, the 42 day follow-up visit is
performed after the short-term period is completed and the subject
enters LTE directly. If the subject opts not to enter LTE then a
follow-up visit is completed 42 days after end of treatment. At the
time of writing there is no defined end date to the long term
extension period, however, the LTE provision may be further
adjusted based on results from the ongoing lulizumab development
program. Subjects discontinuing treatment during the LTE period
complete the follow-up visit approximately 6 weeks after receiving
their last dose of study medication.
[0145] Subjects randomized in either Part 1 or Part 2 are treated
for up to 24 weeks and have the same procedures performed and
follow the same visit schedule.
Example 7
Overall Risk/Benefit Assessment
[0146] This Example summarizes potential risks of treatment with
BMS-931699 and the precautions that are required in clinical
studies. This assessment is based on nonclinical data and the
clinical experience to date with BMS-931699 set forth in the
previous Examples.
[0147] Blocking the function of CD28 is expected to modulate the
immune response. Modulation of immune response may predispose to
infection. In nonclinical studies, there was no evidence that
BMS-913699 treatment resulted in bacterial or viral infection. To
date, single doses and repeat doses of BMS-931699 have been
administered in healthy subjects. Based on results from both
single- and repeat-dose IV and SC nonclinical toxicology studies in
cynomolgus monkeys and the clinical data from studies IM128001 and
IM128003, BMS-931699 has demonstrated an acceptable safety profile,
supporting continued development.
[0148] The expected exposure [AUC(TAU)] at steady state for the
highest proposed dose 12.5 mg weekly is approximately 2.5.times.
lower than the lowest tested dose of 1 mg/kg/week in the 6 month
toxicity study in monkeys where minimal to moderate vacuolation of
macrophages were observed. The highest dose in this Phase 2 study
is one third of the highest dose in the MAD study. Intense safety
monitoring is put in place during Part 1 of Phase 2, allowing early
detection of any safety signals.
Example 8
Dose Adaptation
[0149] As discussed above, the Phase 2 study initiates with the 4
regimens of 1.25 mg every other week, 5 mg every other week, 12.5
mg every other week, and 12.5 mg weekly. Safety/receptor occupancy
interim analysis is performed to ensure the exposures and receptor
occupancy observations approximate the original predictions.
[0150] An interim analysis (IA) for safety and receptor occupancy
is performed in Part 1 of the study, when at least 6 patients per
treatment arm have reached Study Day 29. That receptor occupancy
observations approximate the original predictions and BMS-931699 is
well tolerated in SLE patients is confirmed. The rest of the
patients are enrolled in the study.
[0151] Based on the results of this interim analysis, dosing
regimens originally included in Part 1 may be discontinued and/or
new dosing regimens may be added according to the criteria outlined
below. Enrollment in Part 2 of the study is opened after the IA has
resulted in a decision regarding the dosing regimens to be carried
forward:
[0152] Safety
[0153] The safety analysis focuses on incidence and severity of all
adverse events (AEs), serious AEs and pre-established Events of
Special Interest such as infection AEs and any other safety
analysis requested by DMC. The DMC in conjunction with an unblinded
internal safety monitoring team may require one or more doses to be
discontinued if stopping criteria are met or other safety signals
arise that the Medical Monitor and/or DMC consider of sufficient
concern. The unblinded safety monitoring team is not involved in
the regular study activities.
[0154] Receptor Occupancy
[0155] The median receptor occupancy at Day 29 for each treatment
arm is calculated. [0156] If median receptor occupancy of any dose
is <20%, the sponsor considers dropping that dose. [0157] If the
median receptor occupancy for all doses is >90% the sponsor
considers adding or replacing a dose in Part 2 of the study to
ensure an adequate pharmacodynamic range (dose not to exceed 12.5
mg weekly).
[0158] Dose decrease and/or reduction of frequency of
administration is considered if receptor occupancy results fall
outside the parameters indicated above. This adjustment occurs for
safety reasons or in case unforeseen receptor occupancy profiles
are observed in SLE patients. The decision to adjust dose and/or
frequency is taken after review of the data by the clinical team.
Subjects do not change dose. If an arm is modified or removed,
subjects randomized to that arm and discontinued.
[0159] In Part 2 of the study, an interim analysis for futility and
dose adaption occurs after 30 patients per treatment arm (including
patients from Part 1) have completed at least 85 days of treatment
period or discontinued. One or more treatment arms may be dropped
for lack of efficacy, or a treatment arm may be added to explore a
suboptimal dose. Exposure-response analysis for efficacy and safety
may be conducted in parallel with this interim analysis to
facilitate the dose selection for this additional lower treatment
arm.
[0160] The long-term extension period has interim analyses for dose
adaptation. If a significant safety concern is identified, one or
more dose arms may be dropped. If one or more dose arms are dropped
for safety reasons, all subjects currently receiving that or those
dose(s) are discontinued from receiving study medication.
Example 9
Inclusion Criteria
[0161] Men or women (not nursing or pregnant) between 18 and 70
years of age who meet the American College of Rheumatology criteria
for the classification of Systemic Lupus Erythematosus are eligible
(Table 3). The classification is based on 11 criteria. For the
purpose of identifying patients in clinical studies, a person shall
be said to have systemic lupus erythematosus (SLE) if any 4 or more
of the 11 criteria are present, serially (sequentially) or
simultaneously (coincident), during any interval of observation.
Four criteria must be met prior to dosing on Day 1 for entry into
the study. However, the 4 criteria need not be present at study
entry, but have occurred at some time during the course of the
disease and be documented:
TABLE-US-00005 TABLE 3 The 1982 Revised Criteria for Classification
of Systemic Lupus Erythematosus Criterion Definition 1) Malar rash
Fixed erythema, flat or raised, over the malar eminences, tending
to spare the nasolabial folds 2) Discoid rash Erythematous raised
patches with adherent keratotic scaling and follicular plugging;
atrophic scarring may occur in older lesions 3) Photosensitivity
Skin rash as a result of unusual reaction to sunlight, by patient
history or physician observation 4) Oral ulcers Oral or
nasopharyngeal ulceration, usually painless, observed by physician
5) Arthritis Nonerosive arthritis involving 2 or more peripheral
joints, characterized by tenderness, swelling, or effusion 6)
Serositis a) Pleuritis--convincing history of pleuritic pain or
rubbing heard by a physician or evidence of pleural effusion OR b)
Pericarditis--documented by ECG or rub or evidence of pericardial
effusion 7) Renal disorder a) Persistent proteinuria greater than
0.5 grams per day or greater than 3+ if quantitation not performed
OR b) Cellular casts--may be red cell, hemoglobin, granular,
tubular, or mixed 8) Neurologic a) Seizures--in the absence of
offending drugs or known metabolic disorder derangements; e.g.,
uremia, ketoacidosis, or electrolyte imbalance OR b) Psychosis--in
the absence of offending drugs or known metabolic derangements,
e.g., uremia, ketoacidosis, or electrolyte imbalance 9) Hematologic
a) Hemolytic anemia--with reticulocytosis disorder OR b)
Leukopenia--less than 4,000/mm.sup.3 total on 2 or more occasions
OR c) Lymphopenia--less than 1,500/mm.sup.3 on 2 or more occasions
OR d) Thrombocytopenia--less than 100,000/mm.sup.3 in the absence
of offending drugs 10) Immunologic a) Anti-DNA: antibody to native
DNA in abnormal titer disorder OR b) Anti-Sm: presence of antibody
to Sm nuclear antigen OR c) Positive finding of anti-phospholipid
antibodies based: 1) an abnormal serum level of IgG or IgM
anti-cardiolipin antibodies, 2) a positive test result for lupus
anticoagulant using a standard method, or 3) a false positive
serologic test for syphilis known to be positive for at least 6
months and confirmed by Treponema pallidum immobilization or
fluorescent treponemal antibody absorption test. 11) Antinuclear An
abnormal titer of antinuclear antibody by immunofluorescence
antibody or an equivalent assay at any point in time and in the
absence of drugs known to be associated with "drug-induced lupus"
syndrome
[0162] In addition: [0163] Subjects have elevated anti-nuclear
antibody at screening of .gtoreq.1:80 via immunofluorescent assay
at the central laboratory and/or positive anti-dsDNA and/or anti-Sm
above the normal level as determined by the central laboratory. (If
central laboratory results are negative and positive results are
documented at the site, a single repeat of the central laboratory
values is allowed.) [0164] Subjects also have a Systemic Lupus
Erythematosus Disease Activity Index 2000 (SLEDAI-2K) score at
screening of .gtoreq.6 to be eligible. At least 4 of the points are
attributable to clinical criteria including at least one of the
following clinical parameters: arthritis, rash, myositis, mucosal
ulcers, pleurisy, pericarditis, vasculitis and excluding points
from lupus headache and organic brain syndrome. [0165] On Day 1,
subjects have a SLEDAI-2K score of .gtoreq.4 including points from
at least one of the following clinical components: arthritis, rash,
myositis, mucosal ulcers, pleurisy, pericarditis, vasculitis, and
fever and excluding parameters which require central laboratory
results (hematuria, pyuria, urinary casts, proteinuria, positive
anti-dsDNA, decreased complement, thrombocytopenia and leukopenia).
Points from lupus headache and organic brain syndrome are excluded.
[0166] Subjects have at least one of the following manifestations
of SLE, as defined by the British Isles Lupus Assessment Group
(BILAG) 2004 criteria as modified for use in this study: [0167] (1)
BILAG A or B score in the Mucocutaneous body system [0168] (2)
BILAG A or B score in the Musculoskeletal body system due to active
polyarthritis defined as follows: [0169] (a) "BILAG A": severe
arthritis (BILAG #41) manifested by observed active synovitis in
.gtoreq.2 joints with marked loss of functional range of movements
and significant impairment of basic activities of daily living,
that has been present on several days cumulatively over the past 4
weeks, including at the time of the screening visit. Basic ADLs are
defined as the following activities which require assistance or
assistive devices (at least one must be present and documented in
source): ambulation, toileting, grooming including bathing,
dressing, feeding oneself (not responsive to steroids up to 10
mg/day, antimalarials, NSAIDs). [0170] (b) "BILAG B": Moderate
arthritis or tendonitis or tenosynovitis (BILAG #42) defined as
tendonitis/tenosynovitis or active synovitis in .gtoreq.1 joint
(observed or through history) with some loss of functional range of
movements which lead to some loss of functional range of motion as
manifested by effects on instrumental ADLs (such as cooking,
driving, using the telephone or computer, shopping, cleaning, etc.)
which has been present on several days over the last 4 weeks and is
present at the time of the screening visit. [0171] (3) if only one
"B" and no "A" score is present in the Mucocutaneous body system or
in the Musculoskeletal body system due to arthritis, then at least
one B must be present in the other body systems for a total of 2
"B" BILAG body system scores.
[0172] Unless intolerant, subjects must be currently receiving at
least one of the following steroid-sparing agents for a minimum of
12 weeks, and a stable dose for at least 56 days (8 weeks) prior to
signing consent: azathioprine (AZA), chloroquine,
hydroxychloroquine, methotrexate (MTX), leflunomide, mycophenolate
mofetil/mycophenolic acid. Subjects must remain on stable dose
throughout the study.
[0173] Prednisone (or prednisone-equivalent) is not required;
however, if subject is taking prednisone (or prednisone
equivalent), the dose cannot exceed 30 mg/day at screening for a
subject to be eligible and must be stable at a maximum of 10 mg/day
for at least 5 days prior to Day 1 (randomization). Any other
immunossuppressive or biologic drug requires washout periods prior
to study entry. If subjects are receiving non-steroidal
anti-inflammatory drugs (NSAIDs) (including marketed COX-2
inhibitors), doses must be stable for 14 days prior to first dose
of study medication on Day 1 (randomization) and subject must
remain on the same dose throughout the study. Note: NSAIDS should
be withheld for at least 12 hours prior to visits where BILAG,
SLEDAI 2K, joint counts, Cutaneous Lupus Erthematosus Disease Area
and Severity Index (CLASI) and MDGA is assessed.
[0174] Female patients of childbearing potential have a negative
serum or urine pregnancy test (minimum sensitivity 25 IU/L or
equivalent units of human chorionic gonadotropin) within 24 hours
prior to the start of study drug administration. Female patients
are not breastfeeding. Female patients of childbearing potential
must use contraception for the duration of treatment with study
drug plus 5 half-lives of study drug (7 days) plus 30 days
(duration of ovulatory cycle) for a total of 65 days post-treatment
completion. Males who are sexually active with female patients of
childbearing potential must use contraception for the duration of
treatment with study drug plus 5 half-lives of the study drug (7
days) plus 90 days (duration of sperm turnover) for a total of 125
days post-treatment completion. Azoospermic males and women of
child bearing potential who are continuously not heterosexually
active are exempt from contraceptive requirements. However they
must still undergo pregnancy testing.
Example 10
Exclusion Criteria
[0175] The following subjects are not enrolled in the Phase 2
study:
[0176] a) Subjects with drug-induced SLE, rather than "idiopathic"
SLE.
[0177] b) Subjects with other autoimmune disease [(for example
rheumatoid arthritis (RA), multiple sclerosis (MS)]. (Subjects with
type 1 diabetes mellitus, thyroid autoimmune disease and secondary
Sjogren syndrome are eligible.)
[0178] c) Subjects with primary anti-phospholipid antibody syndrome
as the sole or primary feature of their SLE or SLE-like syndrome
are excluded. However, subjects with secondary anti-phospholipid
syndrome are included in the study, unless they have had a serious
thrombotic event (e.g. pulmonary embolism, stroke, or deep vein
thrombosis) within one year prior to signing consent. Subjects on
chronic coumadin or enoxaparin can be enrolled in the study.
[0179] Subjects with the following medical conditions, concominant
illnesses and medical histories are not enrolled in the Phase 2
study:
[0180] a) Subjects with any major surgery within 6 weeks of study
drug administration (Day 1) or any elective surgery planned during
the course of the study.
[0181] b) Subjects with any history or risk for tuberculosis (TB),
specifically subjects with: [0182] (1) Current clinical,
radiographic or laboratory evidence of active TB. [0183] (2) A
history of active TB within the last 3 years, unless there is
documentation that prior anti-TB treatment was appropriate in
duration and type according to current World Health Organization
Guidelines. [0184] (3) Latent TB defined as Positive quantiferon
(QFG) or other diagnostic test in the absence of clinical
manifestations, unless subject has received at least 1 month
treatment with Isoniazid, or other agents recommended by local
Health Authority guidelines, and an interferon gamma release assay
(IGRA) test, eg, QFG or T-Spot, is negative before Day 1. [0185]
(4) Positive QFG test (or other diagnostic test) at screening or
within 3 months prior to Day 1 is acceptable as long as there is
documentation of a negative result by Day 1.
[0186] c) Subjects with active or unstable lupus neuropsychiatric
manifestations, including but not limited to any condition defined
by BILAG "A" criteria, with the exception of mononeritis multiplex
and polyneuropathy which are allowed.
[0187] d) Subjects with active, severe, lupus nephritis (WHO class
III, IV) which requires or may require treatment with cytotoxic
agents or high dose corticosteroids. Subjects with prior,
controlled renal disease with residual proteinuria up to 3 g/day or
a urine protein/creatinine ratio of 3 mg/mg or 339 mg/mmol are
allowed.
[0188] e) Subjects with herpes zoster that resolved less than 2
months prior to screening.
[0189] f) Subjects with evidence (as assessed by the Investigator)
of active or latent bacterial or viral infections at the time of
potential screening, including subjects with evidence of Human
Immunodeficiency Virus (HIV) infection as defined by positivity of
HIV-1, -2 antibody.
[0190] g) Subjects currently on hydroxychloroquine or chloroquine
with evidence of retinopathy within 6 months of screening or who
have had no ophthalmologic evaluation within one year of screening
and do not have this examination done or who are unwilling or
unable to have regular ophthalmologic examinations while
participating in the study.
[0191] h) Concomitant illness that, in the opinion of the
investigator, is likely to require additional systemic
glucocorticosteroid therapy during the study, (e.g., asthma) is
exclusionary. However, treatment for asthma with inhalational
corticosteroid therapy is allowed.
[0192] i) Female subjects with a breast cancer screening suspicious
for malignancy, and in whom the possibility of malignancy cannot be
reasonably excluded following additional clinical, laboratory or
other diagnostic evaluations.
[0193] j) Subjects with a history of cancer within the last five
years (other than non-melanoma skin cell cancers cured by local
resection). Existing non-melanoma skin cell cancers must be removed
prior to randomization (Day 1 treatment). Carcinoma in situ,
treated with definitive surgical intervention, is allowed.
[0194] k) Subjects with any acute and/or chronic serious bacterial
or viral infection (such as pneumonia, renal infection and
sinusitis). Documentation of resolution must be available in
medical chart prior to Day 1 (randomization).
[0195] g) Donation of blood to a blood bank or in a clinical study
(except a screening visit) within 4 weeks of study drug
administration (within 2 weeks for plasma only).
[0196] h) Blood transfusion within 4 weeks of study drug
administration.
[0197] i) Subjects with an inability to be venipunctured and/or
tolerate venous access.
[0198] j) Subjects with a history of any significant drug allergy
(such as anaphylaxis or hepatotoxicity).
[0199] k) Any other sound medical, psychiatric, and/or social
reason as determined by the investigator.
[0200] Subjects with the following medical conditions, concominant
illnesses and medical histories are not enrolled in the Phase 2
study:
[0201] a) Evidence of organ dysfunction or any clinically
significant deviation from normal in physical examination, vital
signs, ECG or clinical laboratory determinations beyond what is
consistent with the target population.
[0202] b) Positive hepatitis-B surface antigen.
[0203] c) Positive hepatitis-C antibody with positive Recombinant
ImmunoBlot Assay (RIBA) or Polymerase Chain Reaction (PCR).
[0204] d) White blood cells (WBC) <1,200/mm3
(1.2.times.109/L).
[0205] e) Platelets <50,000/mm3 (50.times.109/L).
[0206] f) Hemoglobin <8 g/dL or <7 g/dL if due to hemolytic
anemia related to SLE.
[0207] g) Proteinuria >3.0 g/day (3000 mg/day) or equivalent
level of proteinuria as assessed by protein/creatinine ratio (3
mg/mg or 339 mg/mmol).
[0208] h) Serum creatinine >2.0 mg/dL.
[0209] i) Active urinary sediment defined as red blood cell (RBC)
casts.
[0210] j) Serum alanine aminotransferase (ALT) >2.times. upper
limit of normal (ULN), unless explicitly related to lupus based on
the Investigator's judgment.
[0211] k) Serum aspartate aminotransferase (AST) >2.times.ULN,
unless explicitly related to lupus based on the Investigator's
judgment.
[0212] l) Positive urine screen for illegal drugs of abuse, except
if these drugs are prescribed by the treating physician (must be
documented), and except for other drugs that are not illegal within
the country or the region
[0213] m) Any other laboratory test results that, in the opinion of
the Investigator, might place subject at unacceptable risk for
participating in this study.
[0214] Prohibited and/or restricted medications taken prior to
study drug administration in the study are described below:
[0215] 1) Prior exposure to BMS-931699.
[0216] 2) Use of any other drugs, including over-the-counter
medications and herbal preparations, within 1 week prior to study
drug administration except those medications cleared by the BMS
medical monitor.
[0217] 3) The use of cyclophosphamide, any intravenous, any
intra-articular or biologic agent is prohibited during the
study.
[0218] 4) For subjects who develop neutropenia (absolute neutrophil
count <1.3.times.103/.mu.L), dosing with mycophenolate
mofetil/mycophenolic acid should be interrupted or dose reduces as
per the package insert.
[0219] 5) Subjects who have received any live vaccines within 30
days of screening. (Furthermore, live vaccines should not be used
within the 2 months following last dose and any other inactivated
vaccines such as tetanus etc. should be used according to local
guidelines if at all during treatment period.)
[0220] 6) Subjects who are scheduled or anticipated to have
elective surgery during the course of the study.
[0221] No concomitant medications (prescription, over-the-counter
or herbal) are to be administered during study unless they are
prescribed by the investigator for treatment of specific clinical
events.
Example 11
Administration of BMS-931699 or Placebo
[0222] BMS-931699 or a look alike placebo is administered weekly as
a solution subcutaneously (SC) single injection, dependent on the
dosage panel. The clinical label reflects the product name as
"BMS-931699-01" to be linked with the product description on the
vial. The composition of the BMS-931699-01 injection is 12.5 mg/mL
BMS-931699 in 20 mM phosphate, pH 5.9, with 5% (w/v) sorbitol. The
BMS-931699-01 injection is packaged in a 3 cc vial with a 13 mm
opening, 1-panel, open label. The BMS-931699-1 injection appears
clear to slightly opalescent, colorless to pale yellow solution.
The BMS-931699-01 injection is stored refrigerated 2-8.degree. C.
(36-46.degree. F.).
[0223] Table 4 below indicates the total dose and number of vials
per dose for each dosage panel.
TABLE-US-00006 TABLE 4 Treatment Administration Formulation
Treatment Total Daily Dose Strength Number of Vials 1 1.25 mg SC
EOW 12.5 mg/mL 1 2 5 mg SC EOW 12.5 mg/mL 1 3 12.5 mg SC EOW 12.5
mg/mL 1 4 .sup. 12.5 mg SC Weekly 12.5 mg/mL 1 5 Placebo Placebo
Normal Saline Solution (NSS)
[0224] For subcutaneous (SC) dosing, no dilution of the drug
product solution (12.5 mg/mL) is required for doses of 12.5 mg.
However, doses of 5 mg and 1.25 mg require dilution. A 21-gauge,
1.5 inch (3.8 cm) sterile needle is used for withdrawal of this
product from the vial, and a 27 gauge, 0.5 inch (1.3 cm) sterile
needle is used for SC dosing. A conventional, commercially
available polycarbonate syringe of appropriate size is used for
withdrawal and administration. After withdrawal into an appropriate
sized syringe, the product is administered within 4 hours. If not
dosed immediately, filled syringes are kept at 2.degree.-8.degree.
C. (36.degree.-46.degree. F.) with protection from light prior to
use. The placebo for BMS-931699 injection is a normal saline
solution, which is administered in a similar fashion as described
for the BMS-931699 injection. Study personnel administer the dose
to the subject.
[0225] On Day 1, subjects are randomized to one of the dosing arms
in Table 4 in a 1:1:1:1:1 randomization scheme. In the morning on
Day 1, each subject receives a single SC dose of either BMS-931699
or placebo. The primary point of injection is one of the upper
arms. However other points of injections are acceptable. There are
no restrictions related to food and fluid intake associated with
BMS-931699 known at this point.
[0226] Every randomized subject is required to come to the
clinic/research center weekly to be dosed. This ensures
double-blind is maintained despite the variability of regimens.
Subjects randomized to weekly subcutaneous injections of either
placebo or BMS-931699 are dosed weekly as per schedule and subjects
randomized to one of the every other week arm are alternating
between receiving a subcutaneous injection of BMS-931699 one week
and one of placebo the following week.
Example 12
Biomarkers
[0227] Pharmacodynamic, target engagement and disease related
biomarker assays are incorporated into the study inform dose
selection, monitor efficacy and potentially predict treatment
response. Blood and urine are drawn for the measurement of markers
of target engagement and phamacodynamic effects of BMS-931699
including CD28 receptor occupancy, C3, C4, and auto-antibodies.
[0228] Target engagement, as assessed by CD28 receptor occupancy on
T cells, is incorporated into the study to inform dose selection
for the Phase 3 study, monitor efficacy and potentially predict
treatment response. The relationship between the concentration
levels of BMS-931699 and the CD28 receptor occupancy is
characterized.
[0229] Other biomarkers include: other cytokines and chemokines,
anti-double-stranded deoxyribonucleic acid (anti-dsDNA),
anti-nuclear antibody (anti-ANA), anti-Ro (otherwise known as
anti-SSA, anti-SSA/RO, or anti-Ro/SSA) autoantibodies, anti-Lupus
(anti-La) (otherwise known as anti-Sjogren syndrome type B antigen
(anti-SS-B)) autoantibodies, anti-ribonuclear protein (anti-RNP)
autoantibodies, anti-Sm nuclear antigen autoantibodies, anti-APL
autoantibodies, and other autoantibodies. C-reactive protein (CRP),
total immunoglobulin G (IgG), total immunoglobulin M (IgM), RNA
transcripts in whole blood, proteins in urine (NGAL, TWEAK, MCP-1,
IL-18, IL-1), total soluble CD28, T cell activation, leukocyte
phenotypes in peripheral blood mononuclear cells (PMBCs) and whole
blood (surface CD4, surface CD8, surface CD28, surface CD57 and
intracellular granzyme B), soluble inflammatory mediators (serum
IL-6, IL-18, TNF-.alpha., .alpha.-interferon, BLyS(BAFF), CD154,
sCD28 and other soluble receptors, microvessicles) can also serve
as biomarkers.
[0230] Some pharmacodynamic endpoints relevant to SLE are
characterized below:
Blood-Based (RNA) Assessments
[0231] The whole blood ribonucleic acid (RNA) sample is collected
in PAX gene tubes at times indicated. These samples provide broad
genomic profiling to search for novel pharmacodynamic and efficacy
biomarkers related to inflammatory and/or autoimmune pathways.
Furthermore, these samples are used to search for gene expressions
at baseline that may be predictive of efficacy for BMS-931699
treated subjects.
Leukocyte Phenotyping
[0232] Peripheral blood is collected for immunophenotyping by flow
cytometry. T cells may be characterized for activation and for
subpopulations. Markers may include combinations of, but not
limited to, surface CD4, CD8, CD28, CD57, and intracellular
granzyme B. Other peripheral cells, including B cells,
monocytes/macrophags, dendritic cells, and NK cells may be
analyzed.
Urine Biomarkers
[0233] Urine is collected and analyzed for markers of SLE and other
inflammatory disorders. The samples are analyzed for proteomic
profiles of inflammatory markers (including but not limited to
IL-18, IL-1, NGAL, uTWEAK, MCP-1). Exploratory analysis is carried
out to identify biomarkers to monitor PD and response to therapy in
patients with kidney involvement.
Peripheral Blood Serum and Plasma Biomarkers
[0234] Serum and plasma is collected for the measurement of soluble
inflammatory mediators associated with inflammation, SLE or
co-stimulation blockade (including, but not limited to, serum IL-6,
IL-18, TNF-.alpha., .alpha.-interferon, BLyS(BAFF), CD154, sCD28
and other soluble receptors, microvessicles). Exploratory analysis
is carried out to identify biomarkers of SLE, and to monitor PD and
the impact of BMS-931699 on inflammatory pathways.
Outcomes Research Assessments
[0235] Subjects complete the Functional Assessment of Chronic
Illness Therapy-Fatigue (FACIT-F), the FDA Short Form-36
Questionnaire (SF-36) and the Subject's Global Assessment of
Disease Activity (PGA). These pages are source documents in this
study. All outcome research assessments should be completed prior
to study drug administration at scheduled office visits.
Exploratory Efficacy Outcome Measures
Health-Related Quality of Life
[0236] The SF-36 is used to measure health-related quality of life.
Individual subscale scores and two summary scores are calculated:
(1) physical component summary (PCS) which includes physical
functioning, role-physical, bodily pain, and general health; (2)
mental component summary (MCS) which includes vitality, social
functioning, roleemotional, and mental health. The SF-36 is a
widely recognized tool that is recognized by the FDA as a validated
instrument to measure health-related quality of life across
multiple disease states.
Fatigue
[0237] Fatigue is assessed by the FACIT-F. FACIT-F is a health
related quality of life questionnaire focused on Fatigue. FACIT-F
includes the following components; physical well-being,
social/family well-being, emotional well-being, functional
well-being and additional concerns.
[0238] The disclosure set forth herein has been particularly shown
and described with references to specific 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 encompassed by the appended claims.
Sequence CWU 1
1
15111PRTArtificial SequenceSynthetic Peptide 1Arg Ala Ser Arg Pro
Ile Trp Pro Phe Leu Glu 1 5 10 27PRTArtificial SequenceSynthetic
Peptide 2Phe Thr Ser Arg Leu Arg His 1 5 39PRTArtificial
SequenceSynthetic Peptide 3Leu Gln Asn Val Ala Asn Pro Ala Thr 1 5
4108PRTArtificial SequenceSynthetic Peptide 4Asp Ile Gln Met Thr
Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly 1 5 10 15 Asp Arg Val
Thr Ile Thr Cys Arg Ala Ser Arg Pro Ile Trp Pro Phe 20 25 30 Leu
Glu Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile 35 40
45 Tyr Phe Thr Ser Arg Leu Arg His Gly Val Pro Ser Arg Phe Ser Gly
50 55 60 Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu
Gln Pro 65 70 75 80 Glu Asp Phe Ala Thr Tyr Tyr Cys Leu Gln Asn Val
Ala Asn Pro Ala 85 90 95 Thr Phe Ser Gln Gly Thr Lys Val Glu Ile
Lys Arg 100 105 5108PRTArtificial SequenceSynthetic Peptide 5Asp
Ile Cys Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly 1 5 10
15 Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Arg Pro Ile Trp Pro Phe
20 25 30 Leu Glu Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu
Leu Ile 35 40 45 Tyr Phe Thr Ser Arg Leu Arg His Gly Val Pro Ser
Arg Phe Ser Gly 50 55 60 Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr
Ile Ser Ser Leu Gln Pro 65 70 75 80 Glu Asp Phe Ala Thr Tyr Tyr Cys
Leu Gln Asn Val Ala Asn Pro Ala 85 90 95 Thr Phe Ser Gln Gly Thr
Lys Val Glu Ile Lys Arg 100 105 6108PRTArtificial SequenceSynthetic
Peptide 6Asp Ile Gln Met Thr Gln Ser Pro Cys Ser Leu Ser Ala Ser
Val Gly 1 5 10 15 Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Arg Pro
Ile Trp Pro Phe 20 25 30 Leu Glu Trp Tyr Gln Gln Lys Pro Gly Lys
Ala Pro Lys Leu Leu Ile 35 40 45 Tyr Phe Thr Ser Arg Leu Arg His
Gly Val Pro Ser Arg Phe Ser Gly 50 55 60 Ser Gly Ser Gly Thr Asp
Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro 65 70 75 80 Glu Asp Phe Ala
Thr Tyr Tyr Cys Leu Gln Asn Val Ala Asn Pro Ala 85 90 95 Thr Phe
Ser Gln Gly Thr Lys Val Glu Ile Lys Arg 100 105 7108PRTArtificial
SequenceSynthetic Peptide 7Asp Ile Gln Met Thr Gln Ser Pro Ser Ser
Leu Ser Ala Ser Val Gly 1 5 10 15 Asp Cys Val Thr Ile Thr Cys Arg
Ala Ser Arg Pro Ile Trp Pro Phe 20 25 30 Leu Glu Trp Tyr Gln Gln
Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile 35 40 45 Tyr Phe Thr Ser
Arg Leu Arg His Gly Val Pro Ser Arg Phe Ser Gly 50 55 60 Ser Gly
Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro 65 70 75 80
Glu Asp Phe Ala Thr Tyr Tyr Cys Leu Gln Asn Val Ala Asn Pro Ala 85
90 95 Thr Phe Ser Gln Gly Thr Lys Val Glu Ile Lys Arg 100 105
8108PRTArtificial SequenceSynthetic Peptide 8Asp Ile Gln Met Thr
Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly 1 5 10 15 Asp Arg Val
Thr Ile Thr Cys Arg Ala Ser Arg Pro Ile Trp Pro Phe 20 25 30 Leu
Glu Trp Tyr Gln Gln Lys Pro Cys Lys Ala Pro Lys Leu Leu Ile 35 40
45 Tyr Phe Thr Ser Arg Leu Arg His Gly Val Pro Ser Arg Phe Ser Gly
50 55 60 Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu
Gln Pro 65 70 75 80 Glu Asp Phe Ala Thr Tyr Tyr Cys Leu Gln Asn Val
Ala Asn Pro Ala 85 90 95 Thr Phe Ser Gln Gly Thr Lys Val Glu Ile
Lys Arg 100 105 9108PRTArtificial SequenceSynthetic Peptide 9Asp
Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly 1 5 10
15 Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Arg Pro Ile Trp Pro Phe
20 25 30 Leu Glu Trp Tyr Gln Gln Lys Pro Gly Cys Ala Pro Lys Leu
Leu Ile 35 40 45 Tyr Phe Thr Ser Arg Leu Arg His Gly Val Pro Ser
Arg Phe Ser Gly 50 55 60 Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr
Ile Ser Ser Leu Gln Pro 65 70 75 80 Glu Asp Phe Ala Thr Tyr Tyr Cys
Leu Gln Asn Val Ala Asn Pro Ala 85 90 95 Thr Phe Ser Gln Gly Thr
Lys Val Glu Ile Lys Arg 100 105 10108PRTArtificial
SequenceSynthetic Peptide 10Asp Ile Gln Met Thr Gln Ser Pro Ser Ser
Leu Ser Ala Ser Val Gly 1 5 10 15 Asp Arg Val Thr Ile Thr Cys Arg
Ala Ser Arg Pro Ile Trp Pro Phe 20 25 30 Leu Glu Trp Tyr Gln Gln
Lys Pro Gly Lys Ala Pro Cys Leu Leu Ile 35 40 45 Tyr Phe Thr Ser
Arg Leu Arg His Gly Val Pro Ser Arg Phe Ser Gly 50 55 60 Ser Gly
Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro 65 70 75 80
Glu Asp Phe Ala Thr Tyr Tyr Cys Leu Gln Asn Val Ala Asn Pro Ala 85
90 95 Thr Phe Ser Gln Gly Thr Lys Val Glu Ile Lys Arg 100 105
11108PRTArtificial SequenceSynthetic Peptide 11Asp Ile Gln Met Thr
Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly 1 5 10 15 Asp Arg Val
Thr Ile Thr Cys Arg Ala Ser Arg Pro Ile Trp Pro Phe 20 25 30 Leu
Glu Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile 35 40
45 Tyr Phe Thr Ser Arg Leu Arg His Gly Val Pro Cys Arg Phe Ser Gly
50 55 60 Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu
Gln Pro 65 70 75 80 Glu Asp Phe Ala Thr Tyr Tyr Cys Leu Gln Asn Val
Ala Asn Pro Ala 85 90 95 Thr Phe Ser Gln Gly Thr Lys Val Glu Ile
Lys Arg 100 105 12108PRTArtificial SequenceSynthetic Peptide 12Asp
Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly 1 5 10
15 Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Arg Pro Ile Trp Pro Phe
20 25 30 Leu Glu Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu
Leu Ile 35 40 45 Tyr Phe Thr Ser Arg Leu Arg His Gly Val Pro Ser
Arg Phe Ser Gly 50 55 60 Ser Gly Ser Gly Thr Cys Phe Thr Leu Thr
Ile Ser Ser Leu Gln Pro 65 70 75 80 Glu Asp Phe Ala Thr Tyr Tyr Cys
Leu Gln Asn Val Ala Asn Pro Ala 85 90 95 Thr Phe Ser Gln Gly Thr
Lys Val Glu Ile Lys Arg 100 105 13108PRTArtificial
SequenceSynthetic Peptide 13Asp Ile Gln Met Thr Gln Ser Pro Ser Ser
Leu Ser Ala Ser Val Gly 1 5 10 15 Asp Arg Val Thr Ile Thr Cys Arg
Ala Ser Arg Pro Ile Trp Pro Phe 20 25 30 Leu Glu Trp Tyr Gln Gln
Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile 35 40 45 Tyr Phe Thr Ser
Arg Leu Arg His Gly Val Pro Ser Arg Phe Ser Gly 50 55 60 Ser Gly
Ser Gly Thr Asp Phe Thr Leu Cys Ile Ser Ser Leu Gln Pro 65 70 75 80
Glu Asp Phe Ala Thr Tyr Tyr Cys Leu Gln Asn Val Ala Asn Pro Ala 85
90 95 Thr Phe Ser Gln Gly Thr Lys Val Glu Ile Lys Arg 100 105
14108PRTArtificial SequenceSynthetic Peptide 14Asp Ile Gln Met Thr
Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly 1 5 10 15 Asp Arg Val
Thr Ile Thr Cys Arg Ala Ser Arg Pro Ile Trp Pro Phe 20 25 30 Leu
Glu Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile 35 40
45 Tyr Phe Thr Ser Arg Leu Arg His Gly Val Pro Ser Arg Phe Ser Gly
50 55 60 Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu
Cys Pro 65 70 75 80 Glu Asp Phe Ala Thr Tyr Tyr Cys Leu Gln Asn Val
Ala Asn Pro Ala 85 90 95 Thr Phe Ser Gln Gly Thr Lys Val Glu Ile
Lys Arg 100 105 15108PRTArtificial SequenceSynthetic Peptide 15Asp
Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly 1 5 10
15 Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Arg Pro Ile Trp Pro Phe
20 25 30 Leu Glu Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu
Leu Ile 35 40 45 Tyr Phe Thr Ser Arg Leu Arg His Gly Val Pro Ser
Arg Phe Ser Gly 50 55 60 Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr
Ile Ser Ser Leu Gln Pro 65 70 75 80 Glu Asp Phe Ala Thr Tyr Tyr Cys
Leu Gln Asn Val Ala Asn Pro Ala 85 90 95 Thr Phe Ser Gln Gly Thr
Cys Val Glu Ile Lys Arg 100 105
* * * * *