U.S. patent application number 13/494398 was filed with the patent office on 2013-01-10 for anti-psgl-1 antibodies and uses thereof.
This patent application is currently assigned to AbGenomics Cooperatief U.A.. Invention is credited to Stefan Bassarab, Barbara Enenkel, Patrick Garidel, Tobias Litzenburger, Heidrun Schott, Sanjaya Singh.
Application Number | 20130011391 13/494398 |
Document ID | / |
Family ID | 47357432 |
Filed Date | 2013-01-10 |
United States Patent
Application |
20130011391 |
Kind Code |
A1 |
Bassarab; Stefan ; et
al. |
January 10, 2013 |
ANTI-PSGL-1 ANTIBODIES AND USES THEREOF
Abstract
Provided herein, in one aspect, are antibodies that
immunospecifically bind to PSGL-1, polynucleotides comprising
nucleotide sequences encoding such antibodies, and expression
vectors and host cells for producing such antibodies. Also provided
herein are kits and pharmaceutical compositions comprising
antibodies that specifically bind to PSGL-1, as well as methods of
treating a disorder or disease caused by or associated with
increased proliferation and/or numbers of activated T cells using
the antibodies described herein.
Inventors: |
Bassarab; Stefan; (Biberach
an der Riss, DE) ; Enenkel; Barbara; (Warthausen,
DE) ; Garidel; Patrick; (Norderstedt, DE) ;
Schott; Heidrun; (Schemmerhofen, DE) ; Singh;
Sanjaya; (Sandy Hook, CT) ; Litzenburger; Tobias;
(Mittelbiberach, DE) |
Assignee: |
AbGenomics Cooperatief U.A.
Amsterdam
NL
|
Family ID: |
47357432 |
Appl. No.: |
13/494398 |
Filed: |
June 12, 2012 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
|
|
61496249 |
Jun 13, 2011 |
|
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|
Current U.S.
Class: |
424/133.1 ;
424/153.1; 530/387.1; 530/387.3; 530/388.7 |
Current CPC
Class: |
A61P 11/06 20180101;
C07K 2317/24 20130101; C07K 16/2896 20130101; A61P 37/00 20180101;
C07K 2317/53 20130101; A61P 1/04 20180101; A61P 29/00 20180101;
A61P 37/02 20180101; A61P 37/06 20180101; A61P 37/08 20180101; A61P
17/06 20180101; A61P 1/00 20180101; A61K 2039/505 20130101; A61P
3/10 20180101; A61P 19/02 20180101 |
Class at
Publication: |
424/133.1 ;
530/387.3; 530/388.7; 530/387.1; 424/153.1 |
International
Class: |
C07K 16/28 20060101
C07K016/28; A61P 29/00 20060101 A61P029/00; A61P 1/00 20060101
A61P001/00; A61P 19/02 20060101 A61P019/02; A61P 3/10 20060101
A61P003/10; A61K 39/395 20060101 A61K039/395; A61P 17/06 20060101
A61P017/06 |
Claims
1. A monoclonal antibody which immunospecifically binds to human
PSGL-1 comprising: (i) a variable light ("VL") chain region
comprising the amino acid sequence of SEQ ID NO: 3; (ii) a heavy
chain comprising variable heavy ("VH") chain region comprising the
amino acid sequence of SEQ ID NO: 4; and (iii) a human IgG4
constant region which contains a Serine to Proline substitution at
amino acid 228 of the heavy chain numbered according to the EU
index.
2. A monoclonal antibody which immunospecifically binds to human
PSGL-1 comprising: (i) a light chain comprising the amino acid
sequence of SEQ ID NO: 1; and (ii) a heavy chain comprising the
amino acid sequence of SEQ ID NO: 2.
3. A monoclonal antibody which immunospecifically binds to human
PSGL-1, comprising: (i) a heavy chain consisting of SEQ ID NO: 2;
and (ii) a light chain consisting of SEQ ID NO: 1.
4. A monoclonal antibody which immunospecifically binds to human
PSGL-1 and comprises a heavy chain comprising: (i) a VH chain
region comprising SEQ ID NOs: 8, 9, and 10; and (ii) a human IgG4
heavy chain constant region containing a Serine to Proline amino
acid substitution at amino acid 228 of the heavy chain numbered
according to the EU index.
5. The monoclonal antibody of claim 4, wherein the monoclonal
antibody further comprises a light chain comprising a VL chain
region comprising SEQ ID NOs: 5, 6, and 7.
6. The monoclonal antibody of claim 1, wherein the antibody is
purified.
7. A pharmaceutical composition comprising the monoclonal antibody
of claim 1 and a pharmaceutically acceptable carrier.
8. The pharmaceutical composition of claim 7, wherein the
monoclonal antibody is purified.
9. An antibody heavy chain comprising SEQ ID NO: 2.
10. A kit comprising a first container containing the monoclonal
antibody of claim 1.
11. An injection device containing the monoclonal antibody of claim
1.
12. The injection device of claim 11, wherein the injection device
is a syringe.
13. A method for treating an inflammatory disorder, the method
comprising administering to a subject in need thereof a
therapeutically effective amount of the monoclonal antibody of
claim 1.
14. A method for treating an inflammatory disorder, the method
comprising administering to a subject in need thereof a
therapeutically effective amount of the pharmaceutical composition
of claim 7.
15. The method of claim 13, wherein the inflammatory disorder is an
autoimmune disease.
16. The method of claim 13, wherein the inflammatory disorder is:
psoriasis, plaque psoriasis, chronic plaque psoriasis, guttate
psoriasis, inverse psoriasis, pustular psoriasis, erythrodermic
psoriasis, psoriatic arthritis, rheumatoid arthritis, Crohn's
disease, ankylosing spondylitis, or diabetes.
17. The monoclonal antibody of claim 2, wherein the antibody is
purified.
18. A pharmaceutical composition comprising the monoclonal antibody
of claim and a pharmaceutically acceptable carrier.
19. The monoclonal antibody of claim 4, wherein the antibody is
purified.
20. A pharmaceutical composition comprising the monoclonal antibody
of claim 4 and a pharmaceutically acceptable carrier.
Description
1. RELATED APPLICATIONS
[0001] This application claims the benefit of the filing date of
U.S. provisional patent application 61/496,249, filed Jun. 13, 2011
entitled "ANTI-PSGL-1 ANTIBODIES AND USES THEREOF". The entire
teachings and contents of the referenced provisional application
are incorporated herein by reference.
2. FIELD
[0002] Provided herein are antibodies that specifically bind to
P-selectin glycoprotein ligand-1 (PSGL-1), polynucleotides encoding
such antibodies, expression vectors comprising such
polynucleotides, host cells comprising such expression vectors, and
related compositions. Also provided herein are methods for treating
a disorder or a disorder caused by or associated with increased
proliferation and/or numbers of activated T cells, such as
psoriasis, using anti-PSGL-1 antibodies.
3. BACKGROUND
[0003] Inflammatory responses to infection or injury are initiated
by the adherence of leukocytes to the vascular wall (McEver et al.,
1997, J. Clin. Invest., 100 (3): 485-492). Selectins represents a
family of glycoproteins which mediate the first
leukocyte-endothelial cell and leukocyte-platelet interactions
during inflammation. The selectin family, which consists of
L-selectin, E-selectin, and P-selectin, comprise an
NH.sub.2-terminal lectin domain, followed by an EGF-like domain, a
series of consensus repeats, a transmembrane domain, and a short
cytoplasmic tail. The lectin domains of selectins interact with
specific glycoconjugate ligands in order to facilitate cell
adhesion. L-selectin, expressed on most leukocytes, binds to
ligands on some endothelial cells and other leukocytes. E-selectin,
expressed on cytokine activated endothelial cells, binds to ligands
on most leukocytes. P-selectin, expressed on activated platelets
and endothelial cells, also binds to ligands on most
leukocytes.
[0004] P-selectin glycoprotein ligand-1 ("PSGL-1"), also known as
SELPLG or CD162 (cluster of differentiation 162) is a human
mucin-type glycoprotein ligand for all three selectins (Constantin,
Gabriela, 2004, Drugs News Perspect, 17(9): 579-585; McEver et al.,
1997, J. Clin. Invest., 100 (3): 485-492). PSGL-1 is a
disulfide-bonded homodimer with two 120-kD subunits and is
expressed on the surface of monocytes, lymphocytes, granulocytes,
and in some CD34.sup.+ stem cells. PSGL-1 is likely to contribute
to pathological leukocyte recruitment in many inflammatory
disorders since it facilitates the adhesive interactions of
selectins, suggesting that inhibitors of PSGL-1, such as antibodies
to PSGL-1, are potentially useful anti-inflammatory drugs.
[0005] Several anti-PSGL-1 antibodies have been developed (see,
e.g., International Application Pub. No. WO 2005/110475, published
Nov. 24, 2005; International Application Pub. No. WO 2003/013603,
published Feb. 20, 2003; Constantin, Gabriela, 2004, Drugs News
Perspect, 17(9): 579-585).
4. SUMMARY
[0006] In one aspect, provided herein are antibodies and antibody
derived antigen-binding fragments that specifically bind to PSGL-1.
In one embodiment, provided herein is a monoclonal antibody which
immunospecifically binds to human PSGL-1 comprising: (i) a variable
light ("VL") chain region comprising the amino acid sequence of SEQ
ID NO: 3; (ii) a heavy chain comprising a variable heavy ("VH")
chain region comprising the amino acid sequence of SEQ ID NO: 4;
and (iii) a human IgG4 constant region which contains a Serine to
Proline substitution at amino acid 228 of the heavy chain numbered
according to the EU (gamma-G1 immunoglobulin) index (See, Edelman
et al., 1969, Proc. Natl. Acad. Sci. USA, 63(1): 78-85). In a
specific embodiment, provided herein is a monoclonal antibody which
immunospecifically binds to human PSGL-1 comprising: (i) a light
chain comprising the amino acid sequence of SEQ ID NO: 1; and (ii)
a heavy chain comprising the amino acid sequence of SEQ ID NO: 2.
In another specific embodiment, provided herein is a monoclonal
antibody which immunospecifically binds to human PSGL-1 comprising:
(i) a heavy chain consisting of SEQ ID NO: 2; and (ii) a light
chain consisting of SEQ ID NO: 1. In another specific embodiment,
provided herein is a monoclonal antibody which immunospecifically
binds to human PSGL-1 comprising a heavy chain that comprises the
amino acid sequence of SEQ ID NO: 2, and a complementary light
chain. In a specific embodiment, any one of the foregoing
monoclonal antibodies is purified.
[0007] In another aspect, provided herein is a pharmaceutical
composition comprising any one of the foregoing monoclonal
antibodies and a pharmaceutically acceptable carrier. In a specific
embodiment, the pharmaceutical composition comprises a monoclonal
antibody that is purified.
[0008] In one embodiment, provided herein is a pharmaceutical
preparation comprising any one of the foregoing monoclonal
antibodies in an aqueous solution comprising sodium citrate, sodium
chloride, and citric acid monohydrate. In a specific embodiment,
the pharmaceutical preparation is an aqueous solution comprising
9.1 mM sodium citrate dihydrate, 150 mM sodium chloride, and 0.9 mM
citric acid. In another specific embodiment, the monoclonal
antibody is present at a concentration of 0.267 mM in the
pharmaceutical preparation. In a specific embodiment, the
pharmaceutical preparation is an aqueous solution comprising 2.676
g/L sodium, 8.766 g/L sodium chloride, 0.2 g/L polysorbate 80, and
0.189 g/L citric acid. In another specific embodiment, the
monoclonal antibody is present at a concentration of 40 g/L in the
pharmaceutical preparation. In a specific embodiment, all the
foregoing aqueous solutions are at pH 6.0.
[0009] In another aspect, provided herein are polynucleotides
encoding an antibody described herein or an antigen-binding
fragment thereof. In a specific embodiment, provided herein is an
isolated polynucleotide encoding an antibody heavy chain comprising
SEQ ID NO: 2. In an embodiment, an expression vector comprises the
foregoing isolated polynucleotide. In certain embodiments, the
expression vector further comprises a polynucleotide encoding an
antibody light chain comprising SEQ ID NO:1. In a specific
embodiment, the expression vector is a mammalian expression vector.
In another aspect, provided herein is a host cell comprising a
foregoing expression vector. In a specific embodiment, the host
cell comprises (a) a first nucleic acid encoding SEQ ID NO: 2,
operably linked to a promoter functional in said host cell; and (b)
a second nucleic acid encoding SEQ ID NO: 1 operably linked to a
promoter functional in said host cell. In another specific
embodiment, the first and second nucleic acids of the host cell are
in the same expression vector or in different expression
vectors.
[0010] In another aspect, provided herein is a method of producing
any of the foregoing monoclonal antibodies comprising culturing any
of the foregoing host cells such that said first and second nucleic
acids are expressed by said cell, and said heavy and light chains
assemble together to form said antibody.
[0011] In another aspect, provided herein is an antibody heavy
chain comprising SEQ ID NO: 2 or an antibody heavy chain fragment
comprising amino acids 1 to 228 of SEQ ID NO:2. In a specific
embodiment, provided herein is an antibody heavy chain comprising
SEQ ID NO: 2. In another specific embodiment, provided herein is an
antibody heavy chain fragment comprising amino acids 1 to 228 of
SEQ ID NO:2. Also provided herein is a method of producing the
foregoing heavy chain comprising culturing any of the foregoing
host cells such that said heavy chain is expressed by the cell, and
isolating said heavy chain. In a specific embodiment, a method of
producing any of the foregoing antibodies comprises producing the
heavy chain according to the forgoing method for producing the
heavy chain, isolating said heavy chain, and complexing said heavy
chain to an antibody light chain comprising SEQ ID NO:1.
[0012] In another aspect, provided herein is a kit comprising a
first container containing any of the foregoing monoclonal
antibodies. In a specific embodiment, the first container is a vial
containing said monoclonal antibody as a lyophilized sterile powder
under vacuum, and the kit further comprises a second container
comprising a pharmaceutically acceptable fluid. Also provided
herein is an injection device containing any of the foregoing
monoclonal antibodies. In a specific embodiment, the injection
device is a syringe.
[0013] In another aspect, provided herein are methods for
preventing and/or treating a disease or disorder associated with or
caused (in whole or part) by increased and/or numbers of activated
T cells relative to healthy individuals or individuals not having
the particular disease or disorder. In a specific embodiment,
provided herein is a method for treating an inflammatory disorder,
comprising administering to a subject in need thereof a
therapeutically effective amount of any of the foregoing monoclonal
antibodies. In another specific embodiment, a method for treating
an inflammatory disorder, comprises administering to a subject in
need thereof a therapeutically effective amount of any of the
foregoing pharmaceutical compositions. In a specific embodiment,
the inflammatory disorder is an autoimmune disease. In another
specific embodiment, the inflammatory disorder is psoriasis. In
another specific embodiment, the inflammatory disorder is plaque
psoriasis. In another specific embodiment, the plaque psoriasis is
moderate to severe. In another specific embodiment, the
inflammatory disorder is erythrodermic psoriasis. In another
specific embodiment, the inflammatory disorder is psoriatic
arthritis. In another specific embodiment, the inflammatory
disorder is rheumatoid arthritis. In another specific embodiment,
the inflammatory disorder is Crohn's disease. In another specific
embodiment, the inflammatory disorder is ankylosing spondylitis. In
another specific embodiment, the inflammatory disorder is
diabetes.
5. BRIEF DESCRIPTION OF THE FIGURES
[0014] FIG. 1 depicts non-reducing capillary gel electrophoresis
(CGE) for h15A7 and 15A7H. The arrow indicates the peak for the
half antibody molecule.
[0015] FIG. 2 depicts binding of 15A7H and control antibody to
activated human CD4+ T-cells. EC50 values (nM) were derived using a
one site 4-parameter fit model by plotting the mean fluorescence
intensity (MFI) versus antibody concentration.
[0016] FIG. 3 depicts pooled dose response of 15A7H antibody in
trans-vivo DTH in 4 donor PBMCs. The pooled mean.+-.SEM of 4 donors
after treatment with 0.03, 0.1, 0.3, 1 and 10 mg/kg antibody, or
vehicle. pbmc: PBMC only, V: vehicle, 0.03, 0.1, 0.3, 1 and 10
mg/kg of 15A7H antibody. Percent inhibitions of foot pad thickness
in response to 0.03, 0.1, 0.3, 1, and 10 mg/kg of 15A7H antibody
treatment.
[0017] FIG. 4 depicts twenty four hour plasma concentrations of
15A7H antibody in C57BL/6 mice following a single intraperitoneal
injection. Pooled plasma levels of the 2 antibodies (mean.+-.SEM,
n=4) in the experimental animals are plotted against their %
inhibition of footpad thickness in the trans-vivo DTH assay.
[0018] FIG. 5 depicts 15A7H plasma concentrations versus time in
C57BL/6 mice. Four satellite mice were dosed i.p. with 0.03, 0.1,
0.3, 1, and 10 mg/kg of 15A7H antibody. Blood samples were
collected at 1, 3, 5 and 24 hrs.
[0019] FIG. 6 depicts CDC activity of 15A7H and control antibodies,
which were tested at different concentrations (5, 0.5, 0.05, 0.005
and 0.0005 .mu.g/ml).
[0020] FIG. 7A depicts the heavy chain amino acid sequence of
antibody 15A7H (SEQ ID NO:2). The variable heavy chain region (SEQ
ID NO:4) is in bold. The CDRs (CDR1 (SEQ ID NO:8), CDR2 (SEQ ID
NO:9), and CDR3 (SEQ ID NO:10)) are boxed. The framework regions
(FR1 (SEQ ID NO:17), FR2 (SEQ ID NO:18), FR3 (SEQ ID NO:19), and
FR4 (SEQ ID NO:20)) are also indicated. The constant region is
indicated. The hinge region amino acid sequence (SEQ ID NO:12) is
boxed. The substituted proline at amino acid position 228 in the
hinge region is italicized.
[0021] FIG. 7B depicts the light chain amino acid sequence of
antibody 15A7H (SEQ ID NO:1). The variable light chain region (SEQ
ID NO:3) is in bold. The CDRs (CDR1 (SEQ ID NO:5), CDR2 (SEQ ID
NO:6), and CDR3 (SEQ ID NO:7)) are boxed. The framework regions
(FR1 (SEQ ID NO:13), FR2 (SEQ ID NO:14), FR3 (SEQ ID NO:15), and
FR4 (SEQ ID NO:16)) are also indicated. The constant region is
indicated.
TABLE-US-00001 [0022] TABLE 1 List of SEQ ID NOs and their
corresponding sequences SEQ ID NO. Corresponding Sequence SEQ ID
NO: 1 15A7H Light Chain amino acid sequence SEQ ID NO: 2 15A7H
Heavy Chain amino acid sequence SEQ ID NO: 3 15A7H Variable Light
Chain Region (VL) amino acid sequence SEQ ID NO: 4 15A7H Variable
Heavy Chain Region (VH) amino acid sequence SEQ ID NO: 5 15A7H VL
CDR1 amino acid sequence SEQ ID NO: 6 15A7H VL CDR2 amino acid
sequence SEQ ID NO: 7 15A7H VL CDR3 amino acid sequence SEQ ID NO:
8 15A7H VH CDR1 amino acid sequence SEQ ID NO: 9 15A7H VH CDR2
amino acid sequence SEQ ID NO: 10 15A7H VH CDR3 amino acid sequence
SEQ ID NO: 11 Full length human PSGL-1 amino acid sequence SEQ ID
NO: 12 IgG4 hinge region amino acid sequence SEQ ID NO: 13 15A7H VL
FR1 SEQ ID NO: 14 15A7H VL FR2 SEQ ID NO: 15 15A7H VL FR3 SEQ ID
NO: 16 15A7H VL FR4 SEQ ID NO: 17 15A7H VH FR1 SEQ ID NO: 18 15A7H
VH FR2 SEQ ID NO: 19 15A7H VH FR3 SEQ ID NO: 20 15A7H VH FR4 SEQ ID
NO: 21 IgG4 wild-type hinge region amino acid sequence
6. DETAILED DESCRIPTION
[0023] Provided herein are antibodies that specifically bind to
PSGL-1. Also provided are isolated nucleic acids encoding such
antibodies. Further provided are vectors and host cells comprising
nucleic acids encoding such antibodies or antigen-binding fragments
thereof. Also provided are methods of making such antibodies,
cells, e.g. CHO cells, antibodies produced by such cells and
purification of produced antibodies. Also provided herein is a
method of treating and/or preventing a disorder or disease
described herein (e.g., an inflammatory condition) comprising
administering an antibody or an antibody derived antigen-binding
fragment described herein that immunospecifically binds to PSGL-1.
In a specific embodiment, the antibody is the IgG4 monoclonal
antibody 15A7H described in Examples 1-4, infra.
[0024] 6.1 Antibodies
[0025] Provided herein are monoclonal antibodies that
immunospecifically bind to human P-selectin glycoprotein ligand-1
("PSGL-1"). In a specific embodiment, provided herein is a
monoclonal antibody which immunospecifically binds to human PSGL-1
comprising: (i) a variable light ("VL") chain region comprising the
amino acid sequence of SEQ ID NO: 3; (ii) a heavy chain comprising
a variable heavy ("VH") chain region comprising the amino acid
sequence of SEQ ID NO: 4; and (iii) a human IgG4 constant region
which contains a Serine to Proline amino acid substitution at amino
acid 228 of the heavy chain numbered according to the EU index.
Non-limiting examples of human constant regions are described in
the art, e.g., see Kabat et al. (1991) Sequences of Proteins of
Immunological Interest, Fifth Edition, U.S. Department of Health
and Human Services, NIH Publication No. 91-3242. Preferably, the
antibody binds to PSGL-1 and selectively induces apoptosis of
activated T cells (relative to other cells that express PSGL-1). In
a specific embodiment, binding of the antibody to PSGL-1 does not
interfere with the interaction of P-selectin with PSGL-1, a
function associated with PSGL-1 and a requirement for efficient
localization of activated T cells and neutrophils to target
tissues.
[0026] In a specific embodiment, an antibody which
immunospecifically binds to PSGL-1 is a full-length immunoglobulin
G of class 4 (IgG4), and preferably comprises a heavy chain
sequence of SEQ ID NO:2, and even more preferably comprises a heavy
chain sequence of SEQ ID NO:2 and a light chain sequence of SEQ ID
NO:1 (the latter being monoclonal antibody 15A7H).
[0027] Also provided herein are antigen-binding fragments of an
antibody, comprising the variable region sequences SEQ ID NO:3 and
SEQ ID NO:4 and at least a portion of a human heavy chain constant
region containing the human IgG4 hinge region up through and
including a Serine to Proline substitution at amino acid 228 of a
human heavy chain numbered according to the EU index. In a specific
embodiment, an antibody derived antigen-binding fragment herein is
a F(ab').sub.2 fragment.
[0028] An antibody or an antibody derived antigen-binding fragment
described herein is preferably isolated, most preferably
purified.
[0029] As used herein and unless otherwise specified, the terms
"immunospecifically binds," "immunospecifically recognizes,"
"specifically binds," and "specifically recognizes" are analogous
terms in the context of antibodies and antibody derived
antigen-binding fragments and are used interchangeably herein, and
refer to the binding of an antibody or antibody derived
antigen-binding fragment via its antigen combining site to its
epitope, as would be understood by one skilled in the art. In one
specific embodiment, an antibody or an antibody derived
antigen-binding fragment that specifically binds to an antigen also
can bind to other peptides or polypeptides, albeit generally with
lower affinity as determined by, e.g., immunoassays, Biacore.TM.,
KinExA 3000 instrument (Sapidyne Instruments, Boise, Id.), or other
assays known in the art. In a specific embodiment, an antibody or
an antibody derived antigen-binding fragment that
immunospecifically binds to an antigen binds to the antigen with a
K.sub.a that is at least 2 logs, 2.5 logs, 3 logs, 4 logs or
greater than the K.sub.a when the antibody or the antibody derived
antigen-binding fragment binds to another antigen. In another
specific embodiment, an antibody or an antibody derived
antigen-binding fragment that immunospecifically binds to an
antigen do not cross react by binding with other proteins. In
specific embodiments, an antibody or an antibody derived
antigen-binding fragment described herein specifically binds to a
native isoform or native variant of PSGL-1 (that is a naturally
occurring isoform or variant of PSGL-1 in an animal that can be
isolated from an animal, preferably a human). In particular
embodiments, an antibody or an antibody derived antigen-binding
fragment described herein immunospecifically binds to human PSGL-1
or a fragment thereof. In specific embodiments, an antibody or an
antibody derived antigen-binding fragment described herein
specifically binds to human PSGL-1 and/or cynomologous PSGL-1 or a
fragment thereof.
[0030] The amino acid sequence of SEQ ID NO:11 depicts the full
length human PSGL-1, GenBank.TM. accession number AAA74577.1,
GI:902797. In specific embodiments, an antibody described herein
immunospecifically binds to PSGL-1 as determined, e.g., by ELISA or
other antigen-binding assay known in the art, or described
herein.
[0031] In specific aspects, provided herein is an antibody that
specifically binds human and/or cynomologous PSGL-1 and that is an
immunoglobulin G (having a gamma-heavy region) of class 4 (an IgG4)
that is a tetramer of two identical disulfide-bonded dimers, each
comprising a heavy chain and a light chain. The antibody preferably
comprises a heavy chain of SEQ ID NO:2. More preferably, the
antibody comprises a heavy chain of SEQ ID NO:2 and a light chain
of SEQ ID NO:1. With respect to the light chain, in a specific
embodiment, the light chain of an antibody described herein is a
kappa light chain.
[0032] In specific embodiments, an antibody described herein
comprises a heavy chain comprising or consisting of the amino acid
sequence of SEQ ID NO:2. In specific embodiments, an antibody
described herein comprises a light chain comprising or consisting
of the amino acid sequence of SEQ ID NO:1, and comprises a heavy
chain comprising or consisting of the amino acid sequence of SEQ ID
NO:2. In specific embodiments, an antibody described herein
comprises a light chain comprising or consisting of the amino acid
sequence of SEQ ID NO:1. In specific embodiments, an antibody
described herein comprises a heavy chain comprising or consisting
of the amino acid sequence of SEQ ID NO:2.
[0033] In a specific embodiment, the antibodies provided herein are
IgG4 monoclonal antibodies that specifically bind to PSGL-1. IgG4
antibodies are known to undergo a process called Fab arm exchange,
also known as IgG4 shuffling, in which increased susceptibility of
native IgG4 hinge disulfide bonds to reduction allows the heavy
chains to separate and randomly re-associate to produce a mixed
population of IgG4 molecules with randomized heavy-chain and
light-chain pairs (Aalberse et al., 1999. Int Arch Allergy Immunol
118:187-189; Labrijn, et al., 2009, Nat Biotechnol 27:767-771;
Schuurman et al., 2001. Mol Immunol 38:1-8; van der Neut
Kolfschoten et al., 2007. Science 317:1554-1557).
[0034] It has been demonstrated that a Serine to Proline mutation
at position 241 using Kabat numbering (Kabat et al. 1991, Sequences
of Proteins of Immunological Interest, Fifth Edition, U.S.
Department of Health and Human Services, NIH Publication No.
91-3242) or at position 228 using the EU index (Edelman et al.,
1969, Proc. Natl. Acad. Sci. USA, 63(1): 78-85) in the hinge region
of human IgG4 results in considerable reduction of intra-chain
disulfide bond formation, resulting in the reduction of IgG4
"half-antibody" molecules and reduced heterogeneity/shuffling of
IgG4 molecules (Bloom et al. 1997, Protein Sci, 6:407-415; Angal et
al., 1993, Molecular Immunology, 30(1): 105-108)). There are also
published reports that this hinge mutation may decrease IgG4
shuffling and increase the half-life of the IgG4 molecules in vivo
(Labrijn, et al., 2009, Nat Biotechnol 27:767-771; Stubenrauch, et
al., 2010, Drug Metab Dispos 38:84-91). Van der Neut Kolfschoten et
al., reported that the C.sub.H3 domain of IgG4 and not the core
hinge is predominantly involved in the Fab arm exchange reaction
(see Van der Neut Kolfschoten et al, 2007, Science, 317:1554-1557
("Van der Neut Kolfschoten") at page 1555, col. 2). Van der Neut
Kolfschaten reported that exchanging the C.sub.H3 domain of IgG1
for the C.sub.H3 domain of IgG4 activated Fab arm exchange for the
IgG1, while exchanging the C.sub.H3 domain of IgG4 abrogated Fab
arm exchange for the IgG4 (see, p. 1555 and FIG. 2D).
[0035] In a specific embodiment, provided herein are IgG4
antibodies or antigen-binding fragments thereof, that specifically
bind to PSGL-1, and that contain one or more amino acid
substitutions in the IgG4 hinge region, wherein said antibody or
antigen-binding fragment thereof retains specific binding to said
PSGL-1 and wherein IgG4 shuffling is reduced relative to an
antibody comprising an IgG4 hinge region not comprising said one or
more amino acid substitutions. In a specific embodiment, the IgG4
hinge region only comprises a single amino acid substitution. An
example of a "human IgG4 hinge region," is the region on the heavy
chain of an IgG4 antibody between the C.sub.H1 and C.sub.H2 domains
consisting of the amino acid sequence of SEQ ID NO:12, as set forth
in Angal et al., 1993, Molecular Immunology, 30(1): 105-108.
[0036] In a specific embodiment, a reduction in IgG4 shuffling is
determined by detecting of a lower amount of half antibody
molecules or of arm exchange produced from an antibody described
herein which contains one or more amino acid substitutions in the
hinge region, as compared to the amount of half antibody molecules
or of arm exchange produced from an IgG4 molecule containing an
IgG4 hinge region not comprising said one or more amino acid
substitutions. Any assay well-known in the art can be used to
detect half antibody production and bispecific antibody molecules.
See, e.g., Van der Neut Kolfschoten et al, 2007, Science,
317:1554-1557, for examples of assays to detect production of
bi-specific antibodies.
[0037] In a specific embodiment, provided herein are IgG4
monoclonal antibodies that specifically bind to PSGL-1, comprising
a Serine to Proline amino acid substitution at amino acid position
228 of the heavy chain numbered according to the EU index.
[0038] In a specific embodiment, an antibody described herein
comprises a light chain having the amino acid sequence of SEQ ID
NO:1 and a heavy chain comprising a Proline at position 228 of the
heavy chain numbered according to the EU index.
[0039] In a specific embodiment, a monoclonal antibody, which
immunospecifically binds to human PSGL-1 comprises: (i) a light
chain having the amino acid sequence of SEQ ID NO:1; and (ii) a
heavy chain comprising a human IgG4 constant region containing one
or more amino acid substitutions in the IgG4 hinge region, wherein
said antibody retains specific binding to said PSGL-1 and wherein
IgG4 shuffling is reduced relative to an antibody comprising an
IgG4 hinge region not comprising said one or more amino acid
substitutions.
[0040] In a specific embodiment, a monoclonal antibody, which
immunospecifically binds to human PSGL-1 comprises (i) a light
chain having the amino acid sequence of SEQ ID NO:1; and (ii) a
heavy chain comprising a human IgG4 constant region comprising a
Serine to Proline amino acid substitution at amino acid position
228 of the heavy chain numbered according to the EU index or
position 241 according to the Kabat numbering system (Kabat et al.
1991, Sequences of Proteins of Immunological Interest, Fifth
Edition, U.S. Department of Health and Human Services, NIH
Publication No. 91-3242; and Edelman et al., 1969, Proc. Natl.
Acad. Sci. USA, 63(1): 78-85).
[0041] In a specific embodiment, a monoclonal antibody, which
immunospecifically binds to human PSGL-1 comprises: (i) a VL chain
region comprising the amino acid sequence of SEQ ID NO:3; (ii) a VH
chain region comprising the amino acid sequence of SEQ ID NO:4; and
(iii) a human IgG4 constant region containing an IgG4 hinge region
comprising one or more amino acid substitutions in the hinge
region, wherein said antibody retains specific binding to PSGL-1
and wherein the IgG4 shuffling is reduced relative to an antibody
comprising an IgG4 hinge region not comprising said one or more
amino acid substitutions.
[0042] In a specific embodiment, a monoclonal antibody which
immunospecifically binds to human PSGL-1 comprises: (i) a heavy
chain comprising a VH chain region comprising the amino acid
sequence of SEQ ID NO:4, and (ii) a human IgG4 heavy chain constant
region comprising a Serine to Proline amino acid substitution at
amino acid position 228 of the heavy chain numbered according to
the EU index.
[0043] In a specific embodiment, a monoclonal antibody, which
immunospecifically binds to human PSGL-1 comprises: (i) a VL chain
region comprising the amino acid sequence of SEQ ID NO:3; (ii) a
heavy chain comprising a VH chain region comprising the amino acid
sequence of SEQ ID NO:4; and (iii) a human IgG4 heavy chain
constant region comprising a Serine to Proline amino acid
substitution at amino acid position 228 of the heavy chain numbered
according to the EU index.
[0044] In certain embodiments, an IgG4 monoclonal antibody as
described herein comprises the VH CDRs having the amino acid
sequences described herein (e.g., see Table 3) and VL CDRs having
the amino acid sequences described herein (e.g., see Table 2),
wherein the antibody immunospecifically binds to PSGL-1 and has a
hinge region mutation that reduces IgG4 shuffling (e.g., a Serine
to Proline amino acid substitution at amino acid 228 of the heavy
chain numbered according to the EU index). In a specific
embodiment, the IgG4 monoclonal antibody immunospecifically binds
PSGL-1 and comprises a heavy chain comprising (a) a VH chain region
comprising SEQ ID NOs: 8, 9 and 10; and (b) a human IgG4 heavy
chain constant region containing a Serine to Proline amino acid
substitution at amino acid 228 of the heavy chain numbered
according to the EU index. More preferably, the antibody further
comprises a light chain comprising a VL chain region comprising SEQ
ID NOs: 5, 6, and 7.
[0045] Table 2, below, presents the VL CDRs (in particular, VL
CDR1, VL CDR2, and VL CDR3) of the amino acid sequence of 15A7H.
Table 3, below, presents the VH CDRs (in particular, VH CDR1, VH
CDR2, and VH CDR3) of the amino acid sequence of 15A7H. In specific
embodiments, an antibody described herein which immunospecifically
binds to human PSGL-1 (SEQ ID NO:11) comprises the VL CDR sequences
in Table 2. In specific embodiments, an antibody described herein
which immunospecifically binds to human PSGL-1 (SEQ ID NO:11),
comprises the VH CDR sequences selected from those in Table 3.
TABLE-US-00002 TABLE 2 VL CDR Amino Acid Sequences Ab VL CDR1 VL
CDR2 VL CDR3 15A7H RSSQSIVHNDGNT KVSNRFS FQGSYVPLT YFE (SEQ ID NO:
6) (SEQ ID NO: 7) (SEQ ID NO: 5)
TABLE-US-00003 TABLE 3 VH CDR Amino Acid Sequences Ab VH CDR1 VH
CDR2 VH CDR3 15A7H SFGMH YINGGSSTIFYANA YASYGGGAMDY (SEQ ID NO: 8)
VKG (SEQ ID NO: 10) (SEQ ID NO: 9)
[0046] In a specific embodiment, an antibody described herein
immunospecifically binds to human PSGL-1 and comprises: (i) a
variable light ("VL") chain region comprising a VL CDR1, VL CDR2,
and VL CDR3 having the amino acid sequences of SEQ ID NO: 5, SEQ ID
NO: 6, and SEQ ID NO: 7, respectively; (ii) a variable heavy ("VH")
chain region comprising a VH CDR1, VH CDR2, and VH CDR3 having the
amino acid sequences of SEQ ID NO: 8, SEQ ID NO: 9, and SEQ ID NO:
10, respectively; and (iii) a human IgG4 heavy chain constant
region containing an IgG hinge region comprising one or more amino
acid substitutions in the hinge region, wherein said antibody
retains specific binding to said PSGL-1 and wherein IgG4 shuffling
is reduced relative to an antibody comprising an IgG4 hinge region
not comprising said one or more amino acid substitutions.
[0047] In a specific embodiment, an antibody described herein
immunospecifically binds to human PSGL-1 and comprises: (i) a VL
chain region comprising the amino acid sequence of SEQ ID NO: 3;
(ii) a VH chain region comprising a VH CDR1, VH CDR2, and VH CDR3
having the amino acid sequences of SEQ ID NO: 8, SEQ ID NO: 9, and
SEQ ID NO: 10, respectively; and (iii) a human IgG4 heavy chain
constant region containing an IgG4 hinge region comprising one or
more amino acid substitutions in the hinge region, wherein said
antibody retains specific binding to said PSGL-1 and wherein IgG4
shuffling is reduced relative to an antibody comprising an IgG4
hinge region not comprising said one or more amino acid
substitutions.
[0048] In a specific embodiment, an antibody described herein
immunospecifically binds to human PSGL-1 and comprises: (i) a VL
chain region comprising the amino acid sequence of SEQ ID NO: 3;
(ii) a heavy chain comprising VH chain region comprising a VH CDR1,
VH CDR2, and VH CDR3 having the amino acid sequences of SEQ ID NO:
8, SEQ ID NO: 9, and SEQ ID NO: 10, respectively; and (iii) a human
IgG4 heavy chain constant region comprising a Serine to Proline
amino acid substitution at amino acid position 228 of the heavy
chain numbered according to the EU index.
[0049] In a specific embodiment, an antibody described herein
immunospecifically binds to human PSGL-1 and comprises: (i) a VL
chain region comprising a VL CDR1, VL CDR2, and VL CDR3 having the
amino acid sequences of SEQ ID NO: 5, SEQ ID NO: 6, and SEQ ID NO:
7, respectively; (ii) a VH chain region comprising the amino acid
sequence of SEQ ID NO: 4; and (iii) a human IgG4 heavy chain
constant region containing an IgG4 hinge region comprising one or
more amino acid substitutions in the hinge region, wherein said
antibody retains specific binding to said PSGL-1 and wherein IgG4
shuffling is reduced relative to an antibody comprising an IgG4
hinge region not comprising said one or more amino acid
substitutions.
[0050] In a specific embodiment, an antibody described herein
immunospecifically binds to human PSGL-1 and comprises: (i) a VL
chain region comprising a VL CDR1, VL CDR2, and VL CDR3 having the
amino acid sequences of SEQ ID NO: 5, SEQ ID NO: 6, and SEQ ID NO:
7, respectively; (ii) a heavy chain comprising a VH chain region
comprising the amino acid sequence of SEQ ID NO: 4; and (iii) a
human IgG4 heavy chain constant region comprising a Serine to
Proline amino acid substitution at amino acid position 228 of the
heavy chain numbered according to the EU index.
[0051] In specific embodiments, an antibody described herein, which
immunospecifically binds to PSGL-1, e.g., a human PSGL-1
polypeptide of SEQ ID NO:11, comprises framework regions (e.g.,
framework regions of the VL domain and VH domain). Non-limiting
examples of human framework regions are described in the art, e.g.,
see Kabat et al. (1991) Sequences of Proteins of Immunological
Interest, Fifth Edition, U.S. Department of Health and Human
Services, NIH Publication No. 91-3242).
[0052] Table 4, below, presents the VL framework (FR) amino acid
sequences (in particular, VL FR1, VL FR2, VL FR3, and VL FR4
sequences) of antibody 15A7H. Table 5, below, presents the VH FR
amino acid sequences (in particular, VH FR1, VH FR2, VH FR3, and VH
FR4 sequences) of antibody 15A7H.
TABLE-US-00004 TABLE 4 VL FR Amino Acid Sequences Ab VL FR1 VL FR2
VL FR3 VL FR4 15A7H DIQMTQSPSSLSASVG WYQQKPGKAPKLL
GVPSRFSGSGSGTHFTLTISS FGQGTKVEIKR DRVTITC IY LQPEDFATYYC (SEQ ID
NO: 16) (SEQ ID NO: 13) (SEQ ID NO: 14) (SEQ ID NO: 15)
TABLE-US-00005 TABLE 5 VH FR Amino Acid Sequences Ab VH FR1 VH FR2
VH FR3 VH FR4 15A7H EVQLVESGGGLVQPG WVRQAPGKGLEWVA
RFTISRDNAKNTLYLQMNS WGQGTLVTVSS GSLRLSCAASGFTFS (SEQ ID NO: 18)
LRAEDTAVYYCAR (SEQ ID NO: 20) (SEQ ID NO: 17) (SEQ ID NO: 19)
[0053] In certain embodiments, the above-described IgG4 antibodies
having a mutation in the hinge region that reduces IgG4 shuffling
comprise the VL FRs having the amino acid sequence described herein
(see Table 4). In specific embodiments, an antibody described
herein comprises a VL chain region comprising FR1, CDR1, FR2, CDR2,
FR3, CDR3, and FR4 of Tables 1 and 3.
[0054] In certain embodiments, the above-described IgG4 antibodies
having a mutation in the hinge region that reduces IgG4 shuffling
comprise the VH FRs having the amino acid sequence described herein
(see Table 5). In specific embodiments, an antibody comprises a VH
chain region comprising FR1, CDR1, FR2, CDR2, FR3, CDR3, and FR4,
of Tables 2 and 4.
[0055] In another specific embodiment, an IgG4 monoclonal antibody
described herein comprises (i) a VL chain region comprising VL FR1,
VL FR2, VL FR3, and VL FR4 having the amino acid sequences of SEQ
ID NO: 13, 14, 15, and 16, respectively; and (ii) a VH chain region
comprising VH FR1, VH FR2, VH FR3, and VH FR4 having the amino acid
sequences of SEQ ID NO: 17, 18, 19, and 20, respectively, and has a
mutation in the hinge region that reduces IgG4 shuffling. In a
specific embodiment, an antibody described herein comprises a VL
chain region comprising VL FR1, VL FR2, VL FR3, and VL FR4 having
the amino acid sequences of SEQ ID NOs:13, 14, 15, and 16,
respectively. In another specific embodiment, an antibody described
herein comprises a VH chain region comprising VH FR1, VH FR2, VH
FR3, and VH FR4 having the amino acid sequences of SEQ ID NOs:17,
18, 19, and 20, respectively.
[0056] In particular embodiments, the glycosylation of the constant
region of antibodies described herein can be modified. For example,
an aglycoslated constant region of an antibody can be made (i.e.,
the antibody constant region lacks glycosylation) or a constant
region of an antibody comprising a mutation or substitution at one
or more glycosylation sites to eliminate glycosylation at the one
or more glycosylation sites can be made.
[0057] Glycosylation can occur via N-linked (or asparagine-linked)
glycosylation or O-linked glycosylation. N-linked glycosylation
involves carbohydrate modification at the side-chain NH.sub.2 group
of an asparagine amino acid in a polypeptide. O-linked
glycosylation involves carbohydrate modification at the hydroxyl
group on the side chain of a serine, threonine, or hydroxylysine
amino acid.
[0058] In certain embodiments, aglycosylated antibodies can be
produced in bacterial cells which lack the necessary glycosylation
machinery. Cells with altered glycosylation machinery have been
described in the art and can be used as host cells in which to
express recombinant antibodies described herein to thereby produce
an antibody with altered glycosylation. See, for example, Shields,
R. L. et al. (2002) J. Biol. Chem. 277:26733-26740; Umana et al.
(1999) Nat. Biotech. 17:176-1, as well as, European Patent No: EP
1,176,195; PCT Publications WO 03/035835; WO 99/54342.
[0059] In certain embodiments, one or more modifications can be
made to the Fc region of an antibody described here, generally, to
alter one or more functional properties of the antibody, such as
serum half-life, complement fixation, Fc receptor binding, and/or
antigen-dependent cellular cytotoxicity. These modifications are
known in the art, and are described in for example, International
Patent Application Publication No. WO 2008/153926 A2. Examples of
such modifications include, but are not limited to: 1) altering the
number of cysteine residues in the hinge region to facilitate
assembly of the light and heavy chains or to increase or decrease
the stability of the antibody; 2) mutating one or more amino acid
in the CH2-CH3 domain interface region of the Fc-hinge fragment of
an antibody to decrease the biological half life of the antibody;
3) replacing one or more amino acids selected from amino acid
residues 234, 235, 236, 237, 297, 318, 320 and 322 according to the
EU index of Kabat with a different amino acid residue such that the
antibody has an altered affinity for an effector ligand but retains
the antigen-binding ability of the parent antibody; and/or 4)
modifying one or more amino acids at the following positions: 238,
239, 248, 249, 252, 254, 255, 256, 258, 265, 267, 268, 269, 270,
272, 276, 278, 280, 283, 285, 286, 289, 290, 292, 293, 294, 295,
296, 298, 301, 303, 305, 307, 309, 312, 315, 320, 322, 324, 326,
327, 329, 330, 331, 333, 334, 335, 337, 338, 340, 360, 373, 376,
378, 382, 388, 389, 398, 414, 416, 419, 430, 434, 435, 437, 438 or
439 according to the EU Index of Kabat to increase the ability of
the antibody to mediate antibody dependent cellular cytotoxicity
(ADCC) and/or to increase the affinity of the antibody for an
Fc.gamma. receptor. Provided herein are antibodies and antibody
derived antigen-binding fragments that immunospecifically bind to
PSGL-1 and that can modulate PSGL-1 activity. In certain
embodiments, an antibody and antibody derived antigen-binding
fragment provided herein immunospecifically binds to PSGL-1 without
inhibiting PSGL-1 binding to P-selectin, and induces apoptosis of
activated T cells. PSGL-1 activity can relate to any activity of
PSGL-1 known or described in the art, e.g., activation of T cells
during an inflammatory response. PSGL-1 activity or PSGL-1 function
are used interchangeably herein. In certain aspects, PSGL-1
activity is induced by PSGL-1 ligand (e.g., P-selectin) binding to
PSGL-1.
[0060] In certain embodiments, an anti-PSGL-1 antibody or an
antibody derived antigen-binding fragment described herein does not
block or inhibit binding of P-selectin to PSGL-1.
[0061] In a specific embodiment, an antibody or an antibody derived
antigen-binding fragment described herein reduces leukocyte
recruitment during an inflammatory response.
[0062] In certain aspects, an antibody or an antibody derived
antigen-binding fragment described herein reduces or inhibit
survival of cells that express PSGL-1 and respond to PSGL-1
activity signaling (e.g., cells that proliferate in response to
PSGL-1 ligand stimulation, PSGL-1 signaling or selectin binding),
e.g., induces apoptosis in activated T cells. Cell survival assays
are described in the art and can be readily carried out by one of
skill in the art. For example, cell viability can be assessed by
using trypan-blue staining or other cell death or viability markers
known in the art (e.g., Annexin-1, propidium iodine (PI) or 7-AAD,
see Gerber A, Bohne M, Rasch J, Struy H, Ansorge S, Gollnick H.,
2000. Investigation of annexin V binding to lymphocytes after
extracorporeal photoimmunotherapy as an early marker of apoptosis.
Dermatology. 2000; 201(2):111-7, Coder, D. M. 2001. Assessment of
Cell Viability. Current Protocols in Cytometry. 9.2.1-9.2.14, and
Muppidi, J., Porter, M. and Siegel, R. M. 2004. Measurement of
Apoptosis and Other Forms of Cell Death. Current Protocols in
Immunology. 59:3.17.1-3.17.36.).
[0063] In specific embodiments, antibodies described herein
specifically bind to PSGL-1 and inhibit (e.g., partially or
completely inhibit) activated T cell survival as assessed by
methods described herein or known to one of skill in the art (e.g.,
trypan blue exclusion assay, see Coder, D. M. 2001. Assessment of
Cell Viability. Current Protocols in Cytometry. 9.2.1-9.2.14). In
some embodiments, the term "inhibit" or "inhibition" means the
reduction or prevention of activated T cell survival. Activated T
cell survival can be reduced by about 10%, about 20%, about 30%,
about 40%, about 50%, about 60%, about 70%, about 80%, about 90%,
about 100%, about 125%, about 150% or more compared to a control
(e.g., T cell survival in the absence of the antibodies described
herein or in the presence of a non-specific antibody).
[0064] In certain aspects, an anti-PSGL-1 antibody described herein
is capable of inducing apoptosis (i.e., programmed cell death) of
activated T cells that express PSGL-1. Apoptosis assays are
described in the art and can be readily carried out by one of skill
in the art (see, e.g., Muppidi, J., Porter, M. and Siegel, R. M.
2004. Measurement of Apoptosis and Other Forms of Cell Death.
Current Protocols in Immunology. 59:3.17.1-3.17.36). The term
"induce" or "inducing" means initiation of or an increase of
apoptosis above a control level. Apoptosis of activated T cells can
be induced by about 10%, about 20%, about 30%, about 40%, about
50%, about 60%, about 70%, about 80%, about 90%, about 100%, about
125%, about 150% or more compared to a control (e.g. Apoptosis of
activated T cells in the absence of the antibodies describe herein
or in the presence of a non-specific antibody).
[0065] T cells and T cell lines which are appropriate for use in
the assays described herein relating to PSGL-1 activity are readily
available (e.g., ARR, DU.528, Jurkat, H-SB2, RPMI 8402, CML-T1,
Karpas 45, KE-37/SKW-3, SUP-T1, SUP-T3, MOLT 3/4, P12-Ichikawa,
PF-382, CCRF-CEM, HPB-ALL, K-T1, TALL-1, MOLT 16/17, TALL-104,
DND-41, Loucy, MOLT 13, Peer/Be13, HUT 78/H9, HUT 102, MT-1, DEL,
JB6, Karpas 299, SU-DHL1, 12H5, 3DO54.8, 3DO11.10, 8DO51.15, or
3DO18.3) or can be readily identified using methods known in the
art (see, e.g., Thornton, A. M. 2003. Fractionation of T and B
Cells Using Magnetic Beads. Current Protocols in Immunology.
55:3.5A.1-3.5A.11., Hathcock, K. 2001. T Cell Enrichment by
Cytotoxic Elimination of B Cells and Accessory Cells. Current
Protocols in Immunology. 00:3.3.1-3.3.5., Horgan, K., Shaw, S, and
Boirivant, M. 2009. Immunomagnetic Purification of T Cell
Subpopulations. Current Protocols in Immunology. 85:7.4.1-7.4.9.,
and Kanof, M. E. 2001. Purification of T Cell Subpopulations.
Current Protocols in Immunology. 00:7.3.1-7.3.5). In particular
embodiments, cells or cell lines for use in cell proliferation
assays can express PSGL-1, endogenously or recombinantly. Cells or
cell lines for use in cell viability assays can express PSGL-1,
endogenously or recombinantly, and exert changes in cell viability
in response to PSGL-1 ligand or anti-PSGL-1 antibody binding. Cells
or cell lines for use in apoptosis assays can express PSGL-1,
endogenously or recombinantly, and exert changes in apoptosis in
response to PSGL-1 ligand or anti-PSGL-1 antibody binding.
Preferably the cells or cell lines are human (e.g. ARR, DU.528,
Jurkat, H-SB2, RPMI 8402, CML-T1, Karpas 45, KE-37/SKW-3, SUP-T1,
SUP-T3, MOLT 3/4, P12-Ichikawa, PF-382, CCRF-CEM, HPB-ALL, K-T1,
TALL-1, MOLT 16/17, TALL-104, DND-41, Loucy, MOLT 13, Peer/Be13,
HUT 78/H9, HUT 102, MT-1, DEL, JB6, Karpas 299, or SU-DHL1).
[0066] Methods for determining immunospecific binding of an
antibody to its target antigen are readily available and described
in the art. For example, the affinities and binding properties of
an antibody for its target antigen, can be determined by a variety
of in vitro assay methods (biochemical or immunological based
assays) known in the art such as equilibrium methods (e.g.,
enzyme-linked immunoabsorbent assay (ELISA), or radioimmunoassay
(RIA)), or kinetics (e.g., Biacore.TM. analysis), and other methods
such as indirect binding assays, competitive inhibition assays,
fluorescence resonance energy transfer (FRET), immunoprecipitation,
gel electrophoresis and chromatography (e.g., gel filtration).
These and other methods can utilize a label on one or more of the
components being examined and/or employ a variety of detection
methods including but not limited to chromogenic, fluorescent,
luminescent, or isotopic labels. In certain embodiments, use of
labels is not necessary, e.g., Biacore.TM. systems utilize the
natural phenomenon of surface plasmon resonance (SPR) to deliver
data in real time, without the use of labels.
[0067] In a specific embodiment, the antibodies described herein
are isolated. In a specific embodiment, the antibodies described
herein are purified. In a particular embodiment, an antibody
described herein is a recombinant monoclonal antibody.
[0068] In a particular embodiment, provided herein is an antibody
or antibody derived antigen-binding fragment which has been
modified in a manner suitable for large scale manufacturing. This
can involve cloning polynucleotide sequences encoding the necessary
domains of an anti-PSGL-1 antibody, such as one or more CDRs or
FRs, into a suitable expression vector which also contains
polynucleotide sequences encoding suitable constant regions, so
that an entire antibody is produced. The polynucleotide sequences
provided by the expression vectors are nucleotide sequences which
can be optimized to maximize antibody yield and stability for cell
culture manufacturing conditions and purification processes.
[0069] 6.2 Polynucleotides
[0070] In certain aspects, provided herein are polynucleotides
comprising a nucleotide sequence encoding an antibody or an
antibody derived antigen-binding fragment described herein that
immunospecifically bind to a PSGL-1 antigen, and vectors comprising
such polynucleotides for recombinant expression in host cells
(e.g., microbial organisms, such as E. coli and mammalian cells,
such as murine hybridoma cells, CHO cells, and 3T3 fibroblasts).
Provided herein are polynucleotides comprising nucleotide sequences
encoding any of the antibodies or antibody derived antigen-binding
fragment thereof provided herein, as well as vectors comprising
such polynucleotide sequences, e.g., expression vectors for their
efficient expression in host cells, e.g., mammalian cells. In a
specific embodiment, the polynucleotide comprising nucleotide
sequences encoding any of the antibodies or antibody derived
antigen-binding fragments described herein are isolated or
purified.
[0071] In particular aspects, provided herein are polynucleotides
comprising nucleotide sequences encoding antibodies or antibody
derived antigen-binding fragments, which immunospecifically bind to
PSGL-1 and comprises an amino acid sequence as described
herein.
[0072] In specific embodiments, a polynucleotide described herein
encodes a heavy chain having the amino acid sequence of SEQ ID NO:
2.
[0073] The polynucleotides can comprise nucleotide sequences
encoding a heavy chain comprising the VH FRs and CDRs of antibodies
described herein (see, e.g., Tables 2 and 4) and further comprising
a mutation in the hinge region that reduces IgG4 shuffling.
[0074] Also provided herein are polynucleotides encoding an
antibody heavy chain comprising SEQ ID NO: 2, which are optimized,
e.g., by codon/RNA optimization, replacement with heterologous
signal sequences, and elimination of mRNA instability elements.
Also provided herein are polynucleotides encoding an antibody light
chain comprising SEQ ID NO: 1, which are optimized, e.g., by
codon/RNA optimization, replacement with heterologous signal
sequences, and elimination of mRNA instability elements. Methods to
generate optimized nucleic acids encoding an antibody or an
antibody derived antigen-binding fragment for recombinant
expression by introducing codon changes and/or eliminating
inhibitory regions in the mRNA can be carried out by adapting the
optimization methods described in, e.g., U.S. Pat. Nos. 5,965,726;
6,174,666; 6,291,664; 6,414,132; and 6,794,498, accordingly. For
example, potential splice sites and instability elements (e.g., A/T
or A/U rich elements) within the RNA can be mutated without
altering the amino acids encoded by the nucleic acid sequences to
increase stability of the RNA for recombinant expression. The
alterations utilize the degeneracy of the genetic code, e.g., using
an alternative codon for an identical amino acid. In some
embodiments, it can be desirable to alter one or more codons to
encode a conservative mutation, e.g., a similar amino acid with
similar chemical structure and properties and/or function as the
original amino acid. Such methods can increase expression of an
anti-PSGL-1 antibody or an antigen-binding fragment thereof
relative to the expression of an anti-PSGL-1 antibody encoded by
polynucleotides that have not been optimized. Furthermore, the
polynucleotide sequences can be designed to match the preferred
codon usage in the host cell, e.g. E. coli codon usage or CHO codon
usage.
[0075] An optimized polynucleotide sequence encoding an antibody or
an antibody derived antigen-binding fragment described herein can
hybridize to an unoptimized polynucleotide sequence encoding an
antibody or an antibody derived antigen-binding fragment described
herein. In specific embodiments, an optimized nucleotide sequence
encoding an antibody or an antibody derived antigen-binding
fragment described herein hybridizes under high stringency
conditions to an unoptimized polynucleotide sequence encoding an
antibody or an antibody derived antigen-binding fragment described
herein. In a specific embodiment, an optimized nucleotide sequence
encoding an antibody or an antibody derived antigen-binding
fragment described herein hybridizes under high stringency,
intermediate or lower stringency hybridization conditions to an
unoptimized nucleotide sequence encoding an antibody or an antibody
derived antigen-binding fragment described herein. Information
regarding hybridization conditions have been described, see, e.g.,
U.S. Patent Application Publication No. US 2005/0048549 (e.g.,
paragraphs 72-73), which is incorporated herein by reference in its
entirety.
[0076] The polynucleotides can be obtained, and the nucleotide
sequence of the polynucleotides determined, by any method known in
the art. Nucleotide sequences encoding antibodies or antibody
derived antigen-binding fragments described herein, and modified
versions of these antibodies or antibody derived antigen-binding
fragments can be determined using methods well known in the art,
i.e., nucleotide codons known to encode particular amino acids are
assembled in such a way to generate a nucleic acid that encodes the
antibody or the antibody derived antigen-binding fragment. Such a
polynucleotide encoding the antibody can be assembled from
chemically synthesized oligonucleotides (e.g., as described in
Kutmeier et al., 1994, BioTechniques 17:242), which, for example,
involves the synthesis of overlapping oligonucleotides containing
portions of the sequence encoding the antibody or the antibody
derived antigen-binding fragment, annealing and ligating of those
oligonucleotides, and then amplification of the ligated
oligonucleotides by PCR. Various methods to generate synthetic
genes from oligonucleotides are known in the art.
[0077] Alternatively, a polynucleotide encoding an antibody or an
antibody derived antigen-binding fragment described herein can be
generated from nucleic acid from a suitable source (e.g., a
hybridoma) using methods well known in the art (e.g., PCR and other
molecular cloning methods). For example, PCR amplification using
synthetic primers hybridizable to the 3' and 5' ends of a known
sequence can be performed using genomic DNA obtained from cells
producing the antibody of interest, e.g. hybridoma cells. Such PCR
amplification methods can be used to obtain nucleic acids
comprising the sequence encoding the light chain and/or heavy chain
of an antibody. Such PCR amplification methods can be used to
obtain nucleic acids comprising the sequence encoding the variable
light chain region and/or the variable heavy chain region of an
antibody. The amplified nucleic acids can be cloned into vectors
for expression in host cells and for further cloning, for example,
to generate chimeric and humanized antibodies. The constant chain
is usually kappa or lambda for the antibody light chain, for the
antibody heavy chain it can be, without limitation, any IgG isotype
(e.g. human IgG1, IgG2, IgG3 or IgG4) or other immunoglobulins,
including allelic variants.
[0078] If a clone containing a nucleic acid encoding a particular
antibody is not available, but the sequence of the antibody
molecule is known, a nucleic acid encoding the immunoglobulin can
be chemically synthesized and cloned into replicable cloning
vectors using any method well known in the art.
[0079] DNA encoding antibodies or antibody derived antigen-binding
fragments described herein can be readily isolated and sequenced
using conventional procedures (e.g., by using oligonucleotide
probes that are capable of binding specifically to nucleic acids
encoding the heavy and light chains of the antibodies). Once
isolated, the DNA can be placed into expression vectors, which are
then transfected into prokaryotic or eukaryotic host cells such as
E. coli cells, yeast (Pichia, Saccharomyces) simian COS cells,
Chinese hamster ovary (CHO) cells, myeloma cells (NS0), insect or
plant cells that do not otherwise produce immunoglobulin protein,
to obtain the synthesis of antibodies or antibody derived
antigen-binding fragments in the recombinant host cells.
[0080] 6.3 Host Cells and Recombinant Expression of Antibodies
[0081] In certain aspects, provided herein are host cells
recombinantly expressing the antibodies or antibody derived
antigen-binding fragments described herein and related expression
vectors. Provided herein are expression vectors comprising
polynucleotides comprising nucleotide sequences encoding antibodies
or antibody derived antigen-binding fragments described herein for
recombinant expression in prokaryotic and eukaryotic host cells,
preferably in mammalian cells. Also provided herein are host cells
comprising such expression vectors for recombinantly expressing
antibodies or antibody derived antigen-binding fragments described
herein. In a particular aspect, provided herein are methods for
producing an antibody or an antibody derived antigen-binding
fragment described herein, comprising expressing such antibody or
antibody derived antigen-binding fragment from a host cell.
[0082] Recombinant expression of an antibody or an antibody derived
antigen-binding fragment described herein that immunospecifically
binds to a PSGL-1 antigen involves construction of an expression
vector containing a polynucleotide that encodes the antibody or the
antibody derived antigen-binding fragment. Once the polynucleotide
encoding an antibody or an antibody derived antigen-binding
fragment described herein has been obtained, the vector for the
production of the antibody or the antibody derived antigen-binding
fragment can be produced by recombinant DNA technology using
techniques well-known in the art. Thus, methods for preparing an
antibody or an antibody derived antigen-binding fragment by
expressing a polynucleotide containing an antibody or an antibody
derived antigen-binding fragment encoding nucleotide sequence(s)
are described herein. Methods which are well known to those skilled
in the art can be used to construct expression vectors containing
antibody coding sequences or antibody derived antigen-binding
fragment coding sequences and appropriate transcriptional and
translational control signals. These methods include, for example,
in vitro recombinant DNA techniques, synthetic techniques, and in
vivo genetic recombination. Also provided are replicable vectors
comprising a nucleotide sequence encoding an antibody described
herein, a heavy or light chain of an antibody, a heavy or light
chain variable domain of an antibody, or an antibody derived
antigen-binding fragment, operably linked to a promoter, in
particular, a promoter providing for expression in a mammalian
cell. Such vectors can include the nucleotide sequence encoding the
constant region of the antibody molecule (see, e.g., International
Publication Nos. WO 86/05807 and WO 89/01036; and U.S. Pat. No.
5,122,464) and the variable domain of the antibody can be cloned
into such a vector for expression of the entire heavy, the entire
light chain, or both the entire heavy and light chains. Expression
vectors include plasmids, retroviruses, cosmids, EBV-derived
episomes, artificial chromosomes and the like. The expression
vector and expression control sequences are selected to be
compatible with the host cell. The recombinant expression vector
may also encode a signal peptide that facilitates secretion of the
antibody chains from a host cell. The signal peptide may be an
immunoglobulin signal peptide, a heterologous peptide from a
non-immunoglobulin protein or an artificial peptide.
[0083] The expression vector is transferred to a host cell by
conventional techniques known in the art (e.g. liposome-mediated
transfection, polycation-mediated transfection, protoplast fusion,
microinjections, calcium phosphate precipitation, electroporation,
transfer by viral vectors) and the transfected cells are then
cultured by conventional techniques to produce an antibody or an
antibody derived antigen-binding fragment described herein. Thus,
provided herein are host cells containing a polynucleotide encoding
an antibody or an antibody derived antigen-binding fragment
described herein, operably linked to a heterologous promoter. In
certain embodiments for the expression of double-chained
antibodies, vectors encoding both the heavy and light chains can be
co-expressed in the host cell for expression of the entire
immunoglobulin molecule, as detailed below. In certain embodiments,
a host cell contains a vector comprising a polynucleotide encoding
both the heavy chain and light chain of an antibody described
herein, or antigen-binding fragment thereof. In specific
embodiments, a host cell contains two different vectors, a first
vector comprising a polynucleotide encoding a heavy chain of an
antibody described herein, or a fragment thereof (e.g., an
antigen-binding fragment thereof), and a second vector comprising a
polynucleotide encoding a light chain of an antibody described
herein, or a fragment thereof (e.g., an antigen-binding fragment
thereof). In other embodiments, a first host cell comprises a first
vector comprising a polynucleotide encoding a heavy chain of an
antibody described herein, or a fragment thereof, and a second host
cell comprises a second vector comprising a polynucleotide encoding
a light chain of an antibody described herein.
[0084] A variety of host-expression vector systems can be utilized
to express antibody molecules or antibody derived antigen-binding
fragments described herein (see, e.g., U.S. Pat. No. 5,807,715 and
U.S. Pat. No. 7,604,800). Such host-expression systems represent
vehicles by which the coding sequences of interest can be produced
and subsequently purified, but also represent cells which can, when
transformed or transfected with the appropriate nucleotide coding
sequences, express an antibody molecule or an antibody derived
antigen-binding fragment described herein in situ. These include
but are not limited to microorganisms such as bacteria (e.g., E.
coli and B. subtilis) transformed with recombinant bacteriophage
DNA, plasmid DNA or cosmid DNA expression vectors containing
antibody coding sequences or antibody derived antigen-binding
fragment coding sequences; yeast (e.g., Saccharomyces, Pichia)
transformed with recombinant yeast expression vectors containing
antibody coding sequences or antibody derived antigen-binding
fragment coding sequences; insect cell systems infected with
recombinant virus expression vectors (e.g., baculovirus) containing
antibody coding sequences or antibody derived antigen-binding
fragment coding sequences; plant cell systems (e.g., green algae
such as Chlamydomonas reinhardtii) infected with recombinant virus
expression vectors (e.g., cauliflower mosaic virus, CaMV; tobacco
mosaic virus, TMV) or transformed with recombinant plasmid
expression vectors (e.g., Ti plasmid) containing antibody coding
sequences or antibody derived antigen-binding fragment coding
sequences; or mammalian cell systems (e.g., COS, CHO, BHK, MDCK,
HEK 293, NS0, PER.C6, VERO, CRL7O3O, HsS78Bst, HeLa, and NIH 3T3
cells) harboring recombinant expression constructs containing
promoters and/or enhancers derived from the genome of mammalian
cells (e.g., metallothionein promoter, immunoglobulin promoter,
actin promoter) or from mammalian viruses (e.g., the adenovirus
late promoter; the vaccinia virus 7.5K promoter, CMV, Simian Virus
40). Other regulatory elements for expression in eukaryotic cells
are polyadenylation signals such as BGH polyA, SV40 late or early
polyA. Alternatively, polyadenylation signals of immunoglobulin or
other genes can be used. In another specific embodiment, eukaryotic
cells, especially for the expression of an IgG4 monoclonal antibody
described herein, are used for the expression of an antibody
described herein or antigen-binding fragment thereof. For example,
mammalian cells such as Chinese hamster ovary (CHO) cells, in
conjunction with a vector such as the major intermediate early gene
promoter element from human cytomegalovirus is an effective
expression system for antibodies (Foecking et al., 1986, Gene
45:101; and Cockett et al., 1990, Bio/Technology 8:2). In certain
embodiments, antibodies described herein are produced by CHO cells
or NS0 cells. In a specific embodiment, the expression of
nucleotide sequences encoding antibodies or antibody derived
antigen-binding fragments described herein which immunospecifically
bind to a PSGL-1 antigen is regulated by a constitutive promoter,
inducible promoter or tissue specific promoter.
[0085] In bacterial systems, a number of expression vectors can be
advantageously selected depending upon the use intended for the
antibody molecule being expressed. For example, when a large
quantity of such an antibody or an antibody derived antigen-binding
fragment is to be produced, for the generation of pharmaceutical
compositions of an antibody molecule or an antibody derived
antigen-binding fragment, vectors which direct the expression of
high levels of fusion protein products that are readily purified
can be desirable.
[0086] In addition, a host cell strain can be chosen which
modulates the expression of the inserted sequences, or modifies and
processes the gene product in the specific fashion desired. Such
modifications (e.g., glycosylation) and processing of protein
products can be important for the function of the protein.
Different host cells have characteristic and specific mechanisms
for the post-translational processing and modification of proteins
and gene products. Appropriate cell lines or host cells can be
chosen to ensure the correct modification and processing of the
foreign protein expressed. To this end, eukaryotic host cells which
possess the cellular machinery for proper processing of the primary
transcript, glycosylation, and phosphorylation of the gene product
can be used. Such mammalian host cells include but are not limited
to CHO, VERO, BHK, Hela, COS, MDCK, HEK 293, NIH 3T3, W138, BT483,
Hs578T, HTB2, BT2O and T47D, NS0 (a murine myeloma cell line that
does not endogenously produce any immunoglobulin chains), CRL7O3O
and HsS78Bst cells. In certain embodiments, antibodies or antibody
derived antigen-binding fragments described herein are produced in
mammalian cells, such as CHO cells.
[0087] For long-term, high-yield production of recombinant
antibodies or antibody derived antigen-binding fragments, stable
expression is preferred. For example, mammalian cell lines which
stably express the antibody molecule or the antibody derived
antigen-binding fragment can be engineered. Rather than using
expression vectors which contain viral origins of replication, host
cells can be transformed with DNA controlled by appropriate
expression control elements (e.g., promoter, enhancer, sequences,
transcription terminators, polyadenylation sites, etc.), and a
selectable marker. Following the introduction of the foreign DNA,
engineered cells can be allowed to grow for 1-2 days in
non-selective media, and then are switched to a selective media.
The selectable marker in the recombinant plasmid confers resistance
to the selection and allows cells to stably integrate the plasmid
into their chromosomes. After single cell cloning cells are
expanded into production cell lines. This method can advantageously
be used to engineer cell lines which express the antibody molecule
or the antibody derived antigen-binding fragment. Such engineered
cell lines can be particularly useful in screening and evaluation
of compositions that interact directly or indirectly with the
antibody molecule.
[0088] A number of selection systems can be used, including but not
limited to, the herpes simplex virus thymidine kinase (Wigler et
al., 1977, Cell 11:223), hypoxanthineguanine
phosphoribosyltransferase (Szybalska & Szybalski, 1992, Proc.
Natl. Acad. Sci. USA 48:202), and adenine phosphoribosyltransferase
(Lowy et al., 1980, Cell 22:8-17) genes can be employed in tk-,
hgprt- or aprt-cells, respectively. Also, antimetabolite resistance
can be used as the basis of selection for the following genes:
dhfr, which confers resistance to methotrexate (Wigler et al.,
1980, Natl. Acad. Sci. USA 77:357; O'Hare et al., 1981, Proc. Natl.
Acad. Sci. USA 78:1527); gpt, which confers resistance to
mycophenolic acid (Mulligan & Berg, 1981, Proc. Natl. Acad.
Sci. USA 78:2072); neo, which confers resistance to the
aminoglycoside G-418 (Wu and Wu, 1991, Biotherapy 3:87-95;
Tolstoshev, 1993, Ann. Rev. Pharmacol. Toxicol. 32:573-596;
Mulligan, 1993, Science 260:926-932; and Morgan and Anderson, 1993,
Ann. Rev. Biochem. 62:191-217; May, 1993, TIB TECH 11(5):155-2 15);
and hygro, which confers resistance to hygromycin (Santerre et al.,
1984, Gene 30:147). Methods commonly known in the art of
recombinant DNA technology can be routinely applied to select the
desired recombinant clone, and such methods are described, for
example, in Ausubel et al. (eds.), Current Protocols in Molecular
Biology, John Wiley & Sons, NY (1993); Kriegler, Gene Transfer
and Expression, A Laboratory Manual, Stockton Press, NY (1990); and
in Chapters 12 and 13, Dracopoli et al. (eds.), Current Protocols
in Human Genetics, John Wiley & Sons, NY (1994);
Colberre-Garapin et al., 1981, J. Mol. Biol. 150:1, which are
incorporated by reference herein in their entireties.
[0089] The expression levels of an antibody molecule or an antibody
derived antigen-binding fragment can be increased by vector
amplification (for a review, see Bebbington and Hentschel, The use
of vectors based on gene amplification for the expression of cloned
genes in mammalian cells in DNA cloning, Vol. 3 (Academic Press,
New York, 1987)). When a marker in the vector system expressing
antibody or antibody derived antigen-binding fragment is
amplifiable, increase in the level of inhibitor present in culture
of host cell will increase the number of copies of the marker gene.
Since the amplified region is associated with the antibody or
antibody derived antigen-binding fragment gene, production of the
antibody or the antibody derived antigen-binding fragment will also
increase (Crouse et al., 1983, Mol. Cell. Biol. 3:257).
[0090] The host cell can be co-transfected with two or more
expression vectors described herein, the first vector encoding a
heavy chain derived polypeptide and the second vector encoding a
light chain derived polypeptide. The two vectors can contain
identical selectable or different selections markers which enable
sufficient expression of heavy and light chain polypeptides. The
host cells can be co-transfected with different amounts of the two
or more expression vectors.
[0091] Alternatively, a single vector can be used which encodes,
and is capable of expressing, both heavy and light chain
polypeptides. The coding sequences for the heavy and light chains
can comprise cDNA or genomic DNA. The expression vector can be
monocistronic or multicistronic. For example, a bicistronic nucleic
acid construct can comprise in the following order a promoter, a
heavy chain of an antibody described herein), and a light chain of
an antibody described herein. In such an expression vector, the
transcription of both chains can be driven by the promoter, whereas
the translation of the mRNA from the heavy chain can be by a
cap-dependent scanning mechanism and the translation of the mRNA
from the light chain can be by a cap-independent mechanism, e.g.,
by an IRES.
[0092] In some embodiments, the antibody molecules or antibody
derived antigen-binding fragments are produced by culturing the
host cells for a period of time sufficient to allow for high
expression of the molecules in the host cells. In some embodiments
the molecules are expressed in mammalian cells, for example in CHO
cells in serum-free media or in chemically defined media. In some
embodiments, the antibody molecules or antibody derived
antigen-binding fragments are recovered from the culture medium as
a secreted polypeptide or it can be recovered from the host cell
lysates if for example expressed without a secretory signal.
[0093] Once an antibody molecule or antibody derived
antigen-binding fragment described herein has been produced by
recombinant expression, it can be purified by any method known in
the art for purification of an immunoglobulin molecule, for
example, by chromatography (e.g., ion exchange, affinity,
particularly by affinity for the specific antigen after Protein A,
and sizing column chromatography), centrifugation, differential
solubility, precipitation, filtration, reverse phase HPLC, or by
any other standard technique for the purification of proteins to
obtain substantially homogenous and biologically active
preparations of the molecules. Further, the antibodies or antibody
derived antigen-binding fragments described herein can be fused to
heterologous polypeptide sequences to facilitate purification.
[0094] In specific embodiments, an antibody or an antibody derived
antigen-binding fragment described herein is isolated or purified.
For example, in a particular embodiment, a preparation of an
antibody or an antibody derived antigen-binding fragment described
herein is substantially free of cellular material, media components
and/or chemical precursors. The language "substantially free of
cellular material" includes preparations of an antibody or an
antibody derived antigen-binding fragment in which the antibody or
the antibody derived antigen-binding fragment is separated from
cellular components of the cells from which it is isolated or
recombinantly produced. When the antibody or antibody derived
antigen-binding fragment is recombinantly produced, it is also
generally substantially free of culture medium, i.e., culture
medium represents less than about 20%, 10%, 2%, 1%, 0.5%, or 0.1%
of the volume of the protein preparation. When the antibody or the
antibody derived antigen-binding fragment is produced by chemical
synthesis, it is generally substantially free of chemical
precursors or other chemicals, i.e., it is separated from chemical
precursors or other chemicals which are involved in the synthesis
of the protein. Accordingly, such preparations of the antibody or
the antibody derived antigen-binding fragment have less than about
30%, 20%, 10%, 5% (by dry weight) of chemical precursors or
compounds other than the antibody of interest or the antibody
derived antigen-binding fragment of interest.
[0095] 6.4 Pharmaceutical Compositions
[0096] Provided herein are compositions, pharmaceutical
compositions, comprising an antibody or an antibody derived
antigen-binding fragment described herein. In particular aspects,
compositions described herein can be for in vitro, in vivo, or ex
vivo uses. In specific embodiments, provided herein is a
pharmaceutical composition comprising an antibody or antibody
derived antigen-binding fragment described herein and a
pharmaceutically acceptable carrier or excipient.
[0097] Therapeutic compositions containing an antibody or an
antibody derived antigen-binding fragment provided herein can be
prepared for storage by mixing the antibody having the desired
degree of purity with optional physiologically acceptable carriers,
excipients or stabilizers (Remington's Pharmaceutical Sciences
(1990) Mack Publishing Co., Easton, Pa.; Remington: The Science and
Practice of Pharmacy, 21st ed. (2006) Lippincott Williams &
Wilkins, Baltimore, Md.), in the form of lyophilized formulations
or aqueous solutions. Acceptable carriers, excipients, or
stabilizers are nontoxic to recipients at the dosages and
concentrations employed, and include buffers such as phosphate,
citrate, sodium citrate dehydrate, and other organic acids; and/or
non-ionic surfactants such as TWEEN.TM., PLURONICS.TM. or
polyethylene glycol (PEG).
[0098] Compositions, such as those described herein, can also
contain more than one active compound (for example, molecules,
e.g., antibody or antibodies described herein) as necessary for the
particular indication being treated. In certain embodiments,
formulations comprise an antibody or an antibody derived
antigen-binding fragment provided herein and one or more active
compounds with complementary activities that do not adversely
affect each other. Such molecules are suitably present in
combination in amounts that are effective for the purpose intended.
For example, an antibody or antibody derived antigen-binding
fragment described herein can be combined with one or more other
therapeutic agents. Such combined therapy can be administered to
the patient serially or simultaneously or in sequence.
[0099] The compositions to be used for in vivo administration can
be sterile. This is readily accomplished by filtration through,
e.g., sterile filtration membranes.
[0100] In specific aspects, the pharmaceutical compositions
provided herein contain therapeutically effective amounts of an
antibody or an antibody derived antigen-binding fragment provided
herein, and optionally one or more additional prophylactic or
therapeutic agents, in a pharmaceutically acceptable carrier. Such
pharmaceutical compositions are useful in the prevention and/or
treatment of a disorder or disease described herein, such as
psoriasis, or one or more of the symptoms thereof. The term
"therapeutically effective amount" refers to the amount that is
safe and sufficient to prevent or treat a disease. As used herein,
the term "treat", "treated," "treating" or "treatment" is used
herein to mean provide a beneficial or desired clinical result in a
subject with a disease. Beneficial or desired clinical results
include, but are not limited to, alleviation of symptoms,
diminishment of extent of disease, stabilized (i.e., not worsening)
state of disease, delay or slowing of disease progression,
amelioration or palliation of the disease state, and remission
(whether partial or total), whether detectable or undetectable.
"Treatment" can also mean prolonging survival as compared to
expected survival if not receiving treatment.
[0101] Pharmaceutical carriers suitable for administration of an
antibody or an antibody derived antigen-binding fragment provided
herein include any such carriers known to those skilled in the art
to be suitable for the particular mode of administration. In one
embodiment, an antibody or an antibody derived antigen-binding
fragment is formulated into suitable pharmaceutical preparations,
such as sterile solutions or suspensions for parenteral
administration.
[0102] In addition, an antibody or an antibody derived
antigen-binding fragment described herein can be formulated as the
sole pharmaceutically active ingredient in the composition or can
be combined with other active ingredients (such as one or more
other prophylactic or therapeutic agents).
[0103] In the compositions, an antibody or an antibody derived
antigen-binding fragment provided herein is mixed with a suitable
pharmaceutical carrier. The concentrations of the antibody or
antibody derived antigen-binding fragment in the compositions can,
for example, be effective for delivery of an amount, upon
administration, that prevents and/or treats a disorder or disease
described herein (e.g., an inflammatory disorder) or symptom
thereof.
[0104] In one embodiment, the compositions are formulated for
single dosage administration. To formulate a composition, the
weight fraction of compound is dissolved, suspended, dispersed or
otherwise mixed in a selected carrier at an effective concentration
such that the disorder is relieved, treated or one or more symptoms
are ameliorated.
[0105] In certain aspects, an antibody or an antibody derived
antigen-binding fragment provided herein is included in the
pharmaceutically acceptable carrier in an effective amount
sufficient to exert a therapeutically useful effect in the absence
of, or with minimal or negligible, undesirable side effects on the
patient treated. A therapeutically effective concentration can be
determined empirically by testing the compounds in in vitro and in
vivo systems using routine methods and then extrapolated therefrom
for dosages for humans.
[0106] The concentration of antibody or antibody derived
antigen-binding fragment in the pharmaceutical composition will
depend on, e.g., the physicochemical characteristics of the
antibody or the antibody derived antigen-binding fragment, the
dosage schedule, and amount administered as well as other factors
known to those of skill in the art.
[0107] The pharmaceutical compositions, in another embodiment,
provide a dosage of from about 0.001 mg to about 100 mg of antibody
or antibody derived antigen-binding fragment per kilogram of body
weight per day. Pharmaceutical dosage unit forms can be prepared to
provide from about 0.001 mg to about 100 mg, and/or a combination
of other optional essential ingredients per dosage unit form. In a
specific embodiment, the antibody or antibody derived
antigen-binding fragment is formulated at a concentration of 40
mg/mL.
[0108] The antibody or the antibody derived antigen-binding
fragment can be administered at once, or can be divided into a
number of smaller doses to be administered at intervals of time. It
is understood that the precise dosage and duration of treatment is
a function of the disease or disorder described herein being
treated and can be determined empirically using known testing
protocols or by extrapolation from in vivo or in vitro test data.
It is to be noted that concentrations and dosage values can also
vary with the severity of the disease or disorder to be alleviated.
It is to be further understood that for any particular subject,
specific dosage regimens can be adjusted over time according to the
individual need and the professional judgment of the person
administering or supervising the administration of the
compositions.
[0109] The pharmaceutical compositions are provided for
administration to humans and animals in unit dosage forms, such as
sterile parenteral solutions or suspensions containing suitable
quantities of an antibody or an antibody derived antigen-binding
fragment describe herein. The antibody or the antibody derived
antigen-binding fragment is, in one embodiment, formulated and
administered in unit-dosage forms or multiple-dosage forms.
Unit-dose forms as used herein refers to physically discrete units
suitable for human and animal subjects and packaged individually as
is known in the art. Each unit-dose contains a predetermined
quantity of the antibody or the antibody derived antigen-binding
fragment sufficient to produce the desired therapeutic effect, in
association with the required pharmaceutical carrier, vehicle or
diluent. Examples of unit-dose forms include ampoules and syringes.
Unit-dose forms can be administered in fractions or multiples
thereof. A multiple-dose form is a plurality of identical
unit-dosage forms packaged in a single container to be administered
in segregated unit-dose form. Examples of multiple-dose forms
include vials, or bottles of pints or gallons. Hence, multiple dose
form is a multiple of unit-doses which are not segregated in
packaging.
[0110] In certain embodiments, an antibody or an antibody derived
antigen-binding fragment described herein are in a liquid
pharmaceutical composition. Liquid pharmaceutically administrable
compositions can, for example, be prepared by dissolving,
dispersing, or otherwise mixing an antibody described herein in a
carrier, such as, for example, water, saline, aqueous dextrose,
glycerol, glycols, ethanol, and the like, to thereby form a
solution or suspension. If desired, the pharmaceutical composition
to be administered can also contain minor amounts of nontoxic
auxiliary substances such as wetting agents, emulsifying agents,
solubilizing agents, and pH buffering agents and the like.
[0111] Actual methods of preparing such dosage forms are known, or
will be apparent, to those skilled in this art; for example, see,
e.g., Remington's Pharmaceutical Sciences (1990) Mack Publishing
Co., Easton, Pa.; Remington: The Science and Practice of Pharmacy,
21st ed. (2006) Lippincott Williams & Wilkins, Baltimore,
Md.
[0112] Dosage forms or compositions containing antibody in the
range of 0.005% to 99.9% with the balance made up from non-toxic
carrier can be prepared. Methods for preparation of these
compositions are known to those skilled in the art.
[0113] Parenteral administration, in one embodiment, is
characterized by injection, either subcutaneously, intramuscularly
or intravenously is also contemplated herein. Injectables can be
prepared in conventional forms, either as liquid solutions or
suspensions, solid forms suitable for solution or suspension in
liquid prior to injection, or as emulsions. The injectables,
solutions and emulsions also contain one or more excipients.
Suitable excipients are, for example, water, saline, dextrose,
glycerol or ethanol. In addition, if desired, the pharmaceutical
compositions to be administered can also contain minor amounts of
non-toxic auxiliary substances such as wetting or emulsifying
agents, pH buffering agents, stabilizers, solubility enhancers, and
other such agents. Other routes of administration may include,
enteric administration, intracerebral administration, nasal
administration, intraarterial administration, intracardiac
administration, intraosseous infusion, intrathecal administration,
intravenous infusion, subcutaneous implantation or injection,
intramuslcular administration, intrarectal administration
intravaginal administration, intragastrical administration,
intratracheal administration, intrapulmonary administration and
intraperitoneal administration.
[0114] Preparations for parenteral administration include sterile
solutions ready for injection, sterile dry soluble products, such
as lyophilized powders, ready to be combined with a solvent just
prior to use, including sterile suspensions ready for injection,
sterile dry insoluble products ready to be combined with a vehicle
just prior to use and sterile emulsions. The solutions can be
either aqueous or nonaqueous.
[0115] If administered intravenously, suitable carriers include
physiological saline or phosphate buffered saline (PBS), water, and
solutions containing thickening and solubilizing agents, such as
glucose, polyethylene glycol, and polypropylene glycol and mixtures
thereof.
[0116] Pharmaceutically acceptable carriers used in parenteral
preparations include aqueous vehicles, nonaqueous vehicles,
antimicrobial agents, isotonic agents, buffers, antioxidants, local
anesthetics, suspending and dispersing agents, emulsifying agents,
sequestering or chelating agents and other pharmaceutically
acceptable substances. Pharmaceutical carriers also include ethyl
alcohol, polyethylene glycol and propylene glycol for water
miscible vehicles; and sodium hydroxide, hydrochloric acid, citric
acid or lactic acid for pH adjustment.
[0117] Illustratively, intravenous or intraarterial infusion of a
sterile aqueous solution containing an antibody is an effective
mode of administration. Another embodiment is a sterile aqueous or
oily solution or suspension containing an active material injected
as necessary to produce the desired pharmacological effect.
[0118] In a specific embodiment, a pharmaceutical preparation
comprises sodium citrate, sodium chloride, citric acid, polysorbate
80, and water. In a specific embodiment, a pharmaceutical
preparation comprises sodium citrate, sodium chloride, and citric
acid. In a specific embodiment, a pharmaceutical preparation
comprises 9.1 mM sodium citrate, 150 mM sodium chloride, and 0.9 mM
citric acid. In a specific embodiment, a pharmaceutical preparation
comprises an antibody or conjugate described herein at a
concentration of 0.267 mM. In a specific embodiment, a
pharmaceutical preparation comprises 2.676 g/L sodium citrate,
8.766 g/L sodium chloride, 0.2 g/L polysorbate 80, and 0.189 g/L
citric acid. In a specific embodiment, a pharmaceutical preparation
comprises an antibody or an antibody derived antigen-binding
fragment described herein at a concentration of 40 g/L. Preferably,
the foregoing pharmaceutical preparations are at pH 6.0.
[0119] In other embodiments, the pharmaceutical compositions are
lyophilized powders, which can be reconstituted for administration
as solutions, emulsions and other mixtures. They can also be
reconstituted and formulated as solids or gels.
[0120] The lyophilized powder is prepared by dissolving an antibody
provided herein, in a suitable solvent. In some embodiments, the
lyophilized powder is sterile. The solvent can contain an excipient
which improves the stability or other pharmacological component of
the powder or reconstituted solution, prepared from the powder.
Excipients that can be used include, but are not limited to,
dextrose, sorbital, fructose, corn syrup, xylitol, glycerin,
glucose, sucrose or other suitable agent. The solvent can also
contain a buffer, such as citrate, sodium or potassium phosphate or
other such buffer known to those of skill in the art at, in one
embodiment, about neutral pH. Subsequent sterile filtration of the
solution followed by lyophilization under standard conditions known
to those of skill in the art provides the desired formulation. In
one embodiment, the resulting solution will be apportioned into
vials for lyophilization. Each vial will contain a single dosage or
multiple dosages of the compound. The lyophilized powder can be
stored under appropriate conditions, such as at about 4.degree. C.
to room temperature.
[0121] Reconstitution of this lyophilized powder with water for
injection provides a formulation for use in parenteral
administration. For reconstitution, the lyophilized powder is added
to sterile water or other suitable carrier. The precise amount
depends upon the selected compound. Such amount can be empirically
determined.
[0122] 6.5 Therapeutic Methods
[0123] Antibodies and antibody derived antigen-binding fragments
described herein are useful for treating disorders and diseases
associated with or caused (in whole or in part) by increased
proliferation and/or numbers of activated T cells relative to the
proliferation and/or numbers of activated T cells found in healthy
individuals or individuals not having the particular disorder or
disease. Such diseases and disorders are known to one skilled in
the art or can be ascertained by one of skill in the art. In a
specific embodiment, antibodies and antibody derived
antigen-binding fragments described herein are useful for treating
an inflammatory disease or disorder. In one embodiment, the
inflammatory disease is an autoimmune disease. In a specific
embodiment, the inflammatory disease or disorder is psoriasis,
plaque psoriasis, psoriatic arthritis, rheumatoid arthritis,
Crohn's disease, and ankylotic spondylitis.
[0124] Non-limiting examples of disorders and diseases that can be
treated using the antibodies and antibody derived antigen-binding
fragments described herein include psoriasis, Crohn's disease,
ankylosing spondylitis, arthritis (including rheumatoid arthritis,
juvenile rheumatoid arthritis, osteoarthritis, and psoriatic
arthritis), diabetes mellitus, multiple sclerosis,
encephalomyelitis, myasthenia gravis, systemic lupus erythematosis,
autoimmune thyroiditis, dermatitis (including atopic dermatitis and
eczematous dermatitis), Sjogren's Syndrome, aphthous ulcer, iritis,
conjunctivitis, keratoconjunctivitis, type I diabetes, inflammatory
bowel diseases, ulcerative colitis, asthma, allergic asthma,
cutaneous lupus erythematosus, scleroderma, vaginitis, proctitis,
drug eruptions, leprosy reversal reactions, erythema nodosum
leprosum, autoimmune uveitis, allergic encephalomyelitis, acute
necrotizing hemorrhagic encephalopathy, idiopathic bilateral
progressive sensorineural hearing loss, aplastic anemia, pure red
cell anemia, idiopathic thrombocytopenia, polychondritis, Wegener's
granulomatosis, chronic active hepatitis, Stevens-Johnson syndrome,
idiopathic sprue, lichen planus, Graves' disease, sarcoidosis,
primary biliary cirrhosis, uveitis posterior, interstitial lung
fibrosis, allergies such as atopic allergy, AIDS, and T cell
neoplasms such as leukemias or lymphomas.
[0125] In addition, antibodies and antibody derived antigen-binding
fragments are useful for preventing and/or treating certain
disorders and diseases associated with or caused (in whole or in
part) by increased proliferation and/or numbers of activated T
cells relative to the proliferation and/or numbers of activated T
cells found in healthy individuals or individuals not having the
particular disorder or disease. Non-limiting examples of disorders
and diseases that can be prevented and/or treated using the
antibodies and antibody derived antigen-binding fragments described
herein include graft-versus-host disease and cases of
transplantation rejection (including transplantation rejection
using allogeneic or xenogeneic tissues) such as bone marrow
transplantation, liver transplantation, kidney transplant, or the
transplantation of any organ or tissue.
[0126] Accordingly, provided herein are methods for preventing and
treating diseases and disorders described herein using an antibody
or antibody derived antigen-binding fragment described herein. In a
specific embodiment, such methods comprise administering to a
subject in need thereof a therapeutically effective amount of an
antibody or an antibody derived antigen-binding fragment described
herein. In specific embodiments, the antibody administered to treat
a disorder or disease described herein is 15A7H.
[0127] In one embodiment, "treatment" or "treating" a disorder or
disorder refers to an amelioration of a disease or disorder, or at
least one discernible symptom thereof. In another embodiment,
"treatment" or "treating" refers to an amelioration of at least one
measurable physical parameter associated with a disease or
disorder, not necessarily discernible by the subject. In yet
another embodiment, "treatment" or "treating" refers to inhibiting
the progression of a disease or disorder, either physically, e.g.,
stabilization of a discernible symptom, physiologically, e.g.,
stabilization of a physical parameter, or both.
[0128] In specific embodiments, the treatment methods described
herein provide for the reduction or amelioration of the
progression, severity, and/or duration of a disorder or disease
described herein. In further specific embodiments, the treatment
methods described herein reduce one or more symptoms of a disorder
or disease described herein.
[0129] In a particular embodiment, a method for treating a disorder
or disease described herein, can achieve at least one, two, three,
four or more of the following effects due to administration of a
therapeutically effective amount of an antibody or an antibody
derived antigen-binding fragment described herein: (i) the
reduction or amelioration of the severity of the disorder or
disease and/or one or more symptoms associated therewith; (ii) a
reduction in the duration of one or more symptoms associated with
the disorder or disease; (iii) the prevention of the recurrence of
the disorder or disease; (iv) the regression of the disorder or
disease and/or one or more symptoms associated therewith; (v) a
reduction in hospitalization of a subject; (vi) the reduction in
hospitalization length; (vii) the increase in the survival of a
subject; (viii) the inhibition of the progression of the disorder
or disease and/or one or more symptoms associated therewith; (ix)
the enhancement or improvement of the therapeutic effect of another
therapy; (x) a reduction or elimination of the disorder or disease;
(xi) a reduction in mortality; (xii) a decrease in hospitalization
rate; (xiii) the prevention of the development or onset of one or
more symptoms associated with the disorder or disease.
[0130] In one embodiment, "prevention" or "preventing" a disorder
or disease refers to the completion or partial inhibition of the
onset or development of the disorder or disease.
[0131] In a specific embodiment, the disease or disorder treated in
accordance with the methods described herein is psoriasis. It is
generally accepted that T lymphocytes play a key role in the
pathogenesis of psoriasis. In a specific embodiment, a method for
treating psoriasis comprises administering to a subject in need
thereof a therapeutically effective amount of an antibody or an
antibody derived antigen-binding fragment described herein. In
another specific embodiment, a method for ameliorating or
preventing one or more symptoms of psoriasis comprises
administering to a subject in need thereof a therapeutically
effective amount of an antibody or an antibody derived
antigen-binding fragment described herein. Symptoms of psoriasis
include, but are not limited to, red patches of skin covered with
silvery scales, raised patches of skin, small scaling spots on
skin, dry skin, cracked skin, including pustules, itching skin,
thickened, pitted or ridged nail, swollen and stiff joints.
[0132] In another embodiment, the disease or disorder treated in
accordance with the methods described herein is plaque psoriasis.
Plaque psoriasis or psoriasis vulgaris is the most common form of
psoriais and is characterized by sharply demarcated, raised
erythematous skin plaques covered by silvery scale. There is a
predilection of the lesions to involve the extensor surfaces of the
extremities, the lumbosacral area, and the scalp. The corresponding
histopathological findings include significant inflammatory
cellular infiltration of the dermis and epidermis, increased
numbers of dilated vessels, and a substantial thickening of the
epidermis with disordered differentiation of keratinocytes and
hyperkeratosis. Approximately one third of patients with plaque
psoriasis are categorized as having moderate or severe disease and
are consequently candidates for therapy beyond just topical
treatment.
[0133] In a specific embodiment, a method for treating plaque
psoriasis comprises administering to a subject in need thereof a
therapeutically effective amount of an antibody or an antibody
derived antigen-binding fragment described herein. In another
specific embodiment, a method for ameliorating or preventing one or
more symptoms of plaque psoriasis comprises administering to a
subject in need thereof a therapeutically effective amount of an
antibody or an antibody derived antigen-binding fragment described
herein. Symptoms of plaque psoriasis include, but are not limited
to, red patches of skin covered with silvery scales, raised patches
of skin, small scaling spots on skin, dry skin, cracked skin,
including pustules, itching skin, thickened, pitted or ridged nail,
swollen and stiff joints.
[0134] In another embodiment, the disorder treated in accordance
with the methods described herein is chronic plaque psoriasis. In a
specific embodiment, a method for treating chronic plaque psoriasis
comprises administering to a subject in need thereof a
therapeutically effective amount of an antibody or an antibody
derived antigen-binding fragment described herein. In another
specific embodiment, a method described herein is for ameliorating
or preventing one or more symptoms of chronic plaque psoriasis
comprises administering to a subject in need thereof a
therapeutically effective amount of an antibody or an antibody
derived antigen-binding fragment described herein. Symptoms of
plaque chronic psoriasis include, but are not limited to, single or
multiple raised reddened patches of skin, ranging from coin-sized
to larger, on any part of the body, including but not limited to
the knees, elbows, lumbosacral regions, scalp, and nails.
[0135] In another embodiment, the disorder treated in accordance
with the methods described herein is guttate psoriasis. In another
specific embodiment, a method for treating guttate psoriasis
comprises administering to a subject in need thereof a
therapeutically effective amount of an antibody or an antibody
derived antigen-binding fragment described herein. In another
specific embodiment, a method for preventing or ameliorating one or
more symptoms of guttate psoriasis comprises administering to a
subject in need thereof a therapeutically effective amount of an
antibody or an antibody derived antigen-binding fragment described
herein. Symptoms of guttate psoriasis include, but are not limited
to, flares of water drop shaped scaly plaques on the skin, followed
by an infection, such as a streptococcal throat infection.
[0136] In another embodiment, the disease or disorder treated in
accordance with the methods described herein is inverse psoriasis.
In a specific embodiment, a method for treating inverse psoriasis
(also called intertiginous psoriasis and flexural psoriasis)
comprises administering to a subject in need thereof a
therapeutically effective amount of an antibody or an antibody
derived antigen-binding fragment described herein. In another
specific embodiment, a method for preventing or ameliorating one or
more symptoms of inverse psoriasis comprises administering to a
subject in need thereof a therapeutically effective amount of an
antibody or an antibody derived antigen-binding fragment described
herein. Symptoms of inverse psoriasis include, but are not limited
to, smooth, usually moist areas of skin that are red and inflamed,
unlike the scaling associated with plaque psoriasis, on one or more
of the following body parts: armpits, groin, under the breasts, and
in other skin folds around the genitals and buttocks.
[0137] In another embodiment, the disease or disorder treated in
accordance with the methods described herein is pustular psoriasis.
In a specific embodiment, a method for treating pustular psoriasis
comprises administering to a subject in need thereof a
therapeutically effective amount of an antibody or an antibody
derived antigen-binding fragment described herein. In another
specific embodiment, a method described herein for preventing or
ameliorating one or more symptoms of pustular psoriasis comprises
administering to a subject in need thereof a therapeutically
effective amount of an antibody or an antibody derived
antigen-binding fragment described herein. Symptoms of pustular
psoriasis include, but are not limited to, pus-filled blisters that
vary in size and location, but mostly on the hands and feet.
[0138] In another embodiment, the disease or disorder treated in
accordance with the methods described herein is erythodermic
psoriasis. In a specific embodiment, a method for treating
erythodermic psoriasis comprises administering to a subject in need
thereof a therapeutically effective amount of an antibody or an
antibody derived antigen-binding fragment described herein. In
another specific embodiment, a method described herein for
preventing or ameliorating one or more symptoms of erythodermic
psoriasis comprises administering to a subject in need thereof a
therapeutically effective amount of an antibody or an antibody
derived antigen-binding fragment described herein. Symptoms of
erythodermic psoriasis include, but are not limited to, periodic,
widespread, fiery redness of the skin and the shedding of scales in
sheets, rather than smaller flakes. The reddening and shedding of
the skin are often accompanied by severe itching and pain, heart
rate increase, and fluctuating body temperature.
[0139] In another embodiment, the disease or disorder treated in
accordance with the methods described herein is rheumatoid
arthritis. In a specific embodiment, a method for treating
rheumatoid arthritis comprises administering to a subject in need
thereof a therapeutically effective amount of an antibody or an
antibody derived antigen-binding fragment described herein. In
another specific embodiment, a method described herein is for
preventing or treating one or more symptoms of rheumatoid arthritis
comprises administering to a subject in need thereof a
therapeutically effective amount of an antibody or an antibody
derived antigen-binding fragment described herein. Symptoms of
rheumatoid arthritis, include, but are not limited to, fatigue,
loss of appetite, low fever, swollen glands, weakness, joint pain
in wrists, elbows, shoulders, hips, knees, ankles, toes, jaw,
hands, feet, fingers, and/or neck, morning stiffness, chest pain
when taking a breath (pleurisy), eye burning, itching, and
discharge, nodules under the skin, numbness, tingling, or burning
in the hands and feet.
[0140] In another embodiment, the disease or disorder treated in
accordance with the methods described herein is Crohn's disease. In
a specific embodiment, a method for treating Crohn's disease
comprises administering to a subject in need thereof a
therapeutically effective amount of an antibody or an antibody
derived antigen-binding fragment described herein. In another
specific embodiment, a method described herein is for preventing or
ameliorating one or more symptoms of Crohn's disease comprises
administering to a subject in need thereof a therapeutically
effective amount of an antibody or an antibody derived
antigen-binding fragment described herein. Symptoms of Crohn's
disease, but are not limited to, crampy abdominal (belly area)
pain, fever, fatigue, loss of appetite, pain with passing stool
(tenesmus), persistent, watery diarrhea, unintentional weight loss,
constipation, eye inflammation, fistulas (usually around the rectal
area, may cause draining of pus, mucus, or stools), joint pain,
liver inflammation, mouth ulcers, rectal bleeding and bloody
stools, skin lumps or sores (ulcers), and swollen gums.
[0141] In another embodiment, the disease or disorder treated in
accordance with the methods described herein is ankylosing
spondylitis. In a specific embodiment, a method for treating
ankylosing spondylitis comprises administering to a subject in need
thereof a therapeutically effective amount of an antibody or an
antibody derived antigen-binding fragment described herein. In
another specific embodiment, a method for ameliorating or
preventing one or more symptoms of ankylosing spondylitis comprises
administering to a subject in need thereof a therapeutically
effective amount of an antibody or an antibody derived
antigen-binding fragment described herein. Symptoms of ankylosing
spondylitis include, but are not limited to, frequent pain and
stiffness in the lower back and buttocks, spine, and/or neck; and
pain and tenderness spreading to the ribs, shoulder blades, hips,
thighs and heels; inflammation of the eye (iridocyclitis and
uveitis), causing redness, eye pain, vision loss, floaters and
photophobia; fatigue; and nausea
[0142] In another embodiment, the disease or disorder treated in
accordance with the methods described herein is diabetes mellitus.
In a specific embodiment, a method for treating diabetes mellitus
comprises administering to a subject in need thereof a
therapeutically effective amount of an antibody or an antibody
derived antigen-binding fragment described herein. In another
specific embodiment, a method for ameliorating or preventing one or
more symptoms of diabetes mellitus comprises administering to a
subject in need thereof a therapeutically effective amount of an
antibody or an antibody derived antigen-binding fragment described
herein. Symptoms of diabetes mellitus include, but are not limited
to, loss of weight, polyuria (frequent urination), polydipsia
(increased thirst), polyphagia (increased hunger), cardiovascular
disease, diabetic retinopathy, diabetic neuropathy, hyperosmolar
nonketotic state, and diabetic ketoacidosis.
[0143] In particular embodiments, an antibody or an antibody
derived antigen-binding fragment described herein is administered
to a patient who has previously received, or is currently
receiving, one or more other therapies.
[0144] In particular embodiments, an antibody or an antibody
derived antigen-binding fragment described herein is administered
to a patient who has previously received, or is currently
receiving, one or more other therapies. In other particular
embodiments, an antibody or an antibody derived antigen-binding
fragment described herein is administered to a patient who is, or
is suspected of being, resistant or refractory to an
anti-inflammatory therapy.
[0145] In certain aspects, provided herein are methods for killing
T cells in an individual in need thereof, wherein said method
comprises administering to said individual an effective amount of
an antibody or an antibody derived antigen-binding fragment
described herein. In certain aspects, provided herein are methods
for inducing apoptosis of activated T cells in an individual in
need thereof, wherein said method comprises administering to said
individual an effective amount of an antibody or an antibody
derived antigen-binding fragment described herein.
[0146] In certain embodiments, an antibody or an antibody derived
antigen-binding fragment described herein or pharmaceutical
composition thereof may be administered by any suitable method to a
subject in need thereof. Non-limiting examples of administration
methods include intravenous infusion, subcutaneous injection or
implantation, intramuscularly, intrathecally, intraperitoneally,
intrarectally, intravaginally, intranasally, intragastrically,
intratracheally, or intrapulmonarily delivery and/or any other
method of physical delivery described herein or known in the art.
In one embodiment, an antibody or an antibody derived
antigen-binding fragment or a pharmaceutical composition thereof is
administered systemically (e.g., parenterally) to a subject in need
thereof. In another embodiment, an antibody or a pharmaceutical
composition thereof is administered locally (e.g., intratumorally)
to a subject in need thereof. Each dose may or may not be
administered by an identical route of administration. In some
embodiments, an antibody or an antibody derived antigen-binding
fragment described herein can be administered via multiple routes
of administration simultaneously or subsequently to other doses of
the same or a different an antibody described herein.
[0147] When a disease, or a symptom thereof, is being treated,
administration of the antibody or the antibody derived
antigen-binding fragment typically occurs after the onset of the
disease or symptoms thereof. When a symptom of a disease are being
prevented, administration of the antibody or the antibody derived
antigen-binding fragment typically occurs before the onset of the
symptoms.
[0148] The dosage and frequency of administration of an antibody or
an antibody derived antigen-binding fragment described herein or a
pharmaceutical composition thereof is administered in accordance
with the methods for preventing and/or treating while minimizing
side effects. The exact dosage of an antibody or an antibody
derived antigen-binding fragment described herein to be
administered to a particular subject or a pharmaceutical
composition thereof can be determined by a practitioner, in light
of factors related to the subject that requires treatment. Factors
which can be taken into account include the severity of the disease
state, general health of the subject, age, and weight of the
subject, diet, time and frequency of administration, combination(s)
with other therapeutic agents or drugs, reaction sensitivities, and
tolerance/response to therapy. The dosage and frequency of
administration of an antibody or an antibody derived
antigen-binding fragment described herein or a pharmaceutical
composition thereof can be adjusted over time to provide sufficient
levels of the antibody or an antibody derived antigen-binding
fragment, or to maintain the desired effect.
[0149] The precise dose to be employed in the formulation will also
depend on the route of administration, and the seriousness of an
inflammatory disorder or disease, and should be decided according
to the judgment of the practitioner and each patient's
circumstances.
[0150] Effective doses can be extrapolated from dose-response
curves derived from in vitro or animal model test systems.
[0151] In one embodiment, the dosage of an antibody or an antibody
derived antigen-binding fragment described herein that is
administered to a patient to prevent and/or treat a disease or
disorder described herein is typically 0.001 mg/kg to 100 mg/kg of
the patient's body weight. In another embodiment, a suitable dosage
of an antibody or antibody derived antigen-binding fragment that is
therapeutically effective for the prevention and/or treatment of a
disease or disorder described herein is in the range of 0.01 mg/kg
to 100 mg/kg of a patient's body weight.
[0152] In specific embodiments, an "effective amount" of an
antibody or an antibody derived antigen-binding fragment described
herein refers to an amount of an antibody or an antibody derived
antigen-binding fragment described herein which is sufficient to
achieve at least one, two, three, four or more of the following
effects: (i) the reduction or amelioration of the severity of a
disease or disorder described herein and/or one or more symptoms
associated therewith; (ii) the reduction in the duration of one or
more symptoms associated with disorder or disease described herein;
(iii) the prevention in the recurrence of a disease or disorder;
(iv) the regression of a disease or disorder and/or one or more
symptoms associated therewith; (v) the reduction in hospitalization
of a subject; (vi) the reduction in hospitalization length; (vii)
the increase in the survival of a subject; (viii) the inhibition of
the progression of disease or disorder and/or one or more symptoms
associated therewith; (ix) the enhancement or improvement of the
therapeutic effect of another therapy; (x) a reduction or
elimination in disease or disorder; (xi) a reduction in mortality;
(xii) a decrease in hospitalization rate; (xiii) the prevention of
the development or onset of one or more symptoms associated with a
disease or disorder; and (xiv) improvement in the quality of life
as assessed by methods well known in the art, e.g., questionnaires.
In some embodiments, "effective amount" as used herein refers to
the amount of an antibody described herein to achieve a specified
result described in Section 6, infra. In certain embodiments, an
effective amount of an antibody or an antibody derived
antigen-binding fragment is from about 0.01 mg to about 1,000
mg.
[0153] In some embodiments, a single dose of an antibody or an
antibody derived antigen-binding fragment described herein is
administered one or more times to a patient to prevent and/or treat
a disorder or disease described herein.
[0154] In particular embodiments, an antibody or an antibody
derived antigen-binding fragment, or pharmaceutical composition
thereof is administered to a subject in accordance with the methods
for preventing and/or treating a disorder or disease presented
herein in cycles, wherein the antibody or the antibody derived
antigen-binding fragment, or pharmaceutical composition is
administered for a period of time, followed by a period of rest
(i.e., the antibody or the antibody derived antigen-binding
fragment, or pharmaceutical composition is not administered for a
period of time).
[0155] An antibody or an antibody derived antigen-binding fragment
provided herein can be administered prior to, concurrently with, or
subsequent to the administration of one or more additional
therapies (e.g., agents) for use in preventing and/or treating
disorder or disease described herein. The use of the term "in
combination" does not restrict the order in which an antibody or an
antibody derived antigen-binding fragment and one or more
additional therapies are administered to a subject. In specific
embodiments, the therapies can be administered serially or
sequentially.
[0156] In another specific embodiment, an antibody or an antibody
derived antigen-binding fragment described herein are used in
combination with an amount of another therapy (such as, e.g., an
anti-inflammatory agent) to prevent and/or treat a disease or
disorder described herein. In a specific embodiment, such a
combination therapies has synergistic effect. In other embodiments,
such a combination has an additive effect.
[0157] In a specific embodiment, the antibody or the antibody
derived antigen-binding fragment and additional therapy permits the
use of lower dosages (e.g., sub-optimal doses) of the antibody or
the antibody derived antigen-binding fragment and/or the additional
therapy and/or less frequent administration of the antibody or the
antibody derived antigen-binding fragment described herein or the
additional therapy to a subject. In certain embodiments, the
ability to utilize lower dosages of an antibody or an antibody
derived antigen-binding fragment described herein and/or of an
additional therapy and/or to administer an antibody or an antibody
derived antigen-binding fragment, or said additional therapy less
frequently reduces the toxicity associated with the administration
of an antibody or an antibody derived antigen-binding fragment, or
of said additional therapy, respectively, to a subject without
reducing the efficacy of an antibody or of said additional therapy,
respectively, in the prevention and/or treatment of a disorder or
disease described herein. In some embodiments, the administration
of an antibody or an antibody derived antigen-binding fragment
described herein in combination with an additional therapy results
in improved efficacy of the antibody or an antibody derived
antigen-binding fragment described herein and/or of said additional
therapy in preventing and/or treating a disorder or disease
described herein. In some embodiments, the administration of an
antibody or an antibody derived antigen-binding fragment described
herein and one or more additional therapies avoids or reduces
adverse or unwanted side effects associated with the use of any
single therapy.
[0158] In a specific embodiment, a subject administered an antibody
or an antibody derived antigen-binding fragment described herein is
an animal (e.g., a cynomologous or a human subject). In a preferred
embodiment, a subject administered an antibody or an antibody
derived antigen-binding fragment described herein is a human. In a
specific embodiment, a subject administered an antibody or antibody
derived antigen-binding fragment exhibits one or more symptoms of a
disease or disorder described herein.
[0159] 6.6 Cell-Based Methods
[0160] Provided herein are methods for inducing or enhancing
apoptosis in a cell expressing PSGL-1 comprising contacting the
cell with an effective amount of an antibody described herein. In
one embodiment, the cell is an immune cell. In a specific
embodiment, the cell is a T cell. In a more specific embodiment,
the cell is an activated T cell. Methods for detecting apoptosis
are described in the art and can be readily carried out by one of
skill in the art. In specific embodiments, a method for inducing or
enhancing apoptosis of activated T cells expressing PSGL-1
comprises contacting the cells with an effective amount of an
antibody described herein, wherein the effective amount is
sufficient to induce or enhance apoptosis as assessed by methods
known to one of skill in the art.
[0161] Provided herein are methods for reducing or inhibiting the
survival of cells expressing PSGL-1 comprising contacting the cell
with an effective amount of an antibody described herein. In one
embodiment, the cell is an immune cell. In a specific embodiment,
the cell is a T cell. In a more specific embodiment, the cell is an
activated T cell. Cell survival assays are described in the art and
can be readily carried out by one of skill in the art. For example,
cell viability can be assessed by using trypan-blue staining or
other cell death markers (e.g., Annexin-5 staining) or viability
markers (e.g., propidium iodine or 7-AAD exclusion) known in the
art. In specific embodiments, a method for reducing or inhibiting
survival of activated T cells expressing PSGL-1 comprises
contacting the cells with an effective amount of an antibody
described herein, wherein the effective amount reduces or to
inhibits survival of the cells as assessed by methods known to one
of skill in the art (e.g., trypan blue exclusion assay or propidium
iodine or 7-AAD exclusion).
[0162] 6.7 Kits
[0163] Provided herein is a pharmaceutical pack or kit comprising
one or more containers filled with one or more of the ingredients
of the pharmaceutical compositions described herein, such as an
antibody or an antibody derived antigen-binding fragment provided
herein. Optionally associated with such container(s) can be a
notice in the form prescribed by a governmental agency regulating
the manufacture, use or sale of pharmaceuticals or biological
products, which notice reflects approval by the agency of
manufacture, use or sale for human administration.
[0164] In a specific embodiment, provided herein is a kit
comprising a first container containing an antibody or an antibody
derived antigen-binding fragment described herein. In a specific
embodiment, a kit comprises a first container that is a vial
containing said antibody or said antibody derived antigen-binding
fragment as a lyophilized sterile powder under vacuum, and the kit
further comprises a second container comprising a pharmaceutically
acceptable fluid.
[0165] In a specific embodiment, provided herein is an injection
device containing an antibody or an antibody derived
antigen-binding fragment described herein. In a specific
embodiment, the injection device the antibody in sterile solution.
In a specific embodiment, an injection device provided herein is a
syringe.
[0166] Also provided herein are kits that can be used in the above
methods. In one embodiment, a kit comprises an antibody or an
antibody derived antigen-binding fragment described herein,
preferably a purified antibody, in one or more containers. In a
specific embodiment, the kits described herein contain a
substantially isolated PSGL-1 as a control. In another specific
embodiment, the kits described herein further comprise a control
antibody which does not react with PSGL-1. In another specific
embodiment, the kits described herein contain one or more elements
for detecting the binding of an antibody or an antibody derived
antigen-binding fragment to PSGL-1 (e.g., the antibody or the
antibody derived antigen-binding fragment can be conjugated to a
detectable substrate such as a fluorescent compound, an enzymatic
substrate, a radioactive compound or a luminescent compound, or a
second antibody which recognizes the first antibody or antibody
derived antigen-binding fragment can be conjugated to a detectable
substrate). In specific embodiments, the kit can include a
recombinantly produced or chemically synthesized PSGL-1. PSGL-1
provided in the kit can also be attached to a solid support. In a
more specific embodiment, the detecting means of the above
described kit includes a solid support to which PSGL-1 is attached.
Such a kit can also include a non-attached reporter-labeled
anti-human antibody. In this embodiment, binding of the antibody or
the antibody derived antigen-binding fragment to PSGL-1 can be
detected by binding of the said reporter-labeled antibody.
7. EXAMPLES
[0167] The examples in this section (i.e., section 6) are offered
by way of illustration, and not by way of limitation.
7.1 Example 1
Generating Anti-PSGL-1 Monoclonal Antibodies
[0168] Antibody h15A7 contains the humanized 15A7 heavy and light
chain variable regions described in International Application Pub.
No. WO 2005/110475, published Nov. 24, 2005, which is incorporated
herein by reference in its entirety. Antibody h15A7 is an IgG4
isotype antibody with a kappa-type light chain. A
Ser.sup.228.fwdarw.Pro.sup.228 mutation was introduced by PCR using
mutagenic primers into the hinge region of h15A7. The resulting
hinge mutant of h15A7 is called 15A7H herein. The remaining protein
sequence of 15A7H is identical to h15A7. The amino acid sequence of
15A7H is shown in FIG. 7 A-B and depicted in SEQ ID NOs: 1 and 2.
The antibody encoding gene sequences for the heavy and light chains
were introduced into eukaryotic expression vectors derived from the
vector pAD-CMV1 (described in EP 393 438).
[0169] 15A7H was generated using the methods known in the art.
Briefly, recombinant expression vectors encoding in addition to the
heavy and the light chains for 15A7H also encode dihydrofolate
reductase (DHFR) and neomycin phosphotransferase selection markers,
respectively, were co-transfected into the suspension adapted
CHO-DG44 host cell. The selection of stably transfected cells took
place two days after the transfection in selective
hypoxanthine/thymidine-free medium and addition of the antibiotic
G418. Once cells were recovered from the initial selection, a
DHFR-based gene amplification was induced by the addition of
methotrexate. By single cell deposition a monoclonal production
cell line using a CHO-DG44 host cell was developed for 15A7H. For
the production of the 15A7H antibody, cells were expanded for
inoculum for the production bioreactor. The production bioreactor
was run in a fed batch mode for 8 to 14 days. To support the
production of antibody and to prolong the cell culture production
period, a nutrient feed medium was added during the production
stage. The cell culture broth was harvested by centrifugation and
dead-end filtration to efficiently remove cells, providing cell
free culture fluid (CCF) for further purification of the product.
After cell removal during harvest, the protein was purified through
protein A affinity chromatography and anion and cation exchange
chromatography. In addition, two robust virus clearance steps, a
low pH viral inactivation step and a nanofiltration step for virus
removal were included.
[0170] The purity of 15A7H was determined by a capillary gel
electrophoresis method. Samples were denatured with SDS (sodium
dodecyl sulphate) and separated based on size in a capillary filled
with a gel buffer that acts as a sieving medium. In reduced
samples, disulfide bonds are reduced with 2-mercaptoethanol,
resulting in separate peaks for heavy and light chains of the
antibody. In non-reduced samples, iodoacetamide, an alkylating
agent is added to avoid any fragmentation induced by sample
preparation and to ensure that the main IgG peak remains
intact.
[0171] Samples were injected electrokinetically and the mobilized
proteins were detected by UV-absorbance at 200 nm using an UV
detector. For reduced samples, the time corrected area percents
(TCA %) of the sum of heavy and light chain (% LC+HC) are reported.
The reportable value for non-reduced samples is the TCA % of the
IgG main peak.
[0172] A lower amount of half antibody molecules was detected by
non-reducing capillary gel electrophoresis (CGE) (FIG. 1)
indicating that the Ser.sup.228.fwdarw.Pro.sup.228 mutation
significantly lowered the formation of the intra-chain disulfide
bond in the hinge region. The amount of half antibody molecules was
reduced from .about.8-10% for h15A7 to below 1% for 15A7H.
[0173] A formulation for 15A7H is shown in Table 6.
TABLE-US-00006 TABLE 6 Formulation of 15A7H Concentration
Concentration Purpose of Components [mmol/L] [g/L] component Sodium
citrate 9.1 2.676 Buffer dihydrate component
C.sub.6H.sub.5Na.sub.3O.sub.7 .times. 2 H.sub.2O Sodium chloride
150 8.766 Tonicity agent Polysorbate 80 -- 0.2 Stabilizer Citric
acid 0.9 0.189 Buffer monohydrate component C.sub.6H.sub.8O.sub.7
.times. H.sub.2O 15A7H 0.267 40 Active ingredient Water for
injection -- add WFI to a Solvent (WFI) final volume of 1.0 L The
pH of the formulation is 6.0.
7.2 Example 2
Binding of 15A7H Antibodies on Activated Primary Human T Cells
[0174] 15A7H was examined for binding in primary activated human
CD4.sup.+ T cells.
[0175] 7.2.1. Materials and Methods
[0176] Cell Culture and T cell Activation
[0177] Peripheral blood CD4.sup.+ T cells were cultured at
37.degree. C. at 1.times.10.sup.6 in RPMI complete media and
stimulated for 2 days with 20 ug/ml PHA-L (Sigma, Catalog#L2769)
was added. Cells were incubated for an additional four to five days
at 37.degree. C. with 20 ng/ml IL-2 (R&D systems, Catalog
#RD202-IL) to produce "activated CD4+ T cells".
[0178] Cell Binding Assay
[0179] Activated human CD4.sup.+ T cells were counted and plated at
1.times.10.sup.5 cells/100 .mu.l in FACS buffer in a vbottom 96
well culture plate (Falcon BD, Catalog #353263) and incubated on
ice for 30 minutes.
[0180] An anti-lysozyme IgG4 isotype control, h15A7 and 15A7H were
diluted to 90 ug/ml in FACS buffer in a round bottom dilution plate
(Falcon, Catalog #353077). Eleven 3 fold serial dilutions of
antibody preparations were made for each antibody across the plate
with a starting concentration of 30 ug/ml.
[0181] Duplicate cell samples were pelleted and resuspended in 1000
of each antibody dilution for an initial starting concentration of
30 .mu.g/ml. Cells were incubated on ice for 60 minutes. Cells were
pelleted and washed with the wash buffer followed by addition of
1000 of diluted secondary Goat F(ab).sub.2 anti-human Ig's R-PE
conjugate antibody. Secondary Goat F(ab').sub.2 anti-human Ig's
R-PE conjugate antibody (BD Biosciences, Catalog #554655; stock at
1.05 mg/ml) was previously diluted 1:800 in FACS buffer Cells were
incubated on ice for 30 minutes in dark then pelleted and washed a
couple of times with the wash buffer. Cells were resuspended in 150
.mu.l of wash buffer and fixed with addition of 50 .mu.l of BD
cytofix/Cytoperm fixation buffer (BD Biosciences,
Catalog#554655).
[0182] Samples were analyzed on the BD FACS Array Bioanalyzer.
About 5000 events were acquired using BD FACS Array flow cytometer
for each cell sample with a "lymphocyte" gate set according to SSC
and FSC and mean florescence that was determined for each
sample.
[0183] Data Analysis
[0184] Anti-lysozyme IgG4, h15A7 and 15A7H antibody binding
affinity to activated CD4+ T cells were determined by plotting
antibody concentration versus mean fluorescence index (MFI) for
each sample duplicate using XLfit (model: dose response one site,
205). EC50 values were determined for each binding curve.
[0185] 7.2.2. Results
[0186] Binding to activated Human T cells
[0187] Using flow cytometry, the binding of 15A7H to activated
human CD4+ T-cells was determined. FIG. 2 demonstrates that 15A7H
bound with an EC50 of 0.22 nM.
[0188] Aggregate binding data from multiple donors demonstrates
15A7H bound to activated CD4+ T cells with a mean EC50 of
0.22+/-0.02. EC50 values from 4 donors were used to calculate
Mean+/-SEM. Antibody h15A7 was tested under the same conditions and
bound to activated CD4+ T cells with a mean EC50 of 0.27+/-0.05.
Therefore, 15A7H and h15A7 demonstrated comparable binding to
activated T cells (see Table 7).
TABLE-US-00007 TABLE 7 Binding assay to activated CD4+ T-cells.
EC50 (nM) Mean +/- SEM 15A7H 0.22 +/- 0.02 h15A7 0.27 +/- 0.05
7.3 Example 3
In-Vivo Activity of 15A7H in the Human Trans-Vivo Delayed-Type
Hypersensitivity Mouse Model
[0189] 15A7H was tested in the trans-vivo delayed-type
hypersensitivity (DTH) model in female C57BL/6 mice. Doses of 0.03,
0.10, 0.3, 1 and 10 mg/kg of 15A7H were dosed i.p. in a formulation
buffer. Antibody h15A7 was tested under the same conditions.
[0190] 7.3.1. Materials and Methods
[0191] Trans Vivo Assay
[0192] Female C57BL/6 mice were purchased from Harlan Sprague
Dawley, Inc. (Indianapolis, Ind.) at 6-8 weeks of age and used
within four weeks of arrival. All mice were housed in a pathogen
free environment, and treated according to NIH guidelines. All
animal experiments were reviewed by and approved by the
Institutional Animal Care and Use Committee (IACUC) and were
conducted in conformity with the NIH guideline, "Guiding Principles
for Research Involving Animals and Human Beings."
[0193] Blood was collected by venipuncture from normal human donors
that were known to be good tetanus responders. One hundred ml whole
blood was drawn into CPT Vacutainer tubes (Becton Dickinson,
Franklin Lakes, N.J.) and spun at 1800 RCF for 30 min. The buffy
layer containing mononuclear cells and platelets was separated,
washed three times, and resuspended in phosphate-buffered saline
(PBS) and counted. Platelet contamination was minimized by multiple
washes in PBS. No more than a 1:1 ratio of platelets to PBMCs was
allowed. The cells were immediately injected into the mouse
footpads.
[0194] Aluminium phosphate-adsorbed Tetanus toxoid (TT-Tetguard)
was used at a concentration of 0.25 Lf per injection site (Lf unit
is the flocculation value, the amount of toxoid which when mixed
with one International Unit of antitoxin produces an optimal
flocculating mixture).
[0195] 7-10.times.10.sup.6 PBMCs mixed with 0.25 Lf units of TT in
a total volume of 50 .mu.l, were injected into the hind footpads of
mice. Footpad thickness was measured prior to injection and 24
hours post-injection, using a dial thickness gauge (Mitutoyo,
Aurora, Ill.). Pre-injection thickness was subtracted from
post-injection thickness at 24 hours to obtain the change in paw
thickness. All measurements were made in inches.
[0196] Testing of 15A7H and h15A7:
[0197] 15A7H and h15A7 (10 mg/ml stock) were dissolved in a vehicle
containing 25 mM sodium citrate and 115 mM sodium chloride with
0.04% Tween 80, pH 5.97. Mice were injected i.p. with 0.2 ml of
vehicle or 15A7H or h15A7 at 0.03, 0.1, 0.3, 1, and 10 mg/kg, one
hour before footpad injections with PBMCs with or without TT. Each
dose of 15A7H and h15A7 antibody was tested on PBMCs from four
different donors and one mouse per treatment per donor was used.
15A7H and h15A7 treatment groups were compared to the vehicle
treated group.
[0198] Statistics
[0199] All values are reported as the mean.+-.SEM unless otherwise
specified. The changes in footpad thickness in the drug treated
group were compared to the vehicle treated group. Delta paw
thicknesses from 4 individual donor experiments were pooled and
mean.+-.SEM was calculated. The percent inhibition of paw thickness
was calculated as follows: 100.times.(.DELTA. paw
thickness.sub.veh-.DELTA. paw thickness.sub.drug)/(.DELTA. paw
thickness.sub.veh-.DELTA.paw thickness.sub.PBMC). Significance of
inhibitory effects was tested using Kruskal-Wallis One Way Analysis
of Variance (ANOVA) on Ranks. A p value of less than 0.05 was
considered statistically significant.
[0200] Plasma Levels of 15A7H and h15A7 in C57BL/6 Mice:
[0201] Plasma levels of 15A7H and h15A7 at 0.03, 0.1, 0.3, 1, and
10 mg/kg, 24 hours following intraperitoneal injection in female
C57BL/6 mice were determined. Plasma samples were collected from
trans-vivo DTH experimental animals upon termination of the
experiment. Bioanalysis was performed on all samples.
[0202] Satellite Pharmacokinetic Study of 15A7H
[0203] Satellite female C57BL/6 mice receiving intraperitoneal
doses of 15A7H (same dose levels as above) were used to collect
data that are more intensive with respect to 15A7H plasma
concentrations. These mice were not treated with human PBMCs or
tetanus toxoid. Sampling times for these "PK" mice were 1, 3, 5,
and 24 h after dosing. Each dose group of PK mice consisted of 6
mice. Within each dose group, a subgroup of 3 mice was used for
sampling at 1 and 5 h, and another subgroup of 3 mice was used for
sampling at 3 and 24 h.
[0204] Bioanalysis of h15A7 and 15A7H
[0205] A sandwich ELISA was used to quantitate concentrations of
h15A7 or 15A7H in mouse plasma. Briefly, 96-well microtiter plates
coated with PSGL-1 were used to bind either h15A7 or 15A7H in the
diluted plasma sample. After washing, bound h15A7 or 15A7H was
detected by a biotin-labeled monoclonal anti-human IgG.sub.4
antibody and enzyme (HRP)-labeled streptavidin. The amount of
colored product formed during the substrate reaction was measured
photometrically and increased with increasing concentration of
h15A7 or 15A7H in the sample. The h15A7 or 15A7H concentration
corresponding to the measured optical absorbance was calculated via
data-fitting of the non-linear standard curve. The range of
calibration samples was from 0.04 to 1.2 ng/mL. Samples were
diluted at least 1:100. Thus, the lower limit of quantification of
h15A7 or of 15A7H in whole plasma was 4 ng/mL. By using higher
dilution factors the upper limit of quantification can be increased
up to 2.4 mg/mL (1:2000000).
[0206] Due to slight differences of the reactivity of the h15A7 and
15A7H reference material in the ELISA, two calibration curves and
two sets of quality controls were used. Samples from animals
treated with h15A7 were analyzed versus the h15A7 calibration
curve, and samples from animals treated with 15A7H were analyzed
versus the 15A7H calibration curve.
[0207] 7.3.2. Results
[0208] Trans Vivo DTH
[0209] FIG. 3 shows pooled dose response of 15A7H antibody in trans
vivo DTH in 4 donor PBMCs as shown by percent inhibition of DTH
following treatment with 15A7H. When mice were dosed i.p. at -1
hour, with 0.03, 0.1, 0.3, 1, and 10 mg/kg, both h15A7 and 15A7H
inhibited the trans vivo DTH in a dose dependent manner. Table 8
shows the dose response and percent inhibition of 15A7H and h15A7
in trans-vivo DTH. A single dose of 15A7H or h15A7 at 0.3 mg/kg and
higher, showed significant inhibition of trans vivo DTH. The
ED.sub.50 for h15A7 and 15A7H in this assay was 0.09 and 0.1 mg/kg
respectively, n=4 donors.
TABLE-US-00008 TABLE 8 Dose response and % inhibition of in
trans-vivo DTH Mean SEM n Delta PBMC 0.0005 0.0000 4 (inches)
Vehicle 0.0090 0.0010 4 h15A7 0.03 mpk 0.0061 0.0007 4 h15A7 0.1
mpk 0.0047 0.0008 4 h15A7 0.3 mpk 0.0033 0.0006 4 h15A7 1 mpk
0.0018 0.0003 4 h15A7 10 mpk 0.0009 0.0001 4 15A7H 0.03 mpk 0.0060
0.0007 4 15A7H 0.1 mpk 0.0049 0.0006 4 15A7H 0.3 mpk 0.0040 0.0010
4 15A7H 1 mpk 0.0024 0.0006 4 15A7H 10 mpk 0.0012 0.0004 4 %
Vehicle 0.00 0.00 4 inhibition h15A7 0.03 mpk 33.31 6.67 4 h15A7
0.1 mpk 51.02 2.52 4 h15A7 0.3 mpk 67.83 4.26 4 h15A7 1 mpk 85.14
3.17 4 h15A7 10 mpk 95.43 1.41 4 15A7H 0.03 mpk 35.56 3.25 4 15A7H
0.1 mpk 47.95 2.88 4 15A7H 0.3 mpk 61.20 5.62 4 15A7H 1 mpk 79.15
5.19 4 15A7H 10 mpk 91.32 4.94 4
[0210] Compound Plasma Concentrations
[0211] The plasma concentrations of 15A7H for both satellite PK and
DTH mice are summarized in FIG. 5. FIG. 4 shows 24 hour pooled
plasma concentrations of 15A7H plotted against % inhibition of DTH.
Note that the mean concentrations at 24 hours after dosing for the
PK and DTH mice for a given dose level were very similar. This
suggests that, although the PK and DTH mice were treated
differently (only DTH mice received PBMCs and tetanus toxoid), the
pharmacokinetics of 15A7H in the two cases were very similar.
Plasma concentrations of both h15A7 and 15A7H fluctuated only
within a fairly narrow range over the time course of the study
(concentration measurements between 1 and 24 hr after dosing).
[0212] PK-PD
[0213] Table 9 and Table 10 show twenty four hour plasma
concentrations (PK) of h15A7 and 15A7H antibodies and the
corresponding percent inhibition of trans-vivo DTH (PD) at the same
doses. FIG. 4 compares the PK-PD relationship of 15A7H in the
trans-vivo DTH model. The ED.sub.50 for h15A7 and 15A7H were
calculated to be 687 and 770 ng/ml respectively, n=4 donors.
TABLE-US-00009 TABLE 9 PK-PD relationship in the trans-vivo DTH
model. h15A7 dose mg/kg, i.p. Mean SEM n (A) Phamacokinetics (PK)
ng/ml 0.03 111 12 4 0.1 618 64 4 0.3 2320 172 4 1 9355 642 4 10
67668 23686 4 (B) Pharmacodynamics (PD) % inhibition 0.03 33 7 4
0.1 51 3 4 0.3 68 4 4 1 85 3 4 10 95 1 4
TABLE-US-00010 TABLE 10 PK-PD relationship in the trans-vivo DTH
model. 15A7H dose mg/kg, i.p. Mean SEM n (A) Phamacokinetics (PK)
ng/ml 0.03 228 20 4 0.1 708 79 4 0.3 1968 228 4 1 6163 469 4 10
54725 13612 4 (B) Pharmacodynamics (PD) % inhibition 0.03 36 3 4
0.1 48 3 4 0.3 61 6 4 1 79 5 4 10 91 5 4
[0214] 7.3.3. Conclusion
[0215] A single dose of 15A7H or h15A7 at 0.3 mg/kg and higher,
showed significant percent inhibition of trans vivo DTH. The
ED.sub.50 for h15A7 and 15A7H in the trans-vivo DTH assay was 0.09
and 0.1 mg/kg, respectively. Plasma concentrations of both h15A7
and 15A7H fluctuated only within a fairly narrow range over the
time course of the study.
7.4 Example 4
Assessment of Complement-Dependent Cytotoxicity (CDC)
[0216] In an effort to determine whether 15A7H has CDC activity,
lactate dehydrogenase release assays were used to measure the CDC
activity at various concentrations.
[0217] 7.4.1. Materials and Methods
[0218] Ramos cells (obtained ATCC; cat#CRL-1596) were used as
target cells and were cultivated in complete DMEM medium
(Invitrogen, Carlsbad, Calif.; Catalog number 11995) with 0.5 mg/ml
Geneticin (Invitrogen, Carlsbad, Calif.; Catalog number 10131).
Rabbit complement was used as the source of complement proteins
(Accurate Chemical &Scientific Corp., Westbury, N.Y.; catalog
number: AIC4000-1). Cedarlane Cytotoxicity medium was used as the
assay media (Accurate Chemical &Scientific Corp., Westbury,
N.Y.; Catalog Number: CL95100).
[0219] CDC activity was determined by LDH release of target cells
using Cytotoxicity Detection Kit.sup.PLUS (LDH) (Roche Applied
Science, Indianapolis, Ind.; Catalog Number: 04 744 934 001). Mouse
anti-human CD20 purified from 1F5 hybridoma (ATCC), Birmingham,
Ala.; Catalog Number: 6140-01) was used to activate complement as a
positive control antibody in the assay. Human IgG4 .kappa.
(Sigma-Aldrich, St. Louis, Mo.; Catalog Number: 14639) was used as
an isotype control. Samples and controls were set up as follows
(All the samples have duplicates): [0220] Samples: 100 .mu.l
standard rabbit complement (diluted 1:6 in assay media)+50 .mu.l
target cells (Ramos cells in assay media)+50 .mu.l antibody
dilutions [0221] Background control: 200 .mu.l assay media [0222]
Maximal release control: 50 .mu.l target cells+100 .mu.l standard
rabbit complement+50 .mu.l assay media [0223] Spontaneous release
control: 50 .mu.l target cells+100 .mu.l standard rabbit
complement+50 .mu.l assay media
[0224] The plate was incubated at 37.degree. C. in a humid CO.sub.2
incubator for 3 hrs. 30 min before the end of incubation (after 2.5
hrs incubation), 10 .mu.l Lysis Solution (provided in the
Cytotoxicity Detection Kit) was added to the maximal release
control sample. At the end of the incubation, the plate was
centrifuged at 200 g for 10 min at room temperature, and 100 .mu.l
of cell-free supernatants were transferred into corresponding wells
of a 96-well flat-bottom plate for LDH detection. 100 .mu.l of
Reaction Mixture (provided in the Cytotoxicity Detection Kit) was
added to each well and the plate was incubated at room temperature
for 15-30 min in the dark. At the end of the second incubation, the
reaction was stopped by adding 50 .mu.l of Stop solution (provided
in the Cytotoxicity Detection Kit). The absorbance was measured at
the wavelength of 490-492 nm on SpectraMAX Plus plate reader
(Molecular Devices, Sunnyvale, Calif.). CDC % was calculated using
([Antibody induced release]-[Spontaneous release
control])/([Maximal release]-[spontaneous release
control]).times.100%.
[0225] 7.4.2. Results
[0226] The CDC activity of 15A7H was tested under conditions
optimized for CDC response of anti-CD20 with the Ramos cells. As
shown in FIG. 6 and Table 11, different concentrations of
antibodies with fixed complement dilution of 1:12 were tested for
CDC activity. No CDC activity was detected for 15A7H, h15A7 or for
the IgG4 negative control. Mouse anti-human CD20 demonstrated a
clear antibody dose response.
TABLE-US-00011 TABLE 11 Percent CDC activity of 15A7H at different
concentrations Antibody conc. (.mu.g/ml) h15A7 IgG4 Kappa Mouse
anti-CD20 15A7H 5 1.54 -0.87 na -0.45 0.5 4.25 0.50 68.26 -0.15
0.05 0.84 3.22 59.60 -2.59 0.005 2.08 3.29 31.42 -1.37 0.0005 na na
6.77 na
[0227] 7.4.3. Conclusion
[0228] 15A7H shows no CDC activity up to the concentration of 5
.mu.g/ml antibody at the complement dilution of 1:12 in the assays
performed on Ramos cells.
7.5 Example 5
Clinical Study
[0229] 15A7H is given to subjects by intravenous administration of
single rising doses of 125 .mu.g/kg, 500 .mu.g/kg, 1000 .mu.g/kg
and 2000 .mu.g/kg and in other subjects by subcutaneous
administration of single doses of 500 .mu.g/kg and 1000 .mu.g/kg.
Dosing is adapted. 15A7H resuspended in the formulation described
in Table 6 is used. After administration, clinical laboratory tests
(e.g., haematology, clinical chemistry and urinalysis) are
conducted, for example on a weekly basis, using methods know in the
art. Pharmacokinetic parameters are also determined, for example on
a weekly basis, using methods know in the art.
[0230] All publications, patents, and patent applications cited in
this application are hereby incorporated by reference in their
entireties as if each individual publication or patent application
were specifically and individually indicated to be incorporated by
reference. Although the foregoing invention has been described in
some detail by way of illustration and example for purposes of
clarity of understanding, it will be readily apparent to those of
ordinary skill in the art in light of the teachings of this
invention that certain changes and modifications can be made
thereto without departing from the spirit or scope of the appended
claims.
Sequence CWU 1
1
211219PRTArtificial Sequence15A7H Light Chain amino acid sequence
1Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly1 5
10 15Asp Arg Val Thr Ile Thr Cys Arg Ser Ser Gln Ser Ile Val His
Asn 20 25 30Asp Gly Asn Thr Tyr Phe Glu Trp Tyr Gln Gln Lys Pro Gly
Lys Ala 35 40 45Pro Lys Leu Leu Ile Tyr Lys Val Ser Asn Arg Phe Ser
Gly Val Pro 50 55 60Ser Arg Phe Ser Gly Ser Gly Ser Gly Thr His Phe
Thr Leu Thr Ile65 70 75 80Ser Ser Leu Gln Pro Glu Asp Phe Ala Thr
Tyr Tyr Cys Phe Gln Gly 85 90 95Ser Tyr Val Pro Leu Thr Phe Gly Gln
Gly Thr Lys Val Glu Ile Lys 100 105 110Arg Thr Val Ala Ala Pro Ser
Val Phe Ile Phe Pro Pro Ser Asp Glu 115 120 125Gln Leu Lys Ser Gly
Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe 130 135 140Tyr Pro Arg
Glu Ala Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln145 150 155
160Ser Gly Asn Ser Gln Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser
165 170 175Thr Tyr Ser Leu Ser Ser Thr Leu Thr Leu Ser Lys Ala Asp
Tyr Glu 180 185 190Lys His Lys Val Tyr Ala Cys Glu Val Thr His Gln
Gly Leu Ser Ser 195 200 205Pro Val Thr Lys Ser Phe Asn Arg Gly Glu
Cys 210 2152447PRTArtificial Sequence15A7H Heavy Chain amino acid
sequence 2Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro
Gly Gly1 5 10 15Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe
Ser Ser Phe 20 25 30Gly Met His Trp Val Arg Gln Ala Pro Gly Lys Gly
Leu Glu Trp Val 35 40 45Ala Tyr Ile Asn Gly Gly Ser Ser Thr Ile Phe
Tyr Ala Asn Ala Val 50 55 60Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn
Ala Lys Asn Thr Leu Tyr65 70 75 80Leu Gln Met Asn Ser Leu Arg Ala
Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95Ala Arg Tyr Ala Ser Tyr Gly
Gly Gly Ala Met Asp Tyr Trp Gly Gln 100 105 110Gly Thr Leu Val Thr
Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val 115 120 125Phe Pro Leu
Ala Pro Cys Ser Arg Ser Thr Ser Glu Ser Thr Ala Ala 130 135 140Leu
Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser145 150
155 160Trp Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala
Val 165 170 175Leu Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val
Thr Val Pro 180 185 190Ser Ser Ser Leu Gly Thr Lys Thr Tyr Thr Cys
Asn Val Asp His Lys 195 200 205Pro Ser Asn Thr Lys Val Asp Lys Arg
Val Glu Ser Lys Tyr Gly Pro 210 215 220Pro Cys Pro Pro Cys Pro Ala
Pro Glu Phe Leu Gly Gly Pro Ser Val225 230 235 240Phe Leu Phe Pro
Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr 245 250 255Pro Glu
Val Thr Cys Val Val Val Asp Val Ser Gln Glu Asp Pro Glu 260 265
270Val Gln Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala Lys
275 280 285Thr Lys Pro Arg Glu Glu Gln Phe Asn Ser Thr Tyr Arg Val
Val Ser 290 295 300Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly
Lys Glu Tyr Lys305 310 315 320Cys Lys Val Ser Asn Lys Gly Leu Pro
Ser Ser Ile Glu Lys Thr Ile 325 330 335Ser Lys Ala Lys Gly Gln Pro
Arg Glu Pro Gln Val Tyr Thr Leu Pro 340 345 350Pro Ser Gln Glu Glu
Met Thr Lys Asn Gln Val Ser Leu Thr Cys Leu 355 360 365Val Lys Gly
Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn 370 375 380Gly
Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser385 390
395 400Asp Gly Ser Phe Phe Leu Tyr Ser Arg Leu Thr Val Asp Lys Ser
Arg 405 410 415Trp Gln Glu Gly Asn Val Phe Ser Cys Ser Val Met His
Glu Ala Leu 420 425 430His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu
Ser Leu Gly Lys 435 440 4453113PRTArtificial Sequence15A7H Variable
Light Chain Region (VL) amino acid sequence 3Asp Ile Gln Met Thr
Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly1 5 10 15Asp Arg Val Thr
Ile Thr Cys Arg Ser Ser Gln Ser Ile Val His Asn 20 25 30Asp Gly Asn
Thr Tyr Phe Glu Trp Tyr Gln Gln Lys Pro Gly Lys Ala 35 40 45Pro Lys
Leu Leu Ile Tyr Lys Val Ser Asn Arg Phe Ser Gly Val Pro 50 55 60Ser
Arg Phe Ser Gly Ser Gly Ser Gly Thr His Phe Thr Leu Thr Ile65 70 75
80Ser Ser Leu Gln Pro Glu Asp Phe Ala Thr Tyr Tyr Cys Phe Gln Gly
85 90 95Ser Tyr Val Pro Leu Thr Phe Gly Gln Gly Thr Lys Val Glu Ile
Lys 100 105 110Arg4120PRTArtificial Sequence15A7H Variable Heavy
Chain Region (VH) amino acid sequence 4Glu Val Gln Leu Val Glu Ser
Gly Gly Gly Leu Val Gln Pro Gly Gly1 5 10 15Ser Leu Arg Leu Ser Cys
Ala Ala Ser Gly Phe Thr Phe Ser Ser Phe 20 25 30Gly Met His Trp Val
Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45Ala Tyr Ile Asn
Gly Gly Ser Ser Thr Ile Phe Tyr Ala Asn Ala Val 50 55 60Lys Gly Arg
Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Thr Leu Tyr65 70 75 80Leu
Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys 85 90
95Ala Arg Tyr Ala Ser Tyr Gly Gly Gly Ala Met Asp Tyr Trp Gly Gln
100 105 110Gly Thr Leu Val Thr Val Ser Ser 115 120516PRTArtificial
Sequence15A7H VL CDR1 amino acid sequence 5Arg Ser Ser Gln Ser Ile
Val His Asn Asp Gly Asn Thr Tyr Phe Glu1 5 10 1567PRTArtificial
Sequence15A7H VL CDR2 amino acid sequence 6Lys Val Ser Asn Arg Phe
Ser1 579PRTArtificial Sequence15A7H VL CDR3 amino acid sequence
7Phe Gln Gly Ser Tyr Val Pro Leu Thr1 585PRTArtificial
Sequence15A7H VH CDR1 amino acid sequence 8Ser Phe Gly Met His1
5917PRTArtificial Sequence15A7H VH CDR2 amino acid sequence 9Tyr
Ile Asn Gly Gly Ser Ser Thr Ile Phe Tyr Ala Asn Ala Val Lys Gly1 5
10 151011PRTArtificial Sequence15A7H VH CDR3 amino acid sequence
10Tyr Ala Ser Tyr Gly Gly Gly Ala Met Asp Tyr1 5 1011412PRTHomo
sapiensFull length human PSGL-1 amino acid sequence 11Met Pro Leu
Gln Leu Leu Leu Leu Leu Ile Leu Leu Gly Pro Gly Asn1 5 10 15Ser Leu
Gln Leu Trp Asp Thr Trp Ala Asp Glu Ala Glu Lys Ala Leu 20 25 30Gly
Pro Leu Leu Ala Arg Asp Arg Arg Gln Ala Thr Glu Tyr Glu Tyr 35 40
45Leu Asp Tyr Asp Phe Leu Pro Glu Thr Glu Pro Pro Glu Met Leu Arg
50 55 60Asn Ser Thr Asp Thr Thr Pro Leu Thr Gly Pro Gly Thr Pro Glu
Ser65 70 75 80Thr Thr Val Glu Pro Ala Ala Arg Arg Ser Thr Gly Leu
Asp Ala Gly 85 90 95Gly Ala Val Thr Glu Leu Thr Thr Glu Leu Ala Asn
Met Gly Asn Leu 100 105 110Ser Thr Asp Ser Ala Ala Met Glu Ile Gln
Thr Thr Gln Pro Ala Ala 115 120 125Thr Glu Ala Gln Thr Thr Gln Pro
Val Pro Thr Glu Ala Gln Thr Thr 130 135 140Pro Leu Ala Ala Thr Glu
Ala Gln Thr Thr Arg Leu Thr Ala Thr Glu145 150 155 160Ala Gln Thr
Thr Pro Leu Ala Ala Thr Glu Ala Gln Thr Thr Pro Pro 165 170 175Ala
Ala Thr Glu Ala Gln Thr Thr Gln Pro Thr Gly Leu Glu Ala Gln 180 185
190Thr Thr Ala Pro Ala Ala Met Glu Ala Gln Thr Thr Ala Pro Ala Ala
195 200 205Met Glu Ala Gln Thr Thr Pro Pro Ala Ala Met Glu Ala Gln
Thr Thr 210 215 220Gln Thr Thr Ala Met Glu Ala Gln Thr Thr Ala Pro
Glu Ala Thr Glu225 230 235 240Ala Gln Thr Thr Gln Pro Thr Ala Thr
Glu Ala Gln Thr Thr Pro Leu 245 250 255Ala Ala Met Glu Ala Leu Ser
Thr Glu Pro Ser Ala Thr Glu Ala Leu 260 265 270Ser Met Glu Pro Thr
Thr Lys Arg Gly Leu Phe Ile Pro Phe Ser Val 275 280 285Ser Ser Val
Thr His Lys Gly Ile Pro Met Ala Ala Ser Asn Leu Ser 290 295 300Val
Asn Tyr Pro Val Gly Ala Pro Asp His Ile Ser Val Lys Gln Cys305 310
315 320Leu Leu Ala Ile Leu Ile Leu Ala Leu Val Ala Thr Ile Phe Phe
Val 325 330 335Cys Thr Val Val Leu Ala Val Arg Leu Ser Arg Lys Gly
His Met Tyr 340 345 350Pro Val Arg Asn Tyr Ser Pro Thr Glu Met Val
Cys Ile Ser Ser Leu 355 360 365Leu Pro Asp Gly Gly Glu Gly Pro Ser
Ala Thr Ala Asn Gly Gly Leu 370 375 380Ser Lys Ala Lys Ser Pro Gly
Leu Thr Pro Glu Pro Arg Glu Asp Arg385 390 395 400Glu Gly Asp Asp
Leu Thr Leu His Ser Phe Leu Pro 405 4101213PRTArtificial
SequenceIgG4 hinge region amino acid sequence 12Glu Ser Lys Tyr Gly
Pro Pro Cys Pro Pro Cys Pro Ala1 5 101323PRTArtificial
Sequence15A7H VL FR1 13Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu
Ser Ala Ser Val Gly1 5 10 15Asp Arg Val Thr Ile Thr Cys
201415PRTArtificial Sequence15A7H VL FR2 14Trp Tyr Gln Gln Lys Pro
Gly Lys Ala Pro Lys Leu Leu Ile Tyr1 5 10 151532PRTArtificial
Sequence15A7H VL FR3 15Gly Val Pro Ser Arg Phe Ser Gly Ser Gly Ser
Gly Thr His Phe Thr1 5 10 15Leu Thr Ile Ser Ser Leu Gln Pro Glu Asp
Phe Ala Thr Tyr Tyr Cys 20 25 301611PRTArtificial Sequence15A7H VL
FR4 16Phe Gly Gln Gly Thr Lys Val Glu Ile Lys Arg1 5
101730PRTArtificial Sequence15A7H VH FR1 17Glu Val Gln Leu Val Glu
Ser Gly Gly Gly Leu Val Gln Pro Gly Gly1 5 10 15Ser Leu Arg Leu Ser
Cys Ala Ala Ser Gly Phe Thr Phe Ser 20 25 301814PRTArtificial
Sequence15A7H VH FR2 18Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu
Trp Val Ala1 5 101932PRTArtificial Sequence15A7H VH FR3 19Arg Phe
Thr Ile Ser Arg Asp Asn Ala Lys Asn Thr Leu Tyr Leu Gln1 5 10 15Met
Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys Ala Arg 20 25
302011PRTArtificial Sequence15A7H VH FR4 20Trp Gly Gln Gly Thr Leu
Val Thr Val Ser Ser1 5 102113PRTHomo sapiensIgG4 wild-type hinge
region amino acid sequence 21Glu Ser Lys Tyr Gly Pro Pro Cys Pro
Ser Cys Pro Ala1 5 10
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