U.S. patent application number 17/726262 was filed with the patent office on 2022-08-11 for cd200r agonist antibodies and uses thereof.
The applicant listed for this patent is Eli Lilly and Company. Invention is credited to Stephen J. Demarest, Anja Koester, Payal Mehta, Scott Charles Potter, Diana Isabel Ruiz, Derrick Ryan Witcher, Xiufeng Wu.
Application Number | 20220251198 17/726262 |
Document ID | / |
Family ID | |
Filed Date | 2022-08-11 |
United States Patent
Application |
20220251198 |
Kind Code |
A1 |
Demarest; Stephen J. ; et
al. |
August 11, 2022 |
CD200R AGONIST ANTIBODIES AND USES THEREOF
Abstract
The present invention relates to anti-human CD200R agonist
antibodies, and uses thereof for treating diseases such as atopic
dermatitis, chronic spontaneous urticaria, allergy, asthma,
scleroderma, IBD, SLE, MS, RA, GvHD, or psoriasis.
Inventors: |
Demarest; Stephen J.; (San
Diego, CA) ; Koester; Anja; (San Diego, CA) ;
Potter; Scott Charles; (San Diego, CA) ; Ruiz; Diana
Isabel; (San Diego, CA) ; Witcher; Derrick Ryan;
(Fishers, IN) ; Wu; Xiufeng; (Burlingame, CA)
; Mehta; Payal; (Zionsville, IN) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
Eli Lilly and Company |
Indianapolis |
IN |
US |
|
|
Appl. No.: |
17/726262 |
Filed: |
April 21, 2022 |
Related U.S. Patent Documents
|
|
|
|
|
|
Application
Number |
Filing Date |
Patent Number |
|
|
16567256 |
Sep 11, 2019 |
11319370 |
|
|
17726262 |
|
|
|
|
62731204 |
Sep 14, 2018 |
|
|
|
International
Class: |
C07K 16/28 20060101
C07K016/28; A61K 39/395 20060101 A61K039/395; A61P 1/00 20060101
A61P001/00; A61P 17/00 20060101 A61P017/00; A61P 29/00 20060101
A61P029/00 |
Claims
1. An antibody comprising a heavy chain variable region (HCVR) and
a light chain variable region (LCVR), wherein the HCVR comprises a
HCDR1, HCDR2, and HCDR3, and the LCVR comprises a LCDR1, LCDR2, and
LCDR3, wherein the amino acid sequence of the HCDR1 is given by SEQ
ID NO: 1, the amino acid sequence of the HCDR2 is given by SEQ ID
NO: 2, and the amino acid sequence of the HCDR3 is given by SEQ ID
NO: 3, the amino acid sequence of the LCDR1 is given by SEQ ID NO:
4, the amino acid sequence of the LCDR2 is given by SEQ ID NO: 5,
and the amino acid sequence of the LCDR3 is given by SEQ ID NO: 6,
and wherein the antibody binds human CD200R.
2. The antibody of claim 1, comprising a HCVR and a LCVR, wherein
the amino acid sequence of the HCVR is given by SEQ ID NO: 7 and
the amino acid sequence of the LCVR is given by SEQ ID NO: 8.
3. The antibody of claim 2, wherein Xaa at position 1 of SEQ ID NO:
7 is glutamine.
4. The antibody of claim 2, wherein Xaa at position 1 of SEQ ID NO:
7 is pyroglutamic acid.
5. The antibody of claim 2, comprising a heavy chain (HC) and a
light chain (LC), wherein the amino acid sequence of the HC is
given by SEQ ID NO: 9 and the amino acid sequence of the LC is
given by SEQ ID NO: 10.
6. The antibody of claim 5, wherein Xaa at position 1 of SEQ ID NO:
9 is glutamine.
5. The antibody of claim 5, wherein Xaa at position 446 of SEQ ID
NO: 9 is glycine.
8. The antibody of claim 5, wherein Xaa at position 1 of SEQ ID NO:
9 is glutamine and Xaa at position 446 of SEQ ID NO: 9 is
glycine.
9. The antibody of claim 5, wherein Xaa at position 1 of SEQ ID NO:
9 is glutamine and Xaa at position 446 of SEQ ID NO: 9 is
absent.
10. The antibody of claim 5, wherein Xaa at position 1 of SEQ ID
NO: 9 is pyroglutamic acid and Xaa at position 446 of SEQ ID NO: 9
is absent.
11. A method of treating a patient having a disease, wherein the
disease is an autoimmune disease, allergic disease, asthma, atopic
dermatitis, or other inflammatory disorders, comprising
administering to the patient an effective amount of an antibody
comprising a heavy chain variable region (HCVR) and a light chain
variable region (LCVR), wherein the HCVR comprises a HCDR1, HCDR2,
and HCDR3, and the LCVR comprises a LCDR1, LCDR2, and LCDR3,
wherein the amino acid sequence of the HCDR1 is given by SEQ ID NO:
1, the amino acid sequence of the HCDR2 is given by SEQ ID NO: 2,
and the amino acid sequence of the HCDR3 is given by SEQ ID NO: 3,
the amino acid sequence of the LCDR1 is given by SEQ ID NO: 4, the
amino acid sequence of the LCDR2 is given by SEQ ID NO: 5, and the
amino acid sequence of the LCDR3 is given by SEQ ID NO: 6, and
wherein the antibody binds human CD200R.
12. The method of claim 11 wherein the antibody comprises a heavy
chain (HC) and a light chain (LC), wherein the amino acid sequence
of the HC is given by SEQ ID NO: 9 and the amino acid sequence of
the LC is given by SEQ ID NO: 10.
13. A pharmaceutical composition comprising 1) an antibody
comprising a heavy chain variable region (HCVR) and a light chain
variable region (LCVR), wherein the HCVR comprises a HCDR1, HCDR2,
and HCDR3, and the LCVR comprises a LCDR1, LCDR2, and LCDR3,
wherein the amino acid sequence of the HCDR1 is given by SEQ ID NO:
1, the amino acid sequence of the HCDR2 is given by SEQ ID NO: 2,
and the amino acid sequence of the HCDR3 is given by SEQ ID NO: 3,
the amino acid sequence of the LCDR1 is given by SEQ ID NO: 4, the
amino acid sequence of the LCDR2 is given by SEQ ID NO: 5, and the
amino acid sequence of the LCDR3 is given by SEQ ID NO: 6, and 2)
one or more pharmaceutically acceptable carriers, diluents, or
excipients.
14. The pharmaceutical composition of claim 13 wherein the antibody
comprises a heavy chain (HC) and a light chain (LC), wherein the
amino acid sequence of the HC is given by SEQ ID NO: 9 and the
amino acid sequence of the LC is given by SEQ ID NO: 10.
15. The pharmaceutical composition of claim 14 wherein Xaa at
position 1 of SEQ ID NO: 9 is glutamine and Xaa at position 446 of
SEQ ID NO: 9 is absent.
16. The pharmaceutical composition of claim 14, wherein Xaa at
position 1 of SEQ ID NO: 9 is pyroglutamic acid and Xaa at position
446 of SEQ ID NO: 9 is absent.
17. A DNA molecule comprising a polynucleotide that encodes a HC
whose amino acid sequence is given by SEQ ID NO: 9 or a
polynucleotide that encodes a LC whose amino acid sequence is given
by SEQ ID NO: 10.
18. The DNA molecule of claim 17, in which the sequence of the
polynucleotide that encodes the HC is given by SEQ ID NO: 12 or the
sequence of the polynucleotide that encodes the LC is given by SEQ
ID NO: 13.
19. The DNA molecule of claim 17 comprising 1) a polynucleotide
that encodes the HC whose amino acid sequence is given by SEQ ID
NO: 9, and 2) a polynucleotide that encodes the LC whose amino acid
sequence is given by SEQ ID NO: 10.
20. A mammalian cell transformed with the DNA molecule of claim 19,
which transformed mammalian cell is capable of expressing an
antibody comprising two HCs and two LCs, in which the amino acid
sequence of each HC is given by SEQ ID NO: 9, and the amino acid
sequence of each LC is given by SEQ ID NO: 10.
Description
[0001] The present invention is in the field of medicine. More
particularly, the present invention relates to agonistic antibodies
directed to CD200 Receptor (CD200R), compositions comprising such
CD200R agonistic antibodies, and methods of using such CD200R
agonistic antibodies for the treatment of disorders such as
autoimmune disease, allergic disease, asthma, or other inflammatory
disorders.
[0002] Immune checkpoint pathways may modulate both the autoimmune
response and the anti-cancer immune response. In autoimmune disease
therapy, promoting, i.e., agonizing, the effect of an
immune-inhibitory pathway, such that the immune response is
suppressed, is desirable. Conversely, in cancer therapy,
inhibiting, i.e., antagonizing, the effect of an immune-inhibitory
pathway, such that the immune response is derepressed (stimulated),
is desirable.
[0003] CD200R is an Ig superfamily member and part of a family of
checkpoint receptors that negatively regulate immune cell
activation. Activation of the CD200R pathway leads to decreased
cellular function, such as reduced cellular proliferation and
inhibition of inflammatory cytokines. CD200R is primarily expressed
on the surface of cells of the innate system, specifically of the
monocytic lineage like macrophages, mast cells, dendritic cells,
but also on activated T cell subsets such as T memory cells. The
natural ligand for CD200R is CD200, which is more broadly expressed
on multiple cell types including lymphocytes. CD200R and CD200
knockout mice have a normal phenotype, but are more prone to
induced autoimmune disease (see e.g., Simelyte et al., Clin Exp
Immunol. 162: 163-8 (2010)). Conversely, CD200 overexpression in
mice provides resistance to allogeneic transplantation and
DSS-induced colitis (Chen et al., PLoS One. 2016; 11(2): e0146681.
doi: 10.1371/journal.pone.0146681). Therefore, increasing CD200R
mediated signaling constitutes a potential approach to manage
autoimmune disorders that may lead to profound disease modification
and durability of response along with key safety benefits over
current immunomodulatory therapies. For example, CD200R is highly
expressed in differentiated, tissue-resident cells like
macrophages, mast cells, dendritic cells, and innate lymphoid
cells. These cell types contribute to the pathology of diseases
such as atopic dermatitis, and therefore CD200R agonist antibodies
may attenuate the activity of these cells in diseases such as
atopic dermatitis.
[0004] The field has struggled to deliver therapeutically effective
and safe CD200R agonistic antibodies. The difficulty, at least
partly, is thought to be the result of the complex cellular
interactions required to achieve CD200R agonism with minimal safety
concerns (e.g. without inducing cytokine release) in physiological
settings.
[0005] U.S. Pat. No. 8,212,008 discloses CD200R antibodies, such as
Dx182. Dx182 is a humanized IgG1 antibody that agonizes CD200R and
blocks binding of CD200 to CD200R. However, Dx182 also binds to and
activates cynomolgus monkey CD200RLa (cynomolgus monkey activating
form) expressed in murine mast cells, and thereby induces a mast
cell degranulation response in these cells via the cynomolgus
CD200RLa. WO 2015/057906 also discloses CD200R agonist antibodies,
such as H2RM147. H2RM147 is likely to compete with human CD200 for
binding to human CD200R1.
[0006] Thus, there exists a need for alternative CD200R antibodies
that 1) bind human CD200R with desirable association and
dissociation rates for optimal agonist activity, 2) agonize human
CD200R to achieve immunosuppressive response and in vivo efficacy,
3) display sufficient potency as an monotherapy for the treatment
and/or prevention of disorders such as autoimmune disorders,
allergic disease, asthma, or other inflammatory disorders, 4) do
not cause significant cytokine release, 5) do not block binding of
human CD200 and human CD200R, 6) do not bind CD200RLa or binds
CD200RLa with low affinity. and/or 7) demonstrate low
immunogenicity (i.e., sufficiently non-immunogenic in cynomolgus
monkeys and/or humans) and in vivo stability, physical and chemical
stability including, but not limited to, thermal stability,
solubility, low self-association, and pharmacokinetic
characteristics which are acceptable for development and/or use in
the treatment of autoimmune disorders, allergic disease, asthma, or
other inflammatory disorders.
[0007] Accordingly, the present invention provides novel anti-human
CD200R agonist antibodies. The antibodies of the present invention
are particularly advantageous over prior art CD200R agonist
antibodies in view of at least the following properties: 1)
desirable association and dissociation rates, 2) agonism of human
CD200R to achieve an immunosuppressive response and in vivo
efficacy, 3) sufficiently potent as an monotherapy for the
treatment and/or prevention of autoimmune disorders, allergic
disease, asthma, or other inflammatory disorders 4) no significant
cytokine release, 5) no blocking of binding of human CD200 to human
CD200R, and binding to a different epitope compared to prior art
antibodies, 6) lack of binding, or binding with low affinity, the
cyno CD200RLa compared to binding to human CD200R, and/or 7) low
immunogenicity (i.e., sufficiently non-immunogenic in cynomolgus
monkeys and/or humans) and in vivo stability, physical and chemical
stability including, but not limited to, thermal stability,
solubility, low self-association, and pharmacokinetic
characteristics which are acceptable for development and/or use in
the treatment of autoimmune disorders, allergic disease, asthma, or
other inflammatory disorders.
[0008] The subject invention provides an advance over the prior art
by providing compositions and methods useful in the prevention,
downregulation, or amelioration of autoimmune and/or immune
tolerance related disorders, allergic disease, asthma, or other
inflammatory disorders, through immune checkpoint stimulation using
a significantly engineered anti-human CD200R agonist antibody. The
anti-human CD200R agonist antibodies of the present invention are
capable of improving immune pathology or restoring immune
homeostasis, preferably, through inhibition of the innate arm of
the immune response, abrogation of the antigen specific immune
process, and thereby directly addressing the underlying disease
pathology. The use of such antibodies clinically may lead to
long-term durability of the disease(s) being treated.
[0009] Accordingly, the present invention provides an antibody that
binds human CD200R (SEQ ID NO: 15), comprising a heavy chain
variable region (HCVR) and a light chain variable region (LCVR),
wherein the HCVR comprises a HCDR1, HCDR2, and HCDR3, and the LCVR
comprises a LCDR1, LCDR2, and LCDR3, wherein the amino acid
sequence of the HCDR1 is given by SEQ ID NO: 1, the amino acid
sequence of the HCDR2 is given by SEQ ID NO: 2, and the amino acid
sequence of the HCDR3 is given by SEQ ID NO: 3, the amino acid
sequence of the LCDR1 is given by SEQ ID NO: 4, the amino acid
sequence of the LCDR2 is given by SEQ ID NO: 5, and the amino acid
sequence of the LCDR3 is given by SEQ ID NO: 6.
[0010] In an embodiment, the present invention provides an antibody
that binds human CD200R, comprising a HCVR and a LCVR, wherein the
amino acid sequence of the HCVR is given by SEQ ID NO: 7 and the
amino acid sequence of the LCVR is given by SEQ ID NO: 8. In some
embodiments, Xaa at position 1 of SEQ ID NO: 7 is glutamine. In
other embodiments, Xaa at position 1 of SEQ ID NO: 7 is
pyroglutamic acid.
[0011] In an embodiment, the present invention provides an antibody
that binds human CD200R, comprising a heavy chain (HC) and a light
chain (LC), wherein the amino acid sequence of the HC is given by
SEQ ID NO: 9 and the amino acid sequence of the LC is given by SEQ
ID NO: 10. In some embodiments, Xaa at position 1 of SEQ ID NO: 9
is glutamine. In other embodiments, Xaa at position 1 of SEQ ID NO:
9 is pyroglutamic acid. In some embodiments, Xaa at position 446 of
SEQ ID NO: 9 is glycine. In some embodiments, Xaa at position 446
of SEQ ID NO: 9 is absent. In a particular embodiment, Xaa at
position 1 of SEQ ID NO: 9 is glutamine and Xaa at position 446 of
SEQ ID NO: 9 is glycine. In another particular embodiment, Xaa at
position 1 of SEQ ID NO: 9 is pyroglutamic acid and Xaa at position
446 of SEQ ID NO: 9 is glycine. In a particular embodiment, Xaa at
position 1 of SEQ ID NO: 9 is glutamine and Xaa at position 446 of
SEQ ID NO: 9 is absent. In another particular embodiment, Xaa at
position 1 of SEQ ID NO: 9 is pyroglutamic acid and Xaa at position
446 of SEQ ID NO: 9 is absent.
[0012] In an embodiment, an antibody of the present invention does
not cause significant cytokine release. In another embodiment, the
antibody is a CD200R agonist antibody. In a preferred embodiment,
the antibody does not cause significant cytokine release and the
antibody is a CD200R agonist antibody. In some such embodiments,
the antibody is an IgG4 subtype, preferably, an IgG4P. In another
embodiment, the antibody binds human and cynomolgus monkey
CD200R.
[0013] The present disclosure also provides a mammalian cell
capable of expressing a anti-human CD200R antibody comprising: 1) a
HCVR comprising a HCDR1 having the amino acid sequence of SEQ ID
NO: 1, a HCDR2 having the amino acid sequence of SEQ ID NO: 2, a
HCDR3 having the amino acid sequence of SEQ ID NO: 3; and 2) a LCVR
comprising a LCDR1 having the amino acid sequence of SEQ ID NO: 4,
a LCDR2 having the amino acid sequence of SEQ ID NO: 5, and a LCDR3
having the amino acid sequence of SEQ ID NO: 6. In some
embodiments, the present disclosure provides a mammalian cell
capable of expressing an anti-human CD200R antibody comprising: 1)
a HCVR having the amino acid sequence of SEQ ID NO: 7; and 2) a
LCVR having the amino acid sequence of SEQ ID NO: 8. In some
embodiments, the present disclosure provides a mammalian cell
capable of expressing a CD200R antibody comprising: 1) a heavy
chain having the amino acid sequence of SEQ ID NO: 9; and 2) a
light chain having the amino acid sequence of SEQ ID NO: 10. In
some embodiments, the present disclosure provides that the CD200R
antibody consists of two heavy chains each having the amino acid
sequence of SEQ ID NO: 9, and two light chains each having the
amino acid sequence of SEQ ID NO: 10.
[0014] In an embodiment, an antibody of the present invention does
not cause significant cytokine release. In another embodiment, the
antibody is a CD200R agonist antibody. In a preferred embodiment,
the antibody does not cause significant cytokine release and the
antibody is a CD200R agonist antibody. In some such embodiments,
the antibody is an IgG4 subtype, preferably, an IgG4P. In another
embodiment, the antibody binds human and cynomolgus monkey
CD200R.
[0015] The present disclosure also provides a process for producing
an anti-human CD200R antibody, comprising: a) cultivating a
mammalian cell capable of expressing the antibody, wherein the
antibody comprises: 1) a HCVR comprising a HCDR1 having the amino
acid sequence of SEQ ID NO: 1, a HCDR2 having the amino acid
sequence of SEQ ID NO: 2, a HCDR3 having the amino acid sequence of
SEQ ID NO: 3; and 2) a LCVR comprising a LCDR1 having the amino
acid sequence of SEQ ID NO: 4, a LCDR2 having the amino acid
sequence of SEQ ID NO: 5, and a LCDR3 having the amino acid
sequence of SEQ ID NO: 6; and b) recovering the antibody. In some
embodiments, the present disclosure provides a process for
producing a CD200R antibody, comprising: a) cultivating a mammalian
cell capable of expressing the antibody, wherein the antibody
comprises: 1) a HCVR having the amino acid sequence of SEQ ID NO:
7; and 2) a LCVR having the amino acid sequence of SEQ ID NO: 8;
and b) recovering the antibody. In some embodiments, the present
disclosure provides a process for producing an anti-human CD200R
antibody, comprising: a) cultivating a mammalian cell capable of
expressing the antibody, wherein the antibody comprises: 1) a heavy
chain having the amino acid sequence of SEQ ID NO: 9; and 2) a
light chain having the amino acid sequence of SEQ ID NO: 10; and b)
recovering the antibody. In some embodiments, the present
disclosure provides a process for producing an anti-human CD200R
antibody, comprising: a) cultivating a mammalian cell capable of
expressing the antibody, wherein the antibody consists of two heavy
chains having the amino acid sequence of SEQ ID NO: 9 and two light
chains having the amino acid sequence of SEQ ID NO: 10; and b)
recovering the antibody.
[0016] In an embodiment, an antibody of the present invention does
not cause significant cytokine release. In another embodiment, the
antibody is a CD200R agonist antibody. In a preferred embodiment,
the antibody does not cause significant cytokine release and the
antibody is a CD200R agonist antibody. In some such embodiments,
the antibody is an IgG4 subtype, preferably, an IgG4P. In another
embodiment, the antibody binds human and cynomolgus monkey
CD200R.
[0017] The present disclosure also provides the CD200R antibody
produced by the aforementioned processes. The present disclosure
also provides a pharmaceutical composition comprising the CD200R
antibody produced by the aforementioned processes and an acceptable
carrier, diluent, or excipient.
[0018] The present disclosure also provides a DNA molecule
comprising a polynucleotide having the sequence of SEQ ID NO: 12.
The present disclosure also provides a DNA molecule comprising a
polynucleotide having the sequence of SEQ ID NO: 13. The present
disclosure also provides a DNA molecule comprising a polynucleotide
having the sequence of SEQ ID NO: 12 and SEQ ID NO: 13. The present
disclosure also provides a DNA molecule comprising a polynucleotide
that encodes the antibody HC whose amino acid sequence is the
sequence of SEQ ID NO: 9. In an embodiment, the DNA molecule that
encodes the antibody HC is given by SEQ ID NO: 12. The present
disclosure also provides a DNA molecule comprising a polynucleotide
that encodes the antibody LC whose amino acid sequence is the
sequence of SEQ ID NO: 10. In an embodiment, the DNA molecule that
encodes the antibody LC is given by SEQ ID NO: 13.
[0019] The present disclosure also provides a mammalian cell
comprising a DNA molecule comprising a polynucleotide having the
sequence of SEQ ID NO: 12. The present disclosure also provides a
mammalian cell comprising a DNA molecule comprising a
polynucleotide having the sequence of SEQ ID NO: 13. The present
disclosure also provides a mammalian cell comprising a DNA molecule
comprising a polynucleotide having the sequence of SEQ ID NO: 12
and SEQ ID NO: 13.
[0020] The present invention also provides a pharmaceutical
composition comprising an antibody of the present invention.
[0021] The present invention also provides a method of treating a
patient having a disease, wherein the disease is an autoimmune
disease, allergic disease, asthma, or other inflammatory disorders,
comprising administering to a patient in need thereof, an effective
amount of an antibody of the present invention.
[0022] The present invention also provides an antibody of the
present invention for use in therapy.
[0023] The present invention also provides an antibody of the
present invention for use in treating a disease, wherein the
disease is an autoimmune disease, allergic disease, asthma, or
other inflammatory disorders.
[0024] The present invention also provides use of an antibody of
the present invention for the manufacture of a medicament for the
treatment of a disease, wherein the disease is an autoimmune
disease, allergic disease, asthma, or other inflammatory
disorders.
[0025] In an embodiment, the disease is an autoimmune disease. In
another embodiment, the disease is an allergic disease. In another
embodiment, the disease is asthma. In some embodiments, the disease
is chronic idiopathic urticaria (also referred to herein as chronic
spontaneous urticaria (CSU)), celiac disease (including, but not
limited to, refractory celiac disease type II), allergy, chronic
allergic disease, food allergies, eosinophilic esophagitis,
macrophage activation syndrome (MAS), asthma, scleroderma,
pemphigus, irritable bowel disease (IBD), systemic lupus
erythematosus (SLE), multiple sclerosis (MS), rheumatoid arthritis
(RA), graft versus host disease (GvHD), psoriasis, mastocytosis,
inflammatory skin disease, or atopic dermatitis. In other
embodiments, the disease is allergic contact dermatitis, seasonal
allergies, anaphylaxis treatment and prevention, bullous pemphigoid
and other autoimmune blistering diseases, autoimmune hepatitis,
primary sclerosing cholangitis, primary biliary cirrhosis,
idiopathic pulmonary fibrosis, myasthenia gravis, vasculitis, and
myositis. In a particular embodiment, the chronic allergic disease
is hay fever or allergic rhinitis. In a preferred embodiment, the
disease is atopic dermatitis.
[0026] The present invention provides an antibody that binds human
CD200R, wherein the antibody is a CD200R agonist antibody and
wherein the antibody does not cause significant cytokine release.
In an embodiment, the antibody demonstrates CD200R agonism and lack
of significant cytokine release similar to the CD200R agonism and
lack of significant cytokine release as demonstrated by Antibody
I-4P. In an embodiment, the CD200R agonist antibody does not cause
significant cytokine release compared to a wild-type (no mutations
in the Fc portion) IgG1 antibody (which does cause significant
cytokine release, especially release of IFN-.gamma.). In a
particular embodiment, the present invention provides a CD200R
agonist antibody, wherein the antibody does not cause significant
cytokine release compared to a wild-type IgG1 antibody having the
same CDRs as the CD200R agonist antibody. In an embodiment, a
significant cytokine release is detected by comparing the amount of
cytokine present in blood samples incubated with the antibody and
the amount of cytokine present in blood samples without incubation
with the antibody and determining the presence of significant
cytokine release if the amount of cytokine present in blood sample
incubated with the antibody is at least three-fold higher than the
amount of cytokine present in blood sample with no antibody.
[0027] In an embodiment, the antibody comprises a HCVR and a LCVR,
wherein the HCVR comprises a HCDR1, HCDR2, and HCDR3, and the LCVR
comprises a LCDR1, LCDR2, and LCDR3, wherein the amino acid
sequence of the HCDR1 is given by SEQ ID NO: 1, the amino acid
sequence of the HCDR2 is given by SEQ ID NO: 2, and the amino acid
sequence of the HCDR3 is given by SEQ ID NO: 3, the amino acid
sequence of the LCDR1 is given by SEQ ID NO: 4, the amino acid
sequence of the LCDR2 is given by SEQ ID NO: 5, and the amino acid
sequence of the LCDR3 is given by SEQ ID NO: 6. In an embodiment,
the antibody comprises a HCVR and a LCVR, wherein the amino acid
sequence of the HCVR is given by SEQ ID NO: 7 and the amino acid
sequence of the LCVR is given by SEQ ID NO: 8.
[0028] The present invention provides an antibody of the present
invention that binds at least one, at least two, at least three, at
least four, or all of Fc.gamma. RI, Fc.gamma. RIIA_131H, Fc.gamma.
RIIA_131R, Fc.gamma. RIIb, and Fc.gamma. RIIIA_158V.
[0029] In an embodiment, the antibody binds Fc.gamma. RI with a
binding affinity of about 70 .mu.M to about 500 .mu.M. In another
embodiment, the antibody binds Fc.gamma. RIIA_131H with a binding
affinity of about 2 .mu.M to about 5 .mu.M. In another embodiment,
the antibody binds Fc.gamma. RITA_131R with a binding affinity of
about 1 .mu.M to about 5 .mu.M. In another embodiment, the antibody
binds Fc.gamma. RIIb with a binding affinity of about 1 .mu.M to
about 4 .mu.M. In another embodiment, the antibody binds Fc.gamma.
RIIIA_158V with a binding affinity of about 1 .mu.M to about 6
.mu.M. In another embodiment, the antibody further binds Fc.gamma.
RIIIA 158F with a binding affinity of greater than 9 .mu.M.
[0030] In an embodiment, the binding affinities of the antibody to
the receptor are about 70 .mu.M to about 500 .mu.M to Fc.gamma. RI,
about 2 .mu.M to about 5 .mu.M to Fc.gamma. RIIA_131H, about 1
.mu.M to about 5 .mu.M to Fc.gamma. RIIA_131R, about 1 .mu.M to
about 4 .mu.M to Fc.gamma. RIIb, about 1 .mu.M to about 6 .mu.M to
Fc.gamma. RIIIA_158V, and greater than 9 .mu.M to Fc.gamma.
RIIIA_158F. In a more particular embodiment, the binding affinities
of the antibody to the receptor are about 400 .mu.M to Fc.gamma.
RI, about 4 .mu.M to Fc.gamma. RIIA 131H, about 2 .mu.M to
Fc.gamma. RITA_131R, about 2 .mu.M to Fc.gamma. RIIb, about 4 .mu.M
to Fc.gamma. RIIIA_158V, and greater than 10 .mu.M to Fc.gamma.
RIIIA_158F. In a further embodiment, the antibody does not bind
C1q. In some embodiments, the binding affinity is determined by
Surface Plasmon Resonance at 25.degree. C. In other embodiments,
binding to C1q is determined by ELISA.
[0031] As used herein, "CD200R" refers to the CD200 receptor. As
used herein, "hCD200R" or "human CD200R" refers to a wild-type
human CD200R, and, preferably, to a wild-type human CD200R that has
the amino acid sequence set forth in SEQ ID NO: 15.
[0032] The terms "cyno", "cynomolgus" or "cynomolgus monkey" are
used interchangeably, herein. When used in reference to a CD200R
polypeptide, unless otherwise stated, it is intended that the terms
refer to wild-type cynomolgus monkey CD200R, and, preferably, a
wild-type cynomolgus monkey CD200R that has the amino acid sequence
set forth in SEQ ID NO: 16. The terms "CD200RLa" or "activating
form" refer to a cynomolgus monkey CD200R that has the amino acid
sequence set forth in SEQ ID NO: 17. CD200RLa is a close homologue
of human CD200R but with the opposite (activating) activity.
Therefore, a preferred CD200R agonist antibody binds CD200RLa with
a significantly reduced affinity compared to antibody binding to
CD200R.
[0033] As used herein, "human CD200R agonist antibody" or
"anti-human CD200R agonist antibody" refers to an antibody that
binds to human CD200R, and when administered in vitro or in vivo,
results in an achieved immunosuppressive response such as at least
one significantly lessened desired activity such as a desired
reduction in IL-8 production. As used herein, the terms
"production" and "secretion," as they relate to cytokines, are used
interchangeably.
[0034] The term "antibody" as used herein refers to an engineered,
non-naturally occurring polypeptide complex having two heavy chains
(HC) and two light chains (LC) such that the heavy chains and the
light chains are interconnected by disulfide bonds, wherein the
antibody is an IgG isotype antibody. Each heavy chain is comprised
of an N-terminal HCVR and a heavy chain constant region. Each light
chain is comprised of an N-terminal LCVR and a light chain constant
region. When expressed in certain biological systems, antibodies
are glycosylated in the Fc region. Typically, glycosylation occurs
in the Fc region of the antibody at a highly conserved
N-glycosylation site. N-glycans typically attach to asparagine.
Antibodies may be glycosylated at other positions as well.
[0035] Antibodies of the present invention may be an IgG1 or IgG4
antibody. Preferably, antibodies of the present invention are IgG4
antibodies. An IgG4 antibody may have an S228P mutation within the
HC (i.e., IgG4P), which is known to eliminate half-antibody
formation common for the human IgG4 subclass.
[0036] The constant region of the heavy chains contains CH1, CH2,
and CH3 domains. CH1 comes after the HCVR; the CH1 and HCVR form
the heavy chain portion of an antigen-binding (Fab) fragment, which
is the part of an antibody that binds antigen(s). CH2 comes after
the hinge region and before CH3. CH3 comes after CH2 and is at the
carboxy-terminal end of the heavy chain. The constant region of the
light chains contains one domain, CL. CL comes after the LCVR; the
CL and LCVR form the light chain portion of a Fab.
[0037] The HCVR and LCVR regions of an antibody of the present
invention can be further subdivided into regions of
hyper-variability, termed complementarity determining regions
("CDRs"), interspersed with regions that are more conserved, termed
framework regions ("FR"). Each HCVR and LCVR is composed of three
CDRs and four FRs, arranged from amino-terminus to carboxy-terminus
in the following order: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4.
Herein, the three CDRs of the heavy chain are referred to as
"HCDR1, HCDR2, and HCDR3" and the three CDRs of the light chain are
referred to as "LCDR1, LCDR2 and LCDR3". The CDRs contain most of
the residues which form specific interactions with the antigen. The
Kabat CDR definition (Kabat, et al., Ann. NY Acad. Sci. 190: 382-93
(1971); Kabat et al., Sequences of Proteins of Immunological
Interest, Fifth Edition, U.S. Department of Health and Human
Services, NIH Publication No. 91-3242 (1991)) is based upon
antibody sequence variability. The Chothia CDR definition (Chothia
et al., "Canonical structures for the hypervariable regions of
immunoglobulins", Journal of Molecular Biology, 196, 901-917
(1987); Al-Lazikani et al., "Standard conformations for the
canonical structures of immunoglobulins", Journal of Molecular
Biology, 273, 927-948 (1997)) is based on three-dimensional
structures of antibodies and topologies of the CDR loops. The
Chothia CDR definitions are identical to the Kabat CDR definitions
with the exception of HCDR1 and HCDR2. The North CDR definition
(North et al., "A New Clustering of Antibody CDR Loop
Conformations", Journal of Molecular Biology, 406, 228-256 (2011))
is based on affinity propagation clustering with a large number of
crystal structures. For the purposes of the present invention,
assignment of amino acids to CDR domains within the LCVR and HCVR
regions of the antibodies of the present invention is based on the
well-known Kabat numbering convention and North numbering
convention. In the case of the light chain CDRs of the antibodies
of the present invention, the North CDR definitions are used. In
the heavy chain, both HCDR1 and HCDR3 also use the North
definition. HCDR2 uses a hybrid of North and Kabat definitions. The
North definition is used to identify the starting N-terminal site
while Kabat is used to define the last position.
[0038] The present invention contemplates that the antibodies of
the present invention are human or humanized antibodies. In the
context of monoclonal antibodies, the terms "human" and "humanized"
are well-known to those of ordinary skill in the art (Weiner L J,
J. Immunother. 2006; 29: 1-9; Mallbris L, et al., J. Clin. Aesthet.
Dermatol. 2016; 9: 13-15).
[0039] A DNA molecule of the present invention is a DNA molecule
that comprises a non-naturally occurring polynucleotide sequence
encoding a polypeptide having the amino acid sequence of at least
one of the polypeptides in an antibody of the present invention
(e.g., heavy chain, light chain, variable heavy chain, and variable
light chain).
[0040] An isolated DNA encoding a HCVR region can be converted to a
full-length heavy chain gene by operably linking the HCVR-encoding
DNA to another DNA molecule encoding heavy chain constant regions.
The sequences of human, as well as other mammalian, heavy chain
constant region genes are known in the art. DNA fragments
encompassing these regions can be obtained, e.g., by standard PCR
amplification.
[0041] An isolated DNA encoding a LCVR region may be converted to a
full-length light chain gene by operably linking the LCVR-encoding
DNA to another DNA molecule encoding a light chain constant region.
The sequences of human, as well as other mammalian, light chain
constant region genes are known in the art. DNA fragments
encompassing these regions can be obtained by standard PCR
amplification. The light chain constant region can be a kappa or
lambda constant region. Preferably, for antibodies of the present
invention, the light chain constant region is a kappa constant
region.
[0042] The polynucleotides of the present invention can be
expressed in a host cell after the sequences have been operably
linked to an expression control sequence. The expression vectors
are typically replicable in the host organisms either as episomes
or as an integral part of the host chromosomal DNA. Commonly,
expression vectors will contain selection markers, e.g.,
tetracycline, neomycin, and dihydrofolate reductase, to permit
detection of those cells transformed with the desired DNA
sequences.
[0043] The antibodies of the present invention can readily be
produced in mammalian cells, non-limiting examples of which
includes CHO, NSO, HEK293 or COS cells. The host cells are cultured
using techniques well known in the art.
[0044] The vectors containing the polynucleotide sequences of
interest (e.g., the polynucleotides encoding the polypeptides of
the antibody and expression control sequences) can be transferred
into the host cell by well-known methods, which vary depending on
the type of cellular host.
[0045] Various methods of protein purification may be employed to
purify proteins, including, but not limited to, antibodies and such
methods are known in the art.
[0046] An antibody of the present invention, or a pharmaceutical
composition comprising the same, may be administered by parenteral
routes, non-limiting examples of which are subcutaneous
administration and intravenous administration. An antibody of the
present invention may be administered to a patient with
pharmaceutically acceptable carriers, diluents, or excipients in
single or multiple doses. Pharmaceutical compositions of the
present invention can be prepared by methods well known in the art
(e.g., Remington: The Science and Practice of Pharmacy, 22nd ed.
(2012), A. Loyd et al., Pharmaceutical Press) and comprise an
antibody, as disclosed herein, and one or more pharmaceutically
acceptable carriers, diluents, or excipients.
[0047] As used herein, the term "autoimmune disease" or "autoimmune
disorder" are used interchangeably and refer to undesirable
conditions that arise from an inappropriate or unwanted immune
reaction against self-cells and/or tissues or transplanted cells
and/or tissues. The term "autoimmune disease" or "autoimmune
disorder" is meant to include such conditions, whether they be
mediated by humoral or cellular immune responses. "Allergy" (or
"allergic disease") is a T helper 2 (TH2)-driven disease that
develops primarily from activity of TH2 cells. Exemplary diseases
contemplated to be treated by the antibodies of the invention
described herein include chronic idiopathic urticaria, celiac
disease (including, but not limited to, refractory celiac disease
type II), allergy, chronic allergic disease (such as hay fever or
allergic rhinitis), food allergies, eosinophilic esophagitis, MAS,
asthma, scleroderma, and also pemphigus, IBD, SLE, MS, RA, GvHD,
psoriasis, mastocytosis, inflammatory skin disease, and atopic
dermatitis. In other embodiments, the disease contemplated to be
treated by the antibodies of the invention described herein include
is allergic contact dermatitis, seasonal allergies, anaphylaxis
treatment and prevention, bullous pemphigoid and other autoimmune
blistering diseases, autoimmune hepatitis, primary sclerosing
cholangitis, primary biliary cirrhosis, idiopathic pulmonary
fibrosis, myasthenia gravis, vasculitis, and myositis.
[0048] The terms "chronic idiopathic urticaria" and "chronic
spontaneous urticaria (CSU)" are used interchangeably herein.
[0049] As used herein, the term "innate immunity" includes the arm
of the immune response which, in contrast to the adaptive arm of
the immune response, is required to initiate and maintain an
adaptive immune response (antibody and T cell responses).
[0050] The term "treating" (or "treat" or "treatment") refers to
slowing, interrupting, arresting, alleviating, stopping, reducing,
or reversing the progression or severity of an existing symptom,
disorder, condition, or disease.
[0051] "Effective amount" means the amount of an anti-human CD200R
agonist antibody of the present invention or pharmaceutical
composition comprising such an antibody that will elicit the
biological or medical response of or desired therapeutic effect on
a tissue, system, animal, mammal, or human that is being sought by
the researcher, medical doctor, or other clinician. An effective
amount of the antibody may vary according to factors such as the
disease state, age, sex, and weight of the individual, and the
ability of the antibody to elicit a desired response in the
individual. An effective amount is also one in which any toxic or
detrimental effect of the antibody is outweighed by the
therapeutically beneficial effects. Such benefit includes any one
or more of: an increased immune tolerance of transplanted organs;
stabilized autoimmune disease or disorder; or improving signs or
symptoms of an autoimmune disorder, etc. An effective amount can be
readily determined by one skilled in the art, by the use of known
techniques, and by observing results obtained under analogous
circumstances. An effective amount of an anti-human CD200R agonist
antibody of the present invention may be administered in a single
dose or in multiple doses. Furthermore, an effective amount of an
antibody of the invention may be administered in multiple doses of
amounts that would be less than an effective amount if not
administered more than once. In determining the effective amount
for a patient, a number of factors are considered by the attending
medical practitioner, including, but not limited to: the patient's
size (e.g., weight or mass), body surface area, age, and general
health; the specific disease or disorder involved; the degree of,
or involvement, or the severity of the disease or disorder; the
response of the individual patient; the particular compound
administered; the mode of administration; the bioavailability
characteristics of the preparation administered; the dose regimen
selected; the use of concomitant medication; and other relevant
circumstances known to medical practitioners. A weekly, every two
weeks, monthly, or quarterly parenteral (including, but not limited
to, subcutaneous, intramuscular, and/or intravenous) dose can be,
for example, from about 50 mg to about 500 mg, from about 75 mg to
about 500 mg, from about 100 mg to about 500 mg, from about 125 mg
to about 500 mg, from about 250 mg to about 500 mg, from about 300
mg to about 500 mg, from about 350 mg to about 500 mg, from about
400 mg to about 500 mg, from about 450 mg to about 500 mg, from
about 50 mg to about 400 mg, from about 75 mg to about 400 mg, from
about 100 mg to about 400 mg, from about 125 mg to about 400 mg,
from about 250 mg to about 400 mg, from about 300 mg to about 400
mg, from about 350 mg to about 400 mg, from about 50 mg to about
300 mg, from about 75 mg to about 300 mg, from about 100 mg to
about 300 mg, from about 125 mg to about 300 mg, from about 150 mg
to about 300 mg, from about 175 mg to about 300 mg, from about 200
mg to about 300 mg, from about 250 mg to about 300 mg, from about
50 mg to about 250 mg, from about 75 mg to about 250 mg, from about
100 mg to about 250 mg, from about 125 mg to about 250 mg, from
about 150 mg to about 250 mg, from about 175 mg to about 250 mg,
from about 200 mg to about 250 mg, from about 75 mg to about 250
mg, from about 50 mg to about 200 mg, from about 75 mg to about 200
mg, from about 100 mg to about 200 mg, from about 125 mg to about
200 mg, from about 150 mg to about 200 mg, from about 175 mg to
about 200 mg, from about 50 mg to about 175 mg, from about 75 mg to
about 175 mg, from about 100 mg to about 175 mg, from about 125 mg
to about 175 mg, or from about 150 mg to about 175 mg. A weekly,
every two week, monthly, or quarterly parenteral (including, but
not limited to, subcutaneous, intramuscular, and/or intravenous)
dose can be from about 0.5 mg/kg to about 10 mg/kg, from about 1
mg/kg to about 10 mg/kg, from about 2 mg/kg to about 10 mg/kg, from
about 3 mg/kg to about 10 mg/kg, from about 4 mg/kg to about 10
mg/kg, from about 5 mg/kg to about 10 mg/kg, from about 6 mg/kg to
about 10 mg/kg, from about 7 mg/kg to about 10 mg/kg from about 8
mg/kg to about 10 mg/kg, from about 1 mg/kg to about 8 mg/kg, from
about 2 mg/kg to about 8 mg/kg, from about 3 mg/kg to about 8
mg/kg, from about 4 mg/kg to about 8 mg/kg, from about 5 mg/kg to
about 8 mg/kg, from about 6 mg/kg to about 8 mg/kg, from about 1
mg/kg to about 6 mg/kg, from about 2 mg/kg to about 6 mg/kg, from
about 3 mg/kg to about 6 mg/kg, from about 4 mg/kg to about 6
mg/kg, from about 5 mg/kg to about 6 mg/kg, from about 1 mg/kg to
about 5 mg/kg, from about 2 mg/kg to about 5 mg/kg, from about 3
mg/kg to about 5 mg/kg, from about 4 mg/kg to about 5 mg/kg, from
about 1 mg/kg to about 4 mg/kg, from about 2 mg/kg to about 4
mg/kg, from about 3 mg/kg to about 4 mg/kg, from about 3.5 mg/kg to
about 5 mg/kg, or about 4 mg/kg to about 5 mg/kg.
[0052] However, doses below or above the doses mentioned herein are
also envisioned, especially considering dosage considerations known
to those skilled in the art and/or described herein. Progress of
the patient being treated may be monitored by periodic assessment,
and the dose adjusted accordingly if necessary.
[0053] As used herein, the term "effective response" of a patient
or a patient's responsiveness to treatment refers to the clinical
or therapeutic benefit imparted to a patient upon administration an
antibody of the present disclosure. Such benefit includes any one
or more of the following: an increased immune tolerance of
transplanted organs; stabilized autoimmune disease or disorder; or
improving signs or symptoms of an autoimmune disorder, etc.
[0054] As used herein, "significant cytokine release" refers to a
significant increase in measurable cytokines that can be detected
by methods known to persons of ordinary skill. For example,
significant cytokine release may be detected in human blood samples
by ELISA, wherein cytokine levels from unstimulated blood are
compared to cytokine levels with blood incubated with antibody. In
some such studies, for example, a significant cytokine release may
be detected if the levels of IFN-.gamma. are at least three-fold
higher in blood incubated with antibody compared to levels in
unstimulated blood.
[0055] A potential advantage of methods disclosed herein is the
possibility of producing marked and/or prolonged relief in a
patient suffering from an autoimmune disorder, allergic disease,
asthma, or other inflammatory disorders, with an acceptable safety
profile including acceptable tolerability, toxicities and/or
adverse events, so that the patient benefits from the treatment
method overall. The efficacy of the treatment of the present
disclosure can be measured by various endpoints that are commonly
used in evaluating treatments for various autoimmune disorders
including, but not limited to, American College of Rheumatology
(ACR) 20, ACR50, ACR70, Psoriasis Area and
[0056] Severity Index (PAST) 50, PASI75, PASI90, PASI100, Systemic
Lupus Erythematosus Disease Activity Index (SLEDAI). Various other
approaches to determining efficacy of any particular therapy of the
present invention can be optionally employed, including, for
example, immune cell activation markers, measures of inflammation,
cell-cycle dependent biomarkers measurement visualization, and/or
measurement of response through pain assessments.
EXAMPLE
Antibody Expression and Purification
[0057] Anti-human CD200R agonist antibodies of the present
invention can be expressed and purified essentially as follows. An
appropriate host cell, such as HEK 293 or CHO, can be either
transiently or stably transfected with an expression system for
secreting antibodies using an optimal predetermined HC:LC vector
ratio (such as 1:3 or 1:2) or a single vector system encoding both
the HC and the LC. Clarified media, into which the antibody has
been secreted, may be purified using any of many commonly-used
techniques. For example, the medium may be conveniently applied to
a Mab Select.RTM. column (GE Healthcare), or KappaSelect column (GE
Healthcare) for Fab fragment, that has been equilibrated with a
compatible buffer, such as phosphate buffered saline (pH 7.4). The
column may be washed to remove nonspecific binding components.
[0058] The bound antibody may be eluted, for example, by pH
gradient (such as 20 mM Tris buffer, pH 7.0 to 10 mM sodium citrate
buffer, pH 3.0, or phosphate buffered saline pH 7.4 to 100 mM
glycine buffer, pH 3.0). Antibody fractions may be detected, such
as by SDS-PAGE, and then may be pooled. Further purification is
optional, depending on intended use. The antibody may be
concentrated and or sterile filtered using common techniques.
Soluble aggregate and multimers may be effectively removed by
common techniques, including size exclusion, hydrophobic
interaction, ion exchange, multimodal, or hydroxyapatite
chromatography. The purity of the antibody after these
chromatography steps is between about 95% to about 99%.
[0059] Notably, the C-terminal glycine of Antibody I-4P or the
C-terminal lysine of Antibody I-IgG1 may be truncated
post-translationally. Additionally, the N-terminal glutamine of
Antibody I-4P or Antibody I-IgG1 may be converted to pyroglutamic
acid.
[0060] The product may be held refrigerated, immediately frozen at
-70.degree. C., or may be lyophilized. Amino acid SEQ ID NOs for
exemplified humanized antibodies of the present invention are shown
below in Table 1.
TABLE-US-00001 TABLE 1 Amino acid sequences of exemplified
anti-human CD200R agonist antibodies. Antibody SEQ ID NOs Antibody
HCDR1 HCDR2 HCDR3 LCDR1 LCDR2 LCDR3 Antibody I SEQ ID SEQ ID SEQ ID
SEQ ID SEQ ID SEQ ID NO: 1 NO: 2 NO: 3 NO: 4 NO: 5 NO: 6 Antibody
HCVR LCVR Antibody I SEQ ID SEQ ID NO: 7 NO: 8 Antibody HC LC
Antibody SEQ ID SEQ ID I-4P NO: 9 NO: 10
EXAMPLE
Antibody I-4P Binds Human and Cynomolgus Monkey CD200R
[0061] Surface Plasmon Resonance (SPR) at 37 .degree. C. is
performed to determine the binding kinetics and affinity of
Antibody I-4P to human CD200R, cynomolgus monkey CD200R, and
cynomolgus monkey CD200RLa (herein also referred to as the
cynomolgus monkey "activating form").
[0062] Biacore.RTM. T100 instrument (GE Healthcare, Piscataway,
N.J.), Biacore reagents and Scrubber2 Biacore.RTM. Evaluation
Software (Biologics 2008) are used for the SPR analysis of Antibody
I-4P binding. A CM4 chip (Biacore P/N BR-1006-68) is prepared using
the manufacturer's EDC/NHS amine coupling method (Biacore P/N
BR-1000-50). Briefly, the surfaces of all 4 flow cells (FC) are
activated by injecting a 1:1 mixture of EDC/NHS for 7 minutes at 10
.mu.L/minute. Protein A (Calbiochem P/N 539202) is diluted to 100
.mu.g/mL in 10 mM acetate, pH 4.5 buffer and immobilized for
approximately 400 RU onto all 4 flow cells by 7 minute injection at
a flow rate of 10 .mu.L/minute. Un-reacted sites are blocked with a
7-minute injection of ethanolamine at 10 .mu.L/minute. Injections
of 2.times.10 .mu.L of glycine pH 1.5 are used to remove any
non-covalently associated protein. Running buffer is 1.times. HBS
EP+ (Biacore P/N BR-1006-69).
[0063] Human, cynomolgus monkey (cyno), and cynomolgus monkey
activating CD200 receptors are purified using IMAC and size
exclusion chromatography. Mouse CD200R is generated by Factor Xa
cleavage from a mouse CD200R Fc fusion protein made in house. The
final polishing step for the mouse CD200R receptor is size
exclusion chromatography.
[0064] For human and cyno CD200R binding, antibodies are diluted to
2.5 .mu.g/mL in running buffer, and approximately 150 RU of
Antibody I-4P is captured in flow cells 2 through 4 (RUcaptured).
FC1 is the reference flow cell; therefore, no antibody is captured
in FC1. Human and cyno CD200R are diluted to 500 nM in running
buffer and then two-fold serially diluted in running buffer to 3.9
nM. Duplicate injections of each concentration are injected over
all FC's at 50 .mu.L/minute for 250 seconds followed by a 1200
second dissociation phase. Regeneration is performed by injecting
15 .mu.L of 10 mM glycine pH 1.5 at 30 .mu.L/minute twice over all
FC's. Reference-subtracted data is collected as FC2-FC1, FC3-FC1,
and FC4-FC1. The measurements are obtained at 37.degree. C. The
affinity (K.sub.D) is calculated using a "1:1 (Langmuir) binding"
model in BIA Evaluation.
[0065] For cyno activating CD200R binding, antibodies are diluted
to 2.5 .mu.g/mL in running buffer, and approximately 150 RU of
Antibody I-4P is captured in flow cells 2 through 4 (RUcaptured).
FC1 is the reference flow cell. Cyno activating CD200R is diluted
to 8.1 .mu.M in running buffer and then 2 fold serially diluted in
running buffer to 63.2 nM. Duplicate injections of each
concentration are injected over all FC's at 50 .mu.L/minute for 250
seconds followed by a 1200 second dissociation phase. Regeneration
is performed by injecting 15 .mu.L of 10 mM glycine pH 1.5 at 30
.mu.L/min twice over all FC's. Reference subtracted data is
collected as FC2 FC1, FC3 FC1, and FC4-FC1. The measurements are
obtained at 37.degree. C. The affinity (K.sub.D) is calculated
using the steady state equilibrium analysis with the Scrubber 2
Biacore.RTM. Evaluation Software.
[0066] For mouse CD200R binding, antibodies are diluted to 2.5
.mu.g/mL in running buffer, and approximately 150 RU of Antibody
I-4P is captured in flow cells 2 through 4 (RUcaptured). FC1 is the
reference flow cell. Mouse CD200R is diluted to 10 .mu.M in running
buffer and then 2 fold serially diluted in running buffer to 78 nM.
Duplicate injections of each concentration are injected over all
FC's at 50 .mu.L/minute for 250 seconds followed by a 1200 second
dissociation phase. Regeneration is performed by injecting 15 .mu.L
of 10 mM glycine pH 1.5 at 30 .mu.L/min twice over all FC's.
Reference subtracted data are collected as FC2 FC1, FC3 FC1, and
FC4-FC1. The measurements are obtained at 37.degree. C. The
affinity (K.sub.D) was calculated using the steady state
equilibrium analysis with the Scrubber 2 Biacore.RTM. Evaluation
Software.
[0067] Following procedures essentially as described above, the
following data were obtained. As shown in Table 2, Antibody I-4P
binds human CD200R and cynomolgus monkey CD200R with an affinity in
the nM range, and Antibody I-4P binds the CD200RLa activating
receptor with an affinity in the .mu.M range. Antibody I-4P binds
mouse CD200R with an affinity of >10 .mu.M.
TABLE-US-00002 TABLE 2 Affinity of Antibody I-4P to Human, Cyno,
Cyno Activating, and mouse CD200 Receptors Measured Using Surface
Plasmon Resonance (SPR) at 37.degree. C. Receptor Average K.sub.D
Std. Dev. Antibody Human 5.6 nM 1.2 I-4P Cynomolgus monkey 2.3 nM
0.1 Cynomolgus monkey activating 2.5 .mu.M 0.4 *Mouse CD200R >10
.mu.M n = Assay was performed three times; *n = 1 time assayed
[0068] These data demonstrate that Antibody I-4P binds the CD200RLa
activating receptor and mouse CD200R with reduced affinity compared
to Antibody I-4P affinity to human CD200R and cynomolgus monkey
CD200R.
[0069] Despite substantial engineering to overcome significant
problems associated with lack of cross-reactivity between human and
cyno CD200R, isomerization under stressed conditions (driven by
primarily by an aspartic acid residue in LCDR1 (LC D28)), and a
non-native disulfide bond between HC CDR1 and CDR2, Antibody I-4P
demonstrated a favorable binding profile. For instance, a heavy
chain and light chain CDR residue saturation mutagenesis procedure
using mammalian cell expression was used to determine CDR changes
that closed the affinity gap between human and cyno CD200R. This
procedure was also used to find a residue replacement for LC D28
without compromising affinity. A second CDR library was screened
using a phage-based process, which led to the discovery of
non-predicted and non-germline replacement residues for the
non-native disulfide without compromising antigen binding
affinity.
EXAMPLE
In Vitro Binding of Antibody I-4P to CD200R Expressed in Cells
[0070] CD200R is a member of the "paired receptor family", which
means that a close homologue with opposite, activating activity
exists. This form has not been identified in humans, but low level
mRNA transcripts have been described in whole blood and testis of
cynomolgus monkeys (herein referred to the cynomolgus monkey
"activating form" or "cynomolgus monkey CD200RLa"). Therefore, the
cynomolgus monkey activating form could present a safety concern
during toxicology studies in cynomolgus monkeys.
[0071] To determine if Antibody I-4P binds to cell-expressed,
membrane -bound CD200R from cynomolgus monkey, human, and the
activating form cynomolgus CD200RLa, flow cytometry is used. CHO
cells are transfected with human CD200R (SEQ ID NO: 15), cynomolgus
monkey CD200R (SEQ ID NO: 16), or the cynomolgus monkey activating
form (SEQ ID NO: 17) and are selected for high expression. Cells
(2.sup.e5) are suspended in 1.sup.e6/50 .mu.L in PBS for each cell
line and FL4 dye (MultiCyt.RTM. Proliferation and Encoder FL4 dye)
is added. The FL4 dye is diluted 1:5000 for cells expressing human
and cyno CD200R, 1:700 for cells expressing the cynomolgus monkey
activating form, and 1:50 for untransfected cells. The dye is mixed
with the cells and the mixture is incubated at 4.degree. C. for 30
minutes in the dark. The cells are washed twice with 10 mL of PBS
and spun down at 1200 RPM for 5 minutes. The cells are then mixed
in FACS buffer at 8.sup.e5 cells/50 .mu.L/well.
[0072] The cells are incubated for 30 minutes at room temperature
with antibody titrations made in FACS buffer. The cells are washed
once with FACS buffer and 100 .mu.L of PE-conjugated anti human-Fc
antibody at a 1:1000 dilution is added to each well for 15 minutes
in the dark at 4.degree. C. Cells are washed three times and then
resuspended in 150 .mu.L of FACS buffer. Sytox blue (2 .mu.L/well)
is added, cells are transferred to a FACS plate, and run on a
Fortessa LSRII cytometry instrument (BD Biosciences). Data is
analyzed using FlowJo (FlowJo, LLC) software.
[0073] Following procedures essentially as described above, the
following data were obtained. Antibody I-4P binds to cynomolgus
monkey CD200R and human CD200R. Antibody I-4P binds to the
cynomolgus monkey activating form similar to binding to
untransfected control cells. These data demonstrate that there is
no binding of Antibody I-4P to the cynomolgus monkey activating
form; therefore, there may be a reduced safety concern during
toxicology studies in cynomolgus monkeys.
EXAMPLE
Antibody I-4P is a CD200R Agonist
[0074] To demonstrate the agonist activity of Antibody I-4P, a
human monocyte cell line U937 (ATCC, CRL1539.2), is transfected
with the cDNA for human CD200R. Cytokine production, including
IL-8, from these cells can be induced by immune complexes (IC) that
bind and activate Fc.gamma. Receptors. For IC stimulation, human
IgG1 isotype control antibody is coated to a high-binding plate
overnight. The next day, 4.times.10.sup.5 CD200R-expressing U937
cells/well are incubated with different concentrations of Antibody
I-4P for 1 hour on ice before added to the pre-coated plate for IC
stimulation and incubated at 37.degree. C. for 24 hours. After 24
hours the cells are spun down, the supernatant is removed, and the
IL-8 concentration measured using MSD kit (Mesoscale
Diagnostics).
[0075] Following procedures essentially as described above, the
following data were obtained. As shown in Table 3, the reduction of
IC-induced IL-8 with Antibody I-4P as percent inhibition compared
to isotype control at the corresponding concentration. The relative
IC50 is based on a four parameter logistic fit of the slope of
percent inhibition over concentration. The average IC50 from 3
independent experiments was determined to be 0.2 .mu.g/mL.+-.0.02
.mu.g/mL.
TABLE-US-00003 TABLE 3 Concentration-dependent inhibition of
immune-complex induced IL-8 secretion in cells expressing human
CD200R. Antibody I-4P (.mu.g/ml) average % IL-8 inhibition SEM 0.01
-2.0 3.1 0.03 2.3 2.9 0.1 14.0 6.0 0.3 24.0 4.2 1 47.1 2.8 3 55.7
2.9 10 67.8 3.3 30 76.2 4.0
[0076] These data demonstrate that Antibody I-4P is able to inhibit
IC-induced IL-8 production in a concentration-dependent manner.
[0077] The ability of CD200R agonist antibodies with different
isotype backbones to agonize CD200R and inhibit immune-complex
stimulated IL-8 release from human CD200R-expressing U937 cells is
also examined. For stimulation, human IgG1 isotype control antibody
is coated at 10 .mu.g/ml to a high-binding plate overnight. The
next day, 4.times.10.sup.5 CD200R-expressing U937 cells/well are
incubated with different concentrations of Antibody IgG4PAA (the
two leucine to alanine substitutions (SLL228PAA) are known to
disrupt hydrophobic interactions with Fc.gamma.Rs to eliminate
residual effector function) or Antibody I-4P for 1 hour on ice
before added to the pre-coated plate for IC-stimulation followed by
an incubated at 37.degree. C. for 24 hours. The cells are spun
down, the supernatant is removed, and the IL-8 concentration
measured using MSD kit (Mesoscale Diagnostics) according to
manufacturer's instructions. The IL-8 concentrations are converted
to percent inhibition relative to isotype control. The IL-8
concentration are plotted versus the antibody concentration, and a
4 parameter logistic model is used to fit percent inhibition versus
log concentration using R statistical software. According to
procedures essentially as described above, the following data
(shown in Table 4) were obtained.
TABLE-US-00004 TABLE 4 Concentration-dependent reduction in IL-8
production IgG4PAA IgG4SP Antibody avg % IL-8 avg % IL-8 .mu.g/ml
inhibition SEM* inhibition SEM* 0.01 -3.2 6.0 15.4 3.7 0.03 -5.0
5.6 35.8 3.6 0.1 -10.4 10.1 44.0 3.0 0.3 15.0 5.3 80.0 2.8 1 16.9
3.9 73.8 1.8 3 35.5 4.1 82.0 2.6 10 45.4 1.7 87.1 1.4 30 53.5 3.2
86.4 1.5 *Standard error of the mean
[0078] These data demonstrate that IgG4PAA has weaker inhibitory
activity (IC.sub.50 =1.45 .mu.g/ml) compared to Antibody I-4P
(IC.sub.50=0.07 .mu.g/ml).
EXAMPLE
Fc.gamma. Receptor Binding is Required for Agonism In Vivo
[0079] Clustering through Fc.gamma. Receptor in the lipid raft can
increase the inhibitory potency on inflammatory cells. In order to
identify whether Fc.gamma. Receptor interaction is beneficial for
agonism through CD200R, two mouse CD200R antibodies are engineered;
one to ablate any Fc.gamma. Receptor binding (mIgG2aAA) and one to
have functional Fc.gamma. Receptor binding (mIgG2a). Both molecules
are tested in two independent models of induced inflammatory
disease in mice; contact dermatitis and CD40-induced colon
inflammation model.
[0080] Contact dermatitis model: The ability of anti-human CD200R
agonist antibodies to treat contact dermatitis may be determined by
an in vivo mouse model performed essentially as described as
follows (see e.g. Tolstrup et al., Anti-inflammatory effect of a
retrovirus-derived immunosuppressive peptide in mouse models, BMC
Immunology 2013, 14:51). Male 12 week-old C57B1/6J mice are
anesthetized, their abdomens are shaved, and 100 .mu.L of 3%
oxazalone in ethanol is applied to the shaved area. Seven days
after sensitization, CD200R agonist antibody IgG2a or IgG2aAA is
administered at 0.1, 1, or 10 mg/kg subcutaneously (SC), or an
isotype control mIgG2a is administered at 10 mg/kg SC for
comparison. Four hours after antibody administration, mice are
anesthetized, baseline ear thickness is measured with calipers, and
ears are challenged with 10 .mu.L of 2% oxazalone in ethanol on
each side of both ears. Twenty-four hours post-challenge, ear
thickness is again measured. The hypersensitivity reaction is
assessed by measuring the difference between ear thickness pre- and
24 hours post-challenges. Statistical differences from isotype
control are determined using a 1-way ANOVA with Dunnett's post post
test (GraphPad Prism).
[0081] CD40-induced colon inflammation model: The ability of
anti-human CD200R agonist antibodies to treat CD40-induced colon
inflammation model may be determined by an in vivo mouse model
performed essentially as described as follows. Female 14 week-old
RAG2N12 (B6.129S6-Rag2tm1Fwa N12; Taconic) mice are injected with
100 .mu.g/mouse anti-CD40 antibody (BioXcel clone FGK4.5) to induce
colon inflammation. One hour post-induction of disease, CD200R
agonist antibody IgG2a, IgG2aAA, or isotype control antibody is
administered subcutaneously at 0.1, 1, or 10 mg/kg. Animals are
sacrificed six days later and colon inflammation is determined by
measuring the length and the weight of the colon. The colon
length-to-weight ratio is used to determine colon inflammation.
Statistical differences from isotype control are determined using a
1-way ANOVA with Dunnett's post post test (GraphPad Prism).
[0082] Following procedures essentially as described above, the
following data were obtained.
TABLE-US-00005 TABLE 5 Ear inflammation as measured in the contact
dermatitis model by change in thickness (mm) Ear thickness (mm)
mIgG2a mIgG2aAA Isotype control 0.200 .+-. 0.05 0.200 .+-. 0.05 10
mg/kg 0.118 .+-. 0.03** 0.160 .+-. 0.03* 1.0 mg/kg 0.113 .+-.
0.06** 0.170 .+-. 0.04 0.1 mg/kg 0.176 .+-. 0.05 0.178 .+-. 00.3 *p
< 0.05; **p < 0.001. n = 5/group
TABLE-US-00006 TABLE 6 Colon inflammation as measured in the
CD40-induced colon inflammation model by weight-to-length ratio
(mg/cm) Colon length to weight ratio (mg/cm) mIgG2a mIgG2aAA
Isotype control 37 .+-. 1.1 37 .+-. 1.1 10 mg/kg 26 .+-. 0.8** 33
.+-. 0.7 1.0 mg/kg 29 .+-. 1.6* 36 .+-. 1.9 0.1 mg/kg 32 .+-. 2.6
36 (n = 1) n = 5/group, *p < 0.05; **p < 0.001.
[0083] These data demonstrate that compared to isotype controls,
the antibody with full effector function (mIgG2a) exhibited an
immune suppressive function in both models. However, the Fc.gamma.
Receptor null variant (mIgG2aAA) was much less potent in the
contact dermatitis model and had little to no effect in the colon
inflammation model. The difference in activity was not due to
depletion of CD200R-expressing cells, as the Fc.gamma.
Receptor-competent IgG2a antibody was demonstrated in an
independent experiment not to deplete CD200R expressing cells in
mice (data not shown).
[0084] These data demonstrate that Fc.gamma. Receptor binding is
required to provide optimal agonism to the CD200R to mediate an
anti-inflammatory signal.
EXAMPLE
Antibody I Binding to Fc.gamma. Receptors
[0085] To determine if the antibody Fc affects the binding
characteristics of Antibody I-4P to Fc.gamma. receptors, the
binding of Antibody I-4P, Antibody I-IgG1, and Antibody I-4PAA, to
the human Fc.gamma.RI, Fc.gamma.RIIa, Fc.gamma.RIIb, and
Fc.gamma.RIIIa receptor extracellular domains (ECDs) is measured by
SPR at 25.degree. C. Antibody I-IgG1 and Antibody I-4PAA have the
same CDRs as Antibody I-4P. Antibody I-IgG1 has identical HCVR,
LCVR, and LC as Antibody I-4P, but Antibody I-IgG1 has a HC whose
amino acid sequence is given by SEQ ID NO: 11. Antibody I-4PAA
differs from Antibody I-4P by having a SLL228PAA mutation in the
HC.
[0086] Biacore.RTM. T100 instrument and Biacore.RTM. 3000 (GE
Healthcare, Piscataway, NJ), Biacore.RTM. reagents and Scrubber2
Biacore.RTM. Evaluation Software (Biologics 2008) are used for the
SPR analysis of antibody binding. A CM5 chip (Biacore.RTM. P/N
BR-1006-68) is prepared using the manufacturer's EDC/NHS amine
coupling method (Biacore.RTM. P/N BR-1000-50). Briefly, the
surfaces of all 4 FCs are activated by injecting a 1:1 mixture of
EDC/NHS for 7 minutes at 10 .mu.L/minute. Protein A (Calbiochem P/N
539202) is diluted to 100 .mu.g/mL in 10 mM acetate, pH 4.5 buffer
and immobilized for approximately 400 RU onto all 4 flow cells by 7
minute injection at a flow rate of 10 .mu.L/minute. Un-reacted
sites are blocked with a 7-minute injection of ethanolamine at 10
.mu.L/minute. Injections of 2.times.10 .mu.L of glycine pH 1.5 are
used to remove any non-covalently associated protein.
[0087] The Fc.gamma.R ECDs -Fc.gamma.RI (CD64), Fc.gamma.RIIA_131R,
and Fc.gamma.RIIA_131H (CD32a), Fc.gamma.RIIIA_158V,
Fc.gamma.RIIIA_158F (CD16a), and Fc.gamma.RIIb (CD32b; inhibitory
receptor) (see e.g. Bruhns et al., Blood. 2009 Apr 16; 113(16):
3716-25) are produced from stable CHO cell expression according to
methods well-known in the art and purified using IgG Sepharose and
size exclusion chromatography.
[0088] For Fc.gamma.RI binding, antibodies are diluted to 2.5
.mu.g/mL in running buffer (lx HBS-EP+(Biacore.RTM. P/N
BR-1006-69), and approximately 150 RU of each antibody is captured
in flow cells 2 through 4 (RUcaptured). FC1 is the reference flow
cell, therefore, no antibody is captured in FC1. Fc.gamma.RI ECD is
diluted to 200 nM in running buffer and then two-fold serially
diluted in running buffer to 0.78 nM. Duplicate injections of each
concentration are injected over all FCs at 40 .mu.L/minute for 120
seconds followed by a 1200 second dissociation phase. Regeneration
is performed by injecting 15 .mu.L of 10 mM glycine pH 1.5 at 30
.mu.L/minute over all FCs. Reference-subtracted data is collected
as FC2-FC1, FC3-FC1, and FC4-FC1. The measurements are obtained at
25.degree. C. The affinity (K.sub.D) is calculated using either
steady state equilibrium analysis with the Scrubber 2 Biacore.RTM.
Evaluation Software or a "1:1 (Langmuir) binding" model in BIA
Evaluation.
[0089] For Fc.gamma.RIIa, Fc.gamma.RIIb, and Fc.gamma.RIIIa
binding, antibodies are diluted to 5 m/mL in running buffer, and
approximately 500 RU of each variant is captured in flow cells 2
through 4 (RUcaptured). FC1 is the reference flow cell. Fc.gamma.
receptor ECDs are diluted to 10 .mu.M in running buffer and then 2
fold serially diluted in running buffer to 39 nM. Duplicate
injections of each concentration are injected over all FCs at 40
.mu.L/minute for 60 seconds followed by a 120 second dissociation
phase. Regeneration is performed by injecting 15 .mu.L of 10 mM
glycine pH 1.5 at 30 .mu.L/min over all FCs.
[0090] Reference-subtracted data is collected as FC2-FC1, FC3-FC1,
and FC4-FC1. The measurements are obtained at 25.degree. C. The
affinity (K.sub.D) is calculated using the steady state equilibrium
analysis with the Scrubber 2 Biacore.RTM. Evaluation Software.
[0091] Following procedures essentially as described above, the
following data as shown in Table 7 were obtained.
TABLE-US-00007 TABLE 7 In Vitro Binding Parameters of Antibody
I-4P, Antibody I-IgG1, and Antibody I-4PAA to Human Fc.gamma.
Receptor ECDs Measured Using SPR at 25.degree. C. Average Std
Sample Human Ligand KD Dev* IgG1 Control Antibody Fc.gamma. RI 56.1
pM 2.2 IgG4 PAA Control Antibody Fc.gamma. RI 229.0 nM 11.5
Antibody I-IgG1 Fc.gamma. RI 48.9 pM 2.2 Antibody I-4PAA Fc.gamma.
RI 273.3 nM 12.6 Antibody I-4P Fc.gamma. RI 369.3 pM 9.2 IgG1
Control Antibody Fc.gamma. RIIA_131H 0.5 .mu.M 0.0 IgG4 PAA Control
Antibody Fc.gamma. RIIA_131H >10 .mu.M Antibody I-IgG1 Fc.gamma.
RIIA_131H 0.5 .mu.M 0.0 Antibody I-4PAA Fc.gamma. RIIA_131H >10
.mu.M Antibody I-4P Fc.gamma. RIIA_131H 3.9 .mu.M 0.3 IgG1 Control
Antibody Fc.gamma. RIIA_131R 0.6 .mu.M 0.0 IgG4 PAA Control
Antibody Fc.gamma. RIIA_131R >10 .mu.M Antibody I-IgG1 Fc.gamma.
RIIA_131R 0.6 .mu.M 0.0 Antibody I-4PAA Fc.gamma. RIIA_131R >10
.mu.M Antibody I-4P Fc.gamma. RIIA_131R 1.7 .mu.M 0.1 IgG1 Control
Antibody Fc.gamma. RIIb 2.8 .mu.M 0.1 IgG4 PAA Control Antibody
Fc.gamma. RIIb >10 .mu.M Antibody I-IgG1 Fc.gamma. RIIb 2.8
.mu.M 0.1 Antibody I-4PAA Fc.gamma. RIIb >10 .mu.M Antibody I-4P
Fc.gamma. RIIb 2.2 .mu.M 0.1 IgG1 Control Antibody Fc.gamma.
RIIIA_158V 0.2 .mu.M 0.0 IgG4 PAA Control Antibody Fc.gamma.
RIIIA_158V 8.9 .mu.M 1.1 Antibody I-IgG1 Fc.gamma. RIIIA_158V 0.2
.mu.M 0.0 Antibody I-4PAA Fc.gamma. RIIIA_158V >10 .mu.M
Antibody I-4P Fc.gamma. RIIIA_158V 4.3 .mu.M 0.4 IgG1 Control
Antibody Fc.gamma. RIIIA_158F 1.0 .mu.M 0.1 IgG4 PAA Control
Antibody Fc.gamma. RIIIA_158F >10 .mu.M Antibody I-IgG1
Fc.gamma. RIIIA_158F 0.9 .mu.M 0.1 Antibody I-4PAA Fc.gamma.
RIIIA_158F >10 .mu.M Antibody I-4P Fc.gamma. RIIIA_158F >10
.mu.M Assay was performed three independent times. *Standard
deviation was not determined for measurements >10 .mu.M.
[0092] Table 7 summarizes the affinity (K.sub.D) of Antibody
I-IgG1, Antibody I-4PAA, and Antibody I-4P to the human
Fc.gamma.RI, Fc.gamma.Rlla, Fc.gamma.RIIb, and Fc.gamma.RIIIa
receptor ECDs as measured by SPR. The binding characteristics of
Antibody I-4P demonstrate binding to the Fc.gamma. receptors with
affinities that are substantially in between the binding affinities
of IgG1 control/Antibody I-IgG1 and IgG4 PAA control/Antibody
I-4PAA. For example, the data demonstrate that Antibody I-4P has
reduced binding to Fc.gamma.RIIIa receptor ECD compared to Antibody
I-IgG1 (which can be attributed to cytokine release in the whole
blood assay) but still has a higher binding affinity to Fc.gamma.RI
and Fc.gamma.RIIb receptor ECDs compared to Antibody I-4PAA.
[0093] The binding characteristics demonstrated by Antibody I-4P to
Fc.gamma.Rs are thought to contribute to enhanced in vivo efficacy
without causing significant cytokine release.
EXAMPLE
IgG1 Fc Mutants Binding to Fc.gamma. Receptors
[0094] IgG1 antibodies are known to induce cytokine release. To
determine the mechanism for IgG1-induced cytokine release, IgG1-Fc
mutations are generated. These CD200R antibodies have different
CDRs from Antibody I. The antibodies in Table 8 (IgG1, no
mutations, P331S, P331S+S267G, A330S+P331S+S267G, A330S+S267G,
K322A, K322A+S267G, and N325S+L328F+S267G) have identical CDRs with
one another. The S267G antibody has different CDRs from the other
antibody mutants and Antibody I-4P.
[0095] An S267G mutation is generated to reduce Fc.gamma. RIII
binding (EU numbering: see, e.g., Kabat et al., "Sequences of
Proteins of Immunological Interest," National Institutes of Health,
Bethesda, Md. (1991); and Shields R L et al., High resolution
mapping of the binding site on human IgG1 for Fc gamma RI, Fc gamma
RII, Fc gamma RIII, and FcRn and design of IgG1 variants with
improved binding to Fc gamma R. 2001 J. Biol. Chem. 276,
6591-6604).
[0096] The S267G mutation is also combined with mutations that
reduce C1q binding without significantly impacting
Fc.gamma.R-binding (K322A, A330S, and P331S; see e.g. Oganesyan Vet
al., 2008 Structural characterization of a human Fc fragment
engineered for lack of effector functions. Acta Crystallogr. D
Biol. Chrystallogr. 64, 700-704; Idusogie, E et al., 2000 Mapping
of the C1q Binding Site on Rituxan, a Chimeric Antibody with a
Human IgG1 Fc. J. of Immunology, 164(8) 4178-4184; and Tao M. H.
and Morrison M. L. 1993 Structural features of human immunoglobulin
G that determine isotype- specific differences in complement
activation. J. of Exp. Med., 178(2), 661-667). Additional mutations
that reduce Fc.gamma.RIII and C1q binding while modulating binding
to Fc.gamma.RIIA and Fc.gamma.RIIB are also generated (N325S+L328F;
see e.g. Shang L et al., 2014 Selective antibody intervention of
Toll-like receptor 4 activation through Fc.gamma.R tethering. J.
Biol. Chem. 289, 15309-18; Monnet E et al., 2017 Evidence of
NI-0101 pharmacological activity, an anti-TLR4 antibody, in a
randomized phase I dose escalation study in healthy volunteers
receiving LPS. Clin Pharmacol Ther. 2017 101, 200-208).
[0097] Fc.gamma. receptor binding is determined by Biacore.RTM.,
and IFN.gamma. is determined by a multiplex assay based on the
Mesoscale platform, both as described herein. C1q binding is
determined by ELISA. For the ELISA, a 96 well microplate is coated
with 100 .mu.L/well of each antibody diluted in DPBS (Dulbecco's
HyClone) with a concentration range of 10 .mu.g/mL to 0.19
.mu.g/mL. Testing is performed in duplicate wells. The plate is
sealed and incubated overnight at 4.degree. C. The coating reagent
is removed from each well, and 200 .mu.L/well of casein blocking
reagent (Thermo) is added. The plate is sealed and incubated for 2
hours at room temperature (RT). Each well is washed 3 times with
Wash Buffer (1.times. TBE with 0.05% Tween 20). 100 .mu.L/well of
Human C1q (MS Biomedical) at 10 .mu.g/mL diluted in casein blocking
reagent is added and incubated for 3 hours at RT. The plate is then
washed three times with wash buffer before 100 .mu.L/well of a
1:800 times dilution of Sheep anti-human C1q-HRP (Abcam #ab46191)
in casein blocker is added and incubated for 1 hour at RT. The
plate is washed 6 times with wash buffer, and 100 .mu.L/well of TMB
Substrate (Pierce) is added to each well and incubated for 7
minutes. 100 .mu.L of 1 N HCl is added to each well to stop the
reaction. Optical density is immediately measured using a
colorimetric microplate reader set to 450 nm.
[0098] Following procedures essentially as described above, the
following data were obtained (N=1; Table 8).
TABLE-US-00008 TABLE 8 Fc.gamma.R and C1q binding and whole blood
cytokine release measurements with IgG1 mutants. Whole Fc.gamma.
Fc.gamma. Fc.gamma. Fc.gamma. Fc.gamma. Fc.gamma. Blood RI,
RIIA_131H, RIIA_131R, RIIb, RIIIA_158V, RIIIA_158F, C1q IFN.gamma.
Mutation pM .mu.M .mu.M .mu.M .mu.M .mu.M Elisa Release.sup.a Hu
IgG1 55.2 0.71 1.03 4.2 0.28 2.59 ++ ND IgGl, no 46.4 0.69 1.04
4.23 0.27 2.12 +++ Yes mutations P331S 54.3 1.15 1.14 4.64 0.45
3.11 + Yes P331S + 142.4 5.2 0.77 4.38 2.08 >10 - No S267G A330S
+ 511.4 5.1 0.78 4.3 2.48 9.8 - No P331S + S267G A330S + 167.7 3.31
0.82 4.99 1.66 10.83 - No S267G K322A 30.5 0.98 0.82 3.41 0.28 2.58
- Yes K322A+ 70.5 5.02 0.66 4.43 1.65 9.99 - No S267G N325S + 68.7
2.64 0.06 0.275 7.35 >10 - No L328F S267G 130.7 3.13 0.53 3.41
0.73 4.5 Yes Human 384.7 5.12 2.89 3.31 5.47 >10 - No IgG4P
control antibody .sup.aAny cytokine release significantly above
baseline levels within whole blood is recorded as `Yes`, however,
the exact levels over baseline may vary.
[0099] These data demonstrate that combining mutations that reduce
C1q binding and alter Fc.gamma.R binding leads to a lack of
IFN.gamma. release over baseline, which suggests a more desirable
safety profile when administered to patients. For example, reducing
C1q binding and reducing binding to Fc.gamma.RIII (or Fc.gamma.RI)
results in a lack of IFN.gamma. release over baseline.
EXAMPLE
In Vitro Cytokine Release
[0100] Clinical toxicity, including cytokine release syndrome
(CRS), has been associated with the administration of antibodies.
CRS, one of the most severe adverse events associated with
monoclonal antibodies, is characterized by high levels of immune
cell activation and rapid systemic release of pro-inflammatory
cytokines and can potentially be fatal. Importantly, preclinical
models do not adequately predict the potential risk for CRS.
Consequently, an in vitro cytokine release assay using human blood
cells is developed to mitigate potential risks of CRS after
antibody administration. Antibody, in particular IgG1 antibody,
binding to Fc.gamma. receptors can cause unwanted cytokine
release.
[0101] To determine whether Antibody I-4P or Antibody I-IgG1 induce
cytokine release from unstimulated human whole blood, an in vitro
cytokine release study is performed. Freshly collected whole blood
from six healthy humans are incubated with 100 .mu.g/m1 of Antibody
I-4P, Antibody I-IgG1, or control IgG1 antibody for 24 hours. The
positive control is a homolog of Campath-1H (anti-CD52) IgG1
antibody known to cause cytokine release syndrome in clinic. The
negative control is an hIgG1 antibody that does not cause cytokine
release. Using a commercially available multiplex assay based on
the Mesoscale platform, ten cytokines including IFN-.gamma., IL-2,
IL-6, IL-13, IL-8, IL-12p70, IL-10, and TNF-.alpha. are measured in
cell culture supernatants.
[0102] Following procedures essentially as described above, the
following data were obtained. As shown in Table 9, incubation of
whole blood with 10 .mu.g/m1 positive control antibody resulted in
robust cytokine production for 9 of the 10 cytokines analyzed in
most donors. Incubation of whole blood with Antibody I-IgG1 induced
a significant release of IFN-.gamma.. Incubation of whole blood
with 100 .mu.g/m1 Antibody I-4P or 100 .mu.g/m1 negative control
IgG1 did not result in significant levels of any of the evaluated
cytokines.
TABLE-US-00009 TABLE 9 Fold change relative to baseline (PBS
control sample); MEDIAN .+-. SEM Antibody Antibody Negative
Positive Cytokine I-IgG1 I-4P control control IFN-y 10 .+-. 19 0.9
.+-. 0.08 0.8 .+-. 0.06 612 .+-. 431 IL-1.beta. 1.8 .+-. 3.sup.
1.19 .+-. 2 1.04 .+-. 1.4 3 .+-. 5 IL-2 0.36 .+-. 0.14 1.7 .+-.
0.133 1.9 .+-. 0.86 1.33 .+-. 1.3 IL-4 0.96 .+-. 1.4 1.08 .+-. 0.42
0.83 .+-. 0.73 10 .+-. 24 IL-6 1.25 .+-. 1.8 1.17 .+-. 0.17 1.03
.+-. 0.13 15 .+-. 18 IL-8 1.1 .+-. 0.58 1.2 .+-. 0.08 1.25 .+-.
0.24 8.8 .+-. 5.sup. IL-10 0.88 .+-. 0.11 1.25 .+-. 0.15 1.26 .+-.
0.3 3.9 .+-. 2.6 IL-12p70 0.97 .+-. 0.37 0.63 .+-. 0.19 0.49 .+-.
0.5 7 .+-. 11 IL-13 1.18 .+-. 0.27 1.18 .+-. 0.12 1.1 .+-. 0.24 5.5
.+-. 1.89 TNF-.alpha. 1.37 .+-. 0.4 1.1 .+-. 0.05 0.96 .+-. 0.07 20
.+-. 17
[0103] These data demonstrate that Antibody I-4P does not cause
significant cytokine release, and suggest a low risk of cytokine
release in the clinic following administration of Antibody
I-4P.
EXAMPLE
Antibody I Does Not Block Binding of CD200 to CD200R
[0104] Both CD200 and CD200R are cell-expressed molecules and
contain two Ig-like domains. They interact through their NH2
terminal domains compatible with immunological synapse-like
interactions occurring between myeloid cells and other
CD200-expressing cells. To determine if Antibody I-4P binds CD200R
in the presence of ligand, co-binding experiments on HEL92.7.1
cells, a human erythroblastoma cell line which expresses CD200R,
are performed by flow cytometry. For the study, 2.sup.e5 cells are
incubated (pre-treated) with 300 nM of CD200Fc (RD Systems; fusion
protein of immunoglobulin 1 Fc region with CD200), Antibody I-4P,
isotype control antibody, or PBS for one hour at room temperature.
Cells are washed 3 times and incubated with Fc block (Miltenyi
Biotec) for 20 minutes at room temperature. The cells are stained
with various concentrations of AF647-labeled Antibody I-4P for one
hour at room temperature and cells are then washed and suspended in
FACS buffer for analysis by flow cytometry.
[0105] The median fluorescence intensity (MFI) is determined for
each concentration of AF647-labeled Antibody I-4P, and the MFI
indicates the amount of binding in the presence of ligand.
Following procedures essentially as described above, the data in
Table 10 were obtained.
TABLE-US-00010 TABLE 10 Antibody binding in the presence of CD200.
No Pre- Stain treatment Pre-Treatment Antibody No Pre- Isotype
Antibody I-4P-AF647 treatment Control I-4P CD200-Fc (ug/mL) (MFI)
(MFI) (MFI) (MFI) 0 49.1 49.1 49.1 49.1 0.4 416 399 70.4 230 0.8
694 664 76.3 370 1.6 1184 1154 96.9 630 3.125 1979 1914 133 1068
6.25 3097 2987 200 1728 12.5 4216 4105 319 2641 25 5137 4916 496
3421 50 5651 5515 745 3957
[0106] These data demonstrate that Antibody I-4P does not block
CD200 ligand from binding human CD200R (human CD200-Fc data
compared to isotype control and no pre-treatment data). The
Antibody I-4P pre-treatment data serve as a control and demonstrate
reduced labeled Antibody I-4P binding following pre-treatment with
Antibody I-4P.
[0107] The epitope for Antibody I-4P was determined to be close to
the cell membrane on domain 2 of CD200R (data not shown).
EXAMPLE
Antibody I-4P Inhibits Contact Hypersensitivity in Humanized
Mice
[0108] To demonstrate the anti-inflammatory effects of Antibody
I-4P, female huNOG-EXL mice (NOD.Cg-Prkdc.sup.scid Il2rg.sup.tm1Sug
Tg(SV40/HTLV-IL3,CSF2)10-7JicaicTac) are purchased from Taconic
Biosciences at 20 weeks of age and allowed to acclimate for more
than 1 week. Mice are housed four mice per cage at 22 .degree. C.
under a 12 h light:dark cycle and allowed food and water ad
libitum. On day 0, mice are anesthetized with 5% isoflurane, their
abdomens are shaved, and 100 .mu.L of 3% oxazalone in ethanol is
applied to the shaved area. Five days after sensitization, Antibody
I-4P is administered at 1 or 10 mg/kg subcutaneously (SC); IgG4P
isotype control is administered at 10 mg/kg SC for comparison. Four
hours after antibody administration, mice are anesthetized with 5%
isoflurane, ear thickness is measured with calipers, and ears are
challenged with 10 .mu.L of 2% oxazalone in ethanol on each side of
both ears. The challenge procedure is repeated on days 10 and 14.
The hypersensitivity reaction is assessed by measuring the
difference between ear thickness pre- and 24 hours
post-challenges.
[0109] Statistics: Inflammation is determined by measuring the
differences in ear thickness from pre- to 24 hours post challenges
for each challenge. Percent inhibition is calculated from the mean
ear thickness of the isotype controls set to 0% inhibition.
Statistical differences from isotype control are determined using a
1-way or 2-way ANOVA with Dunnett's test where appropriate
(GraphPad Prism).
[0110] Following procedures essentially as described above, the
following data were obtained. As shown in Table below, a single
treatment with Antibody I-4P at 1 or 10 mg/kg SC 4 hours prior to
the first challenge significantly ameliorated the inflammatory
response after the 3rd challenge compared to isotype-treated
mice.
TABLE-US-00011 TABLE 11 Delta ear thickness % inhibition of
Treatment (mm) .+-. SEM isotype p-value Isotype control 0.108 .+-.
0.005 N/A Antibody I-4P 0.056 .+-. 0.008 47.9 .+-. 7.8 0.0001 10
mg/kg Antibody I-4P 0.064 .+-. 0.007 41.4 .+-. 6.4 0.0001 1
mg/kg
TABLE-US-00012 SEQUENCES HCDR1 of Antibody I-4P and Antibody I-IgG1
(SEQ ID NO: 1) KASGFSFS SGYYMA HCDR2 of Antibody I-4P and Antibody
I-IgG1 (SEQ ID NO: 2) LIGVGSGSLWYAQKFQG HCDR3 of Antibody I-4P and
Antibody I-IgG1 (SEQ ID NO: 3) ARHFALSDPFNL LCDR1 of Antibody I-4P
and Antibody I-IgG1 (SEQ ID NO: 4) QASESIDSYLL LCDR2 of Antibody
I-4P and Antibody I-IgG1 (SEQ ID NO: 5) KQASTLAS LCDR3 of Antibody
I-4P and Antibody I-IgG1 (SEQ ID NO: 6) QNYYDIS SND Antibody HCVR
of Antibody I-4P and Antibody I-IgG1 (SEQ ID NO: 7)
XVQLVQSGAEVKKPGASVKVSCKASGFSFSSGYYMAWVRQAPGQGLEWMGLI
GVGSGSLWYAQKFQGRVTMTRDTSTSTVYMELSSLRSEDTAVYYCARHFALSDP
FNLWGQGTLVTVSS wherein Xaa at position 1 is either glutamine or
pyroglutamic acid Antibody LCVR of Antibody I-4P and Antibody
I-IgG1 (SEQ ID NO: 8)
EIVLTQSPDFQSVTPKEKVTITCQASESIDSYLLWYQQKPDQSPKLLIKQASTLASG
VPSRFSGSGSGTDFTLTINSLEAEDAATYYCQNYYDISSNDFGGGTKVEIK Antibody Heavy
Chain of Antibody I-4P (SEQ ID NO: 9)
XVQLVQSGAEVKKPGASVKVSCKASGFSFSSGYYMAWVRQAPGQGLEWMGLI
GVGSGSLWYAQKFQGRVTMTRDTSTSTVYMELSSLRSEDTAVYYCARHFALSDP
FNLWGQGTLVTVSSASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSW
NSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTKTYTCNVDHKPSNTKVDK
RVESKYGPPCPPCPAPEFLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPE
VQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVS
NKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVE
WESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHN HYTQKSLSLSLX
wherein Xaa at position 1 is either glutamine or pyroglutamic acid;
and Xaa at position 446 is either glycine or absent. Antibody Light
Chain of Antibody I-4P and Antibody I-IgG1 (SEQ ID NO: 10)
EIVLTQSPDFQSVTPKEKVTITCQASESIDSYLLWYQQKPDQSPKLLIKQASTLASG
VPSRFSGSGSGTDFTLTINSLEAEDAATYYCQNYYDISSNDFGGGTKVEIKRTVAA
PSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQD
SKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC Antibody Heavy
Chain of Antibody I-IgG1 (SEQ ID NO: 11)
XVQLVQSGAEVKKPGASVKVSCKASGFSFSSGYYMAWVRQAPGQGLEWMGLI
GVGSGSLWYAQKFQGRVTMTRDTSTSTVYMELSSLRSEDTAVYYCARHFALSDP
FNLWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSW
NSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDK
KVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHE
DPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKC
KVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDI
AVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHE
ALHNHYTQKSLSLSPGX wherein Xaa at position 1 is either glutamine or
pyroglutamic acid; and Xaa at position 450 is either lysine or
absent. DNA Encoding Heavy Chain of Antibody I-4P (SEQ ID NO: 12)
caggtgcagctggtgcagtctggggctgaggtgaagaagcctggggcctcagtgaaggtttcctgcaaggcatc-
tggattctcc
ttcagtagcggctactacatggcatgggtgcggcaggcccctggacaagggcttgagtggatgggactgattgg-
tgttggtagt
ggtagcctatggtacgcgcagaagttccaaggccgggtcaccatgaccagggacacgtccacgagcacagtcta-
catggagct
gagcagcctgagatctgaggacacggccgtgtattactgtgcgagacattttgctctgtctgatccctttaact-
tgtggggccagg
gcacactcgtcaccgtctcctcagctagcaccaagggcccatcggtcttccccctggcaccctgctccaggagc-
acctccgaga
gcacagccgccctgggctgcctggtcaaggactacttccccgaaccggtgacggtgtcgtggaactcaggcgcc-
ctgaccag
cggcgtgcacaccttcccggctgtcctacagtcctcaggactctactccctcagcagcgtggtgaccgtgccct-
ccagcagcttg
ggcacgaagacctacacctgcaacgtagatcacaagcccagcaacaccaaggtggacaagagagttgagtccaa-
atatggtc
ccccatgcccaccctgcccagcacctgagttcctggggggaccatcagtcttcctgttccccccaaaacccaag-
gacactctcat
gatctcccggacccctgaggtcacgtgcgtggtggtggacgtgagccaggaagaccccgaggtccagttcaact-
ggtacgtgg
atggcgtggaggtgcataatgccaagacaaagccgcgggaggagcagttcaacagcacgtaccgtgtggtcagc-
gtcctcac
cgtcctgcaccaggactggctgaacggcaaggagtacaagtgcaaggtctccaacaaaggcctcccgtcctcca-
tcgagaaaa
ccatctccaaagccaaagggcagccccgagagccacaggtgtacaccctgcccccatcccaggaggagatgacc-
aagaacc
aggtcagcctgacctgcctggtcaaaggcttctaccccagcgacatcgccgtggagtgggaaagcaatgggcag-
ccggagaa
caactacaagaccacgcctcccgtgctggactccgacggctccttcttcctctacagcaggctaaccgtggaca-
agagcaggtg
gcaggaggggaatgtcttctcatgctccgtgatgcatgaggctctgcacaaccactacacacagaagagcctct-
ccctgtctctg ggt DNA Encoding Light Chain of Antibody I-4P and
Antibody I-IgG1 (SEQ ID NO: 13)
gaaattgtgctgactcagtctccagactttcagtctgtgactccaaaggagaaagtcaccatcacctgccaggc-
cagtgagtcgat
tgatagctatttactgtggtaccagcagaaaccagatcagtctccaaagctcctcatcaagcaggcatccactc-
tggcatctgggg
tcccctcgaggttcagtggcagtggatctgggacagatttcaccctcaccatcaatagcctggaagctgaagat-
gctgcaacgtat
tactgtcaaaactattatgatattagtagtaatgatttcggcggagggaccaaggtggagatcaaacggaccgt-
ggctgcaccatc
tgtcttcatcttcccgccatctgatgagcagttgaaatctggaactgcctctgttgtgtgcctgctgaataact-
tctatcccagaga
ggccaaagtacagtggaaggtggataacgccctccaatcgggtaactcccaggagagtgtcacagagcaggaca-
gcaaggaca
gcacctacagcctcagcagcaccctgacgctgagcaaagcagactacgagaaacacaaagtctacgcctgcgaa-
gtcaccca tcagggcctgagctcgcccgtcacaaagagcttcaacaggggagagtgc DNA
Encoding Heavy Chain of Antibody I-IgG1 (SEQ ID NO: 14)
caggtgcagctggtgcagtctggggctgaggtgaagaagcctggggcctcagtgaaggtttcctgcaaggcatc-
tggattctcc
ttcagtagcggctactacatggcatgggtgcggcaggcccctggacaagggcttgagtggatgggactgattgg-
tgttggtagt
ggtagcctatggtacgcgcagaagttccaaggccgggtcaccatgaccagggacacgtccacgagcacagtcta-
catggagct
gagcagcctgagatctgaggacacggccgtgtattactgtgcgagacattttgctctgtctgatccctttaact-
tgtggggccagg
gcacactcgtcaccgtctcctcagctagcaccaagggcccatcggtcttccccctggcaccctcctccaagagc-
acctctgggg
gcacagcggccctgggctgcctggtcaaggactacttccccgaaccggtgacggtgtcgtggaactcaggcgcc-
ctgaccag
cggcgtgcacaccttcccggctgtcctacagtcctcaggactctactccctcagcagcgtggtgaccgtgccct-
ccagcagcttg
ggcacccagacctacatctgcaacgtgaatcacaagcccagcaacaccaaggtggacaagaaagttgagcccaa-
atcttgtga
caaaactcacacatgcccaccgtgcccagcacctgaactcctggggggaccgtcagtcttcctcttccccccaa-
aacccaagga
caccctcatgatctcccggacccctgaggtcacatgcgtggtggtggacgtgagccacgaagaccctgaggtca-
agttcaactg
gtacgtggacggcgtggaggtgcataatgccaagacaaagccgcgggaggagcagtacaacagcacgtaccgtg-
tggtcag
cgtcctcaccgtcctgcaccaggactggctgaatggcaaggagtacaagtgcaaggtctccaacaaagccctcc-
cagccccca
tcgagaaaaccatctccaaagccaaagggcagccccgagaaccacaggtgtacaccctgcccccatcccgggat-
gagctgac
caagaaccaggtcagcctgacctgcctggtcaaaggcttctatcccagcgacatcgccgtggagtgggagagca-
atgggcag
ccggagaacaactacaagaccacgcctcccgtgctggactccgacggctccttcttcctctacagcaagctcac-
cgtggacaag
agcaggtggcagcaggggaacgtcttctcatgctccgtgatgcatgaggctctgcacaaccactacacgcagaa-
gagcctctcc ctgtctccgggtaag Human CD200R (SEQ ID NO: 15)
MLCPWRTANLGLLLILTIFLVAEAEGAAQPNNSLMLQTSKENHALASSSLCMDE
KQITQNYSKVLAEVNTSWPVKMATNAVLCCPPIALRNLIIITWEIILRGQPSCTKA
YRKETNETKETNCTDERITWVSRPDQNSDLQIRPVAITHDGYYRCIMVTPDGNFH
RGYHLQVLVTPELTLFQNRNRTAVCKAVAGKPAAQISWIPEGDCATKQEYWSN
GTVTVKSTCHWEVHNVSTVTCHVSHLTGNKSLYIELLPVPGAKKSAKLYIPYIILT
IIILTIVGFIWLLKVNGCRKYKLNKTESTPVVEEDEMQPYASYTEKNNPLYDTTNK
VKASQALQSEVDTDLHTL Cynomolgus monkey CD200R (SEQ ID NO: 16)
MLCPWRTANLGLLLILAVFLVAEAEGAAQ SNNSLMLQTSKENHTLASNSLCMDE
KQITQNHSKVLAEVNISWPVQMARNAVLCCPPIEFRNLIVITWEIILRGQPSCTKT
YRKDTNETKETNCTDERITWVSTPDQNSDLQIHPVAITHDGYYRCIMATPDGNFH
RGYHLQVLVTPEVTLFESRNRTAVCKAVAGKPAAQISWIPAGDCAPTEQEYWGN
GTVTVKSTCHWEGHNVSTVTCHVSHLTGNKSLYIELLPVPGAKKSAKLYMPYVI
LTIIILTIVGFIWLLKISGCRKYNLNKTESTSVVEEDEMQPYASYTEKNNPLYDTTN
KVKASQALQSEVGTDLHTL Cynomolgus monkey CD200RLa (SEQ ID NO: 17)
MHTLGKMSASRLLISIIIMVSASSSSCMDGKQMTQNYSKMSAEGNISQPVLMDTN
AMLCCPPIEFRNLIVIVWEIIIRGQPSCTKAYRKETNETKETNCTDERITWVSTPDQ
NSDLQIHPVAITHDGYYRCIMATPDGNFHRGYHLQVLVTPEVTLFQSRNRTAVCK
AVAGKPAAQISWIPAGDCAPTEHEYWGNGTVTVESMCHWGDHNASTMTCHVS
HLTGNKSLYIKLNSGLRTSGSPALDLLIILYVKLSLFVVILVTTGFVFFQRINYVRK SL
Sequence CWU 1
1
17114PRTArtificial SequenceSynthetic Construct 1Lys Ala Ser Gly Phe
Ser Phe Ser Ser Gly Tyr Tyr Met Ala1 5 10217PRTArtificial
SequenceSynthetic Construct 2Leu Ile Gly Val Gly Ser Gly Ser Leu
Trp Tyr Ala Gln Lys Phe Gln1 5 10 15Gly312PRTArtificial
SequenceSynthetic Construct 3Ala Arg His Phe Ala Leu Ser Asp Pro
Phe Asn Leu1 5 10411PRTArtificial SequenceSynthetic Construct 4Gln
Ala Ser Glu Ser Ile Asp Ser Tyr Leu Leu1 5 1058PRTArtificial
SequenceSynthetic Construct 5Lys Gln Ala Ser Thr Leu Ala Ser1
5610PRTArtificial SequenceSynthetic Construct 6Gln Asn Tyr Tyr Asp
Ile Ser Ser Asn Asp1 5 107120PRTArtificial SequenceSynthetic
ConstructMISC_FEATURE(1)..(1)Xaa at position 1 is either glutamine
or pyroglutamic acid 7Xaa Val Gln Leu Val Gln Ser Gly Ala Glu Val
Lys Lys Pro Gly Ala1 5 10 15Ser Val Lys Val Ser Cys Lys Ala Ser Gly
Phe Ser Phe Ser Ser Gly 20 25 30Tyr Tyr Met Ala Trp Val Arg Gln Ala
Pro Gly Gln Gly Leu Glu Trp 35 40 45Met Gly Leu Ile Gly Val Gly Ser
Gly Ser Leu Trp Tyr Ala Gln Lys 50 55 60Phe Gln Gly Arg Val Thr Met
Thr Arg Asp Thr Ser Thr Ser Thr Val65 70 75 80Tyr Met Glu Leu Ser
Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr 85 90 95Cys Ala Arg His
Phe Ala Leu Ser Asp Pro Phe Asn Leu Trp Gly Gln 100 105 110Gly Thr
Leu Val Thr Val Ser Ser 115 1208108PRTArtificial SequenceSynthetic
Construct 8Glu Ile Val Leu Thr Gln Ser Pro Asp Phe Gln Ser Val Thr
Pro Lys1 5 10 15Glu Lys Val Thr Ile Thr Cys Gln Ala Ser Glu Ser Ile
Asp Ser Tyr 20 25 30Leu Leu Trp Tyr Gln Gln Lys Pro Asp Gln Ser Pro
Lys Leu Leu Ile 35 40 45Lys Gln Ala Ser Thr Leu Ala Ser Gly Val Pro
Ser Arg Phe Ser Gly 50 55 60Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr
Ile Asn Ser Leu Glu Ala65 70 75 80Glu Asp Ala Ala Thr Tyr Tyr Cys
Gln Asn Tyr Tyr Asp Ile Ser Ser 85 90 95Asn Asp Phe Gly Gly Gly Thr
Lys Val Glu Ile Lys 100 1059446PRTArtificial SequenceSynthetic
ConstructMISC_FEATURE(1)..(1)Xaa at position 1 is either glutamine
or pyroglutamic acidMISC_FEATURE(446)..(446)Xaa at position 446 is
either glycine or absent 9Xaa Val Gln Leu Val Gln Ser Gly Ala Glu
Val Lys Lys Pro Gly Ala1 5 10 15Ser Val Lys Val Ser Cys Lys Ala Ser
Gly Phe Ser Phe Ser Ser Gly 20 25 30Tyr Tyr Met Ala Trp Val Arg Gln
Ala Pro Gly Gln Gly Leu Glu Trp 35 40 45Met Gly Leu Ile Gly Val Gly
Ser Gly Ser Leu Trp Tyr Ala Gln Lys 50 55 60Phe Gln Gly Arg Val Thr
Met Thr Arg Asp Thr Ser Thr Ser Thr Val65 70 75 80Tyr Met Glu Leu
Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr 85 90 95Cys Ala Arg
His Phe Ala Leu Ser Asp Pro Phe Asn Leu 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 Xaa 435 440 44510215PRTArtificial
SequenceArtificial Sequences 10Glu Ile Val Leu Thr Gln Ser Pro Asp
Phe Gln Ser Val Thr Pro Lys1 5 10 15Glu Lys Val Thr Ile Thr Cys Gln
Ala Ser Glu Ser Ile Asp Ser Tyr 20 25 30Leu Leu Trp Tyr Gln Gln Lys
Pro Asp Gln Ser Pro Lys Leu Leu Ile 35 40 45Lys Gln Ala Ser Thr Leu
Ala Ser Gly Val Pro Ser Arg Phe Ser Gly 50 55 60Ser Gly Ser Gly Thr
Asp Phe Thr Leu Thr Ile Asn Ser Leu Glu Ala65 70 75 80Glu Asp Ala
Ala Thr Tyr Tyr Cys Gln Asn Tyr Tyr Asp Ile Ser Ser 85 90 95Asn Asp
Phe Gly Gly Gly Thr Lys Val Glu Ile Lys Arg Thr Val Ala 100 105
110Ala Pro Ser Val Phe Ile Phe Pro Pro Ser Asp Glu Gln Leu Lys Ser
115 120 125Gly Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe Tyr Pro
Arg Glu 130 135 140Ala Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln
Ser Gly Asn Ser145 150 155 160Gln Glu Ser Val Thr Glu Gln Asp Ser
Lys Asp Ser Thr Tyr Ser Leu 165 170 175Ser Ser Thr Leu Thr Leu Ser
Lys Ala Asp Tyr Glu Lys His Lys Val 180 185 190Tyr Ala Cys Glu Val
Thr His Gln Gly Leu Ser Ser Pro Val Thr Lys 195 200 205Ser Phe Asn
Arg Gly Glu Cys 210 21511450PRTArtificial SequenceSynthetic
ConstructMISC_FEATURE(1)..(1)Xaa at position 1 is either glutamine
or pyroglutamic acidMISC_FEATURE(450)..(450)Xaa at position 450 is
either lysine or absent 11Xaa Val Gln Leu Val Gln Ser Gly Ala Glu
Val Lys Lys Pro Gly Ala1 5 10 15Ser Val Lys Val Ser Cys Lys Ala Ser
Gly Phe Ser Phe Ser Ser Gly 20 25 30Tyr Tyr Met Ala Trp Val Arg Gln
Ala Pro Gly Gln Gly Leu Glu Trp 35 40 45Met Gly Leu Ile Gly Val Gly
Ser Gly Ser Leu Trp Tyr Ala Gln Lys 50 55 60Phe Gln Gly Arg Val Thr
Met Thr Arg Asp Thr Ser Thr Ser Thr Val65 70 75 80Tyr Met Glu Leu
Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr 85 90 95Cys Ala Arg
His Phe Ala Leu Ser Asp Pro Phe Asn Leu 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 Ser Ser Lys Ser Thr Ser Gly Gly 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 Gln
Thr Tyr Ile Cys Asn Val Asn His Lys 195 200 205Pro Ser Asn Thr Lys
Val Asp Lys Lys Val Glu Pro Lys Ser Cys Asp 210 215 220Lys Thr His
Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly Gly225 230 235
240Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile
245 250 255Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser
His Glu 260 265 270Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly
Val Glu Val His 275 280 285Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln
Tyr Asn Ser Thr Tyr Arg 290 295 300Val Val Ser Val Leu Thr Val Leu
His Gln Asp Trp Leu Asn Gly Lys305 310 315 320Glu Tyr Lys Cys Lys
Val Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu 325 330 335Lys Thr Ile
Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr 340 345 350Thr
Leu Pro Pro Ser Arg Asp Glu Leu Thr Lys Asn Gln Val Ser Leu 355 360
365Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp
370 375 380Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro
Pro Val385 390 395 400Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser
Lys Leu Thr Val Asp 405 410 415Lys Ser Arg Trp Gln Gln Gly Asn Val
Phe Ser Cys Ser Val Met His 420 425 430Glu Ala Leu His Asn His Tyr
Thr Gln Lys Ser Leu Ser Leu Ser Pro 435 440 445Gly Xaa
450121338DNAArtificial SequenceSynthetic Construct 12caggtgcagc
tggtgcagtc tggggctgag gtgaagaagc ctggggcctc agtgaaggtt 60tcctgcaagg
catctggatt ctccttcagt agcggctact acatggcatg ggtgcggcag
120gcccctggac aagggcttga gtggatggga ctgattggtg ttggtagtgg
tagcctatgg 180tacgcgcaga agttccaagg ccgggtcacc atgaccaggg
acacgtccac gagcacagtc 240tacatggagc tgagcagcct gagatctgag
gacacggccg tgtattactg tgcgagacat 300tttgctctgt ctgatccctt
taacttgtgg ggccagggca cactcgtcac cgtctcctca 360gctagcacca
agggcccatc ggtcttcccc ctggcaccct gctccaggag cacctccgag
420agcacagccg ccctgggctg cctggtcaag gactacttcc ccgaaccggt
gacggtgtcg 480tggaactcag gcgccctgac cagcggcgtg cacaccttcc
cggctgtcct acagtcctca 540ggactctact ccctcagcag cgtggtgacc
gtgccctcca gcagcttggg cacgaagacc 600tacacctgca acgtagatca
caagcccagc aacaccaagg tggacaagag agttgagtcc 660aaatatggtc
ccccatgccc accctgccca gcacctgagt tcctgggggg accatcagtc
720ttcctgttcc ccccaaaacc caaggacact ctcatgatct cccggacccc
tgaggtcacg 780tgcgtggtgg tggacgtgag ccaggaagac cccgaggtcc
agttcaactg gtacgtggat 840ggcgtggagg tgcataatgc caagacaaag
ccgcgggagg agcagttcaa cagcacgtac 900cgtgtggtca gcgtcctcac
cgtcctgcac caggactggc tgaacggcaa ggagtacaag 960tgcaaggtct
ccaacaaagg cctcccgtcc tccatcgaga aaaccatctc caaagccaaa
1020gggcagcccc gagagccaca ggtgtacacc ctgcccccat cccaggagga
gatgaccaag 1080aaccaggtca gcctgacctg cctggtcaaa ggcttctacc
ccagcgacat cgccgtggag 1140tgggaaagca atgggcagcc ggagaacaac
tacaagacca cgcctcccgt gctggactcc 1200gacggctcct tcttcctcta
cagcaggcta accgtggaca agagcaggtg gcaggagggg 1260aatgtcttct
catgctccgt gatgcatgag gctctgcaca accactacac acagaagagc
1320ctctccctgt ctctgggt 133813645DNAArtificial SequenceSynthetic
Construct 13gaaattgtgc tgactcagtc tccagacttt cagtctgtga ctccaaagga
gaaagtcacc 60atcacctgcc aggccagtga gtcgattgat agctatttac tgtggtacca
gcagaaacca 120gatcagtctc caaagctcct catcaagcag gcatccactc
tggcatctgg ggtcccctcg 180aggttcagtg gcagtggatc tgggacagat
ttcaccctca ccatcaatag cctggaagct 240gaagatgctg caacgtatta
ctgtcaaaac tattatgata ttagtagtaa tgatttcggc 300ggagggacca
aggtggagat caaacggacc gtggctgcac catctgtctt catcttcccg
360ccatctgatg agcagttgaa atctggaact gcctctgttg tgtgcctgct
gaataacttc 420tatcccagag aggccaaagt acagtggaag gtggataacg
ccctccaatc gggtaactcc 480caggagagtg tcacagagca ggacagcaag
gacagcacct acagcctcag cagcaccctg 540acgctgagca aagcagacta
cgagaaacac aaagtctacg cctgcgaagt cacccatcag 600ggcctgagct
cgcccgtcac aaagagcttc aacaggggag agtgc 645141350DNAArtificial
SequenceSynthetic Construct 14caggtgcagc tggtgcagtc tggggctgag
gtgaagaagc ctggggcctc agtgaaggtt 60tcctgcaagg catctggatt ctccttcagt
agcggctact acatggcatg ggtgcggcag 120gcccctggac aagggcttga
gtggatggga ctgattggtg ttggtagtgg tagcctatgg 180tacgcgcaga
agttccaagg ccgggtcacc atgaccaggg acacgtccac gagcacagtc
240tacatggagc tgagcagcct gagatctgag gacacggccg tgtattactg
tgcgagacat 300tttgctctgt ctgatccctt taacttgtgg ggccagggca
cactcgtcac cgtctcctca 360gctagcacca agggcccatc ggtcttcccc
ctggcaccct cctccaagag cacctctggg 420ggcacagcgg ccctgggctg
cctggtcaag gactacttcc ccgaaccggt gacggtgtcg 480tggaactcag
gcgccctgac cagcggcgtg cacaccttcc cggctgtcct acagtcctca
540ggactctact ccctcagcag cgtggtgacc gtgccctcca gcagcttggg
cacccagacc 600tacatctgca acgtgaatca caagcccagc aacaccaagg
tggacaagaa agttgagccc 660aaatcttgtg acaaaactca cacatgccca
ccgtgcccag cacctgaact cctgggggga 720ccgtcagtct tcctcttccc
cccaaaaccc aaggacaccc tcatgatctc ccggacccct 780gaggtcacat
gcgtggtggt ggacgtgagc cacgaagacc ctgaggtcaa gttcaactgg
840tacgtggacg gcgtggaggt gcataatgcc aagacaaagc cgcgggagga
gcagtacaac 900agcacgtacc gtgtggtcag cgtcctcacc gtcctgcacc
aggactggct gaatggcaag 960gagtacaagt gcaaggtctc caacaaagcc
ctcccagccc ccatcgagaa aaccatctcc 1020aaagccaaag ggcagccccg
agaaccacag gtgtacaccc tgcccccatc ccgggatgag 1080ctgaccaaga
accaggtcag cctgacctgc ctggtcaaag gcttctatcc cagcgacatc
1140gccgtggagt gggagagcaa tgggcagccg gagaacaact acaagaccac
gcctcccgtg 1200ctggactccg acggctcctt cttcctctac agcaagctca
ccgtggacaa gagcaggtgg 1260cagcagggga acgtcttctc atgctccgtg
atgcatgagg ctctgcacaa ccactacacg 1320cagaagagcc tctccctgtc
tccgggtaag 135015348PRTHomo sapiens 15Met Leu Cys Pro Trp Arg Thr
Ala Asn Leu Gly Leu Leu Leu Ile Leu1 5 10 15Thr Ile Phe Leu Val Ala
Glu Ala Glu Gly Ala Ala Gln Pro Asn Asn 20 25 30Ser Leu Met Leu Gln
Thr Ser Lys Glu Asn His Ala Leu Ala Ser Ser 35 40 45Ser Leu Cys Met
Asp Glu Lys Gln Ile Thr Gln Asn Tyr Ser Lys Val 50 55 60Leu Ala Glu
Val Asn Thr Ser Trp Pro Val Lys Met Ala Thr Asn Ala65 70 75 80Val
Leu Cys Cys Pro Pro Ile Ala Leu Arg Asn Leu Ile Ile Ile Thr 85 90
95Trp Glu Ile Ile Leu Arg Gly Gln Pro Ser Cys Thr Lys Ala Tyr Arg
100 105 110Lys Glu Thr Asn Glu Thr Lys Glu Thr Asn Cys Thr Asp Glu
Arg Ile 115 120 125Thr Trp Val Ser Arg Pro Asp Gln Asn Ser Asp Leu
Gln Ile Arg Pro 130 135 140Val Ala Ile Thr His Asp Gly Tyr Tyr Arg
Cys Ile Met Val Thr Pro145 150 155 160Asp Gly Asn Phe His Arg Gly
Tyr His Leu Gln Val Leu Val Thr Pro 165 170 175Glu Leu Thr Leu Phe
Gln Asn Arg Asn Arg Thr Ala Val Cys Lys Ala 180 185 190Val Ala Gly
Lys Pro Ala Ala Gln Ile Ser Trp Ile Pro Glu Gly Asp 195 200 205Cys
Ala Thr Lys Gln Glu Tyr Trp Ser Asn Gly Thr Val Thr Val Lys 210 215
220Ser Thr Cys His Trp Glu Val His Asn Val Ser Thr Val Thr Cys
His225 230 235 240Val Ser His Leu Thr Gly Asn Lys Ser Leu Tyr Ile
Glu Leu Leu Pro 245 250 255Val Pro Gly Ala Lys Lys Ser Ala Lys Leu
Tyr Ile Pro Tyr Ile Ile 260 265 270Leu Thr Ile Ile Ile Leu Thr Ile
Val Gly Phe Ile Trp Leu Leu Lys 275 280 285Val Asn Gly Cys Arg Lys
Tyr Lys Leu Asn Lys Thr Glu Ser Thr Pro 290 295 300Val Val Glu Glu
Asp Glu Met Gln Pro Tyr Ala Ser Tyr Thr Glu Lys305 310 315 320Asn
Asn Pro Leu Tyr Asp Thr Thr Asn Lys Val Lys Ala Ser Gln Ala 325 330
335Leu Gln Ser Glu Val Asp Thr Asp Leu His Thr Leu
340 34516349PRTMacaca fascicularis 16Met Leu Cys Pro Trp Arg Thr
Ala Asn Leu Gly Leu Leu Leu Ile Leu1 5 10 15Ala Val Phe Leu Val Ala
Glu Ala Glu Gly Ala Ala Gln Ser Asn Asn 20 25 30Ser Leu Met Leu Gln
Thr Ser Lys Glu Asn His Thr Leu Ala Ser Asn 35 40 45Ser Leu Cys Met
Asp Glu Lys Gln Ile Thr Gln Asn His Ser Lys Val 50 55 60Leu Ala Glu
Val Asn Ile Ser Trp Pro Val Gln Met Ala Arg Asn Ala65 70 75 80Val
Leu Cys Cys Pro Pro Ile Glu Phe Arg Asn Leu Ile Val Ile Thr 85 90
95Trp Glu Ile Ile Leu Arg Gly Gln Pro Ser Cys Thr Lys Thr Tyr Arg
100 105 110Lys Asp Thr Asn Glu Thr Lys Glu Thr Asn Cys Thr Asp Glu
Arg Ile 115 120 125Thr Trp Val Ser Thr Pro Asp Gln Asn Ser Asp Leu
Gln Ile His Pro 130 135 140Val Ala Ile Thr His Asp Gly Tyr Tyr Arg
Cys Ile Met Ala Thr Pro145 150 155 160Asp Gly Asn Phe His Arg Gly
Tyr His Leu Gln Val Leu Val Thr Pro 165 170 175Glu Val Thr Leu Phe
Glu Ser Arg Asn Arg Thr Ala Val Cys Lys Ala 180 185 190Val Ala Gly
Lys Pro Ala Ala Gln Ile Ser Trp Ile Pro Ala Gly Asp 195 200 205Cys
Ala Pro Thr Glu Gln Glu Tyr Trp Gly Asn Gly Thr Val Thr Val 210 215
220Lys Ser Thr Cys His Trp Glu Gly His Asn Val Ser Thr Val Thr
Cys225 230 235 240His Val Ser His Leu Thr Gly Asn Lys Ser Leu Tyr
Ile Glu Leu Leu 245 250 255Pro Val Pro Gly Ala Lys Lys Ser Ala Lys
Leu Tyr Met Pro Tyr Val 260 265 270Ile Leu Thr Ile Ile Ile Leu Thr
Ile Val Gly Phe Ile Trp Leu Leu 275 280 285Lys Ile Ser Gly Cys Arg
Lys Tyr Asn Leu Asn Lys Thr Glu Ser Thr 290 295 300Ser Val Val Glu
Glu Asp Glu Met Gln Pro Tyr Ala Ser Tyr Thr Glu305 310 315 320Lys
Asn Asn Pro Leu Tyr Asp Thr Thr Asn Lys Val Lys Ala Ser Gln 325 330
335Ala Leu Gln Ser Glu Val Gly Thr Asp Leu His Thr Leu 340
34517278PRTMacaca fascicularis 17Met His Thr Leu Gly Lys Met Ser
Ala Ser Arg Leu Leu Ile Ser Ile1 5 10 15Ile Ile Met Val Ser Ala Ser
Ser Ser Ser Cys Met Asp Gly Lys Gln 20 25 30Met Thr Gln Asn Tyr Ser
Lys Met Ser Ala Glu Gly Asn Ile Ser Gln 35 40 45Pro Val Leu Met Asp
Thr Asn Ala Met Leu Cys Cys Pro Pro Ile Glu 50 55 60Phe Arg Asn Leu
Ile Val Ile Val Trp Glu Ile Ile Ile Arg Gly Gln65 70 75 80Pro Ser
Cys Thr Lys Ala Tyr Arg Lys Glu Thr Asn Glu Thr Lys Glu 85 90 95Thr
Asn Cys Thr Asp Glu Arg Ile Thr Trp Val Ser Thr Pro Asp Gln 100 105
110Asn Ser Asp Leu Gln Ile His Pro Val Ala Ile Thr His Asp Gly Tyr
115 120 125Tyr Arg Cys Ile Met Ala Thr Pro Asp Gly Asn Phe His Arg
Gly Tyr 130 135 140His Leu Gln Val Leu Val Thr Pro Glu Val Thr Leu
Phe Gln Ser Arg145 150 155 160Asn Arg Thr Ala Val Cys Lys Ala Val
Ala Gly Lys Pro Ala Ala Gln 165 170 175Ile Ser Trp Ile Pro Ala Gly
Asp Cys Ala Pro Thr Glu His Glu Tyr 180 185 190Trp Gly Asn Gly Thr
Val Thr Val Glu Ser Met Cys His Trp Gly Asp 195 200 205His Asn Ala
Ser Thr Met Thr Cys His Val Ser His Leu Thr Gly Asn 210 215 220Lys
Ser Leu Tyr Ile Lys Leu Asn Ser Gly Leu Arg Thr Ser Gly Ser225 230
235 240Pro Ala Leu Asp Leu Leu Ile Ile Leu Tyr Val Lys Leu Ser Leu
Phe 245 250 255Val Val Ile Leu Val Thr Thr Gly Phe Val Phe Phe Gln
Arg Ile Asn 260 265 270Tyr Val Arg Lys Ser Leu 275
* * * * *