U.S. patent application number 14/827693 was filed with the patent office on 2016-02-04 for biomarkers of immunomodulatory effects in humans treated with anti-cd200 antibodies.
The applicant listed for this patent is ALEXION PHARMACEUTICALS, INC.. Invention is credited to Roxanne COFIELL, Susan Faas MCKNIGHT, Yan YAN.
Application Number | 20160033514 14/827693 |
Document ID | / |
Family ID | 44305832 |
Filed Date | 2016-02-04 |
United States Patent
Application |
20160033514 |
Kind Code |
A1 |
MCKNIGHT; Susan Faas ; et
al. |
February 4, 2016 |
BIOMARKERS OF IMMUNOMODULATORY EFFECTS IN HUMANS TREATED WITH
ANTI-CD200 ANTIBODIES
Abstract
The present disclosure relates to anti-CD200 antibodies (e.g.,
variant anti-CD200 antibodies having decreased or no effector
function) and to biomarkers for use in a variety of diagnostic and
therapeutic methods, e.g., determining whether a human has been
administered one or more of the antibodies at a dose sufficient to
induce a desired immunomodulatory effect in the human and/or
selecting an appropriate dosing schedule for a patient.
Inventors: |
MCKNIGHT; Susan Faas; (Old
Lyme, CT) ; COFIELL; Roxanne; (Glastonbury, CT)
; YAN; Yan; (Cheshire, CT) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
ALEXION PHARMACEUTICALS, INC. |
Cheshire |
CT |
US |
|
|
Family ID: |
44305832 |
Appl. No.: |
14/827693 |
Filed: |
August 17, 2015 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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13521671 |
Apr 17, 2013 |
9180186 |
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PCT/US2011/020750 |
Jan 11, 2011 |
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14827693 |
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61416974 |
Nov 24, 2010 |
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61337997 |
Feb 11, 2010 |
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61401442 |
Aug 12, 2010 |
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61294066 |
Jan 11, 2010 |
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Current U.S.
Class: |
506/9 ; 435/29;
435/6.11; 435/6.12; 435/7.24 |
Current CPC
Class: |
A61P 11/06 20180101;
A61P 43/00 20180101; C07K 16/2803 20130101; A61P 19/08 20180101;
G01N 2333/70517 20130101; A61K 39/39558 20130101; A61P 17/00
20180101; A61P 25/00 20180101; A61P 1/04 20180101; A61P 11/00
20180101; A61P 21/00 20180101; G01N 2800/52 20130101; A61P 17/04
20180101; A61K 2039/505 20130101; G01N 33/57492 20130101; A61P
19/02 20180101; G01N 33/5094 20130101; G01N 33/57484 20130101; A61P
5/50 20180101; G01N 2333/70596 20130101; A61K 2039/545 20130101;
A61P 3/10 20180101; A61P 11/02 20180101; A61P 13/12 20180101; A61K
2039/57 20130101; A61P 9/00 20180101; G01N 33/68 20130101; G01N
33/5091 20130101; A61P 35/00 20180101; A61P 3/04 20180101; A61P
35/02 20180101; A61P 37/06 20180101; A61P 11/16 20180101; A61P
19/10 20180101; A61P 37/02 20180101; A61P 37/08 20180101; A61P 7/10
20180101; A61P 21/04 20180101; A61P 29/00 20180101 |
International
Class: |
G01N 33/574 20060101
G01N033/574; G01N 33/50 20060101 G01N033/50 |
Claims
1-266. (canceled)
267. A method for determining whether an anti-CD200 antibody or an
antigen-binding fragment thereof has produced a desired anti-CD200
antibody-associated immunomodulatory effect in a human, the method
comprising detecting a change in at least one anti-CD200
antibody-associated immunomodulatory biomarker in a biological
sample obtained from a human after administration of an anti-CD200
antibody or antigen-binding fragment thereof to the human, wherein
the change in at least one anti-CD200 antibody-associated
immunomodulatory biomarker is selected from the group consisting
of: (i) a reduction in the concentration of regulatory T cells in
the biological sample, relative to the concentration of regulatory
T cells of the same histological type in a biological sample
obtained from the human prior to administration of the antibody or
antigen-binding fragment thereof; (ii) an increase in the
concentration of CD8.sup.+ T cells in the biological sample,
relative to the concentration of CD8.sup.+ T cells of the same
histological type in a biological sample obtained from the human
prior to administration of the antibody or antigen-binding fragment
thereof; (iii) an increase in the concentration of CD4.sup.+ T
cells in the biological sample, relative to the concentration of
CD4.sup.+ T cells of the same histological type in a biological
sample obtained from the human prior to administration of the
antibody or antigen-binding fragment thereof; (iv) an increase in
the concentration of activated T cells in the biological sample,
relative to the concentration of activated T cells of the same
histological type in a biological sample obtained from the human
prior to administration of the antibody or antigen-binding fragment
thereof; (v) a reduction in the concentration of CD200.sup.+
leukocytes in the biological sample, relative to the concentration
of CD200.sup.+ leukocytes of the same histological type in a
biological sample obtained from the human prior to administration
of the antibody or antigen-binding fragment thereof; (vi) an
increase in the concentration of CD200R.sup.+ leukocytes in the
biological sample, relative to the concentration of CD200R.sup.+
leukocytes of the same histological type in a biological sample
obtained from the human prior to administration of the antibody or
antigen-binding fragment thereof; (vii) a ratio of percent
activated T cells to percent regulatory T cells of at least 2:1 in
the biological sample obtained from the patient after
administration to the patient of the anti-CD200 antibody or
antigen-binding fragment thereof; (viii) a ratio of percent
activated T cells to percent regulatory T cells of at least 3:1 in
the biological sample obtained from the patient after
administration to the patient of the anti-CD200 antibody or
antigen-binding fragment thereof; (ix) a ratio of percent activated
T cells to percent regulatory T cells of at least 4:1 in the
biological sample obtained from the patient after administration to
the patient of the anti-CD200 antibody or antigen-binding fragment
thereof; (x) a ratio of percent activated T cells to percent
regulatory T cells of at least 5:1 in the biological sample
obtained from the patient after administration to the patient of
the anti-CD200 antibody or antigen-binding fragment thereof; (xi) a
ratio of percent activated T cells to percent regulatory T cells of
at least 6:1 in the biological sample obtained from the patient
after administration to the patient of the anti-CD200 antibody or
antigen-binding fragment thereof; (xii) an increase in the ratio of
percent activated T cells to percent regulatory T cells in the
biological sample, relative to the corresponding ratio of percent
activated T cells to percent regulatory T cells of the same
histological type in a biological sample obtained from the human
prior to administration of the antibody or antigen-binding fragment
thereof; (xiii) a decreased level of CD200 expression by a
plurality of leukocytes in the biological sample, relative to the
level of CD200 expression by a plurality of leukocytes of the same
histological type in a biological sample from the human prior to
administration of the antibody or antigen-binding fragment thereof;
(xiv) an increased level of CD200R expression by a plurality of
leukocytes in the biological sample, relative to the level of
CD200R expression by a plurality of leukocytes in a biological
sample obtained from the human prior to administration of the
anti-CD200 antibody or antigen-binding fragment thereof; (xv) a
decrease in the concentration of one or more CD200.sup.+ bone
marrow subsets in the biological sample, relative to the
concentration of the corresponding one or more CD200.sup.+ bone
marrow subsets in a biological sample obtained from the human prior
to administration of the anti-CD200 antibody or antigen-binding
fragment thereof; (xvi) a decrease in the level of CD200 expression
by a plurality of lymphocytes in the biological sample, relative to
the level of CD200 expression by a plurality of lymphocytes of the
same histological type in a biological sample obtained from the
human prior to administration of the anti-CD200 antibody or
antigen-binding fragment thereof, wherein the lymphocytes are bone
marrow cells or splenic cells; and (xvii) a decrease in tumor
burden as measured by imaging.
268. The method of claim 267, wherein the detecting occurs within
or less than eight weeks, seven weeks, six weeks, five weeks, four
weeks, three weeks, or two weeks after administration of the
antibody or antigen-binding fragment thereof.
269. The method of claim 267, wherein the human is afflicted with a
cancer.
270. The method of claim 267, wherein the human has, is suspected
of having, or is at risk for developing, an inflammatory disorder
or a bone disorder.
271. The method of claim 269, wherein the cancer is chronic
lymphocytic leukemia (CLL).
272. The method of claim 269, wherein the cancer is a solid
tumor.
273. The method of claim 272, wherein the solid tumor is a colon
cancer, a breast cancer, a lung cancer, a renal cancer, a
pancreatic cancer, a thyroid cancer, a skin cancer, a cancer of the
nervous system, a cervical cancer, an ovarian cancer, a testicular
cancer, a head and neck cancer, a cancer of the eye, a stomach
cancer, or a liver cancer.
274. The method according to claim 273, wherein the cancer of the
nervous system is a neuroblastoma.
275. The method according to claim 267, wherein the per-dose amount
of the anti-CD200 antibody or antigen-binding fragment thereof
administered to the patient is at least: (i) 100 mg/m.sup.2 of the
patient; (ii) 200 mg/m.sup.2 of the patient; (iii) 300 mg/m.sup.2
of the patient; (iv) 400 mg/m.sup.2 of the patient, or (v) 500
mg/m.sup.2 of the patient.
276. The method of claim 267, wherein the anti-CD200 antibody or an
antigen-binding fragment thereof is administered to the patient at
least once per week, at least once every two weeks, at least once
every three weeks, or at least once every four weeks.
277. The method of claim 272, wherein the solid tumor is reduced in
mass.
278. The method of claim 277, wherein the solid tumor is reduced in
mass by at least about 57.6%.
279. The method of claim 267, wherein the anti-CD200 antibody is a
murine antibody, a chimeric antibody, a humanized antibody, or a
human antibody.
280. The method of claim 267, wherein the antigen-binding fragment
is selected from the group consisting of an Fab, an F(ab').sub.2,
an Fv, and a single-chain antibody.
281. The method of claim 267, wherein the anti-CD200 antibody or
antigen-binding fragment thereof comprises a variant constant
region that has decreased or no effector function, relative to a
non-variant form of the constant region.
282. The method of claim 267, wherein the anti-CD200 antibody or
antigen-binding fragment thereof inhibits the interaction between
CD200 and CD200R.
283. A method for selecting a cancer patient for treatment with an
anti-CD200 antibody or an antigen-binding fragment thereof, the
method comprising: determining whether the immune system of a
patient with cancer is competent to mount an immune response
against the cancer; and if the patient's immune system is
determined to be competent, selecting the patient for an anti-CD200
antibody or antigen-binding fragment thereof therapy.
284. The method of claim 283, wherein the patient's immune system
is determined to be competent to mount an immune response against
the cancer in the presence of the anti-CD200 antibody or an
antigen-binding fragment thereof.
285. The method of claim 283, wherein the patient's immune system
is determined to be competent to mount an immune response against
the cancer in the absence of the anti-CD200 antibody or
antigen-binding fragment thereof.
286. The method of claim 283, wherein the determining comprises
measuring: (i) the absolute number of CD8.sup.+ T cells per
microliter of blood obtained from the patient prior to
administering the anti-CD200 antibody or antigen-binding fragment
thereof; or (ii) the absolute number per microliter of blood of at
least one immune cell population in the patient prior to
administering the anti-CD200 antibody or antigen-binding fragment
thereof, wherein the at least one immune cell population is
selected from the group consisting of CD4.sup.+ helper T cells,
non-cancer CD45.sup.+ lymphocytes, CD19.sup.+ B cells,
CD16.sup.+CD56.sup.+natural Killer (NK) cells, and CD3.sup.+ cells.
Description
CROSS-REFERENCE TO RELATED APPLICATIONS
[0001] This application claims priority to and the benefit of U.S.
Provisional Patent Application Ser. No. 61/416,974, filed Nov. 24,
2010; 61/401,442, filed Aug. 12, 2010; 61/337,997, filed Feb. 11,
2010; 61/294,066, filed Jan. 11, 2010, all entiled "Biomarkers of
Immunomodulatory Effects in Humans Treated with Anti-CD200
Antibodies," the entire contents of which are incorporated herein
by reference.
TECHNICAL FIELD
[0002] The field of the invention is medicine, immunology,
molecular biology, and protein chemistry.
BACKGROUND
[0003] Human CD200 protein is a type 1a transmembrane glycoprotein
that is normally expressed on thymocytes (e.g., T cells and B
cells), neurons, and endothelial cells. Through engagement with its
cognate receptor, CD200R, CD200 protein transduces an
immunoregulatory signal that can suppress T-cell-mediated immune
responses. CD200 knockout animal studies as well as experiments
using antagonist anti-CD200 antibodies and recombinant CD200-Fc
fusion proteins have demonstrated that CD200 protein functions as
an immunosuppressive agent in autoimmune disorder and during
transplantation. (See, e.g., Hoek et al. (2000) Science
290:1768-1771 and Gorczynski et al. (1999) J Immunol
163:1654-1660.) The interaction between CD200 and CD200R results in
altered cytokine profiles and promotes a T.sub.H2 T cell response
(humoral immune response) over a T.sub.H1 response (cellular immune
response). See, e.g., Kretz-Rommel (2007) J Immunol
178:5595-5605.
[0004] The human immune system employs a variety of
immunosurveillance mechanisms, which can identify malignant cells
within a host organism and kill the cells before a cancer develops.
See, e.g., Geertsen et al. (1999) Int J Mol Med 3(1):49-57;
Kerebijn et al. (1999) Crit Rev Oncol Hematol 31(1):31-53; and
Pardoll (2003) Annu Rev Immunol 21:807-39. However, cancer cells
are known to evade detection by the immune system. One potential
mechanism by which cancer cells escape immunosurveillance is
expression or overexpression of CD200 protein. In fact, CD200
protein has been shown to be expressed or overexpressed on a
variety of human cancer cells including, e.g., B cell chronic
lymphocytic leukemia cells, prostate cancer cells, breast cancer
cells, colon cancer cells, and brain cancer cells. See, e.g.,
Kawasaki et al. (2007) Biochem Biophys Res Commun 364(4):778-782;
Kretz-Rommel et al. (2007), supra; and Siva et al. (2008) Cancer
Immunol Immunother 57(7):987-96.
[0005] Molecular biomarkers are often used in early drug
development studies to determine, for example, whether a drug is
biologically active in a patient--that the drug produced a
measurable biological effect in the patient to which the drug is
administered. For example, biomarkers can be useful during phase I
studies to establish dosing schedules for future phase II studies
and in general to help determine clinically-meaningful and
optimized dosing schedules for treating patients suffering from
disease. Biomarkers can also be useful for identifying the
occurrence of potential side-effects or other non-therapeutic
effects in a human treated with a drug to thereby determine a
safety profile for the drug.
SUMMARY
[0006] The present disclosure is based, at least in part, on the
discovery by the inventors of several biomarkers, a change (e.g.,
an increase or decrease) in one or more of which evidences the
occurrence in a human of a desired immunomodulatory effect as a
result of administration of an anti-CD200 antibody to the human.
For example, the inventors have observed that following
administration of an anti-CD200 antibody to a human, the
concentration of circulating CD200.sup.+ leukocytes (e.g., subsets
of CD200.sup.+ T cells including, e.g., CD200.sup.+/CD4.sup.+ T
cells and/or activated CD200.sup.+/CD4' T cells) is reduced in the
human. While the disclosure is not bound by any particular theory
or mechanism of action, the inventors believe that the observed
loss of CD200.sup.+ leukocytes is due to one or both of: (a) loss
of CD200 expression by the leukocytes and (b) mobilization of the
cells out of the periphery, rather than a deletion of the
CD200.sup.+ leukocytes. Also observed by the inventors was that
upon administration of an anti-CD200 antibody, the expression level
of CD200R by a variety of leukocyte subsets (e.g., CD4.sup.+ T
cells, CD8.sup.+ T cells, activated CD4.sup.+ T cells, NK T cells,
or CD21.sup.+/CD25.sup.+/Fox3P.sup.+ T cells) was increased. In
addition, the inventors further observed that administration of an
anti-CD200 antibody to a human afflicted with a cancer resulted in:
(i) an increased concentration of activated T cells as compared to
the concentration of the cells in the human prior to administration
of the anti-CD200 antibody; (ii) a decreased concentration of
regulatory T cells, as compared to the concentration of the cells
in the human prior to administration of the anti-CD200 antibody;
and (iii) an increase in the ratio of percent activated T cells to
percent regulatory T cells, as compared to the corresponding ratio
in the human prior to administration of the anti-CD200 antibody. In
fact, as elaborated on in the working examples, the concentration
of regulatory T cells decreased in four of seven (57%) patients
whose clinical disease stabilized or improved, whereas only 29% of
patients whose clinical disease progressed clinically experienced a
similar decrease in the concentration of regulatory T cells.
[0007] Anti-CD200 antibodies are currently under investigation as
potential therapeutic agents for treating a variety of diseases
including, but not limited to, cancer, inflammatory disorders
(e.g., graft rejection), and bone disorders. For example, the
humanized anti-CD200 antibody ALXN6000 (samalizumab; Alexion
Pharmaceuticals, Inc., Cheshire, Conn.) is being evaluated
presently in clinical trials for the treatment of cancer. While the
disclosure is not bound by any particular theory or mechanism of
action, the inventors believe that monitoring a patient treated
with an anti-CD200 antibody such as samalizumab for a change (e.g.,
an increase or a decrease) in one or more of the biomarkers
described herein is useful for determining whether the anti-CD200
antibody is capable of producing a desired immunomodulatory effect
in the human to which the antibody is administered. Moreover,
monitoring the extent of the immunomodulatory effect (e.g., by
detecting a change in one or more of the biomarkers described
herein) is also useful for identifying a dose--a threshold dose or
a dosing schedule--of an anti-CD200 antibody (e.g., samalizumab)
that, by virtue of the immunomodulatory effect of the antibody in
the human, is sufficient to achieve a clinically-meaningful effect
on the disease (i.e., sufficient to treat a disease such as
cancer). To wit, seven of twenty-five B-CLL and multiple myeloma
patients administered samalizumab in a phase I safety study
exhibited stable disease as determined by serial assessments of
peripheral blood counts and CT scans. A desired immunomodulatory
effect of the anti-CD200 antibody was observed in treated patients
as reflected in a change (e.g., an increase or reduction) in one or
more of the anti-CD200 antibody-associated biomarkers described
herein.
[0008] Accordingly, in one aspect, the disclosure provides a method
for determining whether an anti-CD200 antibody has produced a
desired immunomodulatory effect in a human (e.g., a cancer
patient). The method includes detecting an increase or decrease of
at least one immunomodulatory biomarker (sometimes referred to
herein as an "anti-CD200 antibody-associated immunomodulatory
biomarker") described herein in a blood sample obtained from a
human who has been administered an anti-CD200 antibody to thereby
determine whether the anti-CD200 antibody has produced an
immunomodulatory effect in the human. The immunomodulatory effect
can be characterized by a change (e.g., an increase or a decrease)
in at least one biomarker, e.g., an anti-CD200 antibody-associated
immunomodulatory biomarker described herein, the change selected
from the group consisting of: (i) a reduced concentration of
regulatory T cells, relative to the concentration of regulatory T
cells of the same histological type in the human prior to the first
administration of the antibody; (ii) an increased concentration of
CD8.sup.+ T cells, relative to the concentration of CD8.sup.+ T
cells of the same histological type in the human prior to the first
administration of the antibody; (iii) an increased concentration of
activated T cells, relative to the concentration of activated T
cells of the same histological type in the human prior to the first
administration of the antibody; (iv) a reduced concentration of
CD200.sup.+ leukocytes (e.g., CD200.sup.+ T cells), relative to the
concentration of CD200.sup.+ leukocytes of the same histological
type in the human prior to the first administration of the
antibody; (v) an increase in the concentration of CD200R.sup.+
leukocytes (e.g., CD200R.sup.+ T cells), relative to the
concentration of CD200R.sup.+ leukocytes of the same histological
type in the human prior to the first administration of the
antibody; (vi) a ratio of percent activated T cells to percent
regulatory T cells (T regs) of at least 2:1 (e.g., at least 3:1, at
least 4:1, at least 5:1, at least 6:1, or at least 7:1), relative
to the ratio of activated T cells to T regs in the human prior to
the first administration of the antibody; (vii) a decreased level
of CD200 expression by a plurality of leukocytes in a biological
sample obtained from a patient prior to administration to the
patient of an anti-CD200 antibody, relative to the level of CD200
expression by a plurality of leukocytes of the same histological
type in a biological sample from the patient prior to
administration of the antibody; and (viii) an increased level of
CD200R expression by a plurality of leukocytes in a biological
sample from a patient administered an anti-CD200 antibody, relative
to the level of CD200R expression by a plurality of leukocytes in a
biological sample from the patient prior to administration of the
anti-CD200 antibody. In some embodiments, a reduction in CD200
expression by a plurality of leukocytes (e.g., bone marrow cells or
splenocytes) in a biological sample obtained from the patient after
administration of the anti-CD200 antibody, as compared to a control
expression level (e.g., the level of CD200 expression in a
plurality of leukocytes of the same histological type in a
biological sample obtained from the patient prior to administration
of the anti-CD200 antibody) indicates that the anti-CD200 antibody
has produced a desired immunomodulatory effect in the human. It is
understood that any of the methods described herein can involve
determining whether there has been a change (e.g., an increase or a
decrease) in two or more (e.g., three, four, five, six, seven,
eight, nine, or 10 or more) of the anti-CD200 antibody-associated
biomarkers described herein. Where interrogation of more than one
of the biomarkers is practiced, any combination of two or more
(e.g., three, four, five, six, seven, eight, nine, or 10 or more)
of the biomarkers can be analyzed.
[0009] It is understood that in some embodiments, a change in
expression can be a change in protein expression or a change in
mRNA expression. That is, for example, the methods can interrogate
a population of leukocytes from a patient to determine if a
reduction in the level of CD200 mRNA and/or CD200 protein
expression has occurred, relative to a control level of mRNA and/or
protein expression. Methods for measuring protein and mRNA
expression are well known in the art and described herein.
[0010] In some embodiments, a reduction in the concentration of one
or more subsets of CD200.sup.+ bone marrow cells in a biological
sample obtained from the patient, as compared to the concentration
of the same subsets of CD200.sup.+ bone marrow cells in a control
sample, indicates that the anti-CD200 antibody has produced a
desired immunomodulatory effect in the human. The CD200.sup.+
leukocytes (e.g., bone marrow cells or splenocytes) or subsets can
be, but are not limited to, any of the CD200.sup.+ leukocytes
(e.g., bone marrow cells or splenocytes) or subsets described
herein (infra).
[0011] It is understood that the detecting can comprise, e.g.,
measuring the concentration of the appropriate selected cell type
(e.g., CD200.sup.+ or CD200R.sup.+ leukocytes) or quantifying the
level of expression of one or more expression markers such as CD200
or CD200R.
[0012] In some embodiments of any of the methods described herein,
the detecting can occur following the first dose of the anti-CD200
antibody. For example, the detecting (i.e., detecting a change
(e.g., an increase or decrease) in at least one of the biomarkers)
can occur within (or less than) two (2) months (e.g., less than
eight weeks, seven weeks, six weeks, five weeks, one month, four
weeks, three weeks, two weeks or 13 days, 12 days, 11 days, 10
days, nine days, eight days, seven days, six days, five days, or
less than 5 days) after the first therapeutic dose of the
anti-CD200 antibody is administered to the human. In some
embodiments of any of the methods described herein, the detecting
does not occur until at least 10 days (e.g., at least 11 days, 12
days, 13 days, 14 days or one week, two weeks, three weeks, four
weeks, a month, five weeks, six weeks, seven weeks, or eight weeks
or more) after the first therapeutic dose of the anti-CD200
antibody is administered to the human. It is understood that, e.g.,
in the following methods described herein, measuring the
concentration of the specified cell types or quantifying the level
of expression of an expression marker (e.g., CD200 or CD200R) can
occur, e.g., within any one of the aforementioned time periods.
[0013] In embodiments in which at least two (e.g., at least three,
four, five, six, seven, eight, nine, 10, 11, 12, 13, or 14 or more)
doses of the anti-CD200 antibody are administered to the human
prior to detecting a change (e.g., an increase or a decrease) in
the at least one biomarker, the detecting can occur, e.g., within
(or less than) two months (e.g., less than eight weeks, seven
weeks, six weeks, five weeks, one month, four weeks, three weeks,
two weeks or 13 days, 12 days, 11 days, 10 days, nine days, eight
days, seven days, six days, five days, or less than 5 days), and/or
not until at least 1 day (e.g., at least two days, three days, four
days, five days, six days, seven days, eight days, nine days, 10
days, 11 days, 12 days, 13 days, 14 days, 15 days, 16 days, 17
days, 18 days, 19 days, 20 days, or three weeks, four weeks, a
month, five weeks, six weeks, seven weeks, or eight weeks or more)
after, the last dose of the multiple dose anti-CD200 antibody
regimen is administered to the human. In some embodiments, the
detecting can occur between dosing (e.g., between the first and
second dose, between the second and third dose, between the third
and fourth dose, between the fifth and six dose, and/or between the
seventh and eighth dose). Such detection can be useful for
determining a dosing schedule for the human that is effective to
maintain the immunomodulatory effect (e.g., the peak or maximum
level of the immunomodulatory effect) in the human over the course
of treatment. It is understood that, e.g., in the following methods
described herein, measuring the concentration of the specified cell
types or quantifying the level of expression of an expression
marker (e.g., CD200 or CD200R) can occur, e.g., within any one of
the aforementioned time periods. In some embodiments, detecting a
change m one or more of the biomarkers described herein can occur
throughout the treatment of the patient (e.g., before and/or after
each dose of the anti-CD200 antibody administered to the patient).
Such detection can be useful for, among other things, a
longitudinal evaluation of the effect of the anti-CD200 antibody on
the physiology of the patient and allowing for a more precise
correlation between the occurrence of immunomodulatory effects and
efficacy of the anti-CD200 antibody treatment.
[0014] In some embodiments, a positive determination that a desired
immunomodulatory effect has occurred in the human results in a
decision by a medical practitioner to continue, or officially
begin, a treatment regimen for the human (e.g., where the human
has, is suspected of having, or at risk for developing, a disease
(e.g., a cancer) which the medical practitioner believes will
benefit from an anti-CD200 antibody immunomodulatory therapy) that
includes administration of an anti-CD200 antibody in an amount and
with a frequency effective to maintain the occurrence in the human
of the desired immunomodulatory effect. In some embodiments, a
positive determination that a desired immunomodulatory effect has
occurred in the human results in the medical practitioner
continuing to prescribe and/or select an anti-CD200 antibody
therapy for the human. In some embodiments, a positive
determination that a desired immunomodulatory effect has occurred
in the human as a result of administration of the anti-CD200
antibody results in a continued monitoring of the human for a
change (e.g., an increase or decrease) in the one or more
biomarkers in the human, e.g., after each dose of the anti-CD200
antibody administered or after every two doses, etc. This practice
can also be useful for determining a dosing schedule for the human
that is effective to maintain the immunomodulatory effect (e.g.,
the peak or maximum level of the desired immunomodulatory effect)
in the human over the course of treatment.
[0015] In another aspect, the disclosure features a method for
determining whether an anti-CD200 antibody has produced a desired
immunomodulatory effect in a human. The method comprises measuring
the concentration of CD200.sup.+ leukocytes in a blood sample
obtained from a human administered an anti-CD200 antibody, wherein
a reduction in the concentration of CD200.sup.+ leukocytes in the
blood sample as compared to the concentration of CD200.sup.+
leukocytes of the same histological type in a control sample
indicates that the anti-CD200 antibody has produced a desired
immunomodulatory effect in the human. The CD200.sup.+ leukocytes
can be, e.g., any of the CD200.sup.+ leukocytes described
herein.
[0016] In another aspect, the disclosure features a method for
determining whether an anti-CD200 antibody has produced a desired
immunomodulatory effect in a human. The method comprises measuring
the concentration of CD200.sup.+ T cells in a blood sample obtained
from a human administered an anti-CD200 antibody, wherein a
reduction in the concentration of CD200.sup.+ T cells in the blood
sample as compared to the concentration of CD200.sup.+ T cells of
the same histological type in a control sample indicates that the
anti-CD200 antibody has produced a desired immunomodulatory effect
in the human.
[0017] In another aspect, the disclosure features a method for
determining whether an anti-CD200 antibody has produced a desired
immunomodulatory effect in a human, which method comprises
measuring the concentration of CD200R.sup.+ leukocytes in a blood
sample obtained from a human administered an anti-CD200 antibody,
wherein an increase in the concentration of CD200R.sup.+ leukocytes
in the blood sample as compared to the concentration of
CD200R.sup.+ leukocytes of the same histological type in a control
sample indicates that the anti-CD200 antibody has produced a
desired immunomodulatory effect in the human.
[0018] In another aspect, the disclosure features a method for
determining whether an anti-CD200 antibody has produced a desired
immunomodulatory effect in a human. The method comprises
quantifying the level of CD200 expression by a plurality of
leukocytes in a biological sample from a human administered an
anti-CD200 antibody, wherein a reduction in CD200 expression by the
plurality as compared to the expression level of a plurality of
leukocytes of the same histological type in a control sample
indicates that the anti-CD200 antibody has produced a desired
immunomodulatory effect in the human.
[0019] In yet another aspect, the disclosure features a method for
determining whether an anti-CD200 antibody has produced a desired
immunomodulatory effect in a human, wherein the method comprises
quantifying the level of CD200R expression by a plurality of
leukocytes in a biological sample from a human administered an
anti-CD200 antibody, wherein an increase in CD200R expression by
the plurality as compared to the expression level of CD200R by a
plurality of leukocytes of the same histological type in a control
sample indicates that the anti-CD200 antibody has produced a
desired immunomodulatory effect in the human.
[0020] In some embodiments, any of the methods described herein
(e.g., the methods for determining whether an anti-CD200 has
produced a desired immunomodulatory effect in a human) can include
administering the anti-CD200 antibody to the human in accordance
with the methods. For example, the disclosure features a method for
determining whether an anti-CD200 antibody has produced a desired
immunomodulatory effect in a human, which method comprises:
administering an anti-CD200 antibody to a human and quantifying the
level of CD200R expression by a plurality of leukocytes in a
biological sample from the human after administration of the
anti-CD200 antibody, wherein an increase in CD200R expression by
the plurality as compared to the expression level of CD200R by a
plurality of leukocytes of the same histological type in a control
sample indicates that the anti-CD200 antibody has produced a
desired immunomodulatory effect in the human.
[0021] In some embodiments, any of the methods described herein
(e.g., the methods for determining whether an anti-CD200 has
produced a desired immunomodulatory effect in a human) can include
measuring the concentration of the specified cell type, or
quantifying the level of expression of a specified expression
marker on a specified cell type, in a biological sample obtained
from the human prior to administration of the antibody. For
example, the disclosure features a method for determining whether
an anti-CD200 antibody has produced a desired immunomodulatory
effect in a human, wherein the method comprises: measuring the
concentration of CD200.sup.+ T cells in a blood sample from a human
prior to administering an anti-CD200 antibody to the human; and
measuring the concentration of CD200.sup.+ T cells in a blood
sample from the human after an anti-CD200 antibody has been
administered to the human (e.g., by the same practitioner or a
different practitioner), wherein a reduction in the concentration
of CD200.sup.+ T cells in the post-treatment blood sample as
compared to the concentration of CD200.sup.+ T cells of the same
histological type in the blood sample obtained prior to treatment
indicates that the anti-CD200 antibody has produced a desired
immunomodulatory effect in the human.
[0022] In some embodiments, any of the methods described herein
includes obtaining the biological sample (e.g., the blood sample)
from the patient before and/or after administration of the
anti-CD200 antibody to the human.
[0023] In another aspect, the disclosure features a method for
determining whether an anti-CD200 antibody has produced a desired
immunomodulatory effect in a human. The method comprises measuring
the concentration of a population of CD200+ leukocytes (e.g.,
CD200.sup.+ T cells) in a blood sample obtained from a human
administered an anti-CD200 antibody; and quantifying the level of
CD200R expression by a plurality of leukocytes in a biological
sample from the human administered an anti-CD200 antibody, wherein
one or both of: (i) a reduction in the concentration of a
population of CD200.sup.+ leukocytes in the blood sample as
compared to the concentration of a corresponding population of
CD200.sup.+ leukocytes of the same histological type in a control
sample and (ii) an increase in CD200R expression by the plurality
as compared to a control expression level, indicates that the
anti-CD200 antibody has produced a desired immunomodulatory effect
in the human.
[0024] In yet another aspect, the disclosure features a method for
determining whether an anti-CD200 antibody has produced a desired
immunomodulatory effect in a human, the method comprising: (i)
measuring the concentration of a population of CD200.sup.+
leukocytes (e.g., CD200.sup.+ T cells) in a biological sample
obtained from a human prior to administration to the human of an
anti-CD200 antibody to thereby obtain a pre-treatment CD200.sup.+
leukocyte population concentration; (ii) administering to the human
the antibody; and (iii) measuring the concentration of a population
of CD200.sup.+ leukocytes of the same histological type in a blood
sample obtained from the human following administration of the
antibody to thereby obtain a post-treatment CD200.sup.+ leukocyte
population concentration, wherein a reduction in the post-treatment
CD200.sup.+ leukocyte concentration as compared to the
pre-treatment CD200.sup.+ leukocyte concentration indicates that
the antibody has produced a desired immunomodulatory effect in the
human.
[0025] In another aspect, the disclosure features a method for
determining whether an anti-CD200 antibody has produced a desired
immunomodulatory effect in a human, the method comprising: (i)
measuring the concentration of CD200R.sup.+ leukocytes (e.g.,
CD200R.sup.+ T cells) in a biological sample obtained from a human
prior to administration to the human of an anti-CD200 antibody to
thereby obtain a pre-treatment CD200R.sup.+ leukocyte
concentration; (ii) administering to the human the antibody; and
(iii) measuring the concentration of CD200R.sup.+ leukocytes (e.g.,
CD200R.sup.+ T cells) of the same histological type in a blood
sample obtained from the human following administration of the
antibody to thereby obtain a post-treatment CD200R.sup.+ leukocyte
concentration, wherein an increase in the post-treatment
CD200R.sup.+ leukocyte concentration as compared to the
pre-treatment CD200R+ leukocyte concentration indicates that the
antibody has produced a desired immunomodulatory effect in the
human.
[0026] In yet another aspect, the disclosure features a method for
determining whether an anti-CD200 antibody has produced a desired
immunomodulatory effect in a human, the method comprising: (i)
quantifying the level of CD200 expression by a plurality of
leukocytes in a biological sample from a human prior to
administration to the human of an anti-CD200 antibody to thereby
obtain a pre-treatment CD200 expression level; (ii) administering
to a human the anti-CD200 antibody; and (iii) quantifying the level
of CD200 expression by a plurality of leukocytes in a biological
sample from the human obtained after the administration of the
antibody to thereby obtain a post-treatment CD200 expression level,
wherein a reduction in post-treatment CD200 expression level as
compared to the pre-treatment CD200 expression level indicates that
the antibody has produced a desired immunomodulatory effect in the
human.
[0027] In another aspect, the disclosure features a method for
determining whether an anti-CD200 antibody has produced a desired
immunomodulatory effect in a human. The method includes: (i)
quantifying the level of CD200R expression by a plurality of
leukocytes in a biological sample from a human prior to
administration to the human of an anti-CD200 antibody to thereby
obtain a pre-treatment CD200R expression level; (ii) administering
to a human the anti-CD200 antibody; and (iii) quantifying the level
of CD200R expression by a plurality of leukocytes in a biological
sample obtained from the human after the administration of the
antibody to thereby obtain a post-treatment CD200R expression
level, wherein an increase in post-treatment CD200R expression
level as compared to the pre-treatment CD200R expression level
indicates that the antibody has produced a desired immunomodulatory
effect in the human.
[0028] With respect to CD200 expression by leukocytes, the
leukocytes can be, e.g., T cells such as CD200.sup.+/CD4.sup.+ T
cells, activated CD200.sup.+/CD4.sup.+ T cells, or
CD200.sup.+/CD8.sup.+ T cells. In some embodiments of any of the
methods described herein, the leukocytes are T cells such as
CD200.sup.+/CD4.sup.+ T cells or activated CD200.sup.+/CD4.sup.+ T
cells.
[0029] In some embodiments of any of the methods described herein,
at least a 5 (e.g., 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18,
19, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, or 90
or more) % reduction of the concentration of CD200.sup.+ leukocytes
indicates that a desired immunomodulatory effect has been produced
in the human. In some embodiments of any of the methods described
herein, at least a 5 (e.g., 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16,
17, 18, 19, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85,
or 90 or more) % reduction of the concentration of CD200.sup.+
leukocytes indicates that the antibody is therapeutically effective
in the human.
[0030] In some embodiments of any of the methods described herein,
at least a 5 (e.g., 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18,
19, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95,
or 100 or more) % increase in the concentration of CD200R.sup.+
leukocytes indicates that a desired immunomodulatory effect has
been produced in the human. In some embodiments of any of the
methods described herein, at least a 5 (e.g., 6, 7, 8, 9, 10, 11,
12, 13, 14, 15, 16, 17, 18, 19, 20, 25, 30, 35, 40, 45, 50, 55, 60,
65, 70, 75, 80, 85, 90, 95, or 100 or more) % increase in the
concentration of CD200R.sup.+ leukocytes indicates that the
antibody is therapeutically effective in the human.
[0031] In some embodiments of any of the methods described herein,
e.g., with respect to CD200R expression by leukocytes, the
leukocytes can be, e.g., CD4.sup.+ T cells, CD8.sup.+ T cells,
activated CD4.sup.+ T cells, CD21.sup.+/CD25.sup.+/Fox3P.sup.+ T
cells, and NK T cells.
[0032] In some embodiments of any of the methods described herein,
at least a 5 (e.g., 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18,
19, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, or 90
or more) % reduction in CD200 expression by the leukocytes (e.g., T
cells) indicates that a desired immunomodulatory effect has been
produced in the human.
[0033] In some embodiments of any of the methods described herein,
at least a 5 (e.g., 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18,
19, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, or 90
or more) % reduction in CD200 expression by the leukocytes (e.g., T
cells) indicates that the antibody is therapeutically effective in
the human.
[0034] In some embodiments of any of the methods described herein,
at least a 1.5 (e.g., 2, 2.5, 3, 3.5, 4, 4.5, 5, 5.5, 6, 6.5, 7,
7.5, 8, 8.5, 9, 9.5, or 10 or more)-fold increase in CD200R
expression by the plurality of leukocytes indicates that a desired
immunomodulatory effect has been produced by the antibody in the
human. In some embodiments of any of the methods described herein,
at least a 1.5 (e.g., 2, 2.5, 3, 3.5, 4, 4.5, 5, 5.5, 6, 6.5, 7,
7.5, 8, 8.5, 9, 9.5, or 10 or more)-fold increase in CD200R
expression by the plurality of leukocytes indicates that the
anti-CD200 antibody is therapeutically effective in the human.
[0035] In some embodiments of any of the methods described herein,
a reduction in the concentration of CD200.sup.+ T cells in the
blood sample as compared to the concentration of CD200 T cells of
the same histological type in the control sample indicates that the
antibody is therapeutically effective in the human.
[0036] In some embodiments of any of the methods described herein,
an increase in the concentration of CD200R.sup.+ T cells in the
blood sample as compared to the concentration of CD200R.sup.+ T
cells of the same histological type in the control sample indicates
that the antibody is therapeutically effective in the human.
[0037] In some embodiments of any of the methods described herein,
a reduction in the level of CD200 expression by the plurality as
compared to the control expression level indicates that the
anti-CD200 antibody is therapeutically effective in the human.
[0038] In some embodiments of any of the methods described herein,
an increase in the level of CD200R expression by the plurality as
compared to the control expression level indicates that the
anti-CD200 antibody is therapeutically effective in the human.
[0039] In another aspect, the disclosure features a method for
determining whether an anti-CD200 antibody has produced a desired
immunomodulatory effect in a human, which method comprises
measuring the concentration of regulatory T cells (T regs) in a
blood sample obtained from a human administered an anti-CD200
antibody. A reduction in the concentration of T regs in the blood
sample, as compared to the concentration of T regs of same
histological type in a control sample, indicates that the
anti-CD200 antibody has produced a desired immunomodulatory effect
in the human. The control sample can be, e.g., a blood sample
obtained from the patient prior to administration of the first
therapeutic dose of the anti-CD200 antibody.
[0040] In another aspect, the disclosure features a method for
determining whether an anti-CD200 antibody has produced a desired
immunomodulatory effect in a human, wherein the method includes:
(i) measuring the concentration of regulatory T cells (T regs) in a
blood sample obtained from a human prior to administration to the
human of an anti-CD200 antibody to thereby obtain a pre-treatment T
regs concentration; (ii) administering to the human the anti-CD200
antibody; and (iii) measuring the concentration of T regs of the
same defined histological type in a blood sample obtained from the
human after administration of the anti-CD200 antibody, wherein a
reduction in the concentration of T regs in the post-treatment
blood sample, as compared to the pre-treatment T regs
concentration, indicates that the anti-CD200 antibody has produced
a desired immunomodulatory effect in the human.
[0041] In some embodiments of the methods described herein, the T
regs can be, e.g., CD3.sup.+CD4.sup.+CD25.sup.+FoxP3.sup.+ T cells
or CD3.sup.+CD4.sup.+FoxP3.sup.+ T cells.
[0042] In some embodiments of any of the methods described herein,
at least a 5 (e.g., 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18,
19, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, or 90
or more) % reduction of the concentration of T regs indicates that
a desired immunomodulatory effect has been produced in the human.
In some embodiments, the T regs (for example, the T regs defined by
the foregoing expression markers) can express CD200 or CD200R.
[0043] In some embodiments of any of the methods described herein,
at least a 5 (e.g., 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18,
19, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, or 90
or more) % reduction of the concentration of T regs indicates that
the antibody is therapeutically effective in the human.
[0044] In yet another aspect, the disclosure features a method for
determining whether an anti-CD200 antibody has produced a desired
immunomodulatory effect in a human, which method comprises
measuring the concentration of activated T cells in a blood sample
obtained from a human administered an anti-CD200 antibody, wherein
an increase in the concentration of activated T cells in the blood
sample, as compared to the concentration of activated T cells of
same histological type in a control sample, indicates that the
anti-CD200 antibody has produced a desired immunomodulatory effect
in the human. The control sample can be, e.g., a blood sample
obtained from the patient prior to administration of the first
therapeutic dose of the anti-CD200 antibody.
[0045] In another aspect, the disclosure features a method for
determining whether an anti-CD200 antibody has produced a desired
immunomodulatory effect in a human, the method comprising: (i)
measuring the concentration of activated T cells in a blood sample
obtained from a human prior to administration to the human of an
anti-CD200 antibody to thereby obtain a pre-treatment activated T
cell concentration; (ii) administering to the human the anti-CD200
antibody; and (iii) measuring the concentration of activated T
cells of the same defined histological type in a blood sample
obtained from the human after administration of the anti-CD200
antibody, wherein an increase in the concentration of activated T
cells in the post-treatment blood sample, as compared to the
pre-treatment activated T cell concentration, indicates that the
anti-CD200 antibody has produced a desired immunomodulatory effect
in the human.
[0046] In some embodiments of any of the methods described herein,
at least a 5 (e.g., 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18,
19, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95,
or 100 or more) % increase in the concentration of activated T
cells indicates that a desired immunomodulatory effect has been
produced in the human. In some embodiments of any of the methods
described herein, at least a 5 (e.g., 6, 7, 8, 9, 10, 11, 12, 13,
14, 15, 16, 17, 18, 19, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70,
75, 80, 85, 90, 95, or 100 or more) % increase in the concentration
of activated T cells indicates that the antibody is therapeutically
effective in the human.
[0047] In yet another aspect, the disclosure features a method for
determining whether an anti-CD200 antibody has produced a desired
immunomodulatory effect in a human, which method comprises
determining the ratio of percent activated T cells to percent
regulatory T cells in a blood sample obtained from a human
administered an anti-CD200 antibody, wherein an increase in the
ratio of percent activated T cells to percent regulatory T cells in
the blood sample, as compared to the ratio of percent activated T
cells to percent regulatory T cells in a control sample, indicates
that the anti-CD200 antibody has produced a desired
immunomodulatory effect in the human.
[0048] In yet another aspect, the disclosure features a method for
determining whether an anti-CD200 antibody has produced a desired
immunomodulatory effect in a human, which method comprises
determining the ratio of percent activated T cells to percent
regulatory T cells in a blood sample obtained from a human
administered an anti-CD200 antibody, wherein a ratio of percent
activated T cells to percent regulatory T cells of at least 2:1
(e.g., at least 3:1, at least 4:1, at least 5:1, at least 6:1, or
at least 7:1) indicates that the anti-CD200 antibody has produced a
desired immunomodulatory effect in the human.
[0049] In another aspect, the disclosure features a method for
determining whether an anti-CD200 antibody has produced a desired
immunomodulatory effect in a human, the method comprising: (i)
determining the ratio of percent activated T cells to percent
regulatory T cells in a blood sample obtained from a human
administered an anti-CD200 antibody to thereby determine a
pre-treatment ratio; (ii) administering to the human the anti-CD200
antibody; and (iii) determining the ratio of percent activated T
cells to percent regulatory T cells in a blood sample obtained from
the human after administration of the anti-CD200 antibody, wherein
an increase in the ratio of percent activated T cells to percent
regulatory T cells in the post-treatment blood sample, as compared
to the pre-treatment ratio, indicates that the anti-CD200 antibody
has produced a desired immunomodulatory effect in the human.
[0050] In some embodiments of any of the methods described herein,
the activated T cells can be, e.g.,
CD3.sup.+CD4.sup.+CD25.sup.+FoxP3.sup.neg T cells or
CD3.sup.+CD4.sup.+FoxP3.sup.neg T cells. In some embodiments, the
activated T cells (for example, the activated T cells defined by
the foregoing expression markers) can express CD200 or CD200R.
[0051] In some embodiments of any of the methods described herein,
the control sample can be or contain a blood sample from the human
obtained prior to administering the anti-CD200 antibody.
[0052] In another aspect, the disclosure features a method for
determining whether an anti-CD200 antibody has produced a desired
immunomodulatory effect in a human. The method includes measuring:
(a): (i) the concentration of CD200.sup.+ leukocytes in a
biological sample obtained from a human following administration to
the human of an anti-CD200 antibody and (ii) the concentration of
CD200.sup.+ leukocytes of the same histological type as in (i) in a
biological sample obtained from the human prior to administration
of the antibody; (b): (iii) the concentration of CD200R.sup.+
leukocytes in a biological sample obtained from a human following
administration to the human of an anti-CD200 antibody and (iv) the
concentration of CD200R.sup.+ leukocytes of the same histological
type as in (iii) in a biological sample obtained from the human
prior to administration of the antibody; (c): (v) the level of
expression of CD200R by a plurality of leukocytes in a biological
sample obtained from the human following administration to the
human of an anti-CD200 antibody and (vi) the level of expression of
CD200R by a plurality of leukocytes of the same histological type
as in (v) in a biological sample obtained from the human prior to
administration of the antibody; (d): (vii) the level of expression
of CD200 by a plurality of leukocytes in a biological sample
obtained from the human following administration to the human of an
anti-CD200 antibody and (viii) the level of expression of CD200 by
a plurality of leukocytes of the same histological type as in (vii)
in a biological sample obtained from the human prior to
administration of the antibody; (e): (ix) the concentration of
regulatory T cells in a biological sample from a human following
administration to the human of an anti-CD200 antibody and (x) the
concentration of regulatory T cells of the same histological type
as in (ix) in a biological sample from the human prior to
administration of the anti-CD200 antibody; (f): (xi) the
concentration of activated T cells in a biological sample from a
human following administration of an anti-CD200 antibody to the
human and (xii) the concentration of activated T cells of the same
histological type as in (xi) in a biological sample from the human
prior to administration of the anti-CD200 antibody; (g): (xiii) the
ratio of percent activated T cells to percent regulatory T cells in
a biological sample from a human following administration of an
anti-CD200 antibody and (xiv) the corresponding ratio of percent
activated T cells to percent regulatory T cells (each of the same
histological type as in (xiii)) in a biological sample from the
human prior to administration of the anti-CD200 antibody; (h): (xv)
the concentration of CD8.sup.+ lymphocytes in a biological sample
from a human following administration of an anti-CD200 antibody to
the human and (xvi) the concentration of CD8.sup.+ lymphocytes of
the same histological type as in (xv) in a biological sample from
the human prior to administration of the antibody; (i): (xvii) the
concentration of CD200.sup.+ T cells in a biological sample
obtained from a human following administration to the human of an
anti-CD200 antibody and (xviii) the concentration of CD200.sup.+ T
cells of the same histological type as in (xvii) in a biological
sample obtained from the human prior to administration of the
antibody; (j): (xix) the concentration of CD200R.sup.+ T cells in a
biological sample obtained from a human following administration to
the human of an anti-CD200 antibody and (xx) the concentration of
CD200R.sup.+ T cells of the same histological type as in (xix) in a
biological sample obtained from the human prior to administration
of the antibody; (k): (xxi) the concentration of one or more
subsets of CD200.sup.+ leukocytes in a biological sample obtained
from a human following administration to the human of an anti-CD200
antibody and (xxii) the concentration of one or more subsets of
CD200.sup.+ leukocytes of the same histological type as in (xxi) in
a biological sample obtained from the human prior to administration
of the antibody; (I): (xxiii) the concentration of one or more
subsets of CD200.sup.+ bone marrow cells in a biological sample
obtained from a human following administration to the human of an
anti-CD200 antibody and (xxiv) the concentration of the one or more
subsets of CD200.sup.+ bone marrow cells of the same histological
type as in (xxiii) in a biological sample obtained from the human
prior to administration of the antibody; and/or (m): (xxv) the
level of expression of CD200 by a plurality of bone marrow cells in
a biological sample obtained from the human following
administration to the human of an anti-CD200 antibody and (xxvi)
the level of expression of CD200 by a plurality of bone marrow
cells of the same histological type as in (xxv) in a biological
sample obtained from the human prior to administration of the
antibody, wherein: (a) a reduction in the post-treatment
CD200.sup.+ leukocyte concentration as compared to the
pre-treatment CD200.sup.+ leukocyte concentration indicates that
the antibody has produced a desired immunomodulatory effect in the
human; (b) an increase in the post-treatment CD200R.sup.+ leukocyte
concentration as compared to the pre-treatment CD200R.sup.+
leukocyte concentration indicates that the antibody has produced a
desired immunomodulatory effect in the human; (c) an increase in
post-treatment CD200R expression level by the plurality of
leukocytes as compared to the pre-treatment CD200R expression level
indicates that the antibody has produced a desired immunomodulatory
effect in the human; (d) a reduction in post-treatment CD200.sup.+
expression level by the plurality of leukocytes as compared to the
pre-treatment CD200.sup.+ expression level indicates that the
antibody has produced a desired immunomodulatory effect in the
human; (e) a reduction in the post-treatment concentration of
regulatory T cells as compared to the pre-treatment concentration
of regulatory T cells indicates that the antibody has produced a
desired immunomodulatory effect in the human; (f) an increase in
the post-treatment concentration of activated T cells as compared
to the pre-treatment activated T cell concentration indicates that
the antibody has produced a desired immunomodulatory effect in the
human; (g) an increase in the post-treatment ratio of percent
activated T cells to percent regulatory T cells as compared to the
pre-treatment ratio indicates that the antibody has produced an
immunomodulatory effect in the human or a post-treatment ratio of
percent activated T cells to percent regulatory T cells of at least
2:1 indicates that the antibody has produced a desired
immunomodulatory effect in the human; (h) an increase in the
post-treatment concentration of CD8.sup.+ lymphocytes as compared
to pre-treatment concentration of CD8.sup.+ lymphocytes indicates
that the antibody has produced a desired immunomodulatory effect in
the human; (i) a decrease in the post-treatment concentration of
CD200.sup.+ T cells as compared to the pre-treatment concentration
of CD200.sup.+ T cells indicates that the antibody has produced a
desired immunomodulatory effect in the human; (j) an increase in
the post-treatment concentration of CD200R.sup.+ T cells as
compared to the pre-treatment concentration of CD200R.sup.+ T cells
indicates that the antibody has produced a desired immunomodulatory
effect in the human; (k) a decrease in the post-treatment
concentration of one or more subsets of CD200.sup.+ leukocytes as
compared to the pre-treatment concentration of the CD200.sup.+
leukocytes indicates that the anti-CD200 antibody has produced a
desired immunomodulatory effect in the human; (1) a decrease in the
post-treatment concentration of one or more subsets of CD200.sup.+
bone marrow cells as compared to the pre-treatment concentration of
the CD200' bone marrow cells indicates that the anti-CD200 antibody
has produced a desired immunomodulatory effect in the human; or (m)
a decrease in the post-treatment CD200 expression by the plurality
of bone marrow cells as compared to the pre-treatment CD200
expression level by the plurality indicates that the anti-CD200
antibody has produced a desired immunomodulatory effect in the
human. In some embodiments, two or more (e.g., three, four, five,
six, seven, eight, or nine or more) of any combination of the
conditions are measured. In some embodiments all of the conditions
are measured.
[0053] In yet another aspect, the disclosure features a computer
readable medium comprising a medical profile of a human, the
profile comprising information on one or more anti-CD200
antibody-associated immunomodulatory biomarkers, the biomarkers
selected from the group consisting of: (a): (i) the concentration
of CD200.sup.+ leukocytes (e.g., CD200.sup.+ T cells) in a
biological sample obtained from a human following administration to
the human of an anti-CD200 antibody and (ii) the concentration of
CD200' leukocytes (e.g., CD200.sup.+ T cells) of the same
histological type as in (i) in a biological sample obtained from
the human prior to administration of the antibody; (b): (iii) the
concentration of CD200R.sup.+ leukocytes (e.g., CD200R.sup.+ T
cells) in a biological sample obtained from a human following
administration to the human of an anti-CD200 antibody and (iv) the
concentration of CD200R.sup.+ leukocytes (e.g., CD200R.sup.+ T
cells) of the same histological type as in (iii) in a biological
sample obtained from the human prior to administration of the
antibody; (c): (v) the level of expression of CD200R by a plurality
of leukocytes in a biological sample obtained from the human
following administration to the human of an anti-CD200 antibody and
(vi) the level of expression of CD200R by a plurality of leukocytes
of the same histological type as in (v) in a biological sample
obtained from the human prior to administration of the antibody;
(d): (vii) the level of expression of CD200 by a plurality of
leukocytes in a biological sample obtained from the human following
administration to the human of an anti-CD200 antibody and (viii)
the level of expression of CD200 by a plurality of leukocytes of
the same histological type as in (vii) in a biological sample
obtained from the human prior to administration of the antibody;
(e): (ix) the concentration of regulatory T cells in a biological
sample from a human following administration to the human of an
anti-CD200 antibody and (x) the concentration of regulatory T cells
of the same histological type as in (ix) in a biological sample
from the human prior to administration of the anti-CD200 antibody;
(0: (xi) the concentration of activated T cells in a biological
sample from a human following administration of an anti-CD200
antibody to the human and (xii) the concentration of activated T
cells of the same histological type as in (xi) in a biological
sample from the human prior to administration of the anti-CD200
antibody; (g): (xiii) the ratio of percent activated T cells to
percent regulatory T cells in a biological sample from a human
following administration of an anti-CD200 antibody and (xiv) the
corresponding ratio of percent activated T cells to percent
regulatory T cells (each of the same histological type as in
(xiii)) in a biological sample from the human prior to
administration of the anti-CD200 antibody; and (h): (xv) the
concentration of CD8.sup.+ lymphocytes (e.g., T cells) in a
biological sample from a human following administration of an
anti-CD200 antibody to the human and (xvi) the concentration of
CD8.sup.+ lymphocytes of the same histological type as in (xv) in a
biological sample from the human prior to administration of the
antibody. The medical profile can also include, e.g., (xvii) the
level of expression of CD200 by a plurality of leukocytes (e.g.,
bone marrow cells or splenocytes) in a biological sample obtained
from a patient following administration of an anti-CD200 antibody
and/or (xviii) the level of CD200 expression by a plurality of
leukocytes (e.g., bone marrow cells or splenocytes) of the same
histological type as in (xvii) in a biological sample obtained from
the patient prior to administration of the anti-CD200 antibody. The
medical profile can also include, e.g., (xix) the concentration of
one or more CD200.sup.+ bone marrow cell subsets in a biological
sample obtained from a patient following administration of an
anti-CD200 antibody and/or (xx) the concentration of one or more
CD200.sup.+ bone marrow cell subsets of the same histological type
in (xix) in a biological sample obtained from the patient prior to
administration of the anti-CD200 antibody. In some embodiments, the
medical profile can include such information of two or more (e.g.,
three, four, five, 10, 15, 20, 30, 40, 50, 60, 70, 80, 90, or 100
or more) patients. In some embodiments, the medical profile is
stored on a computer-readable medium such as a computer harddrive,
flashdrive, DVD, or CD.
[0054] In another aspect, the disclosure provides a computer-based
method for determining whether an anti-CD200 antibody has produced
a desired immunomodulatory effect in a human. The method includes
receiving data including a medical profile of a human, the profile
comprising information on at least one of the anti-CD200
antibody-associated immunomodulatory biomarkers described herein
including: (a): (i) the concentration of CD200.sup.+ leukocytes
(e.g., CD200.sup.+ T cells) in a biological sample obtained from a
human following administration to the human of an anti-CD200
antibody and (ii) the concentration of CD200.sup.+ leukocytes
(e.g., CD200.sup.+ T cells) of the same histological type as in (i)
in a biological sample obtained from the human prior to
administration of the antibody; (b): (iii) the concentration of
CD200R.sup.+ leukocytes (e.g., CD200R.sup.+ T cells) in a
biological sample obtained from a human following administration to
the human of an anti-CD200 antibody and (iv) the concentration of
CD200R.sup.+ leukocytes (e.g., CD200R.sup.+ T cells) of the same
histological type as in (iii) in a biological sample obtained from
the human prior to administration of the antibody; (c): (v) the
level of expression of CD200R by a plurality of leukocytes in a
biological sample obtained from the human following administration
to the human of an anti-CD200 antibody and (vi) the level of
expression of CD200R by a plurality of leukocytes of the same
histological type as in (v) in a biological sample obtained from
the human prior to administration of the antibody; (d): (vii) the
level of expression of CD200 by a plurality of leukocytes in a
biological sample obtained from the human following administration
to the human of an anti-CD200 antibody and (viii) the level of
expression of CD200 by a plurality of leukocytes of the same
histological type as in (vii) in a biological sample obtained from
the human prior to administration of the antibody; (e): (ix) the
concentration of regulatory T cells in a biological sample from a
human following administration to the human of an anti-CD200
antibody and (x) the concentration of regulatory T cells of the
same histological type as in (ix) in a biological sample from the
human prior to administration of the anti-CD200 antibody; (f): (xi)
the concentration of activated T cells in a biological sample from
a human following administration of an anti-CD200 antibody to the
human and (xii) the concentration of activated T cells of the same
histological type as in (xi) in a biological sample from the human
prior to administration of the anti-CD200 antibody; (g): (xiii) the
ratio of percent activated T cells to percent regulatory T cells in
a biological sample from a human following administration of an
anti-CD200 antibody and (xiv) the corresponding ratio of percent
activated T cells to percent regulatory T cells (each of the same
histological type as in (xiii)) in a biological sample from the
human prior to administration of the anti-CD200 antibody; and (h):
(xv) the concentration of CD8.sup.+ lymphocytes (e.g., T cells) in
a biological sample from a human following administration of an
anti-CD200 antibody to the human and (xvi) the concentration of
CD8.sup.+ lymphocytes of the same histological type as in (xv) in a
biological sample from the human prior to administration of the
antibody; and processing at least the portion of the data
containing the information to determine whether the antibody has
produced a desired immunomodulatory effect in the human, wherein: a
reduction in the post-treatment CD200.sup.+ leukocyte (e.g.,
CD200.sup.+ T cells) concentration as compared to the pre-treatment
CD200.sup.+ leukocyte (e.g., CD200.sup.+ T cell) concentration
indicates that the antibody has produced a desired immunomodulatory
effect in the human; an increase in the post-treatment CD200R.sup.+
leukocyte (e.g., CD200R.sup.+ T cell) concentration as compared to
the pre-treatment CD200R.sup.+ leukocyte concentration indicates
that the antibody has produced a desired immunomodulatory effect in
the human; a reduction in post-treatment CD200.sup.+ expression
level by the plurality of leukocytes as compared to the
pre-treatment CD200.sup.+ expression level indicates that the
antibody has produced a desired immunomodulatory effect in the
human; an increase in post-treatment CD200R expression level by the
plurality of leukocytes as compared to the pre-treatment CD200R
expression level indicates that the antibody has produced a desired
immunomodulatory effect in the human; a reduction in the
post-treatment concentration of regulatory T cells as compared to
the pre-treatment concentration of regulatory T cells indicates
that the antibody has produced an immunodulatory effect in the
human; an increase in the post-treatment concentration of activated
T cells as compared to the pre-treatment activated T cell
concentration indicates that the antibody has produced a desired
immunomodulatory effect in the human; an increase in the
post-treatment concentration of CD8.sup.+ lymphocytes (e.g., T
cells) as compared to pre-treatment concentration of CD8.sup.+
lymphocytes indicates that the antibody has produced a desired
immunomodulatory effect in the human; and an increase in the
post-treatment ratio of percent activated T cells to percent
regulatory T cells as compared to the pre-treatment ratio indicates
that the antibody has produced a desired immunomodulatory effect in
the human; and/or a post-treatment ratio of percent activated T
cells to percent regulatory T cells of at least 2:1 (e.g., at least
3:1, 4:1, 5:1, 6:1, or even 7:1 or more) indicates that the
antibody has produced a desired immunomodulatory effect in the
human.
[0055] In another aspect, the disclosure features a computer-based
method for determining whether an anti-CD200 antibody has produced
a desired immunomodulatory effect in a human, which method
includes: providing information on one or both of (a): (i) the
concentration of CD200.sup.+ leukocytes (e.g., CD200.sup.+ T cells)
in a biological sample obtained from a human following
administration to the human of an anti-CD200 antibody and (ii) the
concentration of CD200.sup.+ leukocytes (e.g., CD200.sup.+ T cells)
of the same histological type as in (i) in a biological sample
obtained from the human prior to administration of the antibody;
(b): (iii) the concentration of CD200R.sup.+ leukocytes (e.g.,
CD200R.sup.+ T cells) in a biological sample obtained from a human
following administration to the human of an anti-CD200 antibody and
(iv) the concentration of CD200R.sup.+ leukocytes (e.g.,
CD200R.sup.+ T cells) of the same histological type as in (iii) in
a biological sample obtained from the human prior to administration
of the antibody; (c): (v) the level of expression of CD200R by a
plurality of leukocytes in a biological sample obtained from the
human following administration to the human of an anti-CD200
antibody and (vi) the level of expression of CD200R by a plurality
of leukocytes of the same histological type as in (v) in a
biological sample obtained from the human prior to administration
of the antibody; (d): (vii) the level of expression of CD200 by a
plurality of leukocytes in a biological sample obtained from the
human following administration to the human of an anti-CD200
antibody and (viii) the level of expression of CD200 by a plurality
of leukocytes of the same histological type as in (vii) in a
biological sample obtained from the human prior to administration
of the antibody; (e): (ix) the concentration of regulatory T cells
in a biological sample from a human following administration to the
human of an anti-CD200 antibody and (x) the concentration of
regulatory T cells of the same histological type as in (ix) in a
biological sample from the human prior to administration of the
anti-CD200 antibody; (f): (xi) the concentration of activated T
cells in a biological sample from a human following administration
of an anti-CD200 antibody to the human and (xii) the concentration
of activated T cells of the same histological type as in (xi) in a
biological sample from the human prior to administration of the
anti-CD200 antibody; (g): (xiii) the ratio of percent activated T
cells to percent regulatory T cells in a biological sample from a
human following administration of an anti-CD200 antibody and (xiv)
the corresponding ratio of percent activated T cells to percent
regulatory T cells (each of the same histological type as in
(xiii)) in a biological sample from the human prior to
administration of the anti-CD200 antibody; and (h): (xv) the
concentration of CD8.sup.+ lymphocytes (e.g., T cells) in a
biological sample from a human following administration of an
anti-CD200 antibody to the human and (xvi) the concentration of
CD8.sup.+ lymphocytes of the same histological type as in (xv) in a
biological sample from the human prior to administration of the
antibody; inputting the information into a computer; and
calculating a parameter indicating whether the antibody has
produced a desired immunomodulatory effect in the human using the
computer and the input information, wherein: a reduction in the
post-treatment CD200.sup.+ leukocyte (e.g., CD200.sup.+ T cells)
concentration as compared to the pre-treatment CD200.sup.+
leukocyte (e.g., CD200.sup.+ T cell) concentration indicates that
the antibody has produced a desired immunomodulatory effect in the
human; an increase in the post-treatment CD200R.sup.+ leukocyte
(e.g., CD200R.sup.+ T cell) concentration as compared to the
pre-treatment CD200R.sup.+ leukocyte concentration indicates that
the antibody has produced a desired immunomodulatory effect in the
human; a reduction in post-treatment CD200' expression level by the
plurality of leukocytes as compared to the pre-treatment
CD200.sup.+ expression level indicates that the antibody has
produced a desired immunomodulatory effect in the human; an
increase in post-treatment CD200R expression level by the plurality
of leukocytes as compared to the pre-treatment CD200R expression
level indicates that the antibody has produced a desired
immunomodulatory effect in the human; a reduction in the
post-treatment concentration of regulatory T cells as compared to
the pre-treatment concentration of regulatory T cells indicates
that the antibody has produced an immunodulatory effect in the
human; an increase in the post-treatment concentration of activated
T cells as compared to the pre-treatment activated T cell
concentration indicates that the antibody has produced a desired
immunomodulatory effect in the human; an increase in the
post-treatment ratio of percent activated T cells to percent
regulatory T cells as compared to the pre-treatment ratio indicates
that the antibody has produced a desired immunomodulatory effect in
the human; an increase in the post-treatment concentration of
CD8.sup.+ lymphocytes (e.g., T cells) as compared to pre-treatment
concentration of CD8.sup.+ lymphocytes indicates that the antibody
has produced a desired immunomodulatory effect in the human; and/or
a post-treatment ratio of percent activated T cells to percent
regulatory T cells of at least 2:1 (e.g., at least 3:1, 4:1, 5:1,
6:1, or even 7:1 or more) indicates that the antibody has produced
a desired immunomodulatory effect in the human. The method can also
include outputting the parameter.
[0056] In some embodiments, the information can include: (xvii) the
level of expression of CD200 by a plurality of leukocytes (e.g.,
bone marrow cells or splenocytes) in a biological sample obtained
from a patient following administration of an anti-CD200 antibody
and/or (xviii) the level of CD200 expression by a plurality of
leukocytes (e.g., bone marrow cells or splenocytes) of the same
histological type as in (xvii) in a biological sample obtained from
the patient prior to administration of the anti-CD200 antibody,
wherein a post-treatment decrease in the level of CD200 expression
by the plurality, as compared to the pre-treatment level of
expression by the corresponding plurality, indicates that the
anti-CD200 antibody has produced a desired immunomodulatory effect
in the human.
[0057] In some embodiments, the information can include: (xix) the
concentration of one or more CD200.sup.+ bone marrow cell subsets
in a biological sample obtained from a patient following
administration of an anti-CD200 antibody and/or (xx) the
concentration of one or more CD200.sup.+ bone marrow cell subsets
of the same histological type in (xix) in a biological sample
obtained from the patient prior to administration of the anti-CD200
antibody, wherein a post-treatment decrease in the concentration of
the one or more CD200.sup.+ bone marrow subsets, as compared to the
pre-treatment concentration of the corresponding CD200.sup.+ bone
marrow subsets, indicates that the anti-CD200 antibody has produced
a desired immunomodulatory effect in the human.
[0058] In some embodiments of any of the methods described herein,
the human has, is suspected of having, or is likely to develop, a
cancer. The cancer can be, e.g., CLL (e.g., B-CLL). The cancer can
be, e.g., a solid tumor such as, but not limited to, a colon
cancer, a breast cancer, a lung cancer, a renal cancer, a
pancreatic cancer, a thyroid cancer, a skin cancer, a cancer of the
nervous system, a cervical cancer, an ovarian cancer, a testicular
cancer, a head and neck cancer, a bone cancer, a cancer of the eye,
a stomach cancer, or a liver cancer. The cancer of the nervous
system can be a neuroblastoma.
[0059] In some embodiments of any of the methods described herein,
the human has, is suspected of having, or is at risk for developing
an inflammatory condition and/or a bone disorder. Inflammatory
disorders and bone disorders are well-known in the art of medicine.
Examples of each of these disorders are provided herein.
[0060] In some embodiments, any of the methods described herein can
include administering to the human a therapeutically-effective
amount of the anti-CD200 antibody if the antibody has been
determined to produce a desired immunomodulatory effect in the
human.
[0061] In some embodiments, any of the above methods can include
administering to the subject the anti-CD200 antibody, e.g., in an
amount and with a frequency effective to maintain the
immunomodulatory effect in the human.
[0062] The inventors have also discovered that upon administration
of an anti-CD200 antibody to a patient with a cancer comprising a
plurality of CD200-expressing cancer cells, CD200 expression by the
cancer cells is reduced. Cancer cells have evolved a number of ways
to evade detection by the human immune system, which can identify
malignant cells and kill the cells--a process known as
immunosurveillance--before a potentially life-threatening cancer
develops in the human. See, e.g., Geertsen et al. (1999) Int J Mol
Med 3(1):49-57; Kerebijn et al. (1999) Crit Rev Oncol Hematol
31(1):31-53; and Pardoll (2003) Annu Rev Immunol 21:807-39. One
potential mechanism by which cancer cells escape immunosurveillance
is via expression or overexpression of the immunosuppressive CD200
protein. In fact, CD200 protein has been shown to be expressed or
overexpressed on a variety of human cancer cells including, e.g., B
cell chronic lymphocytic leukemia cells, prostate cancer cells,
breast cancer cells, colon cancer cells, and brain cancer cells.
See, e.g., Kawasaki et al. (2007) Biochem Biophys Res Commun
364(4):778-782; Kretz-Rommel et al. (2007), supra; and Siva et al.
(2008) Cancer Immunol Immunother 57(71:987-96. Thus, while the
disclosure is not bound by any particular theory or mechanism of
action, the inventors believe that the anti-CD200
antibody-dependent downregulation of CD200 on the cancer cells
relieves an inhibition of immunosurveillance and allows the immune
system to more effectively identify and fight the cancer.
Accordingly, it is believed to be beneficial to administer to the
human an anti-CD200 antibody in an amount and with a frequency
sufficient to sustain the reduced expression of CD200 by the cancer
cells in the human. Exemplary anti-CD200 antibody dosing schedules
in accordance with the disclosure are provided herein. Two
non-exhaustive, non-limiting examples of such a dosing schedule are
administration of the anti-CD200 antibody in a higher amount (e.g.,
greater than 200 mg/m.sup.2) and/or at a lower amount (e.g., less
than or equal to 200 mg/m.sup.2), but with an increased frequency
(e.g., at least once every 12 days). Additional examples are
provided herein.
[0063] Furthermore, as elaborated on in the working examples set
forth herein, the inventors further report a discovery based on the
observed pharmacodynamic properties of the anti-CD200 antibody
samalizumab administered to a patient on a once per month dosing
schedule. Specifically, under the once per month dosing schedule at
a dose of between (and inclusive of) 50 mg/m.sup.2 to 200
mg/m.sup.2, the immunomodulatory effect of the antibody in the
patients was transient, with affected cell populations recovering
(or nearly recovering) to pre-treatment levels at around day 14.
Administration of the second dose of samalizumab, however, once
again produced the immunomodulatory effect on the specific cell
populations (e.g., CD200.sup.+ cancer cells, CD200.sup.+ T cells,
etc.). Administration of a higher dose (e.g., 300 mg/m.sup.2 to 500
mg/m.sup.2) resulted in a more sustained immunomodulatory effect in
the human. Thus, the inventors concluded that administration of the
antibody at a higher amount and/or with an increased frequency to
thereby sustain the immunomodulatory effect in patients will be
more effective to treat a disease (e.g., a cancer, a bone disorder,
or an inflammatory disorder) in a human.
[0064] Accordingly, in yet another aspect, the disclosure features
a method for treating a human with cancer, which method includes
administering to the human an anti-CD200 antibody in an amount
and/or with a frequency effective to treat the cancer if the
anti-CD200 antibody is determined to have produced an anti-CD200
antibody-associated immunomodulatory effect in the human. The
determining can include, e.g., any of the methods described herein
for determining whether an anti-CD200 antibody has produced a
desired immunomodulatory effect in a human.
[0065] In another aspect, the disclosure features a method for
treating a patient afflicted with cancer, the method comprising:
administering to a patient in need thereof an anti-CD200 antibody
in an amount and with a frequency effective to maintain an
anti-CD200 antibody-associated immunomodulatory effect in the human
to thereby treat the patient's cancer. The immunomodulatory effect
can be indicated by, e.g., a change in one or more of any of the
anti-CD200 antibody-associated immunomodulatory biomarkers
described herein including the concentration of one or more
CD200.sup.+ bone marrow cell subsets and the level of CD200
expression by splenocytes or bone marrow cells (see below). In some
embodiments, the immunomodulatory biomarkers do not include the
concentration of one or more CD200.sup.+ bone marrow cell subsets
and/or the level of CD200 expression by bone marrow cells.
[0066] In some embodiments of any of the methods described herein,
the antibody can be administered to the patient in an amount and
with a frequency effective to maintain in the patient one or more
of the following conditions (e.g., as determined by an analysis
(e.g., a measurement, detection, or quantitation) of a biological
sample from the patient): (i) a reduced concentration of regulatory
T cells, relative to the concentration of regulatory T cells of the
same histological type in the patient prior to the first
administration of the antibody; (ii) an increased concentration of
CD8.sup.+ lymphocytes (e.g., T cells), relative to the
concentration of CD8.sup.+ lymphocytes of the same histological
type in the patient prior to the first administration of the
antibody; (iii) an increased concentration of activated T cells,
relative to the concentration of activated T cells of the same
histological type in the patient prior to the first administration
of the antibody; (iv) a reduced concentration of CD200.sup.+
lymphocytes (e.g., T cells), relative to the concentration of
CD200.sup.+ lymphocytes of the same histological type in the
patient prior to the first administration of the antibody; (v) an
increase in the concentration of CD200R.sup.+ lymphocytes (e.g., T
cells), relative to the concentration of CD200R.sup.+ lymphocytes
of the same histological type in the patient prior to the first
administration of the antibody; (vi) an increase in the ratio of
percent activated T cells to percent regulatory T cells, relative
to the corresponding ratio of a ratio in the patient prior to the
first administration of the antibody; (vii) a ratio of percent
activated T cells to percent regulatory T cells (T regs) of at
least 2:1 (e.g., at least 3:1, at least 4:1, at least 5:1, at least
6:1, or at least 7:1), relative to the ratio of activated T cells
to T regs in the patient prior to the first administration of the
antibody; (viii) a reduction in the level of CD200 expression by
the plurality of leukocytes as compared to the level of CD200
expression by a plurality of leukocytes of the same histological
type in the patient prior to the first administration of the
antibody; (ix) an increase in the level of CD200R expression by a
plurality of leukocytes as compared to the CD200R expression level
by a plurality of leukocytes of the same histological type in the
patient prior to the first administration of the antibody; and (x),
in embodiments where the cancer comprises a plurality of cells that
express (or overexpress) CD200, a reduction in the level of CD200
expression by a plurality of the CD200.sup.+ cancer cells, relative
to the level of CD200 expression by a corresponding plurality of
CD200.sup.+ cancer cells prior to the first administration of the
anti-CD200 antibody. In embodiments where an anti-CD200 antibody
has been administered to the patient two or more times, it is
understood that evaluation of one or more of the above parameters
can be (but need not necessarily be) relative (or as compared to)
the corresponding value of the parameter prior to the first dose of
the anti-CD200 antibody, the most recent administration of the
anti-CD200 antibody, or between doses of the anti-CD200 antibody
administered to the patient. For example, in embodiments where a
patient has been administered over time five (5) doses of an
anti-CD200 antibody, a decrease in the concentration of CD200.sup.+
lymphocytes (e.g., T cells), relative to the concentration of
CD200.sup.+ lymphocytes of the same histological type in the
patient prior to the fifth administration of the antibody can
indicate that a desired immunomodulatory effect has occurred in the
patient as the result of administration of the antibody.
[0067] In some embodiments, the anti-CD200 antibody is administered
to the patient in an amount and with a frequency to maintain all of
the foregoing conditions in the patient for the course of the
cancer treatment. In some embodiments, the antibody is administered
to the patient for at least four weeks (e.g., at least five weeks,
six weeks, seven weeks, eight weeks, two months, three months, four
months, five months, six months, seven months, eight months, nine
months, 10 months, 11 months, one year, 13 months, 14 months, 15
months, 16 months, one and a half years, two years, three years, or
four years or more).
[0068] In some embodiments, the cancer treatment methods described
herein can include administering an anti-CD200 antibody, e.g., a
whole antibody, to a patient in need thereof at an individual dose
of greater than or equal to 100 (e.g., greater than or equal to
150, 175, 200, 225, 250, 275, 300, 325, 350, 375, 400, 425, 450,
475, 500, 525, 550, 575, or 600) mg/m.sup.2 with a frequency of at
least about once every week (e.g., at least once every seven days,
eight days, nine days, 10 days, 11 days, 12 days, 13 days, 14 days,
15 days, 16 days, 17 days, 18 days, 19 days, or 20 days), depending
on the particular patient. In some embodiments, the anti-CD200
antibody can be administered to the patient at least once per week
(e.g., at least once every two weeks or three weeks). In some
embodiments, an individual dose of the anti-CD200 antibody can be
between (and inclusive of) 100 to 600 (e.g., between (and inclusive
of) 150 to 600, 200 to 600, 300 to 600, 100 to 500, 100 to 400, 100
to 300, 100 to 200, 200 to 500, 200 to 400, 200 to 300, 300 to 500,
400 to 500, or 300 to 500) mg/m.sup.2 and can be, in some
embodiments, administered to a patient, e.g., at least once every
seven days. In some embodiments, a patient can receive one dose of
an anti-CD200 antibody described herein once every day (e.g., every
two days, or every three days). As described above, it is
understood that depending on the individual patient parameters
(e.g., height, weight, gender, severity of disease, age,
co-morbidities, and additional medications), one or both of the
frequency and the amount of the anti-CD200 antibody can be modified
to maintain the immunomodulatory effect in the human. Methods for
determining the appropriate dosing strategy for maintaining one or
more of the immunomodulatory effect conditions in the patient are
described herein (infra).
[0069] In another aspect, the disclosure provides a method for
treating cancer, which includes administering to a patient
afflicted with a cancer an anti-CD200 antibody in an amount and
with a frequency effective to maintain an increased concentration
of activated T cells in the patient, as compared to the
concentration of activated T cells in the patient prior to
administration of the antibody, to thereby treat the cancer. The
method can also include after administering the anti-CD200 antibody
to the human, determining whether the concentration of activated T
cells has been increased in the patient.
[0070] In another aspect, the disclosure also features a method for
treating cancer, wherein the method includes administering to a
patient afflicted with cancer an anti-CD200 antibody in an amount
and with a frequency effective to maintain in the patient a reduced
concentration of regulatory T cells, as compared to the
concentration of regulatory T cells in the patient prior to
administration of the anti-CD200 antibody, to thereby treat the
cancer.
[0071] In another aspect, the disclosure also features a method for
treating cancer, the method comprising administering to a patient
afflicted with a cancer an anti-CD200 antibody in an amount and
with a frequency effective to maintain in the patient (e.g., as
determined by an analysis of a biological sample obtained from the
patient after administration of the antibody) a ratio of percent
activated T cells to percent regulatory T cells (T regs) of at
least 2:1 (e.g., at least 3:1, at least 4:1, at least 5:1, at least
6:1, or at least 7:1).
[0072] In some embodiments of any of the methods described herein,
the regulatory T cells can be, e.g., CD3.sup.+
CD4.sup.+CD25.sup.+FoxP3.sup.+ T cells or
CD3.sup.+CD4.sup.+FoxP3.sup.+ T cells. In some embodiments of any
of the methods described herein, the activated T cells can be,
e.g., CD3.sup.+CD4.sup.+CD25.sup.+FoxP3.sup.neg T cells or
CD3.sup.+CD4.sup.+FoxP3.sup.neg T cells.
[0073] In yet another aspect, the disclosure features a method for
determining an anti-CD200 antibody dosing schedule for treating a
patient determined by a medical practitioner to be one who will, or
is likely to, benefit from an anti-CD200 antibody therapy (e.g., a
patient suffering from a cancer, a bone disease, or an inflammatory
disorder). The method comprises, e.g., establishing a peak level or
maximum level of a desired immunomodulatory effect produced in a
patient following administration of an anti-CD200 antibody and
monitoring the patient (e.g., by way of analysis of a biological
sample from the patient) for a change away from that peak level of
effect, wherein the timing of that change in that peak level of
effect (e.g., the duration of time that the peak level of effect is
maintained in a patient at a given dose before an additional dose
is required to maintain that peak level of effect) determines the
dosing schedule for the patient in that a medical practitioner
determines the amount of the anti-CD200 antibody and/or frequency
of administration of the antibody that is necessary to maintain the
peak level of immunomodulatory effect in the patient for the
duration of treatment. In some embodiments, an additional dose (at
a higher dose and/or sooner than originally predetermined) of the
anti-CD200 antibody is administered to the patient if the level of
immunomodulatory effect changes by at least 5 (e.g., 6, 7, 8, 9,
10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 25, 30, 35, 40, 45, 50,
55, 60, 65, 70, 75, 80, 85, or 90 or more) % from the peak
immunomodulatory effect observed for the patient. For example, a
medical practitioner can observe that upon administration of the
first dose of an anti-CD200 antibody, the concentration of
CD200.sup.+ leukocytes decreases to a post-treatment concentration
X, the concentration X representing the peak level of
immunomodulatory effect in the patient. When the practitioner
observes that the post-treatment concentration of the CD200.sup.+
leukocytes increases by at least 5% (supra) from X, the
practitioner can elect to administer an additional dose of the
anti-CD200 antibody (at a higher amount or at the same amount as
the initial dose, but sooner than the practitioner had anticipated)
to the patient to thereby restore and maintain the concentration of
CD200.sup.+ leukocytes at the concentration X or below.
[0074] Thus, in some embodiments, the methods for determining an
anti-CD200 antibody dosing schedule can include monitoring the
level of a desired anti-CD200 antibody-associated immunomodulatory
effect in a patient who has been administered an anti-CD200
antibody to thereby determine for the patient a dosing schedule of
the antibody, wherein the dosing schedule is sufficient to maintain
the immunomodulatory effect in the patient for the duration of the
treatment with the antibody. The occurrence of a change in the peak
level of immunomodulatory effect in the patient can be the trigger
for administering to the patient a higher dose of the anti-CD200
antibody and/or administering the anti-CD200 antibody to the
patient more frequently to thereby maintain the peak level of
immunomodulatory effect in the patient and thereby determine a
dosing schedule for the antibody that achieves such
maintenance.
[0075] In some embodiments, the methods can include administering
to the patient an anti-CD200 antibody to thereby produce in the
patient an anti-CD200 antibody-associated immunomodulatory effect
(e.g., as indicated by a change (e.g., an increase or decrease) in
one or more of the anti-CD200 antibody-associated biomarkers in a
biological sample from the patient). In some embodiments, one or
more (e.g., two, three, four, five, six, seven, eight, nine, or 10
or more) of the following changes in the biomarkers can be
monitored: (i) a reduced concentration of regulatory T cells,
relative to the concentration of regulatory T cells of the same
histological type in the human prior to the first administration of
the antibody; (ii) an increased concentration of CD8.sup.+
leukocytes (e.g., T cells), relative to the concentration of
CD8.sup.+ leukocytes of the same histological type in the human
prior to the first administration of the antibody; (iii) an
increased concentration of activated T cells, relative to the
concentration of activated T cells of the same histological type in
the human prior to the first administration of the antibody; (iv) a
reduced concentration of CD200.sup.+ leukocytes (e.g., CD200.sup.+
T cells), relative to the concentration of CD200.sup.+ leukocytes
of the same histological type in the human prior to the first
administration of the antibody; (v) an increase in the
concentration of CD200R.sup.+ leukocytes (e.g., CD200R.sup.+ T
cells), relative to the concentration of CD200R.sup.+ leukocytes of
the same histological type in the human prior to the first
administration of the antibody; and (vi) a ratio of percent
activated T cells to percent regulatory T cells (T regs) of at
least 2:1 (e.g., at least 3:1, at least 4:1, at least 5:1, at least
6:1, or at least 7:1), relative to the ratio of activated T cells
to T regs in the human prior to the first administration of the
antibody. Additional changes that can be monitored as described
herein. For example, in some embodiments, a post-treatment decrease
in the concentration of one or more CD200.sup.+ bone marrow
subsets, as compared to the pre-treatment concentration of the
corresponding CD200.sup.+ bone marrow subsets, indicates that the
anti-CD200 antibody has produced a desired immunomodulatory effect
in the human. In some embodiments, a post-treatment decrease in the
level of CD200 expression by a plurality of splenocytes and/or bone
marrow cells (e.g., bone marrow cell subsets), as compared to the
pre-treatment level of expression by the corresponding plurality,
indicates that the anti-CD200 antibody has produced a desired
immunomodulatory effect in the human. It is understood that the
monitoring can comprise, e.g., measuring the concentration of the
appropriate selected cell type (e.g., CD200.sup.+ or CD200R.sup.+
leukocytes); quantifying the level of expression of one or more
expression markers such as CD200; or determining the ratio of
percent activated T cells to percent regulatory T cells. In some
embodiments, even a partial reversal of the status of one or more
of these anti-CD200 antibody-associated biomarkers indicates that a
medical practitioner should increase the amount of the anti-CD200
antibody administered to the patient and/or increase the frequency
of administration of the anti-CD200 antibody to the patient, to
thereby maintain in the patient the anti-CD200 antibody-associated
immunomodulatory effect.
[0076] In another aspect, the disclosure features a method for
determining a dosing schedule for treating a patient suffering from
a cancer using an anti-CD200 antibody, the method comprising:
providing a patient suffering from a cancer comprising a plurality
of cancer cells expressing CD200; administering to the patient an
anti-CD200 antibody to thereby reduce the expression of CD200 by
the cancer cells; and monitoring the CD200 expression level by the
cancer cells to thereby determine for the patient a dosing schedule
of the antibody, wherein the dosing schedule is sufficient to
maintain a reduced CD200 expression level (e.g., as compared to the
pretreatment level) by the cancer cells, e.g., for the duration of
the treatment with the antibody. In some embodiments, the CD200
expression level by the cancer cells can be reduced by at least 10
(e.g., at least 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75,
80, 85, or 90 or more) %.
[0077] In another aspect, the disclosure features a method for
determining the dosing schedule for treating a patient suffering
from a cancer using an anti-CD200 antibody, the method comprising:
administering to a patient suffering from a cancer an anti-CD200
antibody to thereby reduce the concentration of CD200.sup.+
leukocytes (e.g., CD200.sup.+ T cells) as measured in a blood
sample obtained from the patient as compared to the concentration
of CD200.sup.+ T cells in a control sample; and monitoring the
concentration of CD200.sup.+ leukocytes (e.g., CD200.sup.+ T cells)
in the patient to thereby determine for the patient a dosing
schedule of the antibody, wherein the dosing schedule is sufficient
to maintain a reduced concentration of the CD200.sup.+ T cells in
the patient for the duration of the treatment of cancer with the
antibody. In some embodiments, the concentration of CD200.sup.+
leukocytes (e.g., CD200.sup.+ T cells) can be reduced by at least
10 (e.g., at least 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70,
75, 80, 85, or 90 or more) %. In some embodiments, the CD200.sup.+
leukocytes (e.g., CD200.sup.+ T cells) are selected from the group
consisting of CD200.sup.+/CD4.sup.+ T cells, activated
CD200.sup.+/CD4.sup.+ T cells, or CD200.sup.+/CD8.sup.+ T
cells.
[0078] In another aspect, the disclosure features a method for
determining the dosing schedule for treating a patient suffering
from a cancer using an anti-CD200 antibody, the method comprising:
administering to a patient suffering from a cancer an anti-CD200
antibody to thereby reduce the level of expression of CD200 by
leukocytes in a blood sample obtained from the patient as compared
to a control expression level of CD200 by leukocytes of the same
histological type in a control sample; and monitoring the
expression level of CD200 by leukocytes in the patient to thereby
determine for the patient a dosing schedule of the antibody,
wherein the dosing schedule is sufficient to maintain a reduced
level of expression of CD200 by the leukocytes (reduced as compared
to the control sample) in the patient for the duration of the
treatment of cancer with the antibody. In some embodiments, the
level of CD200 expression by the leukocytes can be reduced by at
least 10 (e.g., at least 15, 20, 25, 30, 35, 40, 45, 50, 55, 60,
65, 70, 75, 80, 85, or 90 or more) %. In some embodiments, the
CD200.sup.+ leukocytes are selected from the group consisting of
CD200.sup.+/CD4.sup.+ T cells, activated CD200.sup.+/CD4.sup.+ T
cells, or CD200.sup.+/CD8.sup.+ T cells.
[0079] In another aspect, the disclosure features a method for
determining the dosing schedule for treating a patient suffering
from a cancer using an anti-CD200 antibody, the method comprising:
administering to a patient suffering from a cancer an anti-CD200
antibody to thereby increase the concentration of CD200R.sup.+
leukocytes as measured in a blood sample obtained from the patient
as compared to the concentration of CD200R.sup.+ leukocytes in a
control sample; and monitoring the concentration of CD200R.sup.+
leukocytes in the patient to thereby determine for the patient a
dosing schedule of the antibody, wherein the dosing schedule is
sufficient to maintain an increased concentration of the
CD200R.sup.+ leukocytes (increased as compared to the control
sample) in the patient for the duration of the treatment of cancer
with the antibody. In some embodiments, the concentration of
CD200R.sup.+ leukocytes can be increased by at least 10 (e.g., at
least 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85,
90, 95, or 100 or more) %. In some embodiments, the CD200R.sup.+ T
cells are selected from the group consisting of
CD200R.sup.+/CD4.sup.+ T cells and activated CD200R.sup.+/CD4.sup.+
T cells.
[0080] In yet another aspect, the disclosure features a method for
determining the dosing schedule for treating a patient suffering
from a cancer using an anti-CD200 antibody, the method comprising:
administering to a patient suffering from a cancer an anti-CD200
antibody to thereby increase the level of expression of CD200R by
leukocytes as measured in a blood sample obtained from the patient
as compared to a control expression level of CD200R by leukocytes
of the same histological type in a control sample; and monitoring
the expression level of CD200R by leukocytes in the patient to
thereby determine for the patient a dosing schedule of the
antibody, wherein the dosing schedule is sufficient to maintain an
increased level of expression of CD200R (e.g., as compared to the
pre-treatment expression level) by the leukocytes in the patient
for the duration of the treatment of cancer with the antibody. In
some embodiments, the level of CD200R expression by the leukocytes
can be increased by at least 10 (e.g., at least 15, 20, 25, 30, 35,
40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, or 100 or more) %.
In some embodiments, the leukocytes are T cells such as CD4.sup.+ T
cells or activated CD4.sup.+ T cells.
[0081] In yet another aspect, the disclosure features a method for
treating a human suffering from a cancer comprising a plurality of
cancer cells expressing CD200, the method comprising administering
to a human in need thereof an anti-CD200 antibody in an amount and
with a frequency sufficient to reduce the CD200 expression level
(e.g., as compared to the pre-treatment expression level) by the
cancer cells to thereby treat the human's cancer. The method can
also include monitoring the human for a reduction in the CD200
expression level by the cancer cells.
[0082] In another aspect, the disclosure also features a method for
treating a human suffering from a cancer, the method comprising
administering to a human in need thereof an anti-CD200 antibody in
an amount and with a frequency sufficient to reduce the
concentration of CD200.sup.+ leukocytes (e.g., T cells) in the
blood of a cancer patient to thereby treat the human's cancer. The
method can also include monitoring the human for a reduction in the
CD200.sup.+ leukocytes in the blood of the patient.
[0083] In another aspect, the disclosure features a method for
treating a human suffering from a cancer, the method comprising
administering to a human in need thereof an anti-CD200 antibody in
an amount and with a frequency sufficient to result in an increase
in the concentration of CD200R.sup.+ leukocytes in the blood of a
cancer patient to thereby treat the human's cancer. The method can
include monitoring the human for a reduction in the CD200.sup.+
leukocytes in the blood of the patient.
[0084] In another aspect, the disclosure features a method for
treating a human suffering from a cancer, the method comprising
administering to a human in need thereof an anti-CD200 antibody in
an amount and with a frequency sufficient to reduce the expression
level of CD200 by T cells in the blood of a cancer patient to
thereby treat the human's cancer. The method can also include
monitoring the human for a reduction in the expression level of
CD200 by T cells in the blood of the patient.
[0085] In another aspect, the disclosure features a method for
treating a human suffering from a cancer, the method comprising
administering to a human in need thereof an anti-CD200 antibody in
an amount and with a frequency sufficient to result in an increase
in the expression level of CD200R by leukocytes in the blood of a
cancer patient to thereby treat the human's cancer. The method can
also include monitoring the human for a reduction in the expression
level of CD200R by leukocytes in the blood of the patient.
[0086] In some embodiments of any of the above methods, the cancer
can be one that comprises a plurality of cancer cells expressing
CD200. In some embodiments of any of the methods described herein,
the cancer is one that comprises a plurality of cancer cells that,
relative to non-cancer cells of the same histological type,
overexpresses CD200.
[0087] In some embodiments of any of the methods described herein,
the subject (e.g., the human or the patient) is one who does not
have chronic lymphocytic leukemia (CLL) such as B cell CLL.
[0088] In some embodiments of the methods described herein, a
single dose of an anti-CD200 antibody is sufficient to produce a
desired immunomodulatory effect in a human. In some embodiments, a
single dose of an anti-CD200 antibody is sufficient to produce a
clinically meaningful effect on a patient's cancer. In some
embodiments of the methods for treatment described herein, two or
more (e.g., three, four, five, six, seven, eight, nine, or 10 or
more) doses of the anti-CD200 antibody are administered to a
patient in need thereof, e.g., to treat the patient's cancer,
inflammatory disorder, or bone disorder. In embodiments in which
two or more doses of the antibody are administered to a human
(e.g., a patient), each of the two or more doses can be
administered at least 7 (e.g., 8, 9, 10, 11, 12, 13, 14, 15, 16,
17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30 or more)
days apart. In some embodiments, the two or more doses are
administered to the patient over the course of at least one (e.g.,
at least two, three, four, five, or six) month(s). For example, a
medical practitioner may elect to administer at least four doses of
an anti-CD200 antibody to a cancer patient, each of the doses to be
administered once every two weeks (14 days) for two months. It is
understood that the medical practitioner can elect to continue
treatment under the same or a different dosing schedule.
[0089] In yet another aspect, the disclosure features a method for
treating a patient afflicted with a disorder selected from the
group consisting of a bone disorder and an inflammatory disorder,
the method comprising: administering to a patient in need thereof
an anti-CD200 antibody in an amount and with a frequency effective
to maintain an anti-CD200 antibody-associated immunomodulatory
effect in the human to thereby treat the patient's disorder. The
immunomodulatory effect can be indicated by, e.g., any of the
anti-CD200 antibody-associated immunomodulatory biomarkers
described herein. That is, in some embodiments, the antibody can be
administered to the patient in an amount and with a frequency
effective to maintain in the patient one or more of the following
conditions (e.g., as determined by an analysis (e.g., a
measurement, detection, or quantitation) of a biological sample
from the patient): (i) a reduced concentration of regulatory T
cells, relative to the concentration of regulatory T cells of the
same histological type in the patient prior to the first
administration of the antibody; (ii) an increased concentration of
CD8.sup.+ lymphocytes (e.g., T cells), relative to the
concentration of CD8.sup.+ lymphocytes of the same histological
type in the patient prior to the first administration of the
antibody; (iii) an increased concentration of activated T cells,
relative to the concentration of activated T cells of the same
histological type in the patient prior to the first administration
of the antibody; (iv) a reduced concentration of CD200.sup.+
lymphocytes (e.g., T cells), relative to the concentration of
CD200.sup.+ lymphocytes of the same histological type in the
patient prior to the first administration of the antibody; (v) an
increase in the concentration of CD200R lymphocytes (e.g., T
cells), relative to the concentration of CD200R.sup.+ lymphocytes
of the same histological type in the patient prior to the first
administration of the antibody; (vi) an increase in the ratio of
percent activated T cells to percent regulatory T cells, relative
to the corresponding ratio in the patient prior to the first
administration of the antibody; (vii) a ratio of percent activated
T cells to percent regulatory T cells (T regs) of at least 2:1
(e.g., at least 3:1, at least 4:1, at least 5:1, at least 6:1, or
at least 7:1), relative to the ratio of activated T cells to T regs
in the patient prior to the first administration of the antibody;
(viii) a reduction in the level of CD200 expression by the
plurality of leukocytes as compared to the level of CD200
expression by a plurality of leukocytes of the same histological
type in the patient prior to the first administration of the
antibody; and (ix) an increase in the level of CD200R expression by
a plurality of leukocytes as compared to the CD200R expression
level by a plurality of leukocytes of the same histological type in
the patient prior to the first administration of the antibody. In
some embodiments, a post-treatment decrease in the concentration of
one or more CD200.sup.+ bone marrow subsets, as compared to the
pre-treatment concentration of the corresponding CD200.sup.+ bone
marrow subsets, indicates that the anti-CD200 antibody has produced
a desired immunomodulatory effect in the human. In some
embodiments, a post-treatment decrease in the level of CD200
expression by a plurality of splenocytes and/or bone marrow cells
(e.g., bone marrow cell subsets), as compared to the pre-treatment
level of expression by the corresponding plurality, indicates that
the anti-CD200 antibody has produced a desired immunomodulatory
effect in the human. In embodiments in which an anti-CD200 antibody
has been administered to the patient two or more times, it is
understood that evaluation of one or more of the above parameters
can be (but need not necessarily be) relative (or as compared to)
the corresponding value of the parameter prior to the first dose of
the antibody, the most recent administration of the anti-CD200
antibody, or between two administered doses of the antibody. For
example, in embodiments where a patient has been administered over
time five (5) doses of an anti-CD200 antibody, a decrease in the
concentration of CD200.sup.+ lymphocytes (e.g., T cells), relative
to the concentration of CD200.sup.+ lymphocytes of the same
histological type in the patient prior to the fifth administration
of the antibody can indicate that a desired immunomodulatory effect
has occurred in the patient as the result of administration of the
antibody. For example, in embodiments where a patient has been
administered over time five (5) doses of an anti-CD200 antibody, a
decrease in the concentration of CD200.sup.+ lymphocytes (e.g., T
cells), relative to the concentration of CD200.sup.+ lymphocytes of
the same histological type in the patient after the third
administration of the antibody, but prior to the fourth
administration of the antibody, can indicate that a desired
immunomodulatory effect has occurred in the patient as the result
of administration of the antibody.
[0090] In another aspect, the disclosure also features a method for
treating a human suffering from a bone disorder or an inflammatory
disorder, the method comprising administering to a human in need
thereof an anti-CD200 antibody in an amount and with a frequency
sufficient to reduce the concentration of CD200.sup.+ T cells in
the blood of the human to thereby treat the human's bone disorder
or an inflammatory disorder. The method can also include monitoring
for a reduction in the CD200.sup.+ T cells in the blood of the
human.
[0091] In another aspect, the disclosure features a method for
treating a human suffering from a bone disorder or an inflammatory
disorder, the method comprising administering to a human in need
thereof an anti-CD200 antibody in an amount and with a frequency
sufficient to result in an increase in the concentration of
CD200R.sup.+ leukocytes in the blood of the human to thereby treat
the human's bone disorder or an inflammatory disorder. The method
can include monitoring for a reduction in the CD200.sup.+
leukocytes in the blood of the human.
[0092] In another aspect, the disclosure features a method for
treating a human suffering from a bone disorder or an inflammatory
disorder, the method comprising administering to a human in need
thereof an anti-CD200 antibody in an amount and with a frequency
sufficient to reduce the expression level of CD200 by T cells in
the blood of the human to thereby treat the human's bone disorder
or an inflammatory disorder. The method can also include monitoring
for a reduction in the expression level of CD200 by T cells in the
blood of the human.
[0093] In another aspect, the disclosure features a method for
treating a human suffering from a bone disorder or an inflammatory
disorder, the method comprising administering to a human in need
thereof an anti-CD200 antibody in an amount and with a frequency
sufficient to result in an increase in the expression level of
CD200R by leukocytes in the blood of the human to thereby treat the
human's bone disorder or an inflammatory disorder. The method can
also include monitoring for a reduction in the expression level of
CD200R by leukocytes in the blood of the human.
[0094] In another aspect, the disclosure also features a method for
treating a human suffering from a bone disorder or an inflammatory
disorder, the method comprising administering to a human in need
thereof an anti-CD200 antibody in an amount and with a frequency
sufficient to reduce the concentration of CD200.sup.+ T cells in
the blood of the human to thereby treat the human's bone disorder
or an inflammatory disorder. The method can also include monitoring
for a reduction in the CD200.sup.+ T cells in the blood of the
human.
[0095] In another aspect, the disclosure also features a method
that results in reducing the concentration of CD200.sup.+
leukocytes (e.g., T cells such as CD4.sup.+ T cells) in the blood
of a patient, the method comprising administering to a patient in
need thereof an anti-CD200 antibody in an amount effective to
reduce the concentration of CD200.sup.+ leukocytes in the blood of
the patient. The patient can have, be suspected of having, or be at
risk for developing a cancer, an inflammatory disorder, or a bone
disorder.
[0096] In another aspect, the disclosure also features a method
that results in an increase in the concentration of CD200R.sup.+
leukocytes (e.g., T cells such as CD4.sup.+ T cells) in the blood
of a patient, the method comprising administering to a patient in
need thereof an anti-CD200 antibody in an amount effective to
result in an increase in the concentration of CD200R.sup.+
leukocytes (e.g., T cells such as CD4.sup.+ T cells) in the blood
of the patient. The patient can have, be suspected of having, or be
at risk for developing a cancer, an inflammatory disorder, or a
bone disorder.
[0097] In another aspect, the disclosure also features a method for
reducing the expression of CD200 by leukocytes (e.g., T cells such
as CD4.sup.+ T cells) in the peripheral blood of a patient, the
method comprising administering to a patient in need thereof an
anti-CD200 antibody in an amount effective to reduce the expression
of CD200 by leukocytes in the blood of the patient. The patient can
have, be suspected of having, or be at risk for developing a
cancer, an inflammatory disorder, or a bone disorder.
[0098] In another aspect, the disclosure also features a method
that results in an increase in the expression of CD200R by
leukocytes (e.g., T cells such as CD4.sup.+ T cells) in the blood
of a patient, the method comprising administering to a patient in
need thereof an anti-CD200 antibody in an amount effective to
result in an increase in the expression of CD200R by leukocytes in
the blood of the patient. The patient can have, be suspected of
having, or be at risk for developing a cancer, an inflammatory
disorder, or a bone disorder.
[0099] In yet another aspect, the disclosure features a method for
increasing the concentration of activated T cells in a patient in
need thereof (e.g., a cancer patient), the method comprising
administering to the patient an anti-CD200 antibody in an amount
and with a frequency effective to increase the concentration of
activated T cells in the patient.
[0100] In another aspect, the disclosure features a method for
decreasing the concentration of regulatory T cells in a patient in
need thereof (e.g., a cancer patient), the method comprising
administering an anti-CD200 antibody to the patient in an amount
and with a frequency effective to reduce the concentration of
regulatory T cells in the patient.
[0101] In another aspect, the disclosure features a method for
increasing the ratio of percent activated T cells to percent
regulatory T cells in a patient in need thereof (e.g., a cancer
patient), the method comprising administering to the patient an
anti-CD200 antibody in an amount and with a frequency effective to
increase the ratio of percent activated T cells to percent
regulatory T cells in the patient. In some embodiments, the ratio
of percent activated T cells to percent regulatory T cells is
increased to at least 2:1 (e.g., at least 3:1, 4:1, 5:1, 6:1, or
even 7:1 or higher).
[0102] In some embodiments of any of the above methods, the cancer
(or the patient is afflicted with a cancer that) comprises a
plurality of cancer cells expressing CD200. In some embodiments,
the cancer (or the patient is afflicted with a cancer that)
comprises a plurality of cells that, relative to non-cancer cells
of the same histological type as the cells from which cancer is
derived, overexpress CD200.
[0103] In some embodiments, any of the above treatment methods can
be practiced in conjunction with any of the methods described
herein for determining whether an anti-CD200 antibody has produced
a desired immunomodulatory effect in a human.
[0104] The inventors have also discovered an inverse correlation
between the peripheral tumor load (e.g., B CLL tumor cell load) and
the concentration of T cells present in cancer patients. That is,
the greater the concentration of non-cancer T cells present in a
cancer patient, the lower the tumor burden in the patient. While
the discovery is not limited by any particular theory or mechanism
of action, the inventors believe that a cancer patient may receive
an enhanced benefit from an anti-CD200 antibody therapy if the
cancer patient has normal or elevated levels of normal T cells in
his or her body at the time of therapy. In other words, the
inventors have determined that an anti-CD200 antibody therapy will
likely have more efficacy and/or a stronger immunomodulatory effect
in patients with an intact immune system (or not
immunocompromised), e.g., an immune system that is capable of
mounting an immune response against a cancer present in the
patient. As described below, all four of the cancer patients who
had not received prior chemotherapy before samalizumab treatment
had clinically stable or improved disease after samalizumab
treatment. In fact, patient 102-502, who had not received an
immunosuppressive or chemotherapeutic therapy prior to
administration of the anti-CD200 antibody, exhibited a marked
reduction in tumor burden, which correlated with changes in a
number of the biomarkers described herein, including, a marked
reduction in the concentration of CD45.sup.+ B CLL cells, an
increase in CD8.sup.+ T cells, a decrease in regulatory T cells, an
increase in activated T cells, and an increase in the ratio of
percent activated T cells to percent regulatory T cells.
[0105] Accordingly, in some embodiments of any of the methods
described herein (e.g., the methods for treatment, e.g., methods
for treating cancer described herein), the subject (e.g., the
patient or the human) is one that has not received an
immunosuppressive therapy and/or a chemotherapeutic therapy prior
to administration of the anti-CD200 antibody. Examples of
chemotherapeutic and immunosuppressive therapies are described
herein and known in the art. In some embodiments, the subject or
patient or human has not received an immunosuppressive or
chemotherapeutic therapy less than two months (e.g., less than
eight weeks, seven weeks, six weeks, five weeks, a month, 30 days,
25 days, 20 days, 15 days, or 10 days) prior to administration of
the first dose of the anti-CD200 antibody.
[0106] In some embodiments of any of the methods described herein,
the subject is one that has an immune system that is competent to
mount an immune response against the subject's cancer. That is, the
subject (e.g., the patient) is not immunocompromised. In some
embodiments, the subject has not received a chemotherapeutic agent
or any other agent capable of suppressing the immune system of the
patient less than two months before the first dose of an anti-CD200
antibody is administered to the patient. In some embodiments, the
patient is one who is not infected with HIV as determined by, e.g.,
any of one of several commercially available tests for HIV
infection. In some embodiments, the patient is one who does not
have an active HIV infection.
[0107] In some embodiments, a patient's immune system can be
competent to mount an immune response to a cancer only in the
presence of the anti-CD200 antibody (that is, with the aid of the
immunomodulatory effect produced by the antibody following
administration to the subject). In some embodiments, the subject's
immune system is competent to mount an immune response to the
cancer even in the absence of the anti-CD200 antibody--the antibody
enhancing the ability of the immune system to mount an immune
response against the cancer. One method for determining whether the
subject's immune system is competent to mount an immune response is
to determine the concentration of CD3.sup.+ cells in the subject's
blood. Additional methods are known in the art and described
herein.
[0108] In another aspect, the disclosure features a method for
selecting a cancer patient for treatment with an anti-CD200
antibody, wherein the method comprises determining whether the
patient is immunocompetent, and if the cancer patient is
immunocompetent, administering an anti-CD200 antibody to the cancer
patient. The method can include, e.g., measuring the concentration
or absolute number of one or more subsets of immune cells in a
biological sample obtained from the patient prior to administration
of an anti-CD200 antibody. Exemplary cell types, subsets, and
ranges of concentration and number of cell subsets indicative of
immunocompetence are described herein. See section entitled
"Methods for Treatment" (below). Methods for measuring the
concentration or absolute number of one or more cell subsets in a
biological sample from a patient are known in the art and
exemplified herein in the working examples. In some embodiments,
the method comprises: quantifying the concentration of CD3.sup.+
cells present in a biological sample from a patient suffering from
a cancer and administering to the patient the anti-CD200 antibody
in an amount effective to treat the cancer in the patient if the
concentration of CD3.sup.+ cells in the biological sample is
sufficient to aid the anti-CD200 therapy in the subject (e.g., if
the concentration of CD3.sup.+ cells is greater than 300 per
microliter). In some embodiments, the antibody is administered to
the patient if the concentration of CD3.sup.+/CD4.sup.+ cells in
the biological sample is greater than or equal to 200 cells per
microliter. In some embodiments, the antibody is administered to
the patient if the concentration of CD3.sup.+/CD4.sup.+ cells in
the biological sample is greater than or equal to 400 cells per
microliter. In some embodiments, the antibody is administered to
the patient if the concentration of CD3.sup.+/CD8.sup.+ cells in
the biological sample is greater than or equal to 150 cells per
microliter. In some embodiments, the antibody is administered to
the patient if the concentration of CD3.sup.+/CD8.sup.+ cells in
the biological sample is greater than or equal to 500 cells per
microliter.
[0109] In some embodiments of any of the methods described herein,
the anti-CD200 antibody is an IgG1, IgG2, IgG3, IgG4, IgM, IgA1,
IgA2, IgA, IgD, or IgE antibody. In some embodiments, the
anti-CD200 antibody is a murine antibody, a chimeric antibody, a
humanized antibody, a single chain antibody, or a human
antibody.
[0110] In some embodiments of any of the methods described herein,
the anti-CD200 antibody comprises a variant constant region that
has decreased (reduced) or no effector function. In some
embodiments, the variant constant region has less than 90 (e.g.,
less than 85, 80, 75, 70, 65, 60, 55, 50, 45, 40, 35, 30, 25, 20,
15, or 10) % of the effector function activity of the corresponding
non-variant form of the constant region. In some embodiments, a
variant constant region has between about 0 to 30 (e.g., between
about 5 to 30, 10 to 30, 10 to 20, 0 to 20, 0 to 15, 0 to 10, 0 to
25, or 0 to 5) % of the effector function activity of the
corresponding non-variant form of the constant region. For example,
an antibody described herein can contain an IgG1 variant constant
region that exhibits, e.g., less than 20% (or in another example,
between 0 to 20%) of the effector function activity of the
corresponding non-variant IgG1 constant region. The variant
anti-CD200 antibody constant region, as compared to the
corresponding non-variant constant region, can have one or more of:
reduced or no antibody-dependent cell-mediated cytotoxicity (ADCC)
activity; reduced or no complement dependent cytotoxicity (CDC);
and decreased binding to one or more Fc receptors. In some
embodiments, the anti-CD200 antibody comprises a variant constant
region that has been engineered to comprise at least one amino acid
substitution, insertion, or deletion resulting in the reduced or no
effector function. In some embodiments, the anti-CD200 antibody
comprises a variant constant region comprising one or more of the
following characteristics: (i) altered glycosylation and (ii) an
Ala-Ala mutation. The altered glycosylation comprises one or more
of the following: (i) a change in one or more sugar components;
(ii) presence of one or more additional sugar components; and (iii)
absence of one or more sugar components. In some embodiments, the
anti-CD200 antibody can comprise, e.g., a hybrid IgG2/IgG4 constant
region that has reduced or no effector function.
[0111] In some embodiments, the variant constant region having
reduced or no effector function comprises a substitution at
position 265 (relative to Kabat numbering, infra), e.g., an
aspartate 265 to alanine substitution. See, e.g., Baudino et al.
(2008) J Immunol 181:6664-6669. In some embodiments, the variant
constant region having reduced or no effector function comprises
one, two, or three of the following substitutions: L234F, L235E, or
P331S, which have been shown to reduce substantially ADCC and CDC
activity of variant constant regions in which they are present.
See, e.g., Organesyan et al. (2008) Acta Cryst D64:700-704.
Additional modifications to a constant region, to thereby result in
a variant constant region with reduced or no effector function, are
known in the art and recited herein.
[0112] In some embodiments of any of the methods described herein,
the anti-CD200 antibody inhibits the interaction between CD200 and
CD200R.
[0113] In some embodiments of any of the methods described herein,
the anti-CD200 antibody contains the following paired set of CDRs:
a heavy chain CDR1 (HCDR1) comprising the amino acid sequence:
GFTFSGFAMS (SEQ ID NO:4); a heavy chain CDR2 (HCDR2) comprising the
amino acid sequence: SISSGGTTYYLDSVKG (SEQ ID NO:5); a heavy chain
CDR3 (HCDR3) comprising the amino acid sequence: GNYYSGTSYDY (SEQ
ID NO:6); a light chain CDR1 (LCDR1) comprising the amino acid
sequence: RASESVDSYGNSFMH (SEQ ID NO:7); a light chain CDR2 (LCDR2)
comprising the amino acid sequence: RASNLES (SEQ ID NO:8); and a
light chain CDR3 (LCDR3) comprising the amino acid sequence:
QQSNEDPRT (SEQ ID NO:9).
[0114] In some embodiments of any of the methods described herein,
the anti-CD200 antibody contains the following paired set of CDRs:
a HCDR1 comprising the amino acid sequence: GFNIKDYYMH (SEQ ID
NO:10); a HCDR2 comprising the amino acid sequence:
WIDPENGDTKYAPKFQG (SEQ ID NO:11); a HCDR3 comprising the amino acid
sequence: KNYYVSNYNFFDV (SEQ ID NO:12); a LCDR1 comprising the
amino acid sequence: SASSSVRYMY (SEQ ID NO:13); a LCDR2 comprising
the amino acid sequence: DTSKLAS (SEQ ID NO:14); and a LCDR3
comprising the amino acid sequence: FQGSGYPLT (SEQ ID NO:15).
[0115] In some embodiments of any of the methods described herein,
the anti-CD200 antibody contains the following paired set of CDRs:
a HCDR1 comprising the amino acid sequence: GFNIKDYYIH (SEQ ID
NO:16); a HCDR2 comprising the amino acid sequence:
WIDPEIGATKYVPKFQG (SEQ ID NO:17); a HCDR3 comprising the amino acid
sequence: LYGNYDRYYAMDY (SEQ ID NO:18); a LCDR1 comprising the
amino acid sequence: KASQNVRTAVA (SEQ ID NO:19); a LCDR2 comprising
the amino acid sequence: LASNRHT (SEQ ID NO:20); and a LCDR3
comprising the amino acid sequence: LQHWNYPLT (SEQ ID NO:21).
[0116] In some embodiments of any of the methods described herein,
the anti-CD200 antibody contains the following paired set of CDRs:
a HCDR1 comprising the amino acid sequence: GYSFTDYIIL (SEQ ID
NO:22); a HCDR2 comprising the amino acid sequence:
HIDPYYGSSNYNLKFKG (SEQ ID NO:23); a HCDR3 comprising the amino acid
sequence: SKRDYFDY (SEQ ID NO:24); a LCDR1 comprising the amino
acid sequence: KASQDINSYLS (SEQ ID NO:25); a LCDR2 comprising the
amino acid sequence: RANRLVD (SEQ ID NO:26); and a LCDR3 comprising
the amino acid sequence: LQYDEFPYT (SEQ ID NO:27).
[0117] In some embodiments of any of the methods described herein,
the anti-CD200 antibody contains the following paired set of CDRs:
a HCDR1 comprising the amino acid sequence: GYTFTEYTMH (SEQ ID
NO:28); a HCDR2 comprising the amino acid sequence:
GVNPNNGGALYNQKFKG (SEQ ID NO:29); a HCDR3 comprising the amino acid
sequence: RSNYRYDDAMDY (SEQ ID NO:30); a LCDR1 comprising the amino
acid sequence: KSSQSLLDIDEKTYLN (SEQ ID NO:31); a LCDR2 comprising
the amino acid sequence: LVSKLDS (SEQ ID NO:32); and a LCDR3
comprising the amino acid sequence: WQGTHFPQT (SEQ ID NO:33).
[0118] In some embodiments of any of the methods described herein,
the anti-CD200 antibody contains the following paired set of CDRs:
a HCDR1 comprising the amino acid sequence: AFNIKDHYMH (SEQ ID
NO:34); a HCDR2 comprising the amino acid sequence:
WIDPESGDTEYAPKFQG (SEQ ID NO:35); a HCDR3 comprising the amino acid
sequence: FNGYQALDQ (SEQ ID NO:36); a LCDR1 comprising the amino
acid sequence: TASSSVSSSYLH (SEQ ID NO:37); a LCDR2 comprising the
amino acid sequence: STSNLAS (SEQ ID NO:38); and a LCDR3 comprising
the amino acid sequence: RQYHRSPPIFT (SEQ ID NO:39).
[0119] The inventors have also discovered several biomarkers
evidencing the occurrence in a human of a desired immunomodulatory
effect by an anti-CD200 antibody administered to animals with an
autoimmune disorder. For example, the inventors have observed that
following administration of an anti-CD200 antibody to an animal,
the concentration of several leukocyte (e.g., splenocyte) and bone
marrow cell subsets is reduced in the animals. The inventors have
also discovered that the concentration of, e.g., F4/80.sup.+
lymphocytes in spleen are increased following administration of the
anti-CD200 antibody to the animal. While the disclosure is not
bound by any particular theory or mechanism of action, the
inventors believe that monitoring a patient treated with an
anti-CD200 antibody for a change (e.g., an increase or decrease) in
one or more of these biomarkers is useful for, among other things,
determining whether the anti-CD200 antibody is capable of producing
a desired immunomodulatory effect in the human to which the
antibody is administered. Moreover, one or more of the biomarkers
are also useful for identifying a dose--a threshold dose (or a
therapeutic dosing schedule)--of an anti-CD200 antibody, such as
samalizumab, that by virtue of its immunomodulatory effect in the
human is sufficient to achieve a clinically-meaningful effect on
the disease (i.e., sufficient to treat a disease such as cancer or
an autoimmune disorder). As described in the working examples, an
anti-CD200 antibody was capable of reducing the concentration of
autoimmune antibodies in a mouse model of autoimmune disease.
[0120] Accordingly, the disclosure features a method for
determining whether an anti-CD200 antibody has produced a desired
immunomodulatory effect in a human. The method includes measuring
the concentration of one or more subsets of CD200.sup.+ lymphocytes
in a biological sample obtained from a human administered an
anti-CD200 antibody, wherein a reduction in the concentration of
one or more subsets of CD200.sup.+ lymphocytes in a biological
sample as compared to the concentration of the same subsets of
CD200.sup.+ lymphocytes in a control sample indicates that the
anti-CD200 antibody has produced a desired immunomodulatory effect
in the human. The lymphocytes can be, e.g., splenocytes or bone
marrow cell subsets.
[0121] In some embodiments of any of the methods described herein,
the CD200.sup.+ lymphocyte subsets can be, e.g.,
CD3.sup.+/CD200.sup.+ lymphocytes, CD45R.sup.+/CD200.sup.+
lymphocytes, CD5.sup.+/CD200.sup.+ lymphocytes,
CD19.sup.+/CD200.sup.+ lymphocytes, CD138.sup.+/CD200.sup.+
lymphocytes, or CD200R.sup.+/CD200.sup.+ lymphocytes. In some
embodiments of any of the methods described herein, at least a 5%,
10%, 20%, 30%, 40%, 50%, 60%, 70%, or 80% or more reduction of the
concentration of the one or more subsets of CD200.sup.+ lymphocytes
indicates that a desired immunomodulatory effect has been produced
in the human.
[0122] In some embodiments, a reduction in the concentration of one
or more subsets of CD200.sup.+ lymphocytes in the biological sample
as compared to the concentration of the same subsets of CD200.sup.+
lymphocytes in the control sample indicates that the antibody is
therapeutically effective in the human.
[0123] In some embodiments of any of the methods described herein,
the biological sample is a blood sample. In some embodiments, the
biological sample comprises, or consists of, spleen tissue or bone
marrow tissue.
[0124] In another aspect, the disclosure features a method for
determining whether an anti-CD200 antibody has produced a desired
immunomodulatory effect in a human, which method includes measuring
the concentration of one or more subsets of CD200.sup.+ bone marrow
cells in a biological sample obtained from a human administered an
anti-CD200 antibody, wherein a reduction in the concentration of
one or more subsets of CD200.sup.+ bone marrow cells in a
biological sample as compared to the concentration of the same
subsets of CD200.sup.+ bone marrow cells in a control sample
indicates that the anti-CD200 antibody has produced a desired
immunomodulatory effect in the human. The subsets of CD200.sup.+
bone marrow cells can be, e.g., Igk.sup.+/CD200.sup.+ bone marrow
cells, CD138.sup.+/CD200.sup.+ bone marrow cells,
c-kit.sup.+/CD200.sup.+ bone marrow cells, or
c-kit.sup.+/CD200.sup.+/Lin.sup.-/low bone marrow cells.
[0125] In some embodiments of any of the methods described herein,
at least a 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, or 80% or more
reduction of the concentration of the one or more subsets of
CD200.sup.+ bone marrow cells indicates that a desired
immunomodulatory effect has been produced in the human.
[0126] In some embodiments of any of the methods described herein,
a reduction in the concentration of one or more subsets of
CD200.sup.+ bone marrow cells in the biological sample as compared
to the concentration of the same subsets of CD200.sup.+ bone marrow
cells in the control sample indicates that the antibody is
therapeutically effective in the human.
[0127] In some embodiments of any of the above methods, the control
sample is a biological sample of the same type obtained from the
human prior to administering the anti-CD200 antibody.
[0128] In yet another aspect, the disclosure features a method for
determining whether an anti-CD200 antibody has produced a desired
immunomodulatory effect in a human, which method includes
quantifying the level of CD200 expression by a plurality of
leukocytes in a biological sample obtained from a human
administered an anti-CD200 antibody, wherein a reduction in CD200
expression by the plurality as compared to a control expression
level indicates that the anti-CD200 antibody has produced a desired
immunomodulatory effect in the human. The leukocytes can be, e.g.,
one or more bone marrow cell subsets or splenocytes.
[0129] In some embodiments of any of the methods described herein,
the CD200.sup.+ leukocytes can be, e.g., CD3.sup.+/CD200.sup.+
leukocytes, CD45R.sup.+/CD200.sup.+ leukocytes,
CD5.sup.+/CD200.sup.+ leukocytes, CD19.sup.+/CD200.sup.+
leukocytes, CD138.sup.+/CD200.sup.+ leukocytes, or
CD200R.sup.+/CD200.sup.+ leukocytes.
[0130] In some embodiments of any of the methods described herein,
at least a 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, or 80% or more
reduction of the level of CD200 expression by the plurality
indicates that a desired immunomodulatory effect has been produced
in the human.
[0131] In some embodiments of any of the methods described herein,
a reduction in the level of CD200 expression by the plurality as
compared to the control expression level indicates that the
antibody is therapeutically effective in the human.
[0132] In another aspect, the disclosure features a method for
determining whether an anti-CD200 antibody has produced a desired
immunomodulatory effect in a human. The method includes quantifying
the level of CD200 expression by a plurality of bone marrow cells
in a biological sample obtained from a human administered an
anti-CD200 antibody, wherein a reduction in CD200 expression by the
plurality as compared to a control expression level indicates that
the anti-CD200 antibody has produced a desired immunomodulatory
effect in the human.
[0133] In some embodiments of any of the methods described herein,
the CD200.sup.+ bone marrow cells are, e.g., Igk.sup.+/CD200.sup.+
bone marrow cells, CD138.sup.+/CD200.sup.+ bone marrow cells,
c-kit.sup.+/CD200.sup.+ bone marrow cells, or
c-kit.sup.+/CD200.sup.+/Lin.sup.-/low bone marrow cells.
[0134] In some embodiments of any of the methods described herein,
at least a 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, or 80% or more
reduction of the level of CD200 expression by the plurality
indicates that a desired immunomodulatory effect has been produced
in the human.
[0135] In some embodiments of any of the methods described herein,
a reduction in the level of CD200 expression by the plurality as
compared to the control expression level indicates that the
antibody is therapeutically effective in the human.
[0136] In some embodiments of any of the methods described herein,
the control sample is a biological sample of the same type obtained
from the human prior to administering the anti-CD200 antibody.
[0137] In yet another aspect, the disclosure features a method for
determining whether an anti-CD200 antibody has produced a desired
immunomodulatory effect in a human, the method comprising: (i)
measuring the concentration of CD200.sup.+ leukocytes in a
biological sample obtained from a human prior to administration to
the human of an anti-CD200 antibody to thereby obtain a
pre-treatment CD200.sup.+ leukocyte concentration; (ii)
administering to the human the antibody; and (iii) measuring the
concentration of CD200.sup.+ leukocytes in a biological sample
obtained from the human following administration of the antibody to
thereby obtain a post-treatment CD200.sup.+ leukocyte
concentration, wherein a reduction in the post-treatment
CD200.sup.+ leukocyte concentration as compared to the
pre-treatment CD200.sup.+ leukocyte concentration indicates that
the antibody has produced a desired immunomodulatory effect in the
human.
[0138] In another aspect, the disclosure features a method for
determining whether an anti-CD200 antibody has produced a desired
immunomodulatory effect in a human, the method comprising: (i)
measuring the concentration of CD200.sup.+ bone marrow cells in a
biological sample obtained from a human prior to administration to
the human of an anti-CD200 antibody to thereby obtain a
pre-treatment CD200.sup.+ bone marrow cell concentration; (ii)
administering to the human the antibody; and (iii) measuring the
concentration of CD200.sup.+ bone marrow cells in a biological
sample obtained from the human following administration of the
antibody to thereby obtain a post-treatment CD200.sup.+ bone marrow
cell concentration, wherein a reduction in the post-treatment
CD200.sup.+ bone marrow cell concentration as compared to the
pre-treatment CD200.sup.+ bone marrow cell concentration indicates
that the antibody has produced a desired immunomodulatory effect in
the human.
[0139] In another aspect, the disclosure features a method for
determining whether an anti-CD200 antibody has produced a desired
immunomodulatory effect in a human, the method comprising: (i)
quantifying the level of CD200 expression by a plurality of
leukocytes in a biological sample obtained from a human prior to
administration to the human of an anti-CD200 antibody to thereby
obtain a pre-treatment CD200 expression level; (ii) administering
to the human the antibody; and (iii) quantifying the level of CD200
expression by a plurality of leukocytes in a biological sample
obtained from the human following administration of the antibody to
thereby obtain a post-treatment CD200 expression level, wherein a
reduction in the post-treatment CD200 expression level as compared
to the pre-treatment CD200 expression level indicates that the
antibody has produced a desired immunomodulatory effect in the
human.
[0140] In another aspect, the disclosure features a method for
determining whether an anti-CD200 antibody has produced a desired
immunomodulatory effect in a human, the method comprising: (i)
quantifying the level of CD200 expression by a plurality of bone
marrow cells in a biological sample obtained from a human prior to
administration to the human of an anti-CD200 antibody to thereby
obtain a pre-treatment CD200 expression level; (ii) administering
to the human the antibody; and (iii) quantifying the level of CD200
expression by a plurality of bone marrow cells in a biological
sample obtained from the human following administration of the
antibody to thereby obtain a post-treatment CD200 expression level,
wherein a reduction in the post-treatment CD200 expression level as
compared to the pre-treatment CD200 expression level indicates that
the antibody has produced a desired immunomodulatory effect in the
human.
[0141] In yet another aspect, the disclosure features a computer
readable medium comprising a medical profile of a human, the
profile comprising information on at least one of (a): (i) the
concentration of CD200 leukocytes in a biological sample obtained
from a human following administration to the human of an anti-CD200
antibody and (ii) the concentration of CD200.sup.+ leukocytes of
the same histological type as in (i) in a biological sample
obtained from the human prior to administration of the antibody;
(b): (iii) the concentration of CD200.sup.+ bone marrow cells in a
biological sample obtained from a human following administration to
the human of an anti-CD200 antibody and (iv) the concentration of
CD200.sup.+ bone marrow cells of the same histological type as in
(iii) in a biological sample obtained from the human prior to
administration of the antibody; (c): (v) the level of expression of
CD200 by a plurality of leukocytes in a biological sample obtained
from the human following administration to the human of an
anti-CD200 antibody and (vi) the level of expression of CD200 by a
plurality of leukocytes of the same histological type as in (v) in
a biological sample obtained from the human prior to administration
of the antibody; and (d): (vii) the level of expression of CD200 by
a plurality of bone marrow cells in a biological sample obtained
from the human following administration to the human of an
anti-CD200 antibody and (viii) the level of expression of CD200 by
a plurality of bone marrow cells of the same histological type as
in (vii) in a biological sample obtained from the human prior to
administration of the antibody.
[0142] In another aspect, the disclosure features a computer-based
method for determining whether an anti-CD200 antibody has produced
a desired immunomodulatory effect in a human, the method
comprising: (A) receiving data including a medical profile of a
human, the profile comprising information on at least one of: (a):
(i) the concentration of CD200.sup.+ leukocytes in a biological
sample obtained from a human following administration to the human
of an anti-CD200 antibody and (ii) the concentration of CD200.sup.+
leukocytes of the same histological type as in (i) in a biological
sample obtained from the human prior to administration of the
antibody; (b): (iii) the concentration of CD200.sup.+ bone marrow
cells in a biological sample obtained from a human following
administration to the human of an anti-CD200 antibody and (iv) the
concentration of CD200.sup.+ bone marrow cells of the same
histological type as in (iii) in a biological sample obtained from
the human prior to administration of the antibody; (c): (v) the
level of expression of CD200 by a plurality of leukocytes in a
biological sample obtained from the human following administration
to the human of an anti-CD200 antibody and (vi) the level of
expression of CD200 by a plurality of leukocytes of the same
histological type as in (v) in a biological sample obtained from
the human prior to administration of the antibody; and (d): (vii)
the level of expression of CD200 by a plurality of bone marrow
cells in a biological sample obtained from the human following
administration to the human of an anti-CD200 antibody and (viii)
the level of expression of CD200 by a plurality of bone marrow
cells of the same histological type as in (vii) in a biological
sample obtained from the human prior to administration of the
antibody; and (B) processing at least the portion of the data
containing the information to determine whether the antibody has
produced a desired immunomodulatory effect in the human. (1) A
reduction in the post-treatment CD200.sup.+ leukocyte concentration
as compared to the pre-treatment CD200.sup.+ leukocyte
concentration indicates that the antibody has produced a desired
immunomodulatory effect in the human; (2) a reduction in the
post-treatment CD200.sup.+ bone marrow cell concentration as
compared to the pre-treatment CD200.sup.+ bone marrow cell
concentration indicates that the antibody has produced a desired
immunomodulatory effect in the human; (3) a reduction in
post-treatment CD200 expression level by leukocytes as compared to
the pre-treatment CD200 expression level by leukocytes indicates
that the antibody has produced a desired immunomodulatory effect in
the human; or (4) a reduction in post-treatment CD200 expression
level by bone marrow cells as compared to the pre-treatment CD200
expression level by bone marrow cells indicates that the antibody
has produced a desired immunomodulatory effect in the human.
[0143] In another aspect, the disclosure features a computer-based
method for determining whether an anti-CD200 antibody has produced
a desired immunomodulatory effect in a human, the method
comprising: (I) providing information for at least one of: (a): (i)
the concentration of CD200.sup.+ leukocytes in a biological sample
obtained from a human following administration to the human of an
anti-CD200 antibody and (ii) the concentration of CD200.sup.+
leukocytes of the same histological type as in (i) in a biological
sample obtained from the human prior to administration of the
antibody; (b): (iii) the concentration of CD200.sup.+ bone marrow
cells in a biological sample obtained from a human following
administration to the human of an anti-CD200 antibody and (iv) the
concentration of CD200.sup.+ bone marrow cells of the same
histological type as in (iii) in a biological sample obtained from
the human prior to administration of the antibody; (c): (v) the
level of expression of CD200 by a plurality of leukocytes in a
biological sample obtained from the human following administration
to the human of an anti-CD200 antibody and (vi) the level of
expression of CD200 by a plurality of leukocytes of the same
histological type as in (v) in a biological sample obtained from
the human prior to administration of the antibody; and (d): (vii)
the level of expression of CD200 by a plurality of bone marrow
cells in a biological sample obtained from the human following
administration to the human of an anti-CD200 antibody and (viii)
the level of expression of CD200 by a plurality of bone marrow
cells of the same histological type as in (vii) in a biological
sample obtained from the human prior to administration of the
antibody; (II) inputting the information into a computer; and (III)
calculating a parameter indicating whether the antibody has
produced a desired immunomodulatory effect in the human using the
computer and the input information. (1) A reduction in the
post-treatment CD200.sup.+ leukocyte concentration as compared to
the pre-treatment CD200.sup.+ leukocyte concentration indicates
that the antibody has produced a desired immunomodulatory effect in
the human; (2) a reduction in the post-treatment CD200.sup.+ bone
marrow cell concentration as compared to the pre-treatment
CD200.sup.+ bone marrow cell concentration indicates that the
antibody has produced a desired immunomodulatory effect in the
human; (3) a reduction in post-treatment CD200 expression level by
leukocytes as compared to the pre-treatment CD200 expression level
by leukocytes indicates that the antibody has produced a desired
immunomodulatory effect in the human; or (4) a reduction in
post-treatment CD200 expression level by bone marrow cells as
compared to the pre-treatment CD200 expression level by bone marrow
cells indicates that the antibody has produced a desired
immunomodulatory effect in the human. The method can include
outputting the parameter.
[0144] In some embodiments of any of the methods described herein,
the human has, is suspected of having, or is likely to develop, a
cancer. The cancer can be, e.g., chronic lymphocytic leukemia
(e.g., B cell chronic lymphocytic leukemia). The cancer can be a
solid tumor, e.g., a colon cancer, a breast cancer, a lung cancer,
a renal cancer, a pancreatic cancer, a thyroid cancer, a skin
cancer, a cancer of the nervous system, a cervical cancer, an
ovarian cancer, a testicular cancer, a head and neck cancer, a
cancer of the eye, a stomach cancer, or a liver cancer. The cancer
of the nervous system can be, e.g., a neuroblastoma.
[0145] In some embodiments of any of the methods described herein,
the human has, is suspected of having, or is at risk for
developing, an inflammatory disorder or a bone disorder. The
inflammatory disorder can be, e.g., an autoimmune disorder such as,
e.g., a hemolytic disorder. The autoimmune disorder can be an
autoimmune hemolytic anemia. The autoimmune disorder can be, e.g.,
chronic obstructive pulmonary disease, diabetes mellitus type 1,
Goodpasture's syndrome, Grave's disease, Guillain-Barre syndrome,
IgA nephropathy, scleroderma, Sjogren's syndrome, systemic lupus
erthyematosus, lupus nephritis, glomerulonephritis, Wegener's
granulomatosis, pemphigus vulgaris, rheumatoid arthritis, Chagas
disease, cold agglutinin disease, anti-phospholipid syndrome, warm
autoimmune hemolytic anemia, paroxysmal cold hemoglobinuria,
Hashimoto's disease, idiopathic thrombocytopenic purpura,
myasthenia gravis, pulmonary biliary cirrhosis, or Miller Fisher
syndrome.
[0146] In some embodiments of any of the methods described herein,
the one or more subsets of CD200.sup.+ leukocytes are selected from
the group consisting of CD3.sup.+/CD200' lymphocytes,
CD45R.sup.+/CD200.sup.+ lymphocytes, CD5.sup.+/CD200.sup.+
lymphocytes, CD19.sup.+/CD200.sup.+ lymphocytes,
CD138.sup.+/CD200.sup.+ lymphocytes, and CD200R.sup.+/CD200.sup.+
lymphocytes.
[0147] In some embodiments of any of the methods described herein,
the one or more subsets of CD200.sup.+ bone marrow cells are
selected from the group consisting of Igk.sup.+/CD200.sup.+ bone
marrow cells, CD138.sup.+/CD200.sup.+ bone marrow cells,
c-kit.sup.+/CD200.sup.+ bone marrow cells, and
c-kit.sup.+/CD200.sup.+/Lin.sup.- bone marrow cells.
[0148] In some embodiments, the methods include administering to
the human a therapeutically-effective amount of the anti-CD200
antibody if the antibody has been determined to produce a desired
immunomodulatory effect in the human.
[0149] In yet another aspect, the disclosure features a method for
determining an anti-CD200 antibody dosing schedule for a patient,
the method comprising: administering to a patient an anti-CD200
antibody to thereby reduce the concentration of one or more subsets
of CD200.sup.+ leukocytes in a biological sample obtained from the
patient as compared to the concentration of the same subsets of
CD200.sup.+ leukocytes in a control sample, wherein the patient is
afflicted with a disorder selected from the group consisting of a
cancer, an inflammatory disorder, or a bone disorder; and
monitoring the concentration of the one or more subsets of
CD200.sup.+ leukocytes in the patient to thereby determine for the
patient a dosing schedule of the antibody, wherein the dosing
schedule is sufficient to maintain a reduced concentration of the
one or more subsets of CD200.sup.+ leukocytes in the patient for
the duration of the treatment of the disorder with the antibody. In
some embodiments, the concentration of the one or more subsets of
CD200.sup.+ leukocytes is reduced by at least 10%, 20%, 30%, 40%,
50%, 60%, 70%, 80%, or 90% or more.
[0150] In some embodiments of any of the methods described herein,
the one or more subsets of CD200.sup.+ leukocytes are selected from
the group consisting of CD3.sup.+/CD200.sup.+ lymphocytes,
CD45R.sup.+/CD200.sup.+ lymphocytes, CD5.sup.+/CD200.sup.+
lymphocytes, CD19.sup.+/CD200.sup.+ lymphocytes,
CD138.sup.+/CD200.sup.+ lymphocytes, CD200R.sup.+/CD200.sup.+
lymphocytes, CD200.sup.+/CD4.sup.+ T cells, activated
CD200.sup.+/CD4.sup.+ T cells, and CD200.sup.+/CD8.sup.+ T cells.
In some embodiments, the biological sample comprises spleen tissue
from the patient.
[0151] In yet another aspect, the disclosure features a method for
determining an anti-CD200 antibody dosing schedule for a patient.
The method includes administering to a patient an anti-CD200
antibody to thereby reduce the level of expression of CD200 by one
or more subsets of leukocytes in a biological sample obtained from
the patient as compared to a control expression level of CD200 by
leukocytes of the same histological type in a control sample,
wherein the patient is afflicted with a disorder selected from the
group consisting of a cancer, an inflammatory disorder, or a bone
disorder; and monitoring the expression level of CD200 by the one
or more subsets of leukocytes in the patient to thereby determine
for the patient a dosing schedule of the antibody, wherein the
dosing schedule is sufficient to maintain a reduced level of
expression of CD200 by the one or more subsets of leukocytes in the
patient for the duration of the treatment of cancer with the
antibody.
[0152] In some embodiments of any of the methods described herein,
the expression level of CD200 by the one or more subsets of
leukocytes is reduced by at least 10%, 20%, 30%, 40%, 50%, 60%,
70%, or 80% or more.
[0153] In another aspect, the disclosure features a method for
determining an anti-CD200 antibody dosing schedule for a patient.
The method includes administering to a patient an anti-CD200
antibody to thereby reduce the concentration of one or more subsets
of CD200.sup.+ bone marrow cells in a biological sample obtained
from the patient as compared to the concentration of the same
subsets of CD200.sup.+ bone marrow cells in a control sample,
wherein the patient is afflicted with a disorder selected from the
group consisting of a cancer, an inflammatory disorder, or a bone
disorder; and monitoring the concentration of the one or more
subsets of CD200.sup.+ bone marrow cells in the patient to thereby
determine for the patient a dosing schedule of the antibody,
wherein the dosing schedule is sufficient to maintain a reduced
concentration of the one or more subsets of CD200.sup.+ bone marrow
cells in the patient for the duration of the treatment of the
disorder with the antibody.
[0154] In some embodiments, the concentration of the one or more
subsets of CD200.sup.+ bone marrow cells is reduced by at least
10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, or 90% or more.
[0155] In some embodiments, the one or more subsets of CD200.sup.+
bone marrow cells are selected from the group consisting of
Igk.sup.+/CD200.sup.+ bone marrow cells, CD138.sup.+/CD200.sup.+
bone marrow cells, c-kit.sup.+/CD200.sup.+ bone marrow cells, and
c-kit.sup.+/CD200.sup.+/Lin.sup.-/low bone marrow cells.
[0156] In yet another aspect, the disclosure features a method for
determining an anti-CD200 antibody dosing schedule for a patient,
the method comprising: administering to a patient an anti-CD200
antibody to thereby reduce the level of expression of CD200 by one
or more subsets of bone marrow cells in a biological sample
obtained from the patient as compared to a control expression level
of CD200 by bone marrow cells of the same histological type in a
control sample, wherein the patient is afflicted with a disorder
selected from the group consisting of a cancer, an inflammatory
disorder, or a bone disorder; and monitoring the expression level
of CD200 by the one or more subsets of bone marrow cells in the
patient to thereby determine for the patient a dosing schedule of
the antibody, wherein the dosing schedule is sufficient to maintain
a reduced level of expression of CD200 by the one or more subsets
of bone marrow cells in the patient for the duration of the
treatment of cancer with the antibody.
[0157] In yet another aspect, the disclosure features a method for
treating a human suffering from a disorder, the method comprising
administering to a human in need thereof an anti-CD200 antibody in
an amount and with a frequency sufficient to reduce the
concentration of CD200.sup.+ leukocytes or CD200.sup.+ bone marrow
cells in a cancer patient to thereby treat the human, wherein the
human is afflicted with a disorder selected from the group
consisting of a cancer, an inflammatory disorder, and a bone
disorder. The method can include monitoring the human for a
reduction in the CD200.sup.+ leukocytes or CD200.sup.+ bone marrow
cells in the patient. In some embodiments, the concentration of
CD200.sup.+ leukocytes in the blood of the patient is reduced. In
some embodiments, the concentration of CD200.sup.+ leukocytes in
the spleen of the patient is reduced. The method can include
monitoring the human for a reduction in the expression level of
CD200 by leukocytes or bone marrow cells in the patient.
[0158] In some embodiments of any of the methods described herein,
the anti-CD200 antibody is an IgG1, IgG2, IgG3, IgG4, IgM, IgA1,
IgA2, IgA, IgD, or IgE antibody. The antibody can be, e.g., a
murine antibody, a chimeric antibody, a humanized antibody, a
single chain antibody, or a human antibody. In some embodiments,
the anti-CD200 antibody is a variant antibody that has decreased or
no effector function as described herein.
[0159] In any of the methods, the anti-CD200 antibody can be any
one of the anti-CD200 antibodies described herein such as, but in
no way limited to, samalizumab.
[0160] "Polypeptide," "peptide," and "protein" are used
interchangeably and mean any peptide-linked chain of amino acids,
regardless of length or post-translational modification. The CD200
proteins described herein can contain or be wild-type proteins or
can be variants that have not more than 50 (e.g., not more than
one, two, three, four, five, six, seven, eight, nine, ten, 12, 15,
20, 25, 30, 35, 40, or 50) conservative amino acid substitutions.
Conservative substitutions typically include substitutions within
the following groups: glycine and alanine; valine, isoleucine, and
leucine; aspartic acid and glutamic acid; asparagine, glutamine,
serine and threonine; lysine, histidine and arginine; and
phenylalanine and tyrosine.
[0161] The CD200 proteins described herein also include "antigenic
peptide fragments" of the proteins, which are shorter than
full-length CD200 proteins, but retain at least 10% (e.g., at least
10%, at least 15%, at least 20%, at least 25%, at least 30%, at
least 35%, at least 40%, at least 45%, at least 50%, at least 55%,
at least 60%, at least 70%, at least 80%, at least 90%, at least
95%, at least 98%, at least 99%, at least 99.5%, or 100% or more)
of the ability of the full-length protein to induce an antigenic
response in a mammal (see below under "Methods for Producing an
Antibody"). Antigenic peptide fragments of a CD200 protein include
terminal as well internal deletion variants of the protein.
Deletion variants can lack one, two, three, four, five, six, seven,
eight, nine, ten, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 amino
acid segments (of two or more amino acids) or non-contiguous single
amino acids. Antigenic peptide fragments can be at least 6 (e.g.,
at least 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21,
22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38,
39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 55, 60, 65, 70, 75,
80, 85, 90, 95, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190,
or 200 or more) amino acid residues in length (e.g., at least 6
contiguous amino acid residues in any one of SEQ ID NOs:1 to 3). In
some embodiments, an antigenic peptide fragment of a human CD200
protein is less than 225 (e.g., less than 200, 190, 180, 170, 160,
150, 140, 130, 120, 110, 100, 95, 90, 85, 80, 75, 70, 65, 60, 55,
50, 49, 48, 47, 46, 45, 44, 43, 42, 41, 40, 39, 38, 37, 36, 35, 34,
33, 32, 31, 30, 29, 28, 27, 26, 25, 24, 23, 22, 21, 20, 19, 18, 17,
16, 15, 14, 13, 12, 11, 10, 9, 8, or 7) amino acid residues in
length (e.g., less than 225 contiguous amino acid residues in any
one of SEQ ID NOs:1 to 3). In some embodiments, an antigenic
peptide fragment of a full-length CD200 protein is at least 6, but
less than 225, amino acid residues in length.
[0162] In some embodiments, the human CD200 protein can have an
amino acid sequence that is, or is greater than, 70 (e.g., 71, 72,
73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89,
90, 91, 92, 93, 94, 95, 96, 97, 98, or 99) % identical to the human
CD200 protein having the amino acid sequence depicted in SEQ ID
NO:1 or SEQ ID NO:2 (see below). In some embodiments, the human
CD200 protein has the amino acid sequence depicted in SEQ ID NO:1
or SEQ ID NO:2.
[0163] Percent (%) amino acid sequence identity is defined as the
percentage of amino acids in a candidate sequence that are
identical to the amino acids in a reference sequence, after
aligning the sequences and introducing gaps, if necessary, to
achieve the maximum percent sequence identity. Alignment for
purposes of determining percent sequence identity can be achieved
in various ways that are within the skill in the art, for instance,
using publicly available computer software such as BLAST, BLAST-2,
ALIGN, ALIGN-2 or Megalign (DNASTAR) software. Appropriate
parameters for measuring alignment, including any algorithms needed
to achieve maximal alignment over the full-length of the sequences
being compared can be determined by known methods.
[0164] Amino acid sequences for exemplary human CD200 proteins as
well as antigenic peptide fragments thereof are known in the art
and are set forth below.
[0165] As used herein, the term "antibody" refers to a whole or
intact antibody molecule (e.g., IgM, IgG (including IgG1, IgG2,
IgG3, and IgG4), IgA, IgD, or IgE) or any antigen-binding fragment
thereof. The term antibody includes, e.g., a chimerized or chimeric
antibody, a humanized antibody, a deimmunized antibody, and a fully
human antibody. Antigen-binding fragments of an antibody include,
e.g., a single chain antibody, a single chain Fv fragment (scFv),
an Fd fragment, an Fab fragment, an Fab' fragment, or an
F(ab').sub.2 fragment. An scFv fragment is a single polypeptide
chain that includes both the heavy and light chain variable regions
of the antibody from which the scFv is derived. In addition,
intrabodies, minibodies, triabodies, and diabodies (see, e.g.,
Todorovska et al. (2001) J Immunol Methods 248(1):47-66; Hudson and
Kortt (1999) J Immunol Methods 231(1):177-189; Poljak (1994)
Structure 2(12):1121-1123; Rondon and Marasco (1997) Annual Review
of Microbiology 51:257-283, the disclosures of each of which are
incorporated herein by reference in their entirety) are also
included in the definition of antibody and are compatible for use
in the methods described herein. Bispecific antibodies (including
DVD-Ig antibodies; see below) are also embraced by the term
"antibody." Bispecific antibodies are monoclonal, preferably human
or humanized, antibodies that have binding specificities for at
least two different antigens.
[0166] Unless otherwise defined, all technical and scientific terms
used herein have the same meaning as commonly understood by one of
ordinary skill in the art to which this disclosure pertains. In
case of conflict, the present document, including definitions, will
control. Preferred methods and materials are described below,
although methods and materials similar or equivalent to those
described herein can also be used in the practice or testing of the
presently disclosed methods and compositions. All publications,
patent applications, patents, and other references mentioned herein
are incorporated by reference in their entirety.
[0167] Other features and advantages of the present disclosure,
e.g., methods for determining whether an anti-CD200 antibody has
produced in immunomodulatory effect in a human, will be apparent
from the following description, the examples, and from the
claims.
BRIEF DESCRIPTION OF THE DRAWINGS
[0168] FIG. 1 is a line graph depicting a dose-dependent linear
increase in serum AUC (area under the curve) for the first four
cycles (doses) of treatment with the anti-CD200 antibody
samalizumab. Only subjects that received four doses of samalizumab
were included in the AUC analysis. The X-axis depicts the dose
(mg/m2) of antibody administered to the patient within a particular
cohort (see Examples 1 and 2 below). The Y-axis represents the AUC
(for the first, second, third, and fourth doses) in units of
(mg.times.hours).times.mL.sup.-1. The open circles represent
individual AUC for each patient within the cohort. The box/whisker
represents the mean AUC for each cohort.
[0169] FIG. 2 is a series of flow cytometry dot plots depicting the
observed reduction of CD200.sup.+ T cells in the peripheral blood
of a patient treated with the anti-CD200 antibody samalizumab at
100 mg/m.sup.2. In panel (i), the X-axis represents the relative
fluorescence intensity of the signal produced from an anti-CD19
antibody/PerCP conjugate bound to the evaluated cells and the
Y-axis represents the relative fluorescence intensity of the signal
produced from an anti-CD3 antibody/Alexa Fluor.RTM. 700 bound to
the evaluated cells. The T cells identified in panel (i) were then
interrogated for both the expression of CD200 and for evidence of
bound samalizumab (panels ii to v). In panels (ii) to (v), the
Y-axis represents the relative fluorescence intensity of the signal
produced from an anti-CD200 antibody/FITC conjugate bound to
CD3.sup.+ cells (T cells) in the patient blood sample and the
X-axis represents the relative fluorescence intensity of an
antibody specific for samalizumab (e.g. an anti-idiotypic
antibody)/Alexa 647 conjugate bound to the cells. Panel (i) depicts
the flow cytometry profile of cells in a blood sample obtained from
the patient prior to administration of samalizumab and specifically
the CD3.sup.+ and CD19.sup.+ populations present in the blood
sample. Panel (ii) depicts the flow cytometry profile of the gated
CD3.sup.+ cell from panel (i), showing the concentration of
CD3.sup.+ cells in the population of (i) that are also CD200.sup.+
(see the upper left quadrant of the panel) on day 0 (prior to
samalizumab dosing), and panel (iii) shows the same on day 1
following samalizumab dosing. Panel (iv) depicts the flow cytometry
profile showing the concentration of CD200.sup.+/CD3.sup.+ cells in
a biological sample obtained from the patient seven days after the
patient was administered samalizumab (see the upper left quadrant
of the panel). Panel (v) depicts the flow cytometry profile showing
the concentration of CD200.sup.+/CD3.sup.+ cells in a biological
sample obtained from the patient fourteen days after the patient
was administered samalizumab (see the upper left quadrant of the
panel). The large, filled arrows indicate the relevant cell
population.
[0170] FIG. 3 are a series of line graphs depicting the nature of
the reduction in CD200.sup.+/CD4.sup.+ T cells in different
patients, respectively, following the administration of samalizumab
to the patients. For each row of line graphs, the Y-axis represents
the percent change in the CD200.sup.+/CD4.sup.+ T cell population,
from baseline, as measured in a biological sample from the patient.
The numeric units identified on the Y-axis are, in descending order
from the top of each graph: 75, 50, 25, 0, -25, -50, -75, and -100.
The X-axis for each individual line graph represents the time in
days following initial administration of samalizumab. The vertical
bars represent the days on which samalizumab was administered. Each
row of line graphs corresponds to a particular cohort numbered 1 to
6. As elaborated on below in the working Examples, patients in
cohort 6 received 500 mg/m.sup.2 of samalizumab each dose. Patients
in cohort 5 received 400 mg/m.sup.2 of samalizumab each dose. Each
dose of samalizumab administered to patients in cohort 4 was 300
mg/m.sup.2. Each dose of samalizumab administered to patients in
cohort 3 was 200 mg/m.sup.2. Patients in cohort 2 received 100
mg/m.sup.2 of samalizumab and patients in cohort 1 received 50
mg/m.sup.2 of samalizumab for each dose. Each individual line graph
corresponds to one patient within each cohort. Unevaluable patients
are designated by a once-crossed circle. Only four doses (cycles)
are shown in the figure. Data from the single patient from cohort 7
not included.
[0171] FIG. 4 is a series of flow cytometry histograms depicting
the change in expression of CD200 and CD200R on CD4.sup.+ and
CD8.sup.+ T cell subsets in blood samples obtained from a patient
treated with samalizumab at a dose of 100 mg/m.sup.2. The Y-axis in
all panels represents the number of cells. In panels (i), (ii),
(v), (vi), (ix), and (x), the X-axis represents the relative
fluorescence intensity of an anti-CD200 antibody/FITC conjugate
bound to the cells. In panels (iii), (iv), (vii), and (viii), the
X-axis represents the relative fluorescence intensity of an
anti-CD200R antibody/phycoerythrin conjugate bound to the cells.
Panel (i) depicts the number of CD200.sup.+/CD3.sup.+ T cells in a
blood sample from the patient prior to receiving samalizumab. Panel
(ii) depicts the number of CD200.sup.+/CD3.sup.+ T cells in a blood
sample from the patient obtained seven days after receiving
samalizumab. Panel (iii) depicts the number of
CD200R.sup.+/CD3.sup.+ T cells in a blood sample from the patient
prior to receiving samalizumab. Panel (iv) depicts the number of
CD200R.sup.+/CD3.sup.+ T cells in a blood sample from the patient
obtained seven days after receiving samalizumab. Panel (v) depicts
the number of CD200.sup.+/CD3.sup.+/CD4.sup.+ T cells in a blood
sample from the patient prior to receiving samalizumab. Panel (vi)
depicts the number of CD200.sup.+/CD3.sup.+/CD4.sup.+ T cells in a
blood sample from the patient obtained seven days after receiving
samalizumab. Panel (vii) depicts the number of
CD200R.sup.+/CD3.sup.+/CD4.sup.+T cells in a blood sample from the
patient prior to receiving samalizumab. Panel (viii) depicts the
number of CD200R.sup.+/CD3.sup.+/CD4.sup.+ T cells in a blood
sample from the patient obtained seven days after receiving
samalizumab. Panel (ix) depicts the number of
CD200.sup.+/CD3.sup.+/CD8.sup.+ T cells in a blood sample from the
patient prior to receiving samalizumab. Panel (x) depicts the
number of CD200.sup.+/CD3.sup.+/CD8.sup.+ T cells in a blood sample
from the patient obtained seven days after receiving
samalizumab.
[0172] FIG. 5 is a bar graph depicting the reduction in regulatory
T cells (CD4.sup.+/CD25.sup.+/FoxP3.sup.+) in subsets of
samalizumab-treated patients having stable or improved disease
(".gtoreq.SD"; rightmost grouping of bars) or progressive
disease/adverse events ("PD/AE"; leftmost grouping of bars). Each
bar within the graph represents an individual patient. The Y-axis
represents the percentage change in the concentration of regulatory
T cells at the last visit, as compared to baseline (the
concentration of cells of the same histological type in a
biological sample obtained from the patient prior to administration
of the antibody), in a biological sample obtained from each
patient.
[0173] FIG. 6 is a series of line graphs depicting the reduction in
CD200.sup.+ expression by B-CLL cells in different patients,
respectively, following the administration of different doses of
samalizumab to the patients. The Y-axis represents the percent (%)
change from baseline in mean fluorescence intensity (MFI) of an
anti-CD200 antibody/FITC conjugate bound to B-CLL cells present in
the patient samples. The numeric units identified on the Y-axis
are, in descending order from the top of each graph: 50, 0, -50,
and -100. The X-axis represents the time in days following initial
administration of samalizumab. The vertical bars represent the days
on which samalizumab was administered. Each row of line graphs
corresponds to a particular cohort numbered 1 to 6. As elaborated
on below in the working Examples, patients in cohort 6 received 500
mg/m.sup.2 of samalizumab each dose. Patients in cohort 5 received
400 mg/m.sup.2 of samalizumab each dose. Each dose of samalizumab
administered to patients in cohort 4 was 300 mg/m.sup.2. Each dose
of samalizumab administered to patients in cohort 3 was 200
mg/m.sup.2. Patients in cohort 2 received 100 mg/m.sup.2 of
samalizumab and patients in cohort 1 received 50 mg/m.sup.2 of
samalizumab for each dose. Each individual line graph corresponds
to one patient within each cohort. Unevaluable patients are
designated by a once-crossed circle.
[0174] FIG. 7A is a line graph depicting embodiments of the
immunomodulatory effect of samalizumab as observed in CLL patient
102-502. The X-axis represents time in days. The Y-axis to the left
of FIG. 7A represents the percentage of CD45.sup.+ B-CLL cells as
measured in a blood sample obtained from the patient. The Y-axis to
the right of FIG. 7A represents the absolute lymphocyte count in a
blood sample obtained from the patient. The vertical, hashed lines
represent points at which samalizumab was administered to the
patient. Each dose of samalizumab administered to patient 102-502
was 400 mg/m.sup.2.
[0175] FIG. 7B is a line graph depicting an embodiment of the
immunomodulatory effect of samalizumab as observed in CLL patient
102-502. The X-axis represents time in days. The filled triangles
represent the percentage of circulating CD45.sup.+ B CLL cells. The
non-filled triangles represent the percentage of CD8.sup.+ T cells.
The lines representing the percentage of CD4.sup.+ T cells or
regulatory T cells are indicated by arrows. The Y-axis of FIG. 7B
represents the percentage of lymphocyte cells in the assayed
population. The vertical, hashed lines represent points at which
samalizumab was administered to the patient.
[0176] FIG. 8A is a line graph depicting the reduction in the
percentage of CD200.sup.+/CD4' T cells in patient 102-502 over time
after the first dose. The Y-axis represents the percentage of
CD200.sup.+/CD4.sup.+ T cells. The X-axis represents the time in
days following administration of samalizumab.
[0177] FIG. 8B is a line graph depicting the reduction in the level
of CD200 expression by B CLL cells in patient 102-502 over time
after the first dose. The Y-axis represents the mean fluorescence
intensity (MFI) of an anti-CD200 antibody/FITC conjugate bound to
B-CLL cells present in the patient samples. The X-axis represents
the time in days following administration of samalizumab.
[0178] FIG. 9 is a line graph depicting the change in ratio of
percent activated T cells to percent regulatory T cells in patient
102-502. The X-axis represents time in days. The Y-axis represents
ratio of percent activated T cells to percent regulatory T cells in
the assayed population. The vertical, hashed lines represent points
at which samalizumab was administered to the patient.
[0179] FIG. 10 is a line graph depicting the delay in anti-mouse
RBC autoantibody production in mice with autoimmune hemolytic
disease treated with an anti-CD200 antibody. The Y-axis represents
the incidence (%) of autoantibody production in the mice in each
group. The X-axis represents the time in which the presence of
autoantibodies in each mouse was detected. The seven groups of mice
evaluated included: mice that were immunized with rat RBCs, but not
treated with an antibody (No Rx); mice that were immunized with rat
RBCs and treated with a control antibody (Cntrl Ab); mice that were
immunized with rat RBCs and treated with an anti-CD200 antibody
(Antibody 1); mice that were immunized with rat RBCs and treated
with cyclosporine (CsA); mice that were immunized with rat RBCs and
treated with the control antibody and cyclosporine A (Cntrl
Ab+CsA); mice that were immunized with rat RBCs and treated with an
anti-CD200 antibody and cyclosporine A (Antibody 1+CsA); and mice
that were neither immunized with rat RBCs nor treated with antibody
or cyclosporine (No-imm No Rx).
[0180] FIG. 11 is a line graph depicting the effect of an
anti-CD200 antibody on anti-RBC antibody titer in a mouse model of
autoimmune hemolytic disease. C57BL/6 mice were administered
2.times.10.sup.8 rat RBCs intraperitoneally (i.p.) once on study
day 0 and then once per week thereafter for the remainder of the
study. The rat RBC-immunized mice were then treated with an
anti-CD200 antibody that possessed effector function (Antibody 1;
Ab 1) at 5 mg/kg or 1 mg/kg; an anti-CD200 antibody that did not
possess effector function (Antibody 2; Ab 2) at 5 mg/kg; or a
control antibody (Cntl) at 5 mg/kg. A group of mice was also
treated with vehicle only. A final group of mice received no
immunization or antibody treatment (NC). The Y-axis depicts the
relative fluorescence intensity reflected as the OD405.times. serum
dilution factor and the X-axis represents the number of days
following the start of the study.
[0181] FIG. 12 is a bar graph depicting the reduction in
antigen-induced proliferation of splenocytes isolated from mice
treated with an anti-CD200 antibody. The Y-axis represents the mean
counts per minute of .sup.3H-thymidine radioactivity in nucleic
acid isolated from each cell population. The X-axis represents
individual mice, three (3) depicted in each group. For each mouse,
the four measurements are for proliferation of splenocytes induced
by medium alone, mouse red blood cells (mRBC), rat red blood cells
(rRBC), or bovine serum albumin (BSA). The mice of Group 1 were
treated with an anti-CD200 antibody with effector function
(Antibody 1) at a dose of 5 mg/kg. The mice of Group 2 were treated
with Antibody 1 at a dose of 1 mg/kg. The mice of Group 3 were
treated with a control antibody that does not bind to CD200 and the
mice of Group 4 were not treated with an antibody or immunized with
the rat red blood cells.
[0182] FIG. 13 is a bar graph depicting the reduction in
CD200.sup.+ splenocytes in mice treated with an anti-CD200
antibody. C57BL/6 mice were administered 2.times.10.sup.8 rat RBCs
intraperitoneally (i.p.) once on study day 0 and then once per week
thereafter for the remainder of the study. The rat RBC-immunized
mice were then treated with an anti-CD200 antibody that possessed
effector function (Antibody 1; Ab 1) at 5 mg/kg or 1 mg/kg; an
anti-CD200 antibody that did not possess effector function
(Antibody 2; Ab 2) at 5 mg/kg; or a control antibody (Cntl) at 5
mg/kg. A group of mice was also treated with vehicle only. A final
group of mice received no immunization or antibody treatment
(Un-imm, No-Ab). The Y-axis represents the percentage of
CD200.sup.+ cells in the total population of viable splenocytes.
The X-axis represents individual mice, three (3) depicted in each
group.
DETAILED DESCRIPTION
[0183] The present disclosure relates to anti-CD200 antibodies
(e.g., variant anti-CD200 antibodies having decreased or no
effector function) and to biomarkers for use in determining whether
a human has been administered a dose of one or more of the
antibodies that produces a desired immunomodulatory effect in the
human. Also featured are diagnostic and therapeutic methods that
utilize the antibodies and biomarkers. While in no way intended to
be limiting, exemplary anti-CD200 antibodies and CD200-binding
fragments thereof, conjugates, pharmaceutical compositions and
formulations, biomarkers, and methods employing any of the
foregoing are elaborated on below and are exemplified in the
working Examples.
Compositions
[0184] The disclosure features antibodies that bind to a human
CD200 polypeptide (sometimes the antibodies are referred to herein
as "anti-CD200 antibodies"). Also featured are antigen-binding
(CD200-binding) fragments of the antibodies. In some embodiments,
an anti-CD200 antibody described herein binds to an epitope in the
human CD200 protein. For example, the anti-CD200 antibody can bind
to an epitope in the human CD200 protein comprising, or consisting
of, the following amino acid sequence:
MERLVIRMPFSHLSTYSLVWVMAAVVLCTAQVQVVTQDEREQLYTPASLKC
SLQNAQEALIVTWQKKKAVSPENMVTFSENHGVVIQPAYKDKINITQLGLQN
STITFWNITLEDEGCYMCLFNTFGFGKISGTACLTVYVQPIVSLHYKFSEDHLN
ITCSATARPAPMVFWKVPRSGIENSTVTLSHPNGTTSVTSILHIKDPKNQVGKE
VICQVLHLGTVTDFKQTVNKGYWFSVPLLLSIVSLVILLVLISILLYWKRHRNQ DREP (SEQ ID
NO:1; Genbank Accession No. NP.sub.--005935.2). SEQ ID NO:1 depicts
the amino acid sequence for a full-length, precursor human CD200
isoform A protein. In some embodiments, an anti-CD200 antibody
described herein binds to an epitope in the human CD200 protein
comprising, or consisting of, the following amino acid sequence:
MERLTLTRTIGGPLLTATLLGKTINDYQVIRMPFSHLSTYSLVWVMAAVVLC
TAQVQVVTQDEREQLYTPASLKCSLQNAQEALIVTWQKKKAVSPENMVTFS
ENHGVVIQPAYKDKINITQLGLQNSTITFWNITLEDEGCYMCLFNTFGFGKISG
TACLTVYVQPIVSLHYKFSEDHLNITCSATARPAPMVFWKVPRSGIENSTVTL
SHPNGTTSVTSILHIKDPKNQVGKEVICQVLHLGTVTDFKQTVNKGYWFSVPL
LLSIVSLVILLVLISILLYWKRHRNQDREP (SEQ ID NO:2; Genbank Accession No.
NP.sub.-- 001004196.2). SEQ ID NO:2 depicts the amino acid sequence
of a full-length CD200 isoform B protein. In some embodiments, the
anti-CD200 antibody binds to an epitope present in a human CD200
protein having the following amino acid sequence:
VIRMPFSHLSTYSLVWVMAAVVLCTAQVQVVTQDEREQLYTTASLKCSLQN
AQEALIVTWQKKKAVSPENMVTFSENHGVVIQPAYKDKINITQLGLQNSTITF
WNITLEDEGCYMCLFNTFGFGKISGTACLTVYVQPIVSLHYKFSEDHLNITCS
ATARPAPMVFWKVPRSGIENSTVTLSHPNGTTSVTSILHIKDPKNQVGKEVIC
QVLHLGTVTDFKQTVNKGYWFSVPLLLSIVSLVILLVLISILLYWKRHRNQDR GELSQGVQKMT
(SEQ ID NO:3; Genbank Accession No. CAA28943.1; FIG. 3 of McCaughan
et al. (1987) Immunogenetics 25:329-335). SEQ ID NO:3 is an
exemplary sequence for a full-length human CD200 protein.
[0185] In some embodiments, an anti-CD200 antibody described herein
binds to an epitope within the extracellular portion of a CD200
protein. For example, in some embodiments, the anti-CD200 antibody
can bind to CD200 protein at an epitope within or overlapping with:
(i) amino acids 1 to 233 of the amino acid sequence depicted in SEQ
ID NO:1; (ii) amino acids 1 to 258 of the amino acid sequence
depicted in SEQ ID NO:2; or amino acids 1 to 229 of the amino acid
sequence depicted in SEQ ID NO:3.
[0186] In some embodiments, the anti-CD200 antibody binds to an
epitope in the human CD200 protein lacking the leader sequence. For
example, an anti-CD200 antibody described herein can bind to a
CD200 protein at an epitope within or overlapping with amino acids
31 to 233 of the amino acid sequence depicted in SEQ ID NO:1, which
corresponds to the extracellular portion of the mature form of
human CD200 isoform A less the amino terminal leader sequence. In
some embodiments, an anti-CD200 antibody described herein can bind
to a CD200 protein at an epitope within or overlapping with amino
acids 56 to 258 of the amino acid sequence depicted in SEQ ID NO:2,
which corresponds to the extracellular portion of the mature form
of human CD200 isoform B less the amino terminal leader sequence.
In some embodiments, an anti-CD200 antibody described herein can
bind to a CD200 protein at an epitope within or overlapping with
amino acids 27 to 229 of the amino acid sequence depicted in SEQ ID
NO:3, which corresponds to the extracellular portion of the mature
form of human CD200 less the amino terminal leader sequence.
[0187] An "epitope" refers to the site on a protein (e.g., a human
CD200 protein) that is bound by an antibody. "Overlapping epitopes"
include at least one (e.g., two, three, four, five, or six) common
amino acid residue(s).
[0188] In some embodiments, the anti-CD200 antibody specifically
binds to a human CD200 protein (e.g., the human CD200 protein
having the amino acid sequence depicted in SEQ ID NO:1, SEQ ID
NO:2, SEQ ID NO:3, or the extracellular domains of the mature forms
of the CD200 proteins). The terms "specific binding" or
"specifically binds" refer to two molecules forming a complex
(e.g., a complex between an anti-CD200 antibody and a CD200
protein) that is relatively stable under physiologic conditions.
Typically, binding is considered specific when the association
constant (K.sub.a) is higher than 10.sup.6 M.sup.-1. Thus, an
anti-CD200 antibody can specifically bind to a CD200 protein with a
K.sub.a of at least (or greater than) 10.sup.6 (e.g., at least or
greater than 10.sup.7, 10.sup.8, 10.sup.9, 10.sup.10, 10.sup.11,
10.sup.12, 10.sup.13, 10.sup.14, or 10.sup.15 or higher) M.sup.-1.
Examples of antibodies that specifically bind to a human CD200
protein are described in, e.g., U.S. Pat. Nos. 7,408,041;
7,427,665; 7,435,412; and 7,598,353, the disclosures of each of
which are incorporated herein by reference in their entirety.
[0189] The amino acid sequence for several exemplary anti-CD200
antibodies are described in, e.g., U.S. Pat. No. 7,408,041. For
example, the anti-CD200 antibody can comprise the amino acid
sequence of the heavy and light chain variable regions of one of
the Fab antibodies--d1B10, d1A5, d1B5, c2aB7, c1A10, or
c2aA10--depicted in FIG. 23 of U.S. Pat. No. 7,408,041, the
sequences depicted in FIG. 23 being incorporated herein by
reference in their entirety. In some embodiments, an anti-CD200
antibody described herein contains a paired set of heavy chain CDRs
and light chain CDRs of one of the Fab antibodies depicted in FIG.
23 of U.S. Pat. No. 7,408,041. For example, an anti-CD200 antibody
described herein contains the paired set of CDRs from the d1B10 Fab
antibody: a heavy chain CDR1 (HCDR1) comprising the following
sequence: GFTFSGFAMS (SEQ ID NO:4); a heavy chain CDR2 (HCDR2)
comprising the following sequence: SISSGGTTYYLDSVKG (SEQ ID NO:5);
a heavy chain CDR3 (HCDR3) comprising the following sequence:
GNYYSGTSYDY (SEQ ID NO:6); a light chain CDR1 (LCDR1) comprising
the following sequence: RASESVDSYGNSFMH (SEQ ID NO:7); a light
chain CDR2 (LCDR2) comprising the following sequence: RASNLES (SEQ
ID NO:8); and a light chain CDR3 (LCDR3) comprising the following
sequence: QQSNEDPRT (SEQ ID NO:9).
[0190] In another example, an anti-CD200 antibody described herein
can contain the paired set of CDRs from the d1A5 Fab antibody: (i)
a HCDR1 comprising the following sequence: GFNIKDYYMH (SEQ ID
NO:10); a HCDR2 comprising the following sequence:
WIDPENGDTKYAPKFQG (SEQ ID NO:11); a HCDR3 comprising the following
sequence: KNYYVSNYNFFDV (SEQ ID NO:12); a LCDR1 comprising the
following sequence: SASSSVRYMY (SEQ ID NO:13); a LCDR2 comprising
the following sequence: DTSKLAS (SEQ ID NO:14); and a LCDR3
comprising the following sequence: FQGSGYPLT (SEQ ID NO:15).
[0191] In another example, an anti-CD200 antibody described herein
can comprise the paired set of CDRs from the d1B5 Fab antibody: a
HCDR1 comprising the following amino acid sequence: GFNIKDYYIH (SEQ
ID NO:16); a HCDR2 comprising the following amino acid sequence:
WIDPEIGATKYVPKFQG (SEQ ID NO:17); a HCDR3 comprising the following
amino acid sequence: LYGNYDRYYAMDY (SEQ ID NO:18); a LCDR1
comprising the following amino acid sequence: KASQNVRTAVA (SEQ ID
NO:19); a LCDR2 comprising the following amino acid sequence:
LASNRHT (SEQ ID NO:20); and a LCDR3 comprising the following amino
acid sequence: LQHWNYPLT (SEQ ID NO:21).
[0192] In another example, an anti-CD200 antibody described herein
can contain the paired set of CDRs from the c2aB7 Fab antibody: a
HCDR1 comprising the amino acid sequence: GYSFTDYIIL (SEQ ID
NO:22); a HCDR2 comprising the amino acid sequence:
HIDPYYGSSNYNLKFKG (SEQ ID NO:23); a HCDR3 comprising the amino acid
sequence: SKRDYFDY (SEQ ID NO:24); a LCDR1 comprising the amino
acid sequence: KASQDINSYLS (SEQ ID NO:25); a LCDR2 comprising the
amino acid sequence: RANRLVD (SEQ ID NO:26); and a LCDR3 comprising
the amino acid sequence: LQYDEFPYT (SEQ ID NO:27).
[0193] In yet another example, an anti-CD200 antibody described
herein can contain a paired set of CDRs from the c1A10 Fab
antibody: a HCDR1 comprising the amino acid sequence: GYTFTEYTMH
(SEQ ID NO:28); a HCDR2 comprising the amino acid sequence:
GVNPNNGGALYNQKFKG (SEQ ID NO:29); a HCDR3 comprising the amino acid
sequence: RSNYRYDDAMDY (SEQ ID NO:30); a LCDR1 comprising the amino
acid sequence: KSSQSLLDIDEKTYLN (SEQ ID NO:31); a LCDR2 comprising
the amino acid sequence: LVSKLDS (SEQ ID NO:32); and a LCDR3
comprising the amino acid sequence: WQGTHFPQT (SEQ ID NO:33).
[0194] And in yet another example, an anti-CD200 antibody described
herein can contain a paired set of CDRs from the c2aA10 Fab
antibody: a HCDR1 comprising the amino acid sequence: AFNIKDHYMH
(SEQ ID NO:34); a HCDR2 comprising the amino acid sequence:
WIDPESGDTEYAPKFQG (SEQ ID NO:35); a HCDR3 comprising the amino acid
sequence: FNGYQALDQ (SEQ ID NO:36); a LCDR1 comprising the amino
acid sequence: TASSSVSSSYLH (SEQ ID NO:37); a LCDR2 comprising the
amino acid sequence: STSNLAS (SEQ ID NO:38); and a LCDR3 comprising
the amino acid sequence: RQYHRSPPIFT (SEQ ID NO:39).
[0195] Additional exemplary sets of CDRs of anti-CD200 antibodies
are described in, e.g., U.S. Pat. No. 7,427,665. In some
embodiments, the anti-CD200 antibody is samalizumab.
[0196] Methods for determining whether an antibody binds to a
protein antigen and/or the affinity for an antibody to a protein
antigen are known in the art. For example, the binding of an
antibody to a protein antigen can be detected and/or quantified
using a variety of techniques such as, but not limited to, Western
blot, dot blot, surface plasmon resonance (SPR) method (e.g.,
BIAcore system; Pharmacia Biosensor AB, Uppsala, Sweden and
Piscataway, N.J.), or enzyme-linked immunosorbent assay (ELISA)
assays. See, e.g., Harlow and Lane (1988) "Antibodies: A Laboratory
Manual" Cold Spring Harbor Laboratory Press, Cold Spring Harbor,
N.Y.; Benny K. C. Lo (2004) "Antibody Engineering: Methods and
Protocols," Humana Press (ISBN: 1588290921); Borrebaek (1992)
"Antibody Engineering, A Practical Guide," W.H. Freeman and Co.,
NY; Borrebaek (1995) "Antibody Engineering," 2.sup.nd Edition,
Oxford University Press, NY, Oxford; Johne et al. (1993) J Immunol
Meth 160:191-198; Jonsson et al. (1993) Ann Biol Clin 51:19-26; and
Jonsson et al. (1991) Biotechniques 11:620-627.
[0197] In some embodiments, the anti-CD200 antibody can crossblock
binding of another antibody that binds to an epitope within, or
overlapping with, a human CD200 protein. In some embodiments, the
anti-CD200 antibody can crossblock binding of an antibody that
binds to an epitope within, or overlapping with, a peptide fragment
of a human CD200 protein. The peptide fragment can be a fragment of
a human CD200 protein having the amino acid sequence depicted in,
e.g., any one of SEQ ID NOs:1 to 3. As used herein, the term
"crossblocking antibody" refers to an antibody that lowers the
amount of binding of anti-CD200 antibody to an epitope on a CD200
protein relative to the amount of binding of the anti-CD200
antibody to the epitope in the absence of the antibody. Suitable
methods for determining whether a first antibody crossblocks
binding of a second antibody to an epitope are known in the
art.
[0198] Methods for identifying the epitope to which a particular
antibody (e.g., an anti-CD200 antibody) binds are also known in the
art. For example, the binding epitope of an anti-CD200 antibody can
be identified by measuring the binding of the antibody to several
(e.g., three, four, five, six, seven, eight, nine, 10, 15, 20, or
30 or more) overlapping peptide fragments of a CD200 protein (e.g.,
several overlapping fragments of a protein having the amino acid
sequence depicted in, e.g., any one of SEQ ID NOs:1 to 3). Each of
the different overlapping peptides is then bound to a unique
address on a solid support, e.g., separate wells of a multi-well
assay plate. Next, the anti-CD200 antibody is interrogated by
contacting it to each of the peptides in the assay plate for an
amount of time and under conditions that allow for the antibody to
bind to its epitope. Unbound anti-CD200 antibody is removed by
washing each of the wells. Next, a detectably-labeled secondary
antibody that binds to the anti-CD200 antibody, if present in a
well of the plate, is contacted to each of the wells, and unbound
secondary antibody is removed by washing steps. The presence or
amount of the detectable signal produced by the detectably-labeled
secondary antibody in a well is an indication that the anti-CD200
antibody binds to the particular peptide fragment associated with
the well. See, e.g., Harlow and Lane (supra), Benny K. C. Lo
(supra), and U.S. Patent Application Publication No. 20060153836,
the disclosure of which is incorporated by reference in its
entirety. A particular epitope to which an antibody binds can also
be identified using BIAcore chromatographic techniques (see, e.g.,
Pharmacia BIAtechnology Handbook, "Epitope Mapping," Section 6.3.2,
(May 1994); and Johne et al. (1993)J Immunol Methods
160:191-8).
[0199] In some embodiments, an anti-CD200 antibody, or a
CD200-binding fragment thereof, described herein binds to a human
CD200 polypeptide expressed on the surface of a cell. Methods for
determining whether an antibody binds to a protein expressed on the
surface of a cell are known in the art and described in, e.g.,
Petermann et al. (2007)J Clin Invest 117(12):3922-9; Rijkers et al.
(2008) Mol Immunol 45(4):1126-35; and Kretz-Rommel (2007) J Immunol
178(9):5595-605.
[0200] In some embodiments, an anti-CD200 antibody or CD200-binding
fragment thereof described herein inhibits the interaction between
CD200 protein and the CD200 receptor. Methods for determining
whether an agent (such as an antibody) inhibits the interaction
between CD200 and CD200R are known in the art and described in,
e.g., Hatherley and Barclay (2004) Eur J Immunol 34(6):1688-94.
[0201] In some embodiments, the anti-CD200 antibody or
CD200-binding fragment thereof inhibits the formation of
osteoclasts in vitro and/or in vivo. Suitable methods for
determining whether an antibody inhibits the formation of
osteoclasts are known in the art and described in, e.g., PCT
Publication No. WO 08/089022 and Cui et al. (2007) Proc Natl Acad
Sci USA 104(36):14436-14441. For example, murine bone marrow cells
can be cultured in the presence of, e.g., RANKL and M-CSF in the
presence or absence of an anti-CD200 antibody. A decrease in the
percentage of osteoclasts formed from the bone marrow cells in the
presence of the antibody as compared to the percentage of
osteoclasts formed in the absence of the antibody indicates that
the antibody inhibits osteoclast formation in vitro.
[0202] Since CD200 is expressed on normal cells such as endothelial
cells, albeit at lower levels than on cancer cells, it could be in
some embodiments advantageous to administer a variant anti-CD200
antibody (or CD200-binding fragment thereof) with a constant region
modified so that it does not mediate, or has decreased ability to
mediate, ADCC or CDC. Such a modification would be useful to limit
damage to normal cells. CD200 expression is also upregulated on
some activated normal cells (e.g., activated T cells), rendering
such cells vulnerable to killing by an anti-CD200 antibody with
effector function. It may be advantageous to use an anti-CD200
antibody lacking effector function to avoid killing of these cells
by ADCC or CDC. The effector function of an anti-CD200 antibody can
be eliminated by replacing an immunoglobulin constant region that
has effector function (e.g., the IgG1 constant domain) for a
constant region that does not have effector function (e.g., an
IgG2/IgG4 fusion constant region). Additional methods for
eliminating effector function are described below.
[0203] Effector Functions
[0204] The interaction of antibodies and antibody-antigen complexes
with cells of the immune system affects a variety of responses,
referred to herein as effector functions. Exemplary effector
functions include Fc receptor binding, phagocytosis,
down-regulation of cell surface receptors (e.g. B cell receptor;
BCR), etc. Other effector functions include ADCC, whereby
antibodies bind Fc receptors on natural killer (NK) cells or
macrophages leading to cell death, and CDC, which is cell death
induced via activation of the complement cascade (reviewed in
Daeron (1997) Annu Rev Immunol 15:203-234; Ward and Ghetie (1995)
Therapeutic Immunol 2:77-94; and Ravetch and Kinet (1991) Annu Rev
Immunol 9:457-492). Such effector functions generally require the
Fc region to be combined with a binding domain (e.g., an antibody
variable domain) and can be assessed using various assays as herein
disclosed.
[0205] Several antibody effector functions, including ADCC, are
mediated by Fc receptors (FcRs), which bind the Fc region of an
antibody. In ADCC, NK cells or macrophages bind to the Fc region of
the antibody complex and promote lysis of the target cell. The
cross-linking of FcRs on NK cells triggers
perforin/granzyme-mediated cytotoxicity, whereas in macrophages
this cross-linking promotes the release of mediators such as nitric
oxide (NO), TNF-.alpha., and reactive oxygen species. For
CD200-positive target cells, an anti-CD200 antibody binds to the
target cell and the Fc region directs effector function to the
target cell. The affinity of an antibody for a particular FcR, and
hence the effector activity mediated by the antibody, may be
modulated by altering the amino acid sequence and/or
post-translational modifications of the Fc and/or constant region
of the antibody.
[0206] FcRs are defined by their specificity for immunoglobulin
isotypes; Fc receptors for IgG antibodies are referred to as
Fc.gamma.R, for IgE as Fc.epsilon.R, for IgA as Fc.alpha.R and so
on. Three subclasses of Fc.gamma.R have been identified:
Fc.gamma.RI (CD64), Fc.gamma.RII (CD32) and Fc.gamma.RIII (CD16).
Because each Fc.gamma.R subclass is encoded by two or three genes,
and alternative RNA splicing leads to multiple transcripts, a broad
diversity in Fc.gamma.R isoforms exists. The three genes encoding
the Fc.gamma.RI subclass (Fc.gamma.RIA, Fc.gamma.RIB and
Fc.gamma.RIC) are clustered in region 1q21.1 of the long arm of
chromosome 1; the genes encoding Fc.gamma.RII isoforms
(Fc.gamma.RIIA, Fc.gamma.RIIB and Fc.gamma.RIIC) and the two genes
encoding Fc.gamma.RIII (Fc.gamma.RIIIA and Fc.gamma.RIIIB) are all
clustered in region 1q22. These different FcR subtypes are
expressed on different cell types (reviewed in Ravetch and Kinet
(1991) Annu Rev Immunol 9:457-492). For example, in humans,
Fc.gamma.RIIIB is found only on neutrophils, whereas Fc.gamma.RIIIA
is found on macrophages, monocytes, natural killer (NK) cells, and
a subpopulation of T-cells. Notably, Fc.gamma.RIIIA is the only FcR
present on NK cells, one of the cell types implicated in ADCC.
[0207] Fc.gamma.RI, Fc.gamma.RII and Fc.gamma.RIII are
immunoglobulin superfamily (IgSF) receptors; Fc.gamma.RI has three
IgSF domains in its extracellular domain, while Fc.gamma.RII and
Fc.gamma.RIII have only two IgSF domains in their extracellular
domains. Another type of Fc receptor is the neonatal Fc receptor
(FcRn). FcRn is structurally similar to major histocompatibility
complex (MHC) and consists of an .alpha.-chain noncovalently bound
to .beta.32-microglobulin.
[0208] The binding site on human and murine antibodies for
Fc.gamma.R have been previously mapped to the so-called "lower
hinge region" consisting of residues 233-239 (EU index numbering as
in Kabat et al. (1991) Sequences of Proteins of Immunological
Interest, 5th Ed. Public Health Service, National Institutes of
Health, Bethesda, Md.). Woof et al. (1986) Molec Immunol
23:319-330; Duncan et al. (1988) Nature 332:563; Canfield and
Morrison (1991) J Exp Med 173:1483-1491; Chappel et al. (1991) Proc
Natl Acad Sci USA 88:9036-9040. Of residues 233-239, P238 and S239
have been cited as possibly being involved in binding.
[0209] Other previously cited areas possibly involved in binding to
Fc.gamma.R are: G316-K338 (human IgG) for human Fc.gamma.RI (by
sequence comparison only; no substitution mutants were evaluated)
(Woof et al. (1986) Molec Immunol 23:319-330); K274-R301 (human
IgG1) for human Fc.gamma.RIII (based on peptides) (Sarmay et al.
(1984) Molec Immunol 21:43-51); Y407-R416 (human IgG) for human
Fc.gamma.RIII (based on peptides) (Gergely et al. (1984) Biochem
Soc Trans 12:739-743 (1984)); as well as N297 and E318 (murine
IgG2b) for murine Fc.gamma.RII (Lund et al. (1992) Molec Immunol
29:53-59).
[0210] Human effector cells are leukocytes which express one or
more FcRs and perform effector functions. In certain embodiments,
the cells express at least Fc.gamma.RIII and perform ADCC effector
function. Examples of human leukocytes which mediate ADCC include
peripheral blood mononuclear cells (PBMC), natural killer (NK)
cells, monocytes, cytotoxic T cells and neutrophils. Effector cells
may be isolated from a native source thereof, e.g. from blood or
PBMCs.
[0211] In CDC, the antibody-antigen complex binds complement,
resulting in the activation of the complement cascade and
generation of the membrane attack complex. Activation of the
classical complement pathway is initiated by the binding of the
first component of the complement system (C1q) to antibodies (of
the appropriate subclass) which are bound to their cognate antigen;
thus the activation of the complement cascade is regulated in part
by the binding affinity of the immunoglobulin to C1q protein. C1q
and two serine proteases, C1r and C1s, form the complex C1, the
first component of the CDC pathway. C1q is a hexavalent molecule
with a molecular weight of approximately 460,000 and a structure in
which six collagenous "stalks" are connected to six globular head
regions. Burton and Woof (1992) Advances in Immunol 51:1-84. To
activate the complement cascade, it is necessary for C1q to bind to
at least two molecules of IgG1, IgG2, or IgG3, but only one
molecule of IgM, attached to the antigenic target (Ward and Ghetie
(1995) Therapeutic Immunology 2:77-94). To assess complement
activation, a CDC assay, e.g. as described in Gazzano-Santoro et
al. (1996) J Immunol Methods 202:163, can be performed.
[0212] It has been proposed that various residues of the IgG
molecule are involved in binding to C1q including the Glu318,
Lys320 and Lys322 residues on the CH2 domain, amino acid residue
331 located on a turn in close proximity to the same beta strand,
the Lys235 and Gly237 residues located in the lower hinge region,
and residues 231 to 238 located in the N-terminal region of the CH2
domain. See, e.g., Xu et al. (1993) J Immunol 150:152A; PCT
publication no. WO 94/29351; Tao et al. (1993)J Exp Med
178:661-667; Brekke et al. (1994) Eur J Immunol 24:2542-47; Burton
et al. (1980) Nature 288:338-344; and U.S. Pat. Nos. 5,648,260 and
5,624,821. It has further been proposed that the ability of IgG to
bind C1q and activate the complement cascade also depends on the
presence, absence or modification of the carbohydrate moiety
positioned between the two CH2 domains (which is normally anchored
at Asn297). See, e.g., Ward and Ghetie (1995) Therapeutic
Immunology 2:77-94. In certain embodiments, one or more of these
residues may be modified, substituted, or removed or one or more
amino acid residues may be inserted so as to enhance or decrease
CDC activity of the anti-CD200 antibodies provided herein.
[0213] Methods for Decreasing or Eliminating Effector Function
[0214] Effector functions involving the constant region of the
target-specific antibody may be modulated by altering properties of
the constant or Fc region. Altered effector functions include, for
example, a modulation in one or more of the following activities:
ADCC, CDC, apoptosis, binding to one or more Fc-receptors, and
pro-inflammatory responses. Modulation refers to an increase,
decrease, or elimination of an effector function activity exhibited
by a subject antibody as compared to the activity of a second
antibody. In certain embodiments, the second antibody is an
antibody possessing a naturally-occurring effector function that
has not been modified. In particular embodiments, modulation
includes situations in which an activity is abolished or completely
absent. Further, in some instances, a non-variant antibody may
exhibit effector function activity similar or equivalent to the
activity of the chC2aB7-hG1 or the hB7V3V2-hG1 antibodies disclosed
herein.
[0215] A variant constant region with altered FcR binding affinity
and/or ADCC activity and/or altered CDC activity is a polypeptide
which has either enhanced or diminished FcR binding activity and/or
ADCC activity and/or CDC activity compared to the native or parent
polypeptide or to a polypeptide comprising a native sequence or
constant region. A polypeptide variant which displays increased
binding to an FcR binds at least one FcR with greater affinity than
the parent polypeptide. A polypeptide variant which displays
decreased binding to an FcR binds at least one FcR with lower
affinity than a parent polypeptide. Such variants which display
decreased binding to an FcR may possess little or no appreciable
binding to an FcR, e.g., 0 to 50% (e.g., less than 50, 49, 48, 47,
46, 45, 44, 43, 42, 41, 40, 39, 38, 37, 36, 35, 34, 33, 32, 31, 30,
29, 28, 27, 26, 25, 24, 23, 22, 21, 20, 19, 18, 17, 16, 15, 14, 13,
12, 11, 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1%) of the binding to the
FcR as compared to the level of binding of a native sequence
immunoglobulin constant or Fc region to the FcR. Similarly, a
variant anti-CD200 antibody that displays altered ADCC and/or CDC
activity may exhibit either increased or reduced ADCC and/or CDC
activity compared to the native or parent polypeptide. For example,
in some embodiments, the anti-CD200 antibody comprising a variant
constant region can exhibit approximately 0 to 50% (e.g., less than
50, 49, 48, 47, 46, 45, 44, 43, 42, 41, 40, 39, 38, 37, 36, 35, 34,
33, 32, 31, 30, 29, 28, 27, 26, 25, 24, 23, 22, 21, 20, 19, 18, 17,
16, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1%) of the
ADCC and/or CDC activity of the native form of the constant region.
An anti-CD200 antibody comprising a variant constant region
displaying reduced ADCC and/or CDC may exhibit reduced or no ADCC
and/or CDC activity as shown herein by example.
[0216] A native sequence Fe or constant region comprises an amino
acid sequence identical to the amino acid sequence of a Fc or
constant chain region found in nature. A variant or altered Fc or
constant region comprises an amino acid sequence which differs from
that of a native sequence heavy chain region by virtue of at least
one amino acid modification, insertion, or deletion, for example.
In certain embodiments, the variant or altered constant region has
at least one amino acid substitution, insertion, and/or deletion,
compared to a native sequence constant region or to the constant
region of a parent polypeptide, e.g. from about one to about one
hundred amino acid substitutions, insertions, and/or deletions in a
native sequence constant region or in the constant region of the
parent polypeptide. In some embodiments, the variant or altered
constant region herein will possess at least about 70% homology
(similarity) or identity with a native sequence constant region
and/or with a constant region of a parent polypeptide, and in some
instances at least about 75% and in other instances at least about
80% homology or identity therewith, and in other embodiments at
least about 85%, 90% or 95% homology or identity therewith. The
variant or altered constant region may also contain one or more
amino acid deletions or insertions. Additionally, the variant
constant region may contain one or more amino acid substitutions,
deletions, or insertions that results in altered post-translational
modifications, including, for example, an altered glycosylation
pattern.
[0217] Antibodies or antigen-binding fragments thereof with altered
or no effector functions may be generated by engineering or
producing antibodies with variant constant, Fc, or heavy chain
regions; recombinant DNA technology and/or cell culture and
expression conditions may be used to produce antibodies with
altered function and/or activity. For example, recombinant DNA
technology may be used to engineer one or more amino acid
substitutions, deletions, or insertions in regions (such as, for
example, Fc or constant regions) that affect antibody function
including effector functions. Alternatively, changes in
post-translational modifications, such as, e.g., glycosylation
patterns, may be achieved by manipulating the cell culture and
expression conditions by which the antibody is produced.
[0218] Accordingly, certain aspects and methods of the present
disclosure relate to anti-CD200 antibodies with altered effector
functions that comprise one or more amino acid substitutions,
insertions, and/or deletions. In some embodiments, such a variant
anti-CD200 antibody exhibits reduced or no effector function. In
some embodiments, a variant antibody comprises a hybrid constant
region, or a portion thereof, such as a G2/G4 hybrid constant
region (see e.g., Burton et al. (1992) Adv Immun 51:1-18; Canfield
et al. (1991) J Exp Med 173:1483-1491; and Mueller et al. (1997)
Mol Immunol 34(6):441-452). For example (and in accordance with
Kabat numbering), the IgG1 and IgG4 constant regions contain
G.sub.249G.sub.250 residues whereas the IgG2 constant region does
not contain residue 249, but does contain G.sub.250. In a G2/G4
hybrid constant region, where the 249-250 region comes from the G2
sequence, the constant region can be further modified to introduce
a glycine residue at position 249 to produce a G2/G4 fusion having
G.sub.249/G.sub.250.
[0219] In addition to using a G2/G4 construct as described above,
anti-CD200 antibodies with reduced effector function may be
produced by introducing other types of changes in the amino acid
sequence of certain regions of the antibody. Such amino acid
sequence changes include but are not limited to the Ala-Ala
mutation described in, e.g., PCT Publication nos. WO 94/28027 and
WO 98/47531; and Xu et al. (2000) Cell Immunol 200:16-26. Thus, in
some embodiments, anti-CD200 antibodies with mutations within the
constant region including the Ala-Ala mutation may be used to
reduce or abolish effector function. According to these
embodiments, the constant region of an anti-CD200 antibody
comprises a mutation to an alanine at position 234 or a mutation to
an alanine at position 235. Additionally, the constant region may
contain a double mutation: a mutation to an alanine at position 234
and a second mutation to an alanine at position 235. In one
embodiment, the anti-CD200 antibody comprises an IgG4 framework,
wherein the Ala-Ala mutation would describe a mutation(s) from
phenylalanine to alanine at position 234 and/or a mutation from
leucine to alanine at position 235. In another embodiment, the
anti-CD200 antibody comprises an IgG1 framework, wherein the
Ala-Ala mutation would describe a mutation(s) from leucine to
alanine at position 234 and/or a mutation from leucine to alanine
at position 235. An anti-CD200 antibody may alternatively or
additionally carry other mutations, including the point mutation
K322A in the CH2 domain (Hezareh et al. (2001) J Virol 75:12161-8).
An antibody with said mutation(s) in the constant region may
furthermore be a blocking or non-blocking antibody.
[0220] Changes within the hinge region also affect effector
functions. For example, deletion of the hinge region may reduce
affinity for Fc receptors and may reduce complement activation
(Klein et al. (1981) Proc Natl Acad Sci USA 78: 524-528). The
present disclosure therefore also relates to antibodies with
alterations in the hinge region.
[0221] In some embodiments, anti-CD200 antibodies may be modified
to either enhance or inhibit complement dependent cytotoxicity
(CDC). Modulated CDC activity may be achieved by introducing one or
more amino acid substitutions, insertions, or deletions in an Fc
region of the antibody. See, e.g., U.S. Pat. No. 6,194,551.
Alternatively or additionally, cysteine residue(s) may be
introduced in the Fc region, thereby allowing interchain disulfide
bond formation in this region. The homodimeric antibody thus
generated may have improved or reduced internalization capability
and/or increased or decreased complement-mediated cell killing.
See, e.g., Caron et al. (1992) J Exp Med 176:1191-1195 and Shopes
(1992) Immunol 148:2918-2922; PCT publication nos. WO99/51642 and
WO 94/29351; Duncan and Winter (1988) Nature 322:738-40; and U.S.
Pat. Nos. 5,648,260 and 5,624,821. Homodimeric antibodies with
enhanced anti-tumor activity may also be prepared using
heterobifunctional cross-linkers as described in Wolff et al.
(1993) Cancer Research 53:2560-2565. Alternatively, an antibody can
be engineered which has dual Fc regions and may thereby have
enhanced complement lysis and ADCC capabilities. See, e.g.,
Stevenson et al. (1989) Anti-Cancer Drug Design 3:219-230.
[0222] Another potential means of modulating effector function of
antibodies includes changes in glycosylation, which is summarized
in, e.g., Raju (2003) BioProcess International 1(4):44-53.
According to Wright and Morrison, the microheterogeneity of human
IgG oligosaccharides can affect biological functions such as CDC
and ADCC, binding to various Fc receptors, and binding to C1q
protein. (1997) TIBTECH 15:26-32. Glycosylation patterns of
antibodies can differ depending on the producing cell and the cell
culture conditions (Raju, supra). Such differences can lead to
changes in both effector function and pharmacokinetics. See, e.g.,
Israel et al. (1996) Immunology 89(4):573-578; Newkirk et al.
(1996) Clin Exp Immunol 106(2):259-64. Differences in effector
function may be related to the IgG's ability to bind to the
Fc.gamma. receptors (Fc.gamma.Rs) on the effector cells. Shields et
al. have shown that IgG, with variants in amino acid sequence that
have improved binding to Fc.gamma.R, can exhibit up to 100%
enhanced ADCC using human effector cells. (2001) J Biol Chem
276(9):6591-604. While these variants include changes in amino
acids not found at the binding interface, both the nature of the
sugar component as well as its structural pattern may also
contribute to the differences observed. In addition, the presence
or absence of fucose in the oligosaccharide component of an IgG can
improve binding and ADCC. See, e.g., Shields et al. (2002) J Biol
Chem 277(30):26733-40. An IgG that lacked a fucosylated
carbohydrate linked to Asn.sup.297 exhibited normal receptor
binding to the Fc.gamma.RI receptor. In contrast, binding to the
Fc.gamma.RIIIA receptor was improved 50-fold and accompanied by
enhanced ADCC, especially at lower antibody concentrations.
[0223] Shinkawa et al. demonstrated that an antibody to the human
IL-5 receptor produced in a rat hybridoma showed more than 50%
higher ADCC when compared to the antibody produced in Chinese
hamster ovary cells (CHO) (Shinkawa et al. (2003) J Biol Chem
278(5):3466-73). Monosaccharide composition and oligosaccharide
profiling showed that the rat hybridoma-produced IgG had a lower
content of fucose than the CHO-produced protein. The authors
concluded that the lack of fucosylation of an IgG1 has a critical
role in enhancement of ADCC activity.
[0224] A different approach was taken by Umana et al. who changed
the glycosylation pattern of chCE7, a chimeric IgG1
anti-neuroblastoma antibody. (1999) Nat Biotechnol 17(2):176-80).
Using tetracycline, they regulated the activity of a
glycosyltransferase enzyme (GnTIII) which bisects oligosaccharides
that have been implicated in ADCC activity. The ADCC activity of
the parent antibody was barely above background level. Measurement
of ADCC activity of the chCE7 produced at different tetracycline
levels showed an optimal range of GnTIII expression for maximal
chCE7 in vitro ADCC activity. This activity correlated with the
level of constant region-associated, bisected complex
oligosaccharide. Newly optimized variants exhibited substantial
ADCC activity. Similarly, Wright and Morrison produced antibodies
in a CHO cell line deficient in glycosylation and showed that
antibodies produced in this cell line were incapable of
complement-mediated cytolysis. (1994) J Exp Med 180:1087-1096.
Thus, as known alterations that affect effector function include
modifications in the glycosylation pattern or a change in the
number of glycosylated residues, the present disclosure relates to
a CD200 antibody wherein glycosylation is altered to either enhance
or decrease effector function(s) including ADCC and CDC. Altered
glycosylation includes a decrease or increase in the number of
glycosylated residues as well as a change in the pattern or
location of glycosylated residues.
[0225] Still other approaches exist for altering the effector
function of antibodies. For example, antibody-producing cells can
be hypermutagenic, thereby generating antibodies with randomly
altered polypeptide residues throughout an entire antibody
molecule. See, e.g., PCT publication no. WO 05/011735.
Hypermutagenic host cells include cells deficient in DNA mismatch
repair. Antibodies produced in this manner may be less antigenic
and/or have beneficial pharmacokinetic properties. Additionally,
such antibodies may be selected for properties such as enhanced or
decreased effector function(s).
[0226] It is further understood that effector function may vary
according to the binding affinity of the antibody. For example,
antibodies with high affinity may be more efficient in activating
the complement system compared to antibodies with relatively lower
affinity (Marzocchi-Machado et al. (1999) Immunol Invest
28:89-101). Accordingly, an antibody may be altered such that the
binding affinity for its antigen is reduced (e.g., by changing the
variable regions of the antibody by methods such as substitution,
addition, or deletion of one or more amino acid residues). An
anti-CD200 antibody with reduced binding affinity may exhibit
reduced effector functions, including, for example, reduced ADCC
and/or CDC.
[0227] Pharmaceutical Compositions and Formulations
[0228] The compositions containing an anti-CD200 antibody can be
formulated as a pharmaceutical composition, e.g., for
administration to a human to treat cancer. The pharmaceutical
compositions will generally include a pharmaceutically acceptable
carrier. As used herein, a "pharmaceutically acceptable carrier"
refers to, and includes, any and all solvents, dispersion media,
coatings, antibacterial and antifungal agents, isotonic and
absorption delaying agents, and the like that are physiologically
compatible. The compositions can include a pharmaceutically
acceptable salt, e.g., an acid addition salt or a base addition
salt. See, e.g., Berge et al. (1977) J Pharm Sci 66:1-19.
[0229] The compositions can be formulated according to standard
methods. Pharmaceutical formulation is a well-established art, and
is further described in, e.g., Gennaro (2000) "Remington: The
Science and Practice of Pharmacy," 20.sup.th Edition, Lippincott,
Williams & Wilkins (ISBN: 0683306472); Ansel et al. (1999)
"Pharmaceutical Dosage Forms and Drug Delivery Systems," 7.sup.th
Edition, Lippincott Williams & Wilkins Publishers (ISBN:
0683305727); and Kibbe (2000) "Handbook of Pharmaceutical
Excipients American Pharmaceutical Association," 3.sup.rd Edition
(ISBN: 091733096X). In some embodiments, a composition can be
formulated, for example, as a buffered solution at a suitable
concentration and suitable for storage at 2-8.degree. C. In some
embodiments, a composition can be formulated for storage at a
temperature below 0.degree. C. (e.g., -20.degree. C. or -80.degree.
C.).
[0230] The pharmaceutical compositions can be in a variety of
forms. These forms include, e.g., liquid, semi-solid and solid
dosage forms, such as liquid solutions (e.g., injectable and
infusible solutions), dispersions or suspensions, tablets, pills,
powders, liposomes and suppositories. The preferred form depends,
in part, on the intended mode of administration and therapeutic
application. For example, compositions containing an anti-CD200
antibody intended for systemic or local delivery can be in the form
of injectable or infusible solutions. Accordingly, the compositions
can be formulated for administration by a parenteral mode (e.g.,
intravenous, subcutaneous, intraperitoneal, or intramuscular
injection). "Parenteral administration," "administered
parenterally," and other grammatically equivalent phrases, as used
herein, refer to modes of administration other than enteral and
topical administration, usually by injection, and include, without
limitation, intravenous, intranasal, intraocular, pulmonary,
intramuscular, intraarterial, intrathecal, intracapsular,
intraorbital, intracardiac, intradermal, intrapulmonary,
intraperitoneal, transtracheal, subcutaneous, subcuticular,
intraarticular, subcapsular, subarachnoid, intraspinal, epidural,
intracerebral, intracranial, intracarotid and intrasternal
injection and infusion (see below).
[0231] The compositions can be formulated as a solution,
microemulsion, dispersion, liposome, or other ordered structure
suitable for stable storage at high concentration. Sterile
injectable solutions can be prepared by incorporating an antibody
described herein in the required amount in an appropriate solvent
with one or a combination of ingredients enumerated above, as
required, followed by filtered sterilization. Generally,
dispersions are prepared by incorporating an anti-CD200 antibody
described herein into a sterile vehicle that contains a basic
dispersion medium and the required other ingredients from those
enumerated above. In the case of sterile powders for the
preparation of sterile injectable solutions, methods for
preparation include vacuum drying and freeze-drying that yield a
powder of the antibody described herein plus any additional desired
ingredient from a previously sterile-filtered solution thereof. The
proper fluidity of a solution can be maintained, for example, by
the use of a coating such as lecithin, by the maintenance of the
required particle size in the case of dispersion and by the use of
surfactants. Prolonged absorption of injectable compositions can be
brought about by including in the composition a reagent that delays
absorption, for example, monostearate salts and gelatin.
[0232] In certain embodiments, the anti-CD200 antibody can be
prepared with a carrier that will protect the compound against
rapid release, such as a controlled release formulation, including
implants and microencapsulated delivery systems. Biodegradable,
biocompatible polymers can be used, such as ethylene vinyl acetate,
polyanhydrides, polyglycolic acid, collagen, polyorthoesters, and
polylactic acid. Many methods for the preparation of such
formulations are known in the art. (See, e.g., J. R. Robinson
(1978) "Sustained and Controlled Release Drug Delivery Systems,"
Marcel Dekker, Inc., New York.)
[0233] In some embodiments, an antibody described herein can be
formulated in a composition suitable for intrapulmonary
administration (e.g., for administration via nebulizer) to a mammal
such as a human. Methods for preparing such compositions are well
known in the art and described in, e.g., U.S. Patent Application
Publication No. 20080202513; U.S. Pat. Nos. 7,112,341 and
6,019,968; and PCT Publication Nos. WO 00/061178 and WO 06/122257,
the disclosures of each of which are incorporated herein by
reference in their entirety. Dry powder inhaler formulations and
suitable systems for administration of the formulations are
described in, e.g., U.S. Patent Application Publication No.
20070235029, PCT Publication No. WO 00/69887; and U.S. Pat. No.
5,997,848.
[0234] In some embodiments, an anti-CD200 antibody described herein
can be modified, e.g., with a moiety that improves its
stabilization and/or retention in circulation, e.g., in blood,
serum, or other tissues. The stabilization moiety can improve the
stability, or retention of, the antibody by at least 1.5 (e.g., at
least 2, 5, 10, 15, 20, 25, 30, 40, or 50 or more) fold.
[0235] In some embodiments, an anti-CD200 antibody described herein
can be formulated with one or more additional active agents useful
for treating cancer or ameliorating a symptom thereof. For example,
an anti-CD200 antibody can be formulated with a genotoxic agent or
a chemotherapeutic agent, or one or more kinase inhibitors. The
genotoxic or chemotherapeutic agent can be, but is not limited to:
carboplatin, procarbazine, mechlorethamine, cyclophosphamide,
camptothecin, ifosfamide, melphalan, chlorambucil, busulfan,
nitrosurea, dactinomycin, daunorubicin, doxorubicin, bleomycin,
plicomycin, mitomycin, etoposide, podophyllotoxin, taxol,
satraplatinum, 5-fluorouracil, vincristin, vinblastin,
methotrexate, ara-C, taxotere, gemcitabine, cisplatin (CDDP),
adriamycin (ADR), or an analog of any of the aforementioned. Kinase
inhibitors include, e.g., one or more of: trastuzumab, gefitinib,
erlotinib, imatinib mesylate, or sunitinib malate. Additional
agents are known in the art and described herein.
[0236] When the anti-CD200 antibody is to be used in combination
with a second active agent, or when two or more different
anti-CD200 antibodies are to be used, the agents can be formulated
separately or together. For example, the respective pharmaceutical
compositions can be mixed, e.g., just prior to administration, and
administered together or can be administered separately, e.g., at
the same or different times (see below).
[0237] As described above, a composition can be formulated such
that it includes a therapeutically effective amount of an
anti-CD200 antibody or the composition can be formulated to include
a sub-therapeutic amount of the antibody and a sub-therapeutic
amount of one or more additional active agents such that the
components in total are therapeutically effective for treating a
cancer. In some embodiments, a composition can be formulated to
include two or more anti-CD200 antibodies, each at sub-therapeutic
doses, such that the antibodies in combination are at a
concentration that is therapeutically effective for treating a
cancer in a human. Methods for determining a therapeutically
effective dose of an anti-CD200 antibody are known in the art and
described herein.
Methods for Producing an Anti-CD200 Antibody
[0238] Suitable methods for producing an anti-CD200 antibody, or
CD200-binding fragments thereof, in accordance with the disclosure
are known in the art (see, e.g., U.S. Pat. Nos. 7,427,665;
7,435,412; and 7,408,041, the disclosures of each of which are
incorporated herein by reference in their entirety) and described
herein. For example, monoclonal anti-CD200 antibodies may be
generated using human CD200-expressing cells, a human CD200
polypeptide, or an antigenic fragment of a human CD200 polypeptide
as an immunogen, thus raising an immune response in animals from
which antibody-producing cells and in turn monoclonal antibodies
may be isolated. The sequence of such antibodies may be determined
and the antibodies or variants thereof produced by recombinant
techniques. Recombinant techniques may be used to produce chimeric,
CDR-grafted, humanized and fully human antibodies based on the
sequence of the monoclonal antibodies as well as polypeptides
capable of binding to CD200 or a fragment thereof.
[0239] Moreover, antibodies derived from recombinant libraries
("phage antibodies") may be selected using CD200-expressing cells,
or polypeptides derived therefrom, as bait to isolate the
antibodies or polypeptides on the basis of target specificity. The
production and isolation of non-human and chimeric anti-CD200
antibodies are well within the purview of the skilled artisan.
[0240] Recombinant DNA technology can be used to modify one or more
characteristics of the antibodies produced in non-human cells.
Thus, chimeric antibodies can be constructed in order to decrease
the immunogenicity thereof in diagnostic or therapeutic
applications. Moreover, immunogenicity can be minimized by
humanizing the antibodies by CDR grafting and, optionally,
framework modification. See, U.S. Pat. Nos. 5,225,539 and
7,393,648, the contents of each of which are incorporated herein by
reference.
[0241] Antibodies can be obtained from animal serum or, in the case
of monoclonal antibodies or fragments thereof, produced in cell
culture. Recombinant DNA technology can be used to produce the
antibodies according to established procedure, including procedures
in bacterial or preferably mammalian cell culture. The selected
cell culture system preferably secretes the antibody product.
[0242] In another embodiment, a process for the production of an
antibody disclosed herein includes culturing a host, e.g. E. coli
or a mammalian cell, which has been transformed with a hybrid
vector. The vector includes one or more expression cassettes
containing a promoter operably linked to a first DNA sequence
encoding a signal peptide linked in the proper reading frame to a
second DNA sequence encoding the antibody protein. The antibody
protein is then collected and isolated. Optionally, the expression
cassette may include a promoter operably linked to a polycistronic
(e.g., bicistronic) DNA sequence encoding antibody proteins each
individually operably linked to a signal peptide in the proper
reading frame.
[0243] Multiplication of hybridoma cells or mammalian host cells in
vitro is carried out in suitable culture media, which include the
customary standard culture media (such as, for example Dulbecco's
Modified Eagle Medium (DMEM) or RPMI 1640 medium), optionally
replenished by a mammalian serum (e.g. fetal calf serum), or trace
elements and growth sustaining supplements (e.g. feeder cells such
as normal mouse peritoneal exudate cells, spleen cells, bone marrow
macrophages, 2-aminoethanol, insulin, transferrin, low density
lipoprotein, oleic acid, or the like). Multiplication of host cells
which are bacterial cells or yeast cells is likewise carried out in
suitable culture media known in the art. For example, for bacteria
suitable culture media include medium LE, NZCYM, NZYM, NZM,
Terrific Broth, SOB, SOC, 2.times.YT, or M9 Minimal Medium. For
yeast, suitable culture media include medium YPD, YEPD, Minimal
Medium, or Complete Minimal Dropout Medium.
[0244] In vitro production provides relatively pure antibody
preparations and allows scale-up production to give large amounts
of the desired antibodies. Techniques for bacterial cell, yeast,
plant, or mammalian cell cultivation are known in the art and
include homogeneous suspension culture (e.g. in an airlift reactor
or in a continuous stirrer reactor), and immobilized or entrapped
cell culture (e.g. in hollow fibers, microcapsules, on agarose
microbeads or ceramic cartridges).
[0245] Large quantities of the desired antibodies can also be
obtained by multiplying mammalian cells in vivo. For this purpose,
hybridoma cells producing the desired antibodies are injected into
histocompatible mammals to cause growth of antibody-producing
tumors. Optionally, the animals are primed with a hydrocarbon,
especially mineral oils such as pristane (tetramethyl-pentadecane),
prior to the injection. After one to three weeks, the antibodies
are isolated from the body fluids of those mammals. For example,
hybridoma cells obtained by fusion of suitable myeloma cells with
antibody-producing spleen cells from Balb/c mice, or transfected
cells derived from hybridoma cell line Sp2/0 that produce the
desired antibodies are injected intraperitoneally into Balb/c mice
optionally pre-treated with pristane. After one to two weeks,
ascitic fluid is taken from the animals.
[0246] The foregoing, and other, techniques are discussed in, for
example, Kohler and Milstein, (1975) Nature 256:495-497; U.S. Pat.
No. 4,376,110; Harlow and Lane, Antibodies: a Laboratory Manual,
(1988) Cold Spring Harbor, the disclosures of which are all
incorporated herein by reference. Techniques for the preparation of
recombinant antibody molecules are described in the above
references and also in, e.g.:WO97/08320; U.S. Pat. No. 5,427,908;
U.S. Pat. No. 5,508,717; Smith (1985) Science 225:1315-1317;
Parmley and Smith (1988) Gene 73:305-318; De La Cruz et al. (1988)
J Biol Chem 263:4318-4322; U.S. Pat. No. 5,403,484; U.S. Pat. No.
5,223,409; WO88/06630; WO92/15679; U.S. Pat. No. 5,780,279; U.S.
Pat. No. 5,571,698; U.S. Pat. No. 6,040,136; Davis et al. (1999)
Cancer Metastasis Rev 18(4):421-5; and Taylor et al. (1992) Nucleic
Acids Res 20: 6287-6295; Tomizuka et al. (2000) Proc Natl Acad Sci
USA 97(2): 722-727, the contents of each of which are incorporated
herein by reference in their entirety.
[0247] The cell culture supernatants are screened for the desired
antibodies, preferentially by immunofluorescent staining of
CD200-expressing cells, by immunoblotting, by an enzyme
immunoassay, e.g. a sandwich assay or a dot-assay, or a
radioimmunoassay.
[0248] For isolation of the antibodies, the immunoglobulins in the
culture supernatants or in the ascitic fluid may be concentrated,
e.g., by precipitation with ammonium sulfate, dialysis against
hygroscopic material such as polyethylene glycol, filtration
through selective membranes, or the like. If necessary and/or
desired, the antibodies are purified by the customary
chromatography methods, for example gel filtration, ion-exchange
chromatography, chromatography over DEAF-cellulose and/or (immuno-)
affinity chromatography, e.g., affinity chromatography with one or
more surface polypeptides derived from a CD200-expressing cell line
or synthetic CD200 fragment peptides, or with Protein-A or -G.
[0249] Another embodiment provides a process for the preparation of
a bacterial cell line secreting antibodies directed against a human
CD200 protein in a suitable mammal. For example a rabbit is
immunized with pooled samples from CD200-expressing tissue or cells
or CD200 polypeptide or fragments thereof. A phage display library
produced from the immunized rabbit is constructed and panned for
the desired antibodies in accordance with methods well known in the
art (such as, e.g., the methods disclosed in the various references
incorporated herein by reference).
[0250] Hybridoma cells secreting the monoclonal antibodies are also
disclosed. The preferred hybridoma cells are genetically stable,
secrete monoclonal antibodies described herein of the desired
specificity, and can be expanded from deep-frozen cultures by
thawing and propagation in vitro or as ascites in vivo.
[0251] In another embodiment, a process is provided for the
preparation of a hybridoma cell line secreting monoclonal
antibodies against a human CD200 protein. In that process, a
suitable mammal, for example a Balb/c mouse, is immunized with one
or more polypeptides or antigenic fragments of CD200 or with one or
more polypeptides or antigenic fragments derived from a
CD200-expressing cell, the CD200-expressing cell itself, or an
antigenic carrier containing a purified polypeptide as described.
Antibody-producing cells of the immunized mammal are grown briefly
in culture or fused with cells of a suitable myeloma cell line. The
hybrid cells obtained in the fusion are cloned, and cell clones
secreting the desired antibodies are selected. For example, spleen
cells of Balb/c mice immunized with a protein fragment of human
CD200 are fused with cells of the myeloma cell line PAI or the
myeloma cell line Sp2/0-Ag 14. The obtained hybrid cells are then
screened for secretion of the desired antibodies and positive
hybridoma cells are cloned.
[0252] Methods for preparing a hybridoma cell line include
immunizing Balb/c mice by injecting subcutaneously and/or
intraperitoneally a peptide fragment of human CD200 several times,
e.g., four to six times, over several months, e.g., between two and
four months. Spleen cells from the immunized mice are taken two to
four days after the last injection and fused with cells of the
myeloma cell line PAI in the presence of a fusion promoter,
preferably polyethylene glycol. Preferably, the myeloma cells are
fused with a three- to twenty-fold excess of spleen cells from the
immunized mice in a solution containing about 30% to about 50%
polyethylene glycol of a molecular weight around 4000. After the
fusion, the cells are expanded in suitable culture media as
described supra, supplemented with a selection medium, for example
HAT medium, at regular intervals in order to prevent normal myeloma
cells from overgrowing the desired hybridoma cells.
[0253] The antibodies and fragments thereof can be "chimeric."
Chimeric antibodies and antigen-binding fragments thereof comprise
portions from two or more different species (e.g., mouse and
human). Chimeric antibodies can be produced with mouse variable
regions of desired specificity spliced into human constant domain
gene segments (for example, U.S. Pat. No. 4,816,567). In this
manner, non-human antibodies can be modified to make them more
suitable for human clinical application (e.g., methods for treating
or preventing a cancer in a human subject).
[0254] The monoclonal antibodies of the present disclosure include
"humanized" forms of the non-human (e.g., mouse) antibodies.
Humanized or CDR-grafted mAbs are particularly useful as
therapeutic agents for humans because they are not cleared from the
circulation as rapidly as mouse antibodies and do not typically
provoke an adverse immune reaction. Generally, a humanized antibody
has one or more amino acid residues introduced into it from a
non-human source. These non-human amino acid residues are often
referred to as "import" residues, which are typically taken from an
"import" variable domain. Methods of preparing humanized antibodies
are generally well known in the art. For example, humanization can
be essentially performed following the method of Winter and
co-workers (see, e.g., Jones et al. (1986) Nature 321:522-525;
Riechmann et al. (1988) Nature 332:323-327; and Verhoeyen et al.
(1988) Science 239:1534-1536), by substituting rodent CDRs or CDR
sequences for the corresponding sequences of a human antibody. Also
see, e.g., Staelens et al. (2006) Mol Immunol 43:1243-1257. In some
embodiments, humanized forms of non-human (e.g., mouse) antibodies
are human antibodies (recipient antibody) in which hypervariable
(CDR) region residues of the recipient antibody are replaced by
hypervariable region residues from a non-human species (donor
antibody) such as a mouse, rat, rabbit, or non-human primate having
the desired specificity, affinity, and binding capacity. In some
instances, framework region residues of the human immunoglobulin
are also replaced by corresponding non-human residues (so called
"back mutations"). In addition, phage display libraries can be used
to vary amino acids at chosen positions within the antibody
sequence. The properties of a humanized antibody are also affected
by the choice of the human framework. Furthermore, humanized and
chimerized antibodies can be modified to comprise residues that are
not found in the recipient antibody or in the donor antibody in
order to further improve antibody properties, such as, for example,
affinity or effector function.
[0255] Fully human antibodies are also provided in the disclosure.
The term "human antibody" includes antibodies having variable and
constant regions (if present) derived from human germline
immunoglobulin sequences. Human antibodies can include amino acid
residues not encoded by human germline immunoglobulin sequences
(e.g., mutations introduced by random or site-specific mutagenesis
in vitro or by somatic mutation in vivo). However, the term "human
antibody" does not include antibodies in which CDR sequences
derived from the germline of another mammalian species, such as a
mouse, have been grafted onto human framework sequences (i.e.,
humanized antibodies). Fully human or human antibodies may be
derived from transgenic mice carrying human antibody genes
(carrying the variable (V), diversity (D), joining (J), and
constant (C) exons) or from human cells. For example, it is now
possible to produce transgenic animals (e.g., mice) that are
capable, upon immunization, of producing a full repertoire of human
antibodies in the absence of endogenous immunoglobulin production.
See, e.g., Jakobovits et al. (1993) Proc Natl Acad Sci USA 90:2551;
Jakobovits et al. (1993) Nature 362:255-258; Bruggemann et al.
(1993) Year in Immunol 7:33; and Duchosal et al. (1992) Nature
355:258. Transgenic mouse strains can be engineered to contain gene
sequences from unrearranged human immunoglobulin genes. The human
sequences may code for both the heavy and light chains of human
antibodies and would function correctly in the mice, undergoing
rearrangement to provide a wide antibody repertoire similar to that
in humans. The transgenic mice can be immunized with the target
protein (e.g., a human CD200 protein, fragments thereof, or cells
expressing CD200 protein) to create a diverse array of specific
antibodies and their encoding RNA. Nucleic acids encoding the
antibody chain components of such antibodies may then be cloned
from the animal into a display vector. Typically, separate
populations of nucleic acids encoding heavy and light chain
sequences are cloned, and the separate populations then recombined
on insertion into the vector, such that any given copy of the
vector receives a random combination of a heavy and a light chain.
The vector is designed to express antibody chains so that they can
be assembled and displayed on the outer surface of a display
package containing the vector. For example, antibody chains can be
expressed as fusion proteins with a phage coat protein from the
outer surface of the phage. Thereafter, display packages can be
screened for display of antibodies binding to a target.
[0256] In addition, human antibodies can be derived from
phage-display libraries (Hoogenboom et al. (1991)J Mol Biol
227:381; Marks et al. (1991) J Mol Biol 222:581-597; and Vaughan et
al. (1996) Nature Biotech 14:309 (1996)). Synthetic phage libraries
can be created which use randomized combinations of synthetic human
antibody V-regions. By selection on antigen fully human antibodies
can be made in which the V-regions are very human-like in nature.
See, e.g., U.S. Pat. Nos. 6,794,132, 6,680,209, 4,634,666, and
Ostberg et al. (1983) Hybridoma 2:361-367, the contents of each of
which are incorporated herein by reference in their entirety.
[0257] For the generation of human antibodies, also see Mendez et
al. (1998) Nature Genetics 15:146-156, and Green and Jakobovits
(1998) J Exp Med 188:483-495, the disclosures of which are hereby
incorporated by reference in their entirety. Human antibodies are
further discussed and delineated in U.S. Pat. Nos. 5,939,598;
6,673,986; 6,114,598; 6,075,181; 6,162,963; 6,150,584; 6,713,610;
and 6,657,103 as well as U.S. Patent Application Publication Nos.
20030229905 A1, 20040010810 A1, 20040093622 A1, 20060040363 A1,
20050054055 A1, 20050076395 A1, and 20050287630 A1. See also
International Patent Application Publication Nos. WO 94/02602, WO
96/34096, and WO 98/24893, and European Patent No. EP 0 463 151 B1.
The disclosures of each of the above-cited patents, applications,
and references are hereby incorporated by reference in their
entirety.
[0258] In an alternative approach, others, including GenPharm
International, Inc., have utilized a "minilocus" approach. In the
minilocus approach, an exogenous Ig locus is mimicked through the
inclusion of pieces (individual genes) from the Ig locus. Thus, one
or more V.sub.H genes, one or more D.sub.H genes, one or more
J.sub.H genes, a mu constant region, and a second constant region
(preferably a gamma constant region) are formed into a construct
for insertion into an animal. This approach is described in, e.g.,
U.S. Pat. Nos. 5,545,807; 5,545,806; 5,625,825; 5,625,126;
5,633,425; 5,661,016; 5,770,429; 5,789,650; 5,814,318; 5,591,669;
5,612,205; 5,721,367; 5,789,215; 5,643,763; 5,569,825; 5,877,397;
6,300,129; 5,874,299; 6,255,458; and 7,041,871, the disclosures of
which are hereby incorporated by reference. See also European
Patent No. 0 546 073 B1, International Patent
Application-Publication Nos. WO 92/03918, WO 92/22645, WO 92/22647,
WO 92/22670, WO 93/12227, WO 94/00569, WO 94/25585, WO 96/14436, WO
97/13852, and WO 98/24884, the disclosures of each of which are
hereby incorporated by reference in their entirety. See further
Taylor et al. (1992) Nucleic Acids Res 20: 6287; Chen et al. (1993)
Int Immunol 5: 647; Tuaillon et al. (1993) Proc Natl Acad Sci USA
90: 3720-4; Choi et al. (1993) Nature Genetics 4: 117; Lonberg et
al. (1994) Nature 368: 856-859; Taylor et al. (1994) International
Immunology 6: 579-591; Tuaillon et al. (1995) J. Immunol. 154:
6453-65; Fishwild et al. (1996) Nature Biotechnology 14: 845; and
Tuaillon et al. (2000) Eur J Immunol 10: 2998-3005, the disclosures
of each of which are hereby incorporated by reference in their
entirety.
[0259] In certain embodiments, de-immunized anti-CD200 antibodies
or antigen-binding fragments thereof are provided. De-immunized
antibodies or antigen-binding fragments thereof are those modified
so as to render the antibody or antigen-binding fragment thereof
non-immunogenic, or less immunogenic, to a given species.
De-immunization can be achieved by modifying the antibody or
antigen-binding fragment thereof utilizing any of a variety of
techniques known to those skilled in the art (see, e.g., PCT
Publication Nos. WO 04/108158 and WO 00/34317). For example, an
antibody or antigen-binding fragment thereof may be de-immunized by
identifying potential T cell epitopes and/or B cell epitopes within
the amino acid sequence of the antibody or antigen-binding fragment
thereof and removing one or more of the potential T cell epitopes
and/or B cell epitopes from the antibody or antigen-binding
fragment thereof, for example, using recombinant techniques. The
modified antibody or antigen-binding fragment thereof may then
optionally be produced and tested to identify antibodies or
antigen-binding fragments thereof that have retained one or more
desired biological activities, such as, for example, binding
affinity, but have reduced immunogenicity. Methods for identifying
potential T cell epitopes and/or B cell epitopes may be carried out
using techniques known in the art, such as, for example,
computational methods (see e.g., PCT Publication No. WO 02/069232),
in vitro or in silico techniques, and biological assays or physical
methods (such as, for example, determination of the binding of
peptides to MHC molecules, determination of the binding of
peptide:MHC complexes to the T cell receptors from the species to
receive the antibody or antigen-binding fragment thereof, testing
of the protein or peptide parts thereof using transgenic animals
with the MHC molecules of the species to receive the antibody or
antigen-binding fragment thereof, or testing with transgenic
animals reconstituted with immune system cells from the species to
receive the antibody or antigen-binding fragment thereof, etc.). In
various embodiments, the de-immunized anti-CD200 antibodies
described herein include de-immunized antigen-binding fragments,
Fab, Fv, scFv, Fab' and F(ab').sub.2, monoclonal antibodies, murine
antibodies, engineered antibodies (such as, for example, chimeric,
single chain, CDR-grafted, humanized, fully human antibodies, and
artificially selected antibodies), synthetic antibodies and
semi-synthetic antibodies.
[0260] In some embodiments, a recombinant DNA comprising an insert
coding for a heavy chain variable domain and/or for a light chain
variable domain of an anti-CD200 antibody or a CD200
protein-expressing cell line is produced. The term DNA includes
coding single stranded DNAs, double stranded DNAs consisting of
said coding DNAs and of complementary DNAs thereto, or these
complementary (single stranded) DNAs themselves.
[0261] Furthermore, a DNA encoding a heavy chain variable domain
and/or a light chain variable domain of anti-CD200 antibodies, or
the CD200-expressing cell line, can be enzymatically or chemically
synthesized to contain the authentic DNA sequence coding for a
heavy chain variable domain and/or for the light chain variable
domain, or a mutant thereof. A mutant of the authentic DNA is a DNA
encoding a heavy chain variable domain and/or a light chain
variable domain of the above-mentioned antibodies in which one or
more amino acids are deleted, inserted, or exchanged with one or
more other amino acids. Preferably said modification(s) are outside
the CDRs of the heavy chain variable domain and/or of the light
chain variable domain of the antibody in humanization and
expression optimization applications. The term mutant DNA also
embraces silent mutants wherein one or more nucleotides are
replaced by other nucleotides with the new codons coding for the
same amino acid(s). The term mutant sequence also includes a
degenerate sequence. Degenerate sequences are degenerate within the
meaning of the genetic code in that an unlimited number of
nucleotides are replaced by other nucleotides without resulting in
a change of the amino acid sequence originally encoded. Such
degenerate sequences may be useful due to their different
restriction sites and/or frequency of particular codons which are
preferred by the specific host, particularly E. coli, to obtain an
optimal expression of the heavy chain murine variable domain and/or
a light chain murine variable domain.
[0262] The term mutant is intended to include a DNA mutant obtained
by in vitro mutagenesis of the authentic DNA according to methods
known in the art.
[0263] For the assembly of complete tetrameric immunoglobulin
molecules and the expression of chimeric antibodies, the
recombinant DNA inserts coding for heavy and light chain variable
domains are fused with the corresponding DNAs coding for heavy and
light chain constant domains, then transferred into appropriate
host cells, for example after incorporation into hybrid
vectors.
[0264] Recombinant DNAs including an insert coding for a heavy
chain murine variable domain of an anti-CD200 antibody or a
CD200-expressing cell line fused to a human constant domain IgG,
for example .gamma.1, .gamma.2, .gamma.3 or .gamma.4, in particular
embodiments .gamma.1 or .gamma.4, may be used. Recombinant DNAs
including an insert coding for a light chain murine variable domain
of an antibody fused to a human constant domain .kappa. or .lamda.,
preferably .kappa., are also provided.
[0265] Another embodiment pertains to recombinant DNAs coding for a
recombinant polypeptide wherein the heavy chain variable domain and
the light chain variable domain are linked by way of a spacer
group, optionally comprising a signal sequence facilitating the
processing of the antibody in the host cell and/or a DNA sequence
encoding a peptide facilitating the purification of the antibody
and/or a cleavage site and/or a peptide spacer and/or an agent. The
DNA coding for an agent is intended to be a DNA coding for the
agent useful in diagnostic or therapeutic applications. Thus, agent
molecules which are toxins or enzymes, especially enzymes capable
of catalyzing the activation of prodrugs, are particularly
indicated. The DNA encoding such an agent has the sequence of a
naturally occurring enzyme or toxin encoding DNA, or a mutant
thereof, and can be prepared by methods well known in the art.
[0266] Accordingly, the monoclonal antibodies or antigen-binding
fragments of the disclosure can be naked antibodies or
antigen-binding fragments that are not conjugated to other agents,
for example, a therapeutic agent or detectable label.
Alternatively, the monoclonal antibody or antigen-binding fragment
can be conjugated to an agent such as, for example, a cytotoxic
agent, a small molecule, a hormone, an enzyme, a growth factor, a
cytokine, a ribozyme, a peptidomimetic, a chemical, a prodrug, a
nucleic acid molecule including coding sequences (such as
antisense, RNAi, gene-targeting constructs, etc.), or a detectable
label (e.g., an NMR or X-ray contrasting agent, fluorescent
molecule, etc.). In certain embodiments, an anti-CD200 antibody or
antigen-binding fragment (e.g., Fab, Fv, single-chain scFv, Fab',
and F(ab').sub.2) is linked to a molecule that increases the
half-life of the antibody or antigen-binding fragment (see
above).
[0267] Several possible vector systems are available for the
expression of cloned heavy chain and light chain genes in mammalian
cells. One class of vectors relies upon the integration of the
desired gene sequences into the host cell genome. Cells which have
stably integrated DNA can be selected by simultaneously introducing
drug resistance genes such as E. coli gpt (Mulligan and Berg (1981)
Proc Natl Acad Sci USA, 78:2072-2076) or Tn5 neo (Southern and Berg
(1982)J Mol Appl Genet 1:327-341). The selectable marker gene can
be either linked to the DNA gene sequences to be expressed, or
introduced into the same cell by co-transfection (Wigler et al.
(1979) Cell 16:777-785). A second class of vectors utilizes DNA
elements which confer autonomously replicating capabilities to an
extrachromosomal plasmid. These vectors can be derived from animal
viruses, such as bovine papillomavirus (Sarver et al. (1982) Proc
Natl Acad Sci USA, 79:7147-7151), polyoma virus (Deans et al.
(1984) Proc Natl Acad Sci USA 81:1292-1296), or SV40 virus (Lusky
and Botchan (1981) Nature 293:79-81).
[0268] Since an immunoglobulin cDNA is comprised only of sequences
representing the mature mRNA encoding an antibody protein,
additional gene expression elements regulating transcription of the
gene and processing of the RNA are required for the synthesis of
immunoglobulin mRNA. These elements may include splice signals,
transcription promoters, including inducible promoters, enhancers,
and termination signals. cDNA expression vectors incorporating such
elements include those described by Okayama and Berg (1983) Mol
Cell Biol 3:280-289; Cepko et al. (1984) Cell 37:1053-1062; and
Kaufman (1985) Proc Natl Acad Sci USA 82:689-693.
[0269] As is evident from the disclosure, the anti-CD200 antibodies
can be used in therapies (e.g., therapies for treating a cancer),
including combination therapies, as well as in the monitoring of
disease progression.
[0270] In the therapeutic embodiments of the present disclosure,
bispecific antibodies are contemplated. Bispecific antibodies are
monoclonal, preferably human or humanized, antibodies that have
binding specificities for at least two different antigens. In the
present case, one of the binding specificities is for the CD200
antigen on a cell (such as, e.g., an immune cell), the other one is
for any other antigen, and preferably for a cell-surface protein or
receptor or receptor subunit.
[0271] Methods for making bispecific antibodies are within the
purview of those skilled in the art. Traditionally, the recombinant
production of bispecific antibodies is based on the co-expression
of two immunoglobulin heavy-chain/light-chain pairs, where the two
heavy chains have different specificities (Milstein and Cuello
(1983) Nature 305:537-539). Antibody variable domains with the
desired binding specificities (antibody-antigen combining sites)
can be fused to immunoglobulin constant domain sequences. The
fusion preferably is with an immunoglobulin heavy-chain constant
domain, including at least part of the hinge, C.sub.H2, and
C.sub.H3 regions. DNAs encoding the immunoglobulin heavy-chain
fusions and, if desired, the immunoglobulin light chain, are
inserted into separate expression vectors, and are co-transfected
into a suitable host organism. For further details of illustrative
currently known methods for generating bispecific antibodies see,
e.g., Suresh et al. (1986) Methods Enzymol 121:210-228; PCT
Publication No. WO 96/27011; Brennan et al. (1985) Science
229:81-83; Shalaby et al. J Exp Med (1992) 175:217-225; Kostelny et
al. (1992) J Immunol 148(5):1547-1553; Hollinger et al. (1993) Proc
Natl Acad Sci USA 90:6444-6448; Gruber et al. (1994) J Immunol
152:5368-5474; and Tutt et al. (1991)J Immunol 147:60-69.
Bispecific antibodies also include cross-linked or heteroconjugate
antibodies. Heteroconjugate antibodies may be made using any
convenient cross-linking methods. Suitable cross-linking agents are
well known in the art, and are disclosed in U.S. Pat. No.
4,676,980, along with a number of cross-linking techniques.
[0272] Various techniques for making and isolating bispecific
antibody fragments directly from recombinant cell culture have also
been described. For example, bispecific antibodies have been
produced using leucine zippers. See, e.g., Kostelny et al. (1992) J
Immunol 148(5):1547-1553. The leucine zipper peptides from the Fos
and Jun proteins may be linked to the Fab' portions of two
different antibodies by gene fusion. The antibody homodimers may be
reduced at the hinge region to form monomers and then re-oxidized
to form the antibody heterodimers. This method can also be utilized
for the production of antibody homodimers. The "diabody" technology
described by Hollinger et al. (1993) Proc Natl Acad Sci USA
90:6444-6448 has provided an alternative mechanism for making
bispecific antibody fragments. The fragments comprise a heavy-chain
variable domain (VH) connected to a light-chain variable domain
(VL) by a linker which is too short to allow pairing between the
two domains on the same chain. Accordingly, the VH and VL domains
of one fragment are forced to pair with the complementary VL and VH
domains of another fragment, thereby forming two antigen-binding
sites. Another strategy for making bispecific antibody fragments by
the use of single-chain Fv (scFv) dimers has also been reported.
See, e.g., Gruber et al. (1994)J Immunol 152:5368-5374.
Alternatively, the antibodies can be "linear antibodies" as
described in, e.g., Zapata et al. (1995) Protein Eng
8(10):1057-1062. Briefly, these antibodies comprise a pair of
tandem Fd segments (V.sub.H-C.sub.H1-V.sub.H-C.sub.H1) which form a
pair of antigen binding regions. Linear antibodies can be
bispecific or monospecific.
[0273] The disclosure also embraces variant forms of bispecific
antibodies such as the tetravalent dual variable domain
immunoglobulin (DVD-Ig) molecules described in Wu et al. (2007) Nat
Biotechnol 25(11):1290-1297. The DVD-Ig molecules are designed such
that two different light chain variable domains (VL) from two
different parent antibodies are linked in tandem directly or via a
short linker by recombinant DNA techniques, followed by the light
chain constant domain. Methods for generating DVD-Ig molecules from
two parent antibodies are further described in, e.g., PCT
Publication Nos. WO 08/024188 and WO 07/024715, the disclosures of
each of which are incorporated herein by reference in their
entirety.
[0274] In some embodiments, anti-CD200 antibodies can be modified,
e.g., with a moiety that improves the stabilization and/or
retention of the antibodies themselves in circulation, e.g., in
blood, serum, or other tissues. For example, an anti-CD200 antibody
described herein can be PEGylated as described in, e.g., Lee et al.
(1999) Bioconjug Chem 10(6): 973-8; Kinstler et al. (2002) Advanced
Drug Deliveries Reviews 54:477-485; and Roberts et al. (2002)
Advanced Drug Delivery Reviews 54:459-476. The stabilization moiety
can improve the stability, or retention of, the antibody in a
subject's body (e.g., blood or tissue) by at least 1.5 (e.g., at
least 2, 5, 10, 15, 20, 25, 30, 40, or 50 or more) fold.
Biological Samples and Sample Collection
[0275] Suitable biological samples for use in the methods described
herein include any biological fluid, population of cells, or tissue
or fraction thereof, which includes one or more white blood cells
and/or one or more red blood cells. A biological sample can be, for
example, a specimen obtained from a subject (e.g., a mammal such as
a human) or can be derived from such a subject. For example, a
sample can be a tissue section obtained by biopsy, or cells that
are placed in or adapted to tissue culture. A biological sample can
also be a biological fluid such as urine, whole blood or a fraction
thereof (e.g., plasma), saliva, semen, sputum, cerebral spinal
fluid, tears, or mucus. A biological sample can be further
fractionated, if desired, to a fraction containing particular cell
types. For example, a whole blood sample can be fractionated into
serum or into fractions containing particular types of blood cells
such as red blood cells or white blood cells (leukocytes). If
desired, a biological sample can be a combination of different
biological samples from a subject such as a combination of a tissue
and fluid sample.
[0276] The biological samples can be obtained from a subject, e.g.,
a subject having, suspected of having, or at risk of developing, a
cancer (e.g., B-CLL), an inflammatory condition, or a bone disorder
(e.g., a CD200-associated bone disorder). Any suitable methods for
obtaining the biological samples can be employed, although
exemplary methods include, e.g., phlebotomy, swab (e.g., buccal
swab), lavage, or fine needle aspirate biopsy procedure.
Non-limiting examples of tissues susceptible to fine needle
aspiration include lymph node, lung, thyroid, breast, and liver.
Biological samples can also be obtained from bone marrow. Samples
can also be collected, e.g., by microdissection (e.g., laser
capture microdissection (LCM) or laser microdissection (LMD)),
bladder wash, smear (PAP smear), or ductal lavage.
[0277] Methods for obtaining and/or storing samples that preserve
the activity or integrity of cells in the biological sample are
well known to those skilled in the art. For example, a biological
sample can be further contacted with one or more additional agents
such as appropriate buffers and/or inhibitors, including protease
inhibitors, the agents meant to preserve or minimize changes in the
cells (e.g., changes in osmolarity or pH) or denaturation of cell
surface proteins (e.g., GPI-linked proteins) or GPI moieties on the
surface of the cells. Such inhibitors include, for example,
chelators such as ethylenediamine tetraacetic acid (EDTA), ethylene
glycol tetraacetic acid (EGTA), protease inhibitors such as
phenylmethylsulfonyl fluoride (PMSF), aprotinin, and leupeptin.
Appropriate buffers and conditions for storing or otherwise
manipulating whole cells are described in, e.g., Pollard and Walker
(1997), "Basic Cell Culture Protocols," volume 75 of Methods in
molecular biology, Humana Press; Masters (2000) "Animal cell
culture: a practical approach," volume 232 of Practical approach
series, Oxford University Press; and Jones (1996) "Human cell
culture protocols," volume 2 of Methods in molecular medicine,
Humana Press.
[0278] A sample also can be processed to eliminate or minimize the
presence of interfering substances. For example, a biological
sample can be fractionated or purified to remove one or more
materials (e.g., cells) that are not of interest. Methods of
fractionating or purifying a biological sample include, but are not
limited to, flow cytometry, fluorescence activated cell sorting,
and sedimentation.
Biomarkers and Applications
[0279] The inventors have identified and provided herein several
biomarkers consistent with the production in a human of a desired
immunomodulatory effect by an anti-CD200 antibody administered to
the human. A "desired immunomodulatory effect," an "anti-CD200
antibody-associated immunomodulatory effect," and grammatically
similar terms, as used herein, refer to a measurable, desirable
immunological effect in a human attributable to the biological
activity of an anti-CD200 antibody administered to the human. For
example, the inventors have observed that following administration
of an anti-CD200 antibody to a human, the concentration of
circulating CD200.sup.+ lymphocytes (e.g., subsets of CD200.sup.+ T
cells including, e.g., CD200.sup.+/CD4.sup.+ T cells and/or
activated CD200.sup.+/CD4.sup.+ T cells) is reduced in the human as
measured by a reduction in the concentration of such cells in the
blood. Also observed was that upon administration of an anti-CD200
antibody, the expression level of CD200R by at least one leukocyte
subset (e.g., CD4.sup.+ T cells) is increased. While not being
bound by any particular theory or mechanism of action, the
inventors believe that monitoring a patient treated with an
anti-CD200 antibody for a change (e.g., an increase or decrease) in
one or more of these biomarkers is useful for, among other things,
determining whether the anti-CD200 antibody is capable of producing
a biological effect in the human to whom the antibody is
administered. Moreover, monitoring changes in one or more of the
biomarkers is also useful for identifying a dose--a threshold dose
(or a dosing schedule)--of an anti-CD200 antibody, such as
samalizumab, that by virtue of its immunomodulatory effect in the
human, is sufficient to achieve a clinically-meaningful effect in
the disease (i.e., sufficient to treat a disease such as cancer).
Several B-CLL patients administered samalizumab exhibited
clinically stable or improved disease as determined by serial
assessments of peripheral blood counts and CT scans. A desired
immunomodulatory effect of the antibody was observed in all of
these patients as reflected in a change (e.g., an increase or
decrease) in one or more of the biomarkers described herein.
[0280] Thus, in accordance with the present disclosure, to
determine whether an anti-CD200 antibody (e.g., a variant
anti-CD200 antibody that has reduced or no effector function) has
produced a desired immunomodulatory effect (e.g., an anti-CD200
antibody-associated immunomodulatory effect) in the human (and
thereby the human has been administered a dose of the antibody
sufficient to affect the treatment of the patient via, among other
things, its immunomodulatory activity), a practitioner can measure
the concentration of CD200.sup.+ leukocytes (e.g., T cells) in a
blood sample from a human administered an anti-CD200 antibody. A
reduction in the concentration of CD200.sup.+ leukocytes (e.g., T
cells) in the blood sample as compared to the concentration of
CD200.sup.+ leukocytes (e.g., T cells) in a control blood sample
indicates that the anti-CD200 antibody has produced a desired
immunomodulatory effect in the human. In some embodiments, the
practitioner need not measure first hand the concentration of
CD200.sup.+ leukocytes (e.g., T cells) in the blood sample. For
example, a practitioner (e.g., a medical professional or a
diagnostic scientist or technician) provided with information
regarding: (i) the concentration of CD200.sup.+ leukocytes (e.g., T
cells) in a blood sample from the human administered the antibody
and (ii) a control CD200.sup.+ leukocyte concentration can
determine whether the antibody has produced a desired
immunomodulatory effect in the human using the information, e.g.,
comparing the concentration of CD200.sup.+ leukocytes (e.g., T
cells) in the blood sample with the concentration of such cells in
the control sample, wherein reduction in the concentration of
CD200.sup.+ leukocytes (e.g., T cells) in the blood sample as
compared to a control concentration of CD200.sup.+ leukocytes
(e.g., T cells) indicates that the anti-CD200 antibody has produced
a desired immunomodulatory effect in the human.
[0281] Methods for measuring the concentration of CD200.sup.+ cells
(e.g., CD200.sup.+ T cells) are well known in the art and include,
among other methods, flow cytometry. See, e.g., Chen et al. (2009)
Mol Immunol 46(10):1951-1963. A suitable method for detecting
and/or measuring the concentration of CD200.sup.+ T cells is also
set forth in the working examples. In some embodiments, a
practitioner can interrogate a biological sample obtained from a
post-treatment patient (a patient to which an anti-CD200 antibody
has already been administered) for the concentration of cells of a
particular subset of CD200.sup.+ leukocytes (e.g., T cells). For
example, a practitioner can determine the concentration of
CD200.sup.+/CD4.sup.+ T cells and/or the concentration of activated
CD200.sup.+/CD4.sup.+ T cells present in a biological sample from a
post-treatment patient. In some embodiments, a practitioner can
determine the concentration of CD200.sup.+/CD8.sup.+ cells. In each
case, a reduction in the concentration of CD200.sup.+ T cells of a
given subset, as compared to control concentration of CD200.sup.+ T
cells of the same histological type, indicates that the anti-CD200
antibody has produced in the human a desired immunomodulatory
effect.
[0282] As described above, determining whether an anti-CD200
antibody (e.g., a variant anti-CD200 antibody with decreased or no
effector function) has produced a desired immunomodulatory effect
in a human can be performed by comparing the concentration of
CD200.sup.+ T cells in a biological sample obtained from a patient
following administration of the anti-CD200 antibody (the
post-treatment CD200.sup.+ T cell concentration) to the
concentration of CD200.sup.+ cells in a control sample. In some
embodiments, control sample is obtained from the patient prior to
administering to the patient the anti-CD200 antibody. In some
embodiments, the control sample can be (or can be based on), e.g.,
a collection of samples obtained from one or more (e.g., two,
three, four, five, six, seven, eight, nine, 10, 15, 20, 25, 30, 35,
or 40 or more) healthy individuals that have not been administered
an anti-CD200 antibody (e.g., a control concentration of
CD200.sup.+ cells of the same histological type can be an average
of the concentration of the cells in one or more control samples
obtained from patients who have not been administered an anti-CD200
antibody. In some embodiments, the control sample can be or can be
based on, e.g., a collection of samples obtained from one or more
(e.g., two, three, four, five, six, seven, eight, nine, 10, 15, 20,
25, 30, 35, or 40 or more) individuals suffering from the same
cancer or different types of cancers, but who have not been
administered an anti-CD200 antibody. For example, to determine
whether an anti-CD200 antibody has produced a desired
immunomodulatory effect in a human administered the antibody, a
practitioner can compare the post-treatment CD200.sup.+ T cell
concentration to the typical concentration, or average
concentration, of CD200.sup.+ T cells of the same histological type
present in humans who have not been administered an anti-CD200
antibody or at least do not have a detectable level of an
anti-CD200 antibody in a biological sample obtained from the
humans.
[0283] In some embodiments, a post-treatment CD200.sup.+ T cell
concentration that is at least 5% less than the control
concentration indicates that a desired immunomodulatory effect has
occurred in the human administered the anti-CD200 antibody. In some
embodiments, a post-treatment CD200.sup.+ T cell concentration that
is at least 10 (e.g., 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21,
22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38,
39, 40, 41, 42, 43, 44, 45, 50, 55, 60, 65, 70, 75, 80, or more
than 80) % less than the control concentration indicates that a
desired immunomodulatory effect has occurred in the human
administered the anti-CD200 antibody.
[0284] In some embodiments, determining whether an anti-CD200
antibody (e.g., a variant anti-CD200 antibody having reduced or no
effector function) has produced a desired immunomodulatory effect
in a human can be performed by querying whether the post-treatment
CD200.sup.+ T cell concentration falls within a predetermined range
indicative of the occurrence of a desired immunomodulatory effect
by an anti-CD200 antibody in a human. In some embodiments,
determining whether an anti-CD200 antibody has produced a desired
immunomodulatory effect in a human can include querying if the
post-treatment CD200.sup.+ T cell concentration for a given
histological type of CD200.sup.+ T cell falls above or below a
predetermined cut-off value. A cut-off value is typically the
concentration of CD200.sup.+ T cells of a given histological type
above or below which is considered indicative of a certain
phenotype--namely the occurrence of a desired immunomodulatory
effect in a human produced by an anti-CD200 antibody.
[0285] In some embodiments, to determine whether an anti-CD200
antibody (e.g., a variant anti-CD200 antibody that has reduced or
no effector function) has produced a desired immunomodulatory
effect in the human (and thereby the human has been administered a
dose of the antibody sufficient to affect the treatment of the
patient via, among other things, its immunomodulatory activity), a
practitioner can quantify the expression of CD200 by T cells (e.g.,
CD4.sup.+ T cells, CD8.sup.+ T cells, or activated CD4.sup.+ T
cells) in a blood sample from a human administered an anti-CD200
antibody. A reduction in the expression level of CD200 by T cells
in the blood sample as compared to the expression level of CD200 by
T cells of the same histological type in a control blood sample
indicates that the anti-CD200 antibody has produced a desired
immunomodulatory effect in the human. As described above, the
practitioner need not measure first hand the expression level of
CD200 by the T cells in the blood sample. For example, a
practitioner provided with information regarding: (i) the
expression level of CD200 by T cells in a blood sample from the
human administered the antibody and (ii) the expression level of
CD200 by T cells in a control blood sample can determine whether
the antibody has produced a desired immunomodulatory effect in the
human using the information, e.g., comparing the expression level
of CD200 by T cells in the blood sample with the expression level
of CD200 by such cells in the control sample, wherein reduction in
the level of CD200 expression by the T cells in the blood sample as
compared to expression level of CD200 by T cells of the same
histological type in the control sample indicates that the
anti-CD200 antibody has produced a desired immunomodulatory effect
in the human. Suitable methods for quantifying the expression level
of CD200 by cells (e.g., leukoctyes such as T cells) are known in
the art and described herein.
[0286] The inventors also observed that upon administration of an
anti-CD200 antibody, the expression level of CD200R by a variety of
leukocyte subsets is increased. While not being bound by any
particular theory or mechanism of action, the inventors believe
that an increase in CD200R expression by leukocytes is potentially
a compensatory response by these cells to the reduction of cellular
CD200 expression induced by the anti-CD200 antibody. Thus, CD200R
expression by leukocytes serves as an indirect biomarker to monitor
(or detect) the immunomodulatory effect of an anti-CD200 antibody
on CD200 expression by leukocytes in the human to which the
anti-CD200 antibody is administered. In some embodiments, to
determine whether an anti-CD200 antibody has produced a desired
immunomodulatory effect in the human (and thereby the human has
been administered a dose of the antibody sufficient to affect the
treatment of the patient via, among other things, its
immunomodulatory activity), a practitioner can measure the
expression level of CD200R by a plurality of leukocytes (e.g., a
plurality of leukocytes of a given histological type) in a
biological sample (e.g., a blood sample) obtained from a human
following administration of the anti-CD200 antibody (the
post-treatment CD200R expression level), wherein an increase in
post-treatment CD200R expression level as compared to the CD200R
expression level by leukocytes of the same histological type in a
control sample indicates that the anti-CD200 antibody has produced
in the human a desired immunomodulatory effect. In some
embodiments, the practitioner need not measure first hand the
expression level of CD200R by leukocytes in the biological sample.
For example, a practitioner (e.g., a medical professional or a
diagnostic scientist or technician) provided with information
regarding: (i) the expression level of CD200R by a plurality of
leukocytes in a blood sample from the human administered the
antibody and (ii) a control expression level (e.g., the expression
level of CD200R by leukocytes of the same histological type in a
control sample) can determine whether the antibody has produced a
desired immunomodulatory effect in the human using the information,
e.g., comparing the CD200R expression level by the leukocytes in
the biological sample with the control expression level, wherein an
increase in the CD200R expression level by the leukocytes, as
compared to the control expression level, indicates that the
anti-CD200 antibody has produced a desired immunomodulatory effect
in the human.
[0287] Methods for quantifying the expression level of CD200 and/or
CD200R by a cell or a population of cells are well known in the art
and include, among other methods, Western blotting, dot blotting,
and flow cytometry, which are useful for quantifying expression of
protein, or reverse transcriptase polymerase chain reaction
(RT-PCR) and Northern blotting analysis for quantifying expression
of mRNA. See, e.g., Walker et al. (2009) Exp Neurol 215(1):5-19;
Rijkers et al. (2008) Mol Immunol 45(4):1126-1135; and Voehringer
et al. (2004)J Biol Chem 279(52):54117-54123. See generally
Sambrook et al. (1989) "Molecular Cloning: A Laboratory Manual,
2.sup.ndEdition," Cold Spring Harbor Laboratory Press, Cold Spring
Harbor, N.Y. and Ausubel et al. (1992) "Current Protocols in
Molecular Biology," Greene Publishing Associates. A suitable method
for detecting and/or quantifying the expression of CD200 or CD200R
by leukocytes is also set forth in the working examples. In some
embodiments, a practitioner can interrogate a biological sample
(e.g., a blood sample) obtained from a post-treatment patient (a
patient to which an anti-CD200 antibody has been administered) for
the CD200 and/or CD200R expression level (e.g., the average
expression level) by a plurality of leukocytes of a given
histological type. For example, a practitioner can determine the
expression level or average expression level of CD200R by a
plurality of CD4.sup.+ T cells, CD8.sup.+ T cells, activated
CD4.sup.+ T cells, NK T cells, or CD21.sup.+/CD25.sup.+/Fox3P.sup.+
T cells. In one instance, an increase in CD200R expression by a
given subset of leukocytes, as compared to control expression level
(e.g., the average level of expression of leukocytes of the same
histological type in a biological sample obtained from the patient
prior to administration of the antibody), indicates that the
anti-CD200 antibody has produced in the human a desired
immunomodulatory effect.
[0288] In some embodiments, a post-treatment CD200R expression
level that is at least 1.5-fold greater than the control expression
level (that is, the level of expression of CD200R by leukocytes of
the same histological type in a control sample) indicates that a
desired immunomodulatory effect has occurred in the human
administered the anti-CD200 antibody. In some embodiments, a
post-treatment CD200R expression level that is at least 2 (e.g., at
least 2.5, 3, 3.5, 4, 4.5, 5, 5.5, 6, 6.5, 7, 7.5, 8, 8.5, 9, or 10
or more)-fold greater than the control expression level indicates
that a desired immunomodulatory effect has occurred in the human
administered the anti-CD200 antibody. In some embodiments, a
post-treatment CD200R expression level that is at least 5 (e.g., 6,
7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 25, 30, 35,
40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 150, 200, or
250 or more) % greater than the control expression level indicates
that a desired immunomodulatory effect has occurred in the human
administered the anti-CD200 antibody.
[0289] In some embodiments, a post-treatment CD200 expression level
that is at least 5 (e.g., 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16,
17, 18, 19, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85,
or 90 or more) % lower than the control expression level indicates
that a desired immunomodulatory effect has occurred in the human
administered the anti-CD200 antibody.
[0290] In some embodiments, the control sample is a biological
sample obtained from the subject human prior to administering to
the subject human the anti-CD200 antibody. (That is, e.g., the
control CD200R expression level can be the expression level of
CD200R by leukocytes of the same histological type in a biological
sample obtained from the subject human prior to administering to
the subject human the anti-CD200 antibody.) In some embodiments,
the control CD200 or CD200R expression level can be based on, e.g.,
the average expression level of CD200 or CD200R by leukocytes of
the same histological type obtained from one or more (e.g., two,
three, four, five, six, seven, eight, nine, 10, 15, 20, 25, 30, 35,
or 40 or more) healthy individuals that have not been administered
an anti-CD200 antibody. The control CD200 or CD200R expression
level can be based on, e.g., the average expression level of CD200
or CD200R by leukocytes of the same histological type obtained from
one or more (e.g., two, three, four, five, six, seven, eight, nine,
10, 15, 20, 25, 30, 35, or 40 or more) individuals suffering from
the same cancer or different types of cancers, but who have not
been administered an anti-CD200 antibody. For example, to determine
whether an anti-CD200 antibody has produced a desired
immunomodulatory effect in a human administered the antibody, a
practitioner can compare the post-treatment CD200R expression level
to the typical expression level, or average expression level, of
CD200R by leukocytes of the same histological type in a biological
sample obtained from humans who have not been administered an
anti-CD200 antibody or at least do not have a detectable level of
an anti-CD200 antibody in the biological sample.
[0291] In some embodiments, determining whether an anti-CD200
antibody (e.g., a variant anti-CD200 antibody having decreased or
no effector function) has produced a desired immunomodulatory
effect in a human can be performed by querying whether the
post-treatment CD200 or CD200R expression level falls within a
predetermined range indicative of the occurrence of an
immunomodulatory effect by an anti-CD200 antibody in a human. In
some embodiments, determining whether an anti-CD200 antibody has
produced a desired immunomodulatory effect in a human can include
querying if the post-treatment CD200 or CD200R expression level by
a given histological type of leukocytes falls above or below a
predetermined cut-off value. In this case, the cut-off value is
typically the level of expression (e.g., mRNA or protein
expression) by leukocytes of a given histological type above or
below which is considered indicative of a certain phenotype--namely
the occurrence of a desired immunomodulatory effect in a human
produced by an anti-CD200 antibody.
[0292] In some embodiments, to determine whether an anti-CD200
antibody (e.g., a variant anti-CD200 antibody that has reduced or
no effector function) has produced a desired immunomodulatory
effect in the human (and thereby the human has been administered a
dose of the antibody sufficient to affect the treatment of the
patient via, among other things, its immunomodulatory activity), a
practitioner can measure the concentration of CD200R.sup.+
leukocytes in a blood sample from a human administered an
anti-CD200 antibody. An increase in the concentration of
CD200R.sup.+ leukocytes in the blood sample as compared to the
concentration of CD200R.sup.+ leukocytes of the same histological
type in a control blood sample indicates that the anti-CD200
antibody has produced a desired immunomodulatory effect in the
human. A practitioner (e.g., a medical professional or a diagnostic
scientist or technician) provided with information regarding: (i)
the concentration of CD200R.sup.+ leukocytes in a blood sample from
the human administered the antibody and (ii) a control CD200R.sup.+
leukocyte concentration can determine whether the antibody has
produced a desired immunomodulatory effect in the human using the
information, e.g., comparing the concentration of CD200R.sup.+
leukocytes in the blood sample with the concentration of such cells
in the control sample, wherein an increase in the concentration of
CD200R.sup.+ leukocytes in the blood sample as compared to a
control concentration of CD200R.sup.+ leukocytes indicates that the
anti-CD200 antibody has produced a desired immunomodulatory effect
in the human.
[0293] In some embodiments of any of the methods described herein,
the same practitioner may administer the antibody to the human
prior to determining whether a desired immunomodulatory effect has
occurred in the human, whereas in some embodiments, the
practitioner who administers the antibody to the patient is
different from the practioner who determines whether a desired
immunomodulatory effect has occurred in the human. In some
embodiments, the practitioner may obtain a biological sample (e.g.,
the blood sample) from the human prior to administration of the
antibody. In some embodiments, the practitioner may obtain a
biological sample (e.g., a blood sample) from the human following
the administration of the antibody to the human. In some
embodiments, the post-treatment sample can be obtained from the
human less than 48 (e.g., less than 47, 46, 45, 44, 43, 42, 41, 40,
39, 38, 37, 36, 35, 34, 33, 32, 31, 30, 29, 28, 27, 26, 25, 24, 23,
22, 21, 20, 19, 18, 17, 16, 15, 14, 13, 12, 11, 10, nine, eight,
seven, six, five, four, three, two, or even less than one) hour
following administration of the anti-CD200 antibody to the human.
In some embodiments, the post-treatment sample can be obtained from
the human less than 20 (e.g., less than 19, 18, 17, 16, 15, 14, 13,
12, 11, 10, nine, eight, seven, six, five, four, three, two, or
one) day(s) after administering to the human the anti-CD200
antibody. In some embodiments, the biological sample is obtained
from the human no more than 20 (e.g., no more than 19, 18, 17, 16,
15, 14, 13, 12, 11, 10, nine, eight, seven, six, five, four, three,
two, or one) day(s) after the antibody is administered to the
human.
[0294] In some embodiments, determining whether an anti-CD200
antibody has produced a desired immunomodulatory effect in a human
can include (i) measuring the concentration of CD200.sup.+ T cells
in a biological sample obtained from a human prior to
administration to the human of an anti-CD200 antibody to thereby
obtain a pre-treatment CD200 T cell concentration; (ii)
administering to the human the antibody; and (iii) measuring the
concentration of CD200.sup.+ T cells in a blood sample obtained
from the human to thereby obtain a post-treatment CD200.sup.+ T
cell concentration, wherein a reduction in the post-treatment
CD200.sup.+ T cell concentration as compared to the pre-treatment
CD200.sup.+ T cell concentration indicates that the antibody has
produced a desired immunomodulatory effect in the human. In some
embodiments, determining whether an anti-CD200 antibody has
produced a desired immunomodulatory effect in a human can include
(i) measuring the concentration of CD200R.sup.+ leukocytes in a
biological sample obtained from a human prior to administration to
the human of an anti-CD200 antibody to thereby obtain a
pre-treatment CD200R.sup.+ leukocyte concentration; (ii)
administering to the human the antibody; and (iii) measuring the
concentration of CD200R.sup.+ leukocytes in a blood sample obtained
from the human to thereby obtain a post-treatment CD200R.sup.+
leukocyte concentration, wherein an increase in the post-treatment
CD200R.sup.+ leukocyte concentration as compared to the
pre-treatment CD200R.sup.+ leukocyte concentration indicates that
the antibody has produced a desired immunomodulatory effect in the
human. In some embodiments, determining whether an anti-CD200
antibody is biologically active in a human includes: (i)
quantifying the level of CD200R expression by a plurality of
leukocytes in a biological sample from a human prior to
administration to the human of an anti-CD200 antibody to thereby
obtain a pre-treatment CD200R expression level; (ii) administering
to the human the anti-CD200 antibody; and (iii) quantifying the
level of CD200R expression by a plurality of leukocytes in a
biological sample from the human obtained after the administration
of the antibody to thereby obtain a post-treatment CD200R
expression level, wherein an increase in post-treatment CD200R
expression level as compared to the pre-treatment CD200R expression
level indicates that the antibody has produced a desired
immunomodulatory effect in the human. In some embodiments,
determining whether an anti-CD200 antibody is biologically active
in a human includes: (i) quantifying the level of CD200 expression
by a plurality of leukocytes in a biological sample from a human
prior to administration to the human of an anti-CD200 antibody to
thereby obtain a pre-treatment CD200 expression level; (ii)
administering to the human the anti-CD200 antibody; and (iii)
quantifying the level of CD200 expression by a plurality of
leukocytes in a biological sample from the human obtained after the
administration of the antibody to thereby obtain a post-treatment
CD200 expression level, wherein a decrease in post-treatment CD200
expression level as compared to the pre-treatment CD200 expression
level indicates that the antibody has produced a desired
immunomodulatory effect in the human. In some embodiments,
determining whether an anti-CD200 antibody is biologically active
in a human includes: (i) measuring the concentration of activated T
cells in a biological sample from a human prior to administration
to the human of an anti-CD200 antibody to thereby determine a
pre-treatment activated T cell concentration; (ii) administering to
the human the anti-CD200 antibody; and (iii) measuring the
concentration of activated T cells of same histological type as in
(i) to thereby determine a post-treatment activated T cell
concentration, wherein an increase in the post-treatment activated
T cell concentration, as compared to the pre-treatment activated T
cell concentration, indicates that the antibody has produced a
desired immunomodulatory effect in the human. In some embodiments,
determining whether an anti-CD200 antibody is biologically active
in a human includes: (i) measuring the concentration of regulatory
T cells in a biological sample from a human prior to administration
to the human of an anti-CD200 antibody to thereby determine a
pre-treatment regulatory T cell concentration; (ii) administering
to the human the anti-CD200 antibody; and (iii) measuring the
concentration of regulatory T cells of same histological type as in
(i) to thereby determine a post-treatment regulatory T cell
concentration, wherein a decrease in the post-treatment regulatory
T cell concentration, as compared to the pre-treatment regulatory T
cell concentration, indicates that the antibody has produced a
desired immunomodulatory effect in the human. In some embodiments,
determining whether an anti-CD200 antibody is biologically active
in a human includes: (i) determining the ratio of percent activated
T cells to percent regulatory T cells in a biological sample from a
human prior to administration to the human of an anti-CD200
antibody to thereby determine a pre-treatment ratio; (ii)
administering to the human the anti-CD200 antibody; and (iii)
measuring the ratio of percent activated T cells to percent
regulatory T cells of same histological type as in (i) to thereby
determine a post-treatment ratio, wherein an increase in the
post-treatment ratio, as compared to the pre-treatment ratio,
indicates that the antibody has produced a desired immunomodulatory
effect in the human. The ratio can be increased to, e.g., at least
2:1 (e.g., at least 3:1, 4:1, 5:1, 6:1, or even 7:1 or more).
[0295] In some embodiments, the above method steps can be performed
by more than one practitioner. For example, one practitioner may
analyze (e.g., measure the concentration of CD200.sup.+ T cells or
quantify the expression level of CD200R by leukocytes in) the pre-
and post-treatment samples obtained from the human. Another
practitioner may receive information regarding the analysis of the
samples by the first practitioner to thereby determine whether the
anti-CD200 antibody has produced a desired immunomodulatory effect
in the human. In some embodiments, yet another practitioner may
obtain a pre-treatment biological sample from a patient and a
fourth practitioner may obtain a post-treatment biological sample
from the patient. In some embodiments, all steps are carried out by
the same practitioner.
[0296] Further observed was that administration of an anti-CD200
antibody to a human results in one or more of: (a) an increase in
the concentration of activated T cells; (b) a decrease in the
concentration of regulatory T cells; and (c) an increase in the
ratio of percent activated T cells to percent regulatory T cells,
or a ratio of percent activated T cells to percent regulatory T
cells of at least 2:1 (e.g., at least 3:1, 4:1, 5:1, 6:1, or even
7:1 or more). Thus, in accordance with the present disclosure, to
determine whether an anti-CD200 antibody (e.g., a variant
anti-CD200 antibody that has reduced or no effector function) has
produced a desired immunomodulatory effect (e.g., an anti-CD200
antibody-associated immunomodulatory effect) in the human (and
thereby the human has been administered a dose of the antibody
sufficient to affect the treatment of the patient via, among other
things, its immunomodulatory activity), a practitioner can measure
the concentration of activated T cells in a biological sample from
a human administered an anti-CD200 antibody. An increase in the
concentration of activated T cells in the blood sample as compared
to the concentration of activated T cells in a control blood sample
indicates that the anti-CD200 antibody has produced a desired
immunomodulatory effect in the human. In accordance with the
disclosure, to determine whether an anti-CD200 antibody has
produced a desired immunomodulatory effect in the human, a
practitioner can measure the concentration of regulatory T cells in
a biological sample obtained from the human, wherein a decrease in
the concentration of regulatory T cells in the biological sample,
as compared to the concentration of regulatory T cells in a control
sample, indicates that a desired immunomodulatory effect has
occurred in the human. As described above, the practitioner need
not measure first hand the concentration of activated T cells in
the blood sample.
[0297] Methods for measuring the concentration of activated T cells
(e.g., activated CD4.sup.+ T cells) or regulatory T cells are well
known in the art and include, among other methods, flow cytometry.
As described above, determining whether an anti-CD200 antibody
(e.g., a variant anti-CD200 antibody with decreased or no effector
function) has produced a desired immunomodulatory effect in a human
can be performed by comparing the concentration of activated T
cells and/or regulatory T cells in a biological sample obtained
from a patient following administration of the anti-CD200 antibody
(the post-treatment activated T cell concentration) to the
concentration of activated T cells in a control sample. The control
sample can be, e.g., a biological sample obtained from the subject
human prior to administering to the subject human the anti-CD200
antibody. The control sample can be (or can be based on), e.g., a
collection of samples obtained from one or more (e.g., two, three,
four, five, six, seven, eight, nine, 10, 15, 20, 25, 30, 35, or 40
or more) healthy individuals that have not been administered an
anti-CD200 antibody (e.g., a control concentration of activated
cells of the same histological type can be an average of the
concentration of the cells in one or more control samples obtained
from patients who have not been administered an anti-CD200
antibody). For example, to determine whether an anti-CD200 antibody
has produced a desired immunomodulatory effect in a human
administered the antibody, a practitioner can compare the
post-treatment activated T cell concentration to the typical
concentration, or average concentration, of activated T cells of
the same histological type present in humans who have not been
administered an anti-CD200 antibody or at least do not have a
detectable level of an anti-CD200 antibody in a biological sample
obtained from the humans.
[0298] In some embodiments, a post-treatment activated T cell
concentration that is at least 5% greater than the control
concentration indicates that a desired immunomodulatory effect has
occurred in the human administered the anti-CD200 antibody. In some
embodiments, a post-treatment activated T cell concentration that
is at least 10 (e.g., 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21,
22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38,
39, 40, 41, 42, 43, 44, 45, 50, 55, 60, 65, 70, 75, 80, or more
than 80) % greater than the control concentration indicates that a
desired immunomodulatory effect has occurred in the human
administered the anti-CD200 antibody. In some embodiments,
determining whether an anti-CD200 antibody (e.g., a variant
anti-CD200 antibody having reduced or no effector function) has
produced a desired immunomodulatory effect in a human can be
performed by querying whether the post-treatment activated T cell
concentration falls within a predetermined range indicative of the
occurrence of a desired immunomodulatory effect by an anti-CD200
antibody in a human or if the post-treatment activated T cell
concentration for a given histological type of activated T cell
falls above or below a predetermined cut-off value.
[0299] As described above, a comparison of the percent activated T
cells to percent regulatory T cells can also be used to determine
whether a desired immunomodulatory effect has occurred in a human
administered an anti-CD200 antibody. For example, a practitioner
can determine the ratio of the percent activated T cells to percent
regulatory T cells in a biological sample obtained from a human
administered an anti-CD200 antibody, wherein a ratio of at least
2:1 (e.g., at least 3:1, at least 4:1, at least 5:1, at least 6:1,
or at least 7:1 or more) indicates that a desired immunomodulatory
effect has occurred in the patient. In some embodiments, an
increase in the ratio of the percent activated T cells to percent
regulatory T cells in a biological sample obtained from a patient
after administration of the anti-CD200 antibody, relative to the
corresponding ratio determined in a biological sample obtained from
the patient prior to administration of the antibody, indicates that
the anti-CD200 antibody has produced a desired immunomodulatory
effect in the human.
[0300] In some embodiments, the methods are performed using a
computer. For example, the method can include receiving data
including a medical profile of a human by way of, e.g., an internet
communication or directly inputting the information into the
computer. The profile contains information on at least one of: (a):
(i) the concentration of CD200.sup.+ T cells in a biological sample
obtained from a human following administration to the human of an
anti-CD200 antibody and (ii) the concentration of CD200.sup.+ T
cells of the same histological type as in (i) in a biological
sample obtained from the human prior to administration of the
antibody; (b): (iii) the concentration of CD200R.sup.+ T cells in a
biological sample obtained from a human following administration to
the human of an anti-CD200 antibody and (iv) the concentration of
CD200R.sup.+ T cells of the same histological type as in (iii) in a
biological sample obtained from the human prior to administration
of the antibody; (c): (v) the level of expression of CD200R by a
plurality of leukocytes in a biological sample obtained from the
human following administration to the human of an anti-CD200
antibody and (vi) the level of expression of CD200R by a plurality
of leukocytes of the same histological type as in (v) in a
biological sample obtained from the human prior to administration
of the antibody; and (d): (vii) the level of expression of CD200 by
a plurality of leukocytes in a biological sample obtained from the
human following administration to the human of an anti-CD200
antibody and (viii) the level of expression of CD200 by a plurality
of leukocytes of the same histological type as in (vii) in a
biological sample obtained from the human prior to administration
of the antibody. Next, the computer processes at least the portion
of the data containing the information to determine whether the
antibody has produced a desired immunomodulatory effect in the
human.
[0301] Computer-based methods can also include providing
information on at least one of: (a): (i) the concentration of
CD200.sup.+ T cells in a biological sample obtained from a human
following administration to the human of an anti-CD200 antibody and
(ii) the concentration of CD200 T cells of the same histological
type as in (i) in a biological sample obtained from the human prior
to administration of the antibody; (b): (iii) the concentration of
CD200R.sup.+ T cells in a biological sample obtained from a human
following administration to the human of an anti-CD200 antibody and
(iv) the concentration of CD200R.sup.+ T cells of the same
histological type as in (iii) in a biological sample obtained from
the human prior to administration of the antibody; (c): (v) the
level of expression of CD200R by a plurality of leukocytes in a
biological sample obtained from the human following administration
to the human of an anti-CD200 antibody and (vi) the level of
expression of CD200R by a plurality of leukocytes of the same
histological type as in (v) in a biological sample obtained from
the human prior to administration of the antibody; d): (vii) the
level of expression of CD200 by a plurality of leukocytes in a
biological sample obtained from the human following administration
to the human of an anti-CD200 antibody and (viii) the level of
expression of CD200 by a plurality of leukocytes of the same
histological type as in (vii) in a biological sample obtained from
the human prior to administration of the antibody; (e): (ix) the
concentration of regulatory T cells in a biological sample from a
human following administration to the human of an anti-CD200
antibody and (x) the concentration of regulatory T cells of the
same histological type as in (ix) in a biological sample from the
human prior to administration of the anti-CD200 antibody; (f): (xi)
the concentration of activated T cells in a biological sample from
a human following administration of an anti-CD200 antibody to the
human and (xii) the concentration of activated T cells of the same
histological type as in (xi) in a biological sample from the human
prior to administration of the anti-CD200 antibody; (g): (xiii) the
ratio of percent activated T cells to percent regulatory T cells in
a biological sample from a human following administration of an
anti-CD200 antibody and (xiv) the corresponding ratio of percent
activated T cells to percent regulatory T cells (each of the same
histological type as in (xiii)) in a biological sample from the
human prior to administration of the anti-CD200 antibody; and (h):
(xv) the concentration of CD8.sup.+ lymphocytes (e.g., T cells) in
a biological sample from a human following administration of an
anti-CD200 antibody to the human and (xvi) the concentration of
CD8.sup.+ lymphocytes of the same histological type as in (xv) in a
biological sample from the human prior to administration of the
antibody. The information is input into a computer and a parameter
is calculated, the parameter indicating whether the antibody has
produced a desired immunomodulatory effect in the human using the
computer and the input information. The method can also include
outputting the parameter and/or recording the parameter or result
on a computer-readable medium or a physical file such as a patient
record or chart.
[0302] As detailed in the working examples, the inventors have also
discovered that following administration of an anti-CD200 antibody
to an animal afflicted with an autoimmune disease, the
concentration of CD200.sup.+ leukocytes (e.g., subsets of
CD200.sup.+ leukocytes) and CD200.sup.+ bone marrow cells (e.g.,
subsets of CD200.sup.+ bone marrow cells) is reduced in the animal
as measured by a reduction in the concentration of such cells in
spleen tissue. A marked reduction in the concentration of
autoimmune disorder-associated autoantibodies was observed in
animals treated with the anti-CD200 antibody and in which the
immunomodulatory effect occurred. Thus, while not being bound by
any particular theory or mechanism of action, the inventors believe
that monitoring a patient treated with an anti-CD200 antibody for
the occurrence of one or more of these biomarkers is useful for,
among other things, determining whether the anti-CD200 antibody is
capable of producing a biological effect in the human to whom the
antibody is administered. Moreover, monitoring for changes in one
or more of the biomarkers is also useful for identifying a dose--a
threshold dose--of an anti-CD200 antibody, such as samalizumab,
that by virtue of its immunomodulatory effect in the human is
sufficient to achieve a clinically-meaningful effect in the disease
(i.e., sufficient to treat a disease such as an autoimmune
disease). Since a similar immunomodulatory effect on CD200.sup.+
cell populations was observed in cancer patients treated with an
anti-CD200 antibody, the inventors believe that the anti-CD200
antibody-induced immunomodulatory effects on CD200.sup.+ leukocytes
and CD200.sup.+ bone marrow cells is very likely to occur in humans
as well.
[0303] Thus, in accordance with the present disclosure, to
determine whether an anti-CD200 antibody (e.g., a variant
anti-CD200 antibody that has reduced or no effector function) has
produced a desired immunomodulatory effect in a human (and thereby
the human has been administered a dose of the antibody sufficient
to affect the treatment of the patient via, among other things, its
immunomodulatory activity), a practitioner can measure the
concentration of CD200.sup.+ leukocytes (e.g., one or more
CD200.sup.+ bone marrow cell subsets and/or CD200.sup.+
splenocytes) in a biological sample (e.g., a blood sample or a
spleen sample) from a human administered an anti-CD200 antibody. A
reduction in the concentration of CD200.sup.+ leukocytes in the
sample as compared to the concentration of CD200.sup.+ leukocytes
in a control sample indicates that the anti-CD200 antibody has
produced a desired immunomodulatory effect in the human. Similarly,
to determine whether an anti-CD200 antibody (e.g., a variant
anti-CD200 antibody that has reduced or no effector function) has
produced a desired immunomodulatory effect in a human, a
practitioner can also measure the concentration of CD200.sup.+ bone
marrow cells in a biological sample from a human administered an
anti-CD200 antibody. A reduction in the concentration of
CD200.sup.+ bone marrow cells in the sample as compared to the
concentration of CD200.sup.+ bone marrow cells (of the same
histological type) in a control sample indicates that the
anti-CD200 antibody has produced a desired immunomodulatory effect
in the human.
[0304] As described above, determining whether an anti-CD200
antibody (e.g., a variant anti-CD200 antibody with decreased or no
effector function) has produced a desired immunomodulatory effect
in a human can be performed by comparing the concentration of
CD200.sup.+ leukocytes (e.g., CD200.sup.+ splenocytes or
CD200.sup.+ bone marrow cells) in a biological sample obtained from
a patient following administration of the anti-CD200 antibody (the
post-treatment CD200.sup.+ leukocyte or CD200.sup.+ bone marrow
cell concentration) to the concentration of CD200.sup.+ cells in a
control sample.
[0305] In some embodiments, control sample is obtained from the
subject human prior to administering to the subject human the
anti-CD200 antibody. In some embodiments, the control sample can be
(or can be based on), e.g., a collection of samples obtained from
one or more (e.g., two, three, four, five, six, seven, eight, nine,
10, 15, 20, 25, 30, 35, or 40 or more) healthy individuals that
have not been administered an anti-CD200 antibody (e.g., a control
concentration of CD200.sup.+ cells of the same histological type
can be an average of the concentration of the cells in one or more
control samples obtained from patients who have not been
administered an anti-CD200 antibody).
[0306] In some embodiments, a post-treatment CD200.sup.+ leukocyte
or CD200.sup.+ bone marrow cell concentration that is at least 5%
less than the control concentration indicates that a desired
immunomodulatory effect has occurred in the human administered the
anti-CD200 antibody. In some embodiments, a post-treatment
CD200.sup.+ leukocyte or CD200.sup.+ bone marrow cell concentration
that is at least 10 (e.g., 11, 12, 13, 14, 15, 16, 17, 18, 19, 20,
21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37,
38, 39, 40, 41, 42, 43, 44, 45, 50, 55, 60, 65, 70, 75, 80, or more
than 80) % less than the control concentration indicates that a
desired immunomodulatory effect has occurred in the human
administered the anti-CD200 antibody.
[0307] In some embodiments, determining whether an anti-CD200
antibody (e.g., a variant anti-CD200 antibody having reduced or no
effector function) has produced a desired immunomodulatory effect
in a human can be performed by querying whether the post-treatment
CD200.sup.+ leukocyte or CD200.sup.+ bone marrow cell concentration
falls within a predetermined range indicative of the occurrence of
a desired immunomodulatory effect by an anti-CD200 antibody in a
human. In some embodiments, determining whether an anti-CD200
antibody has produced a desired immunomodulatory effect in a human
can include querying if the post-treatment CD200.sup.+ leukocyte or
CD200.sup.+ bone marrow cell concentration for a given histological
type of CD200.sup.+ leukocytes or CD200.sup.+ bone marrow cells
falls above or below a predetermined cut-off value. A cut-off value
is typically the concentration of CD200+ leukocytes or CD200.sup.+
bone marrow cells of a given histological type above or below which
is considered indicative of a certain phenotype--namely the
occurrence of a desired immunomodulatory effect in a human produced
by an anti-CD200 antibody.
[0308] Methods for Treatment
[0309] The disclosure also features methods for treating a variety
of disorders including, e.g., cancers, inflammatory conditions, and
disorders associated with bone loss (also referred to herein as a
"bone disorder"). For example, after it is determined that an
anti-CD200 antibody has produced a desired immunomodulatory effect
in a human suffering from a cancer (e.g., using any of the
diagnostic methods described herein), a medical practitioner may
elect to administer to the human the anti-CD200 antibody in an
amount and with a frequency sufficient to maintain the occurrence
of the immunomodulatory effect to thereby treat the patient's
cancer. Similarly, after it has been determined that an anti-CD200
antibody has produced a desired immunomodulatory effect in a human
suffering from an inflammatory condition, a medical practitioner
may elect to administer to the human the anti-CD200 antibody in an
amount and with a frequency sufficient to maintain the
immunomodulatory effect in the patient to thereby treat the
patient's inflammatory condition. Methods for therapeutically
administering an anti-CD200 antibody to a human are well known in
the art and described in, e.g., U.S. Pat. No. 7,408,041.
[0310] Cancer is a class of diseases or disorders characterized by
uncontrolled division of cells and the ability of these to spread,
either by direct growth into adjacent tissue through invasion, or
by implantation into distant sites by metastasis (where cancer
cells are transported through the bloodstream or lymphatic system).
Cancer can affect people at all ages, but risk tends to increase
with age. Types of cancers can include, e.g., lung cancer, breast
cancer, colon cancer, pancreatic cancer, renal cancer, stomach
cancer, liver cancer, bone cancer, hematological cancer, neural
tissue cancer (e.g., neuroblastoma), melanoma, thyroid cancer,
ovarian cancer, testicular cancer, prostate cancer, cervical
cancer, vaginal cancer, or bladder cancer. Hematological cancers
(liquid tumors) include, e.g., leukemias (e.g., chronic lymphocytic
leukemia such as B cell or T cell type chronic lymphocytic
leukemia) and multiple myeloma. Bone cancers include, without
limitation, osteosarcoma and osteocarcinomas.
[0311] As used herein, a human "at risk of developing a cancer" is
a human that has a predisposition to develop a cancer, i.e., a
genetic predisposition to develop cancer such as a mutation in a
tumor suppressor gene (e.g., mutation in BRCA1, p53, RB, or APC) or
has been exposed to conditions that can result in cancer. Thus, a
human can also be one "at risk of developing a cancer" when the
human has been exposed to mutagenic or carcinogenic levels of
certain compounds (e.g., carcinogenic compounds in cigarette smoke
such as acrolein, arsenic, benzene, benz{a}anthracene,
benzo{a}pyrene, polonium-210 (radon), urethane, or vinyl chloride).
Moreover, the human can be "at risk of developing a cancer" when
the human has been exposed to, e.g., large doses of ultraviolet
light or X-irradiation, or infected by a tumor-causing/associated
virus such as a papillomavirus, Epstein-Barr virus, hepatitis B
virus, or human T-cell leukemia-lymphoma virus. From the above it
will be clear that humans "at risk of developing a cancer" are not
all the humans within a species of interest.
[0312] A human "suspected of having a cancer" is one having one or
more symptoms of a cancer. Symptoms of cancer are well-known to
those of skill in the art and include, without limitation, breast
lumps, pain, weight loss, weakness, excessive fatigue, difficulty
eating, loss of appetite, chronic cough, worsening breathlessness,
coughing up blood, blood in the urine, blood in stool, nausea,
vomiting, liver metastases, lung metastases, bone metastases,
abdominal fullness, bloating, fluid in peritoneal cavity, vaginal
bleeding, constipation, abdominal distension, perforation of colon,
acute peritonitis (infection, fever, pain), pain, vomiting blood,
heavy sweating, fever, high blood pressure, anemia, diarrhea,
jaundice, dizziness, chills, muscle spasms, and difficulty
swallowing. Symptoms of a primary cancer (e.g., a large primary
cancer) can include, e.g., any one of colon metastases, lung
metastases, bladder metastases, liver metastases, bone metastases,
kidney metastases, and pancreas metastases.
[0313] An anti-CD200 antibody or an antigen-binding fragment
thereof described herein can be co-administered to a human with
cancer along with one or more additional therapeutic anti-cancer
agents. Anti-cancer agents include, e.g., chemotherapeutic agents,
ionizing radiation, immunotherapy agents, or hyperthermotherapy
agents. Chemotherapeutic agents include, but are not limited to,
aminoglutethimide, amsacrine, anastrozole, asparaginase, bcg,
bicalutamide, bleomycin, buserelin, busulfan, camptothecin,
capecitabine, carboplatin, carmustine, chlorambucil, cisplatin,
cladribine, clodronate, colchicine, cyclophosphamide, cyproterone,
cytarabine, dacarbazine, dactinomycin, daunorubicin, dienestrol,
diethylstilbestrol, docetaxel, doxorubicin, epirubicin, estradiol,
estramustine, etoposide, exemestane, filgrastim, fludarabine,
fludrocortisone, fluorouracil, fluoxymesterone, flutamide,
gemcitabine, genistein, goserelin, hydroxyurea, idarubicin,
ifosfamide, imatinib, interferon, irinotecan, letrozole,
leucovorin, leuprolide, levamisole, lomustine, mechlorethamine,
medroxyprogesterone, megestrol, melphalan, mercaptopurine, mesna,
methotrexate, mitomycin, mitotane, mitoxantrone, nilutamide,
nocodazole, octreotide, oxaliplatin, paclitaxel, pamidronate,
pentostatin, plicamycin, porfimer, procarbazine, raltitrexed,
rituximab, streptozocin, suramin, tamoxifen, taxol, temozolomide,
teniposide, testosterone, thioguanine, thiotepa, titanocene
dichloride, topotecan, trastuzumab, tretinoin, vinblastine,
vincristine, vindesine, and vinorelbine. In some embodiments, a
pharmaceutical composition comprising an anti-CD200 antibody or
CD200-binding fragment thereof can be co-formulated with one or
more of any of the foregoing agents or any other anti-cancer agent
described herein.
[0314] These chemotherapeutic anti-tumor compounds may be
categorized by their mechanism of action into groups, including,
for example, the following: anti-metabolites/anti-cancer agents,
such as pyrimidine analogs (5-fluorouracil, floxuridine,
capecitabine, gemcitabine and cytarabine) and purine analogs,
folate antagonists and related inhibitors (mercaptopurine,
thioguanine, pentostatin and 2-chlorodeoxyadenosine (cladribine));
antiproliferative/antimitotic agents including natural products
such as vinca alkaloids (vinblastine, vincristine, and
vinorelbine), microtubule disruptors such as taxane (paclitaxel,
docetaxel), vincristine, vinblastine, nocodazole, epothilones and
navelbine, epidipodophyllotoxins (etoposide, teniposide), DNA
damaging agents (actinomycin, amsacrine, anthracyclines, bleomycin,
busulfan, camptothecin, carboplatin, chlorambucil, cisplatin,
cyclophosphamide, cytoxan, dactinomycin, daunorubicin, doxorubicin,
epirubicin, hexamethylmelamineoxaliplatin, iphosphamide, melphalan,
mechlorethamine, mitomycin, mitoxantrone, nitrosourea, plicamycin,
procarbazine, taxol, taxotere, teniposide,
triethylenethiophosphoramide and etoposide (VP 16)); antibiotics
such as dactinomycin (actinomycin D), daunorubicin, doxorubicin
(adriamycin), idarubicin, anthracyclines, mitoxantrone, bleomycins,
plicamycin (mithramycin) and mitomycin; enzymes (L-asparaginase
which systemically metabolizes L-asparagine and deprives cells
which do not have the capacity to synthesize their own asparagine);
antiplatelet agents; antiproliferative/antimitotic alkylating
agents such as nitrogen mustards (mechlorethamine, cyclophosphamide
and analogs, melphalan, chlorambucil), ethylenimines and
methylmelamines (hexamethylmelamine and thiotepa), alkyl
sulfonates-busulfan, nitrosoureas (carmustine (BCNU) and analogs,
streptozocin), trazenes-dacarbazinine (DTIC);
antiproliferative/antimitotic antimetabolites such as folic acid
analogs (methotrexate); platinum coordination complexes (cisplatin,
carboplatin), procarbazine, hydroxyurea, mitotane,
aminoglutethimide; hormones, hormone analogs (estrogen, tamoxifen,
goserelin, bicalutamide, nilutamide) and aromatase inhibitors
(letrozole, anastrozole); anticoagulants (heparin, synthetic
heparin salts and other inhibitors of thrombin); fibrinolytic
agents (such as tissue plasminogen activator, streptokinase and
urokinase), aspirin, dipyridamole, ticlopidine, clopidogrel,
abciximab; antimigratory agents; antisecretory agents (breveldin);
immunosuppressives (cyclosporine, tacrolimus (FK-506), sirolimus
(rapamycin), azathioprine, mycophenolate mofetil); immunomodulatory
agents (thalidomide and analogs thereof such as lenalidomide
(Revlimid, CC-5013) and CC-4047 (Actimid)), cyclophosphamide;
anti-angiogenic compounds (TNP-470, genistein) and growth factor
inhibitors (vascular endothelial growth factor (VEGF)-inhibitors,
fibroblast growth factor (FGF) inhibitors); angiotensin receptor
blocker; nitric oxide donors; anti-sense oligonucleotides;
antibodies (trastuzumab); cell cycle inhibitors and differentiation
inducers (tretinoin); mTOR inhibitors, topoisomerase inhibitors
(doxorubicin (adriamycin), amsacrine, camptothecin, daunorubicin,
dactinomycin, eniposide, epirubicin, etoposide, idarubicin and
mitoxantrone, topotecan, irinotecan), corticosteroids (cortisone,
dexamethasone, hydrocortisone, methylprednisolone, prednisone, and
prednisolone); growth factor signal transduction kinase inhibitors;
mitochondrial dysfunction inducers and caspase activators; and
chromatin disruptors.
[0315] As described above, in some embodiments of the methods
described herein (e.g., in some embodiments of the methods for
treating cancer), the anti-CD200 antibody is not administered to
the human in combination with a chemotherapeutic compound or any
other compound that has or may have an immunosuppressive effect in
the human. That is, in some embodiments, a patient is selected for
treatment with a therapeutic anti-CD200 antibody if the patient has
not already been administered (within a specified period of time
prior to starting the anti-CD200 antibody therapy) a
chemotherapeutic agent (such as any of those described herein) or
any other agent that can (or did) result in an immunosuppression in
the patient. In some embodiments, the human is one who has not
received a chemotherapeutic treatment prior to administration of
the first dose of the anti-CD200 antibody and/or continues to not
receive a chemotherapeutic treatment as long as the patient is
being administered the anti-CD200 antibody. "Prior to
administration of the first dose of the anti-CD200 antibody" can
include, e.g., within a time-period that is less than four months
(e.g., less than 16 weeks, 15 weeks, 14 weeks, 13 weeks, three
months, 12 weeks, 11 weeks, 10 weeks, 9 weeks, two months, eight
weeks, seven weeks, six weeks, five weeks, one month, 30, 29, 28,
27, 26, 25, 24, 23, 22, 21, 20, 19, 18, 17, 16, 15, 14, 13, 12, 11,
or less than 10 days) prior to administration of the first dose of
the anti-CD200 antibody.
[0316] In some embodiments, the methods described herein can
include determining whether the human has a cancer. In some
embodiments, the methods described herein can include the step of
determining whether one or more cancer cells of a human's cancer
express CD200. In some embodiments, the methods can include
determining whether one or more cancer cells of the human's cancer
overexpress CD200, relative to a control sample. In some
embodiments, the control sample is obtained from the same human and
comprises normal cells of the same tissue type as the human's
cancer. For example, a skilled artisan could measure the level of
CD200 protein present on colon cancer cells from a patient as
compared to normal colon cells from the patient. In some
embodiments, the control sample can be the expression level (or
average expression level) of cells obtained from one or more humans
who do not have cancer. In some embodiments, the cancer comprises
cells (e.g., a plurality or even a majority of cells) that express
or overexpress CD200 (e.g., CD200 protein and/or CD200 mRNA). In
some embodiments, at least (or greater than) 10 (e.g., 15, 20, 25,
30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, or 95) % of the
cancer cells of the human's cancer overexpress CD200. In some
embodiments, all assayed cancer cells overexpress CD200 relative to
normal cells. In some embodiments, a cancer cell (e.g., a plurality
of cancer cells, at least 10% of cancer cells, or all assayed
cancer cells) can express CD200 protein at levels at least about
1.4 (e.g., at least about 1.5, 1.6, 1.7, 1.8, 1.9, 2.0, 2.2., 2.5,
3.0, 3.5, 4.0, 4.5, or 5 or more)-fold higher than the expression
levels found on normal cells of the same histological type or
higher than the average expression of normal cells from one or more
patients who do not have cancer.
[0317] In some embodiments, an anti-CD200 antibody is only
administered to a human if the human's cancer comprises a plurality
of cancer cells that express or overexpress CD200. Methods for
detecting expression of CD200 are well known in the art and
include, e.g., Western blot, immunohistochemistry, and flow
cytometry techniques. Suitable methods for detecting CD200
expression are described in detail in, e.g., Kretz-Rommel et al.
(2007) J Immunol 178:5595-5605 and Kretz-Rommel et al. (2008)J
Immunol 180:699-705.
[0318] In some embodiments, an anti-CD200 antibody blocks immune
suppression in cancer by targeting cancer cells that express CD200.
Eradication, or inhibition, of these cancer cells can stimulate the
immune system and allow further eradication of cancer cells.
[0319] In some embodiments, the combination of direct cancer cell
killing and driving the immune response towards a Th1 profile
provides enhanced efficacy in cancer treatment. Thus, in one
embodiment, a cancer treatment is provided wherein an antibody or
antibody fragment, which binds to CD200 and both a) blocks the
interaction between CD200 and its receptor and b) directly kills
the cancer cells expressing CD200, is administered to a cancer
patient. The mechanism by which the cancer cells are killed can
include, but are not limited to, ADCC or CDC; fusion with a toxin;
fusion with a toxic radioactive agent; fusion with a toxic
polypeptide such as granzyme B or perforin; fusion with a cytotoxic
virus (e.g., cytotoxic reovirus such as Reolysin.RTM.); or fusion
with a cytokine such as TNF-.alpha. or IFN-.alpha.. In an
alternative embodiment, a cancer treatment involves administering
an antibody that both a) blocks the interaction between CD200 and
its receptor and b) enhances cytotoxic T cell or NK cell activity
against the tumor. Such enhancement of the cytotoxic T cell or NK
cell activity may, for example, be combined by fusing the antibody
with cytokines such as, e.g., IL-2, IL-12, IL-18, IL-13, and IL-5.
In addition, such enhancement may be achieved by administration of
an anti-CD200 antibody in combination with inhibitors such as
IMiDs, thalidomide, or thalidomide analogs.
[0320] In yet another embodiment, the cancer treatment involves
administering an antibody that both a) blocks the interaction
between CD200 and its receptor and b) attracts T cells to the tumor
cells. T cell attraction can be achieved by fusing the Ab with
chemokines such as MIG, 1P-10, 1-TAC, CCL21, CCL5 or LIGHT. Also,
treatment with chemotherapeutics can result in the desired
upregulation of LIGHT. The combined action of blocking immune
suppression and killing directly through antibody targeting of the
tumor cells is a unique approach that provides increased
efficacy.
[0321] An "inflammatory condition," as used herein, refers to a
process in which one or more substances (e.g., substances not
naturally occurring in the human), via the action of white blood
cells (e.g., B cells, T cells, macrophages, monocytes, or dendritic
cells) inappropriately trigger a pathological response, e.g., a
pathological immune response. Accordingly, such immune cells
involved in the inflammatory response are referred to as
"inflammatory cells." The inappropriately triggered inflammatory
response can be one where no foreign substance (e.g., an antigen, a
virus, a bacterium, a fungus) is present in or on the human. The
inappropriately triggered response can be one where a
self-component (e.g., a self-antigen) is targeted (e.g., an
autoimmune disorder such as multiple sclerosis) by the inflammatory
cells. The inappropriately triggered response can also be a
response that is inappropriate in magnitude or duration, e.g.,
anaphylaxis. Thus, the inappropriately targeted response can be due
to the presence of a microbial infection (e.g., viral, bacterial,
or fungal). Types of inflammatory condition (e.g., autoimmune
disease) can include, but are not limited to, osteoarthritis;
rheumatoid arthritis; spondyloarthropathies; respiratory distress
syndrome (including adult respiratory distress syndrome; ARDS),
POEMS syndrome; inflammatory bowel disease; Crohn's disease,
graft-versus host disease (e.g., rejections of skin grafts, kidney
grafts, heart grafts, lung grafts, liver grafts, or bone marrow
grafts); multicentric Castleman's disease; systemic lupus
erythematosus (SLE); multiple sclerosis; muscular dystrophy;
insulin-dependent diabetes mellitus; dermatomyositis; polymyositis;
inflammatory neuropathies such as Guillain Barre syndrome;
vasculitis such as Wegener's granulomatosus; lupus nephritis (LN);
glomerulonephritis; polyarteritis nodosa; polymyalgia rheumatica;
temporal arteritis; Sjogren's syndrome; Behcet's disease;
Churg-Strauss syndrome; or Takayasu's arteritis. Also included in
inflammatory disorders are certain types of allergies such as
rhinitis, sinusitis, urticaria, hives, angioedema, atopic
dermatitis, food allergies (e.g., a nut allergy), drug allergies
(e.g., penicillin), insect allergies (e.g., allergy to a bee
sting), or mastocytosis. Inflammatory conditions can also include
ulcerative colitis and asthma.
[0322] A human "at risk of developing an inflammatory condition"
refers to a human with a family history of one or more inflammatory
conditions (e.g., a genetic predisposition to one or more
inflammatory disorders) or one exposed to one or more
inflammation-inducing conditions. For example, a human can have
been exposed to a viral or bacterial superantigen such as, but not
limited to, Staphylococcal enterotoxins (SEs), a Streptococcus
pyogenes exotoxin (SPE), a Staphylococcus aureus toxic
shock-syndrome toxin (TSST-1), a Streptococcal mitogenic exotoxin
(SME) and a Streptococcal superantigen (SSA). From the above it
will be clear that humans "at risk of developing an inflammatory
condition" are not all the humans within a species of interest.
[0323] A human "suspected of having an inflammatory condition" is
one who presents with one or more symptoms of an inflammatory
condition. Symptoms of inflammatory disorders are well known in the
art and include, but are not limited to, redness, swelling (e.g.,
swollen joints), joints that are warm to the touch, joint pain,
stiffness, loss of joint function, fever, chills, fatigue, loss of
energy, headaches, loss of appetite, muscle stiffness, insomnia,
itchiness, stuffy nose, sneezing, coughing, one or more neurologic
symptoms such as dizziness, seizures, or pain. From the above it
will be clear that humans "suspected of having an inflammatory
condition" are not all the humans within a species of interest.
[0324] An "autoimmune disorder," as used herein, refers to a
disease state in which, via the action of white blood cells (e.g.,
B cells, T cells, macrophages, monocytes, or dendritic cells), a
pathological immune response (e.g., pathological in duration and/or
magnitude) has been generated in a host organism against a
substance or a tissue that is normally present within the host
organism. Types of autoimmune diseases include, but are not limited
to, chronic obstructive pulmonary disease, diabetes mellitus type
1, Goodpasture's syndrome, SLE, LN, Grave's disease, Guillain-Barre
syndrome, IgA nephropathy, scleroderma, Sjogren's syndrome,
Wegener's granulomatosis, pemphigus vulgaris, Chagas disease,
rheumatoid arthritis, Crohn's disease, Hashimoto's disease,
idiopathic thrombocytopenic purpura, myasthenia gravis, pulmonary
biliary cirrhosis, and Miller Fisher syndrome. Autoimmune disorders
also include certain autoimmune hemolytic disorders such as cold
agglutinin disease (CAD), antiphospholipid syndrome (APS),
autoimmune hemolytic disease (e.g., autoimmune hemolytic anemia;
AIHA), catastrophic anti-phospholipid syndrome (CAPS), warm
autoimmune hemolytic anemia, and paroxysmal cold hemoglobinuria
(PCH).
[0325] A human "at risk of developing autoimmune disorder" refers
to a human with a family history of autoimmune disorders (e.g., a
genetic predisposition to one or more autoimmune disorders) or one
exposed to one or more autoimmune disorder/autoantibody-inducing
conditions. Humans with certain cancers (e.g., liquid tumors such
as multiple myeloma or chronic lymphocytic leukemia) can
pre-dispose patients to developing certain autoimmune hemolytic
diseases. For example, PCH can follow a variety of infections
(e.g., syphilis) or neoplasms such as non-Hodgkin's lymphoma. In
another example, CAD can be associated with HIV infection,
Mycoplasma pneumonia infection, non-Hodgkin's lymphoma, or
Waldenstrom's macroglobulinemia. In yet another example, autoimmune
hemolytic anemia is a well-known complication of human chronic
lymphocytic leukemia, approximately 11% of CLL patients with
advanced disease will develop AIHA. As many as 30% of CLL patients
may be at risk for developing AIHA. See, e.g., Diehl et al. (1998)
Semin Oncol 25(1):80-97 and Gupta et al. (2002) Leukemia
16(10):2092-2095. From the above it will be clear that humans "at
risk of developing an autoimmune disorder" are not all the humans
within a species of interest.
[0326] A human "suspected of having an autoimmune disorder" is one
who presents with one or more symptoms of an autoimmune disorder.
Symptoms of autoimmune disorders can vary in severity and type with
the particular autoimmune disorder and include, but are not limited
to, redness, swelling (e.g., swollen joints), joints that are warm
to the touch, joint pain, stiffness, loss of joint function, fever,
chills, fatigue, loss of energy, pain, fever, pallor, icterus,
urticarial dermal eruption, hemoglobinuria, hemoglobinemia, and
anemia (e.g., severe anemia), headaches, loss of appetite, muscle
stiffness, insomnia, itchiness, stuffy nose, sneezing, coughing,
one or more neurologic symptoms such as dizziness, seizures, or
pain. From the above it will be clear that humans "suspected of
having an autoimmune disorder" are not all the humans within a
species of interest.
[0327] An anti-CD200 antibody described herein can be
co-administered with one or more additional therapeutic agents
useful for treating or preventing an inflammatory condition. The
one or more agents include, e.g., a non-steroidal anti-inflammatory
drug (NSAID), a disease-modifying anti-rheumatic drug (DMARD), a
biological response modifier, or a corticosteroid. Biological
response modifiers include, e.g., an anti-TNF agent (e.g., a
soluble TNF receptor or an antibody specific for TNF such as
adulimumab, infliximab, or etanercept). In some embodiments, the
one or more additional therapeutic agents can be, e.g., steroids,
anti-malarials, aspirin, non-steroidal anti-inflammatory drugs,
immunosuppressants, cytotoxic drugs, corticosteroids (e.g.,
prednisone, dexamethasone, and prednisolone), methotrexate,
methylprednisolone, macrolide immunosuppressants (e.g., sirolimus
and tacrolimus), mitotic inhibitors (e.g., azathioprine,
cyclophosphamide, and methotrexate), fungal metabolites that
inhibit the activity of T lymphocytes (e.g., cyclosporine),
mycophenolate mofetil, glatiramer acetate, and cytotoxic and
DNA-damaging agents (e.g., chlorambucil or any other DNA-damaging
agent described herein or known in the art).
[0328] The anti-CD200 antibodies described herein can also be used
to treat a variety of disorders associated with bone loss
including, e.g., osteoporosis and periodontal disease. Bone loss
can result from a number of disorders such as, but not limited to,
hypercalciuria, nutritional disorders (e.g., eating disorders such
as bulimia or anorexia), menopause, premature ovarian failure,
hypogonadal conditions such as Turner syndrome, Klinefelter
syndrome, Kallmann syndrome, andropause, hypothalamic amenorrhea,
or hyperprolactinemia. Osteoporotic bone loss can also result from
a number of cancers and inflammatory disorders. For example, bone
loss can result from multiple myeloma (MM), rheumatoid arthritis
(RA), and systemic lupus erythematosus (SLE). A human "at risk for
developing a disorder associated with bone loss" is one with a
family history of osteoporosis or a human having a disorder that is
associated with osteoporosis. For example, a human at risk for
developing osteoporosis can be one who has multiple myeloma, a
nutritional disorder, or an osteoporosis-associated inflammatory
disorder such as RA or SLE. A human at risk for developing
osteoporosis can be, e.g., a menopausal woman. From the above it
will be clear that humans "at risk of developing a disorder
associated with bone loss" are not all the humans within a species
of interest.
[0329] A human "suspected of having a disorder associated with bone
loss" is one who presents with one or more symptoms of the
disorder. Symptoms of osteoporosis include, e.g., fragility
fractures, pain (e.g., neck pain or lower back), and stooped
posture resulting from spinal compression fractures.
[0330] In addition to the administration of one or more anti-CD200
antibodies, or CD200-binding fragments thereof, described herein, a
disorder associated with bone loss can be treated with a
bisphosphonate, recombinant parathyroid hormone, hormone
replacement therapy (e.g., estrogen therapy in women), and a
selective estrogen receptor modulator.
[0331] CD200 has been shown in animal models to play a role in
pregnancy. For example, increased CD200 expression, by way of a
soluble CD200-Fc fusion protein, has been shown to decrease the
rate of spontaneous abortion in mice. (See, e.g., Clark et al.
(2001) Mol Human Reprod 7:185-194 and Gorczynski et al. (2001)
Graft 4:338-345.) Thus, prior to administering an anti-CD200
antibody or CD200-binding fragment thereof to a woman, a medical
practitioner can determine if the woman is pregnant. If the woman
is pregnant, the medical practitioner may opt not to administer the
anti-CD200 antibody to the woman. The medical practitioner can
optionally select an alternative therapy for the woman.
[0332] In some embodiments, the therapeutic efficacy of
myeloablative therapies followed by bone marrow transplantation, or
adoptive transfer of T cells reactive with CLL cells, is enhanced
by anti-CD200 therapy. Furthermore, anti-CD200 treatment can
substantially enhance efficacy of cancer vaccines such as dendritic
cells loaded with CLL cell proteins, peptides or RNA derived from
such cells, patient-derived heat-shock proteins (HSPs), tumor
peptides or protein. In other embodiments, an anti-CD200 antibody
or CD200-binding fragment thereof can be used in combination with
an immuno-stimulatory compound, such as CpG, toll-like receptor
agonists or any other adjuvant, anti-CTLA-4 antibodies, and the
like. In some embodiments, efficacy of anti-CD200 antibody (or
CD200-binding fragment) treatment can be improved by blocking of
immunosuppressive mechanisms using anti-PDL1 and/or anti-PDL2
antibodies, anti-IL-10 antibodies, anti-IL-6 antibodies, and the
like. In some embodiments, the efficacy of an anti-CD200 antibody
treatment is improved by administration of agents that increase NK
cell number or T-cell activity such as the small molecule inhibitor
IMiDs, thalidomide, or thalidomide analogs.
[0333] In some embodiments, it can be advantageous to eliminate
plasmacytoid dendritic cells, shown to be immunosuppressive in the
cancer environment. In these embodiments in which delivery of an
anti-CD200 antibody or CD200-binding fragment thereof is intended
to augment an immune response, an anti-CD200 antibody lacking
effector function is advantageous.
[0334] In some embodiments, the methods described herein can
include, after administering the anti-CD200 antibody, monitoring
the human for an improvement in the disorder and/or one or more
symptoms thereof. Monitoring a human for an improvement in a
disorder (e.g., a cancer, an inflammatory condition, or a disorder
associated with bone loss), as defined herein, means evaluating the
subject for a change in a disease parameter, e.g., an improvement
in one or more symptoms of the disease. In some embodiments, the
evaluation is performed at least 1 hour, e.g., at least 2, 4, 6, 8,
12, 24, or 48 hours, or at least 1 day, 2 days, 4 days, 10 days, 13
days, 20 days or more, or at least 1 week, 2 weeks, 4 weeks, 10
weeks, 13 weeks, 20 weeks or more, after an administration. The
human can be evaluated in one or more of the following periods:
prior to beginning of treatment; during the treatment; or after one
or more elements of the treatment have been administered.
Evaluating can include evaluating the need for further treatment,
e.g., evaluating whether a dosage, frequency of administration, or
duration of treatment should be altered. It can also include
evaluating the need to add or drop a selected therapeutic modality,
e.g., adding or dropping any of the treatments for a disorder
described herein.
[0335] In some embodiments, monitoring the progress and/or
effectiveness of a therapeutic treatment includes monitoring the
level of CD200 expression before and after treatment. For example,
pre-treatment levels of CD200 can be ascertained and, after at
least one administration of the therapy, levels of CD200 can again
be determined. A decrease in CD200 levels can be indicative of an
effective treatment (see below). Measurement of CD200 levels can be
used by the practitioner as a guide for increasing dosage amount or
frequency of the therapy. It should of course be understood that
CD200 levels can be directly monitored or, alternatively, any
marker that correlates with CD200 can be monitored.
[0336] The inventors have also discovered that upon administration
of an anti-CD200 antibody to a patient with a cancer comprising
cells expressing CD200, CD200 expression by the cancer cells is
reduced. As noted above, cancer cells have evolved a number of ways
to evade detection by the immune system, which can identify
malignant cells within a host organism and kill the cells before a
cancer develops. See, e.g., Geertsen et al. (1999) Int J Mol Med
3(1):49-57; Kerebijn et al. (1999) Crit Rev Oncol Hematol
31(1):31-53; and Pardoll (2003) Annu Rev Immunol 21:807-39. One
potential mechanism by which cancer cells escape immunosurveillance
is expression or overexpression of the immunosuppressive CD200
protein. In fact, CD200 protein has been shown to be expressed or
overexpressed on a variety of human cancer cells including, e.g., B
cell chronic lymphocytic leukemia cells, prostate cancer cells,
breast cancer cells, colon cancer cells, and brain cancer cells.
See, e.g., Kawasaki et al. (2007) Biochem Biophys Res Commun
364(4):778-782; Kretz-Rommel et al. (2007), supra; and Siva et al.
(2008) Cancer Immunol Immunother 57(7):987-96. Thus, while the
disclosure is not bound by any particular theory or mechanism of
action, the inventors believe that the anti-CD200
antibody-dependent downregulation of CD200 on the cancer cells
relieves an inhibition of immunosurveillance and allows the immune
system to more effectively identify and fight the cancer.
[0337] Accordingly, it is believed to be beneficial to administer
to the human an anti-CD200 antibody in an amount and with a
frequency sufficient to sustain the reduced expression of CD200 by
the cancer cells in the human. Methods for detecting expression or
a change in expression of CD200 by cancer cells are well known in
the art (e.g., Western blot, immunohistochemistry, and flow
cytometry techniques) and described herein. For example, following
the administration of an anti-CD200 antibody to a human, the level
of expression of CD200 by cancer cells can be determined by flow
cytometry analysis of the cancer cells present in a biological
sample obtained from a patient. The CD200 expression level of the
cancer cells post-treatment can be compared to a control expression
level and/or the level of expression of the patient's cancer cells
prior to treatment with the antibody, wherein a reduction in the
level of CD200 expression by the cancer cells indicates that the
anti-CD200 antibody has been administered to the human in an amount
and with a frequency sufficient to reduce CD200 expression by the
cancer cells.
[0338] Through an iterative process, a medical practitioner can
determine the appropriate dose amount, and frequency of
administration of each dose, required to maintain a reduced level
of CD200 expression by the cancer cells in the patient. For
example, a medical practitioner can administer to a cancer patient
at least two (e.g., at least three, four, five, six, seven, or
eight or more) times an anti-CD200 antibody in an amount that
reduces (or is at least expected to reduce) the level of expression
of CD200 by the cancer cells. The at least two doses should be
spaced apart in time by at least one (e.g., at least two, three,
four, five, six, seven, eight, nine, 10, 11, 12, 13, or even 14)
day(s). Biological samples (e.g., blood samples) containing cancer
cells are obtained from the patient at various times, e.g., prior
to the first anti-CD200 antibody administration, between the first
dose and at least one additional dose, and at least one biological
sample collection following the second dose. In some embodiments,
biological samples may be collected at least two times between
doses and/or at least one time after the final dose administered to
the patient. The cancer cells in each biological sample obtained
are then interrogated for CD200 expression to determine whether the
amount and/or the frequency of administration of the anti-CD200
antibody are sufficient to maintain a reduced level of CD200
expression by the cancer cells. Armed with information on CD200
expression by the patient's cancer cells over time and the effect
on CD200 expression by the cells over time by administering the
anti-CD200 antibody to the patient, a medical practitioner (and/or
a computer) can determine an anti-CD200 antibody dosing schedule
for the patient that is sufficient to maintain a reduced level of
CD200 expression by the patient's cancer cells over the course of
the treatment.
[0339] As described above, the inventors have also observed that
upon administration of an anti-CD200 antibody to a patient with a
cancer comprising cells expressing CD200: (i) the level of
expression of CD200 by leukocytes is reduced as compared to the
expression level of CD200 by leukocytes of the same histological
type in a control sample; (ii) the level of expression of CD200R by
leukocytes is increased as compared to the expression level of
CD200R by leukocytes of the same histological type in a control
sample; (iii) the concentration of CD200.sup.+ T cells, as compared
to the concentration of CD200.sup.+ T cells of the same
histological type in a control sample, is reduced; and (iv) the
concentration of CD200R.sup.+ leukocytes, as compared to the
concentration of CD200R.sup.+ leukocytes of the same histological
type in a control sample, is increased. Similarly, the inventors
have also observed that upon administration of an anti-CD200
antibody to an animal with an autoimmune disease the concentration
of CD200.sup.+ leukocytes and CD200.sup.+ bone marrow cells is
reduced as compared to the concentration of such cells in an animal
not treated with the antibody. Accordingly, the disclosure also
features methods for determining the appropriate dose amount, and
frequency of administration of each dose, required to maintain,
e.g., a reduced level of CD200 expression by leukocytes in the
patient; an increased level of CD200R expression by leukocytes in
the patient; a reduced concentration of CD200.sup.+ leukocytes
and/or CD200.sup.+ bone marrow cells in the patient; and/or an
increased concentration of CD200R.sup.+ leukocytes in the patient,
for the duration of the treatment of the patient with an anti-CD200
antibody.
[0340] Using the information provided herein on the
immunomodulatory effect(s) of an anti-CD200 antibody (e.g., the
reduction in the expression level of CD200 by cancer cells or the
increase in CD200R expression by leukocytes in a patient treated
with an anti-CD200 antibody), it would be a matter of routine
experimentation for a skilled artisan in the field of medicine to
determine an appropriate dosing schedule of an anti-CD200 antibody
for a patient that maintains in the patient the presence of at
least one of the immunomodulatory effects disclosed herein.
[0341] For example, an antibody described herein can be
administered as a fixed dose, or in a milligram per kilogram
(mg/kg) dose. In some embodiments, the dose can also be chosen to
reduce or avoid production of antibodies or other host immune
responses against one or more of the active antibodies in the
composition. While in no way intended to be limiting, exemplary
dosages of an antibody include, e.g., 1-100 .mu.g/kg, 0.5-50
.mu.g/kg, 0.1-100 .mu.g/kg, 0.5-25 .mu.g/kg, 1-20 .mu.g/kg, and
1-10 .mu.g/kg, 1-100 mg/kg, 0.5-50 mg/kg, 0.1-100 mg/kg, 0.5-25
mg/kg, 1-20 mg/kg, and 1-10 mg/kg. Exemplary dosages of an antibody
described herein include, without limitation, 0.1 .mu.g/kg, 0.5
.mu.g/kg, 1.0 .mu.g/kg, 2.0 .mu.g/kg, 4 .mu.g/kg, and 8 .mu.g/kg,
0.1 mg/kg, 0.5 mg/kg, 1.0 mg/kg, 2.0 mg/kg, 4 mg/kg, and 8 mg/kg.
Exemplary doses (e.g., of a whole anti-CD200 antibody such as
samalizumab) also include, e.g., greater than or equal to 50
mg/m.sup.2, 75 mg/m.sup.2, 100 mg/m.sup.2, 150 mg/m.sup.2, 200
mg/m.sup.2, 250 mg/m.sup.2, 300 mg/m.sup.2, 350 mg/m.sup.2, 400
mg/m.sup.2, 450 mg/m.sup.2, 500 mg/m.sup.2, 550 mg/m.sup.2, 600
mg/m.sup.2, and/or 700 mg/m.sup.2.
[0342] A pharmaceutical composition can include a therapeutically
effective amount of an antibody described herein. Such effective
amounts can be readily determined by one of ordinary skill in the
art based, in part, on the effect of the administered antibody, or
the combinatorial effect of the antibody and one or more additional
active agents, if more than one agent is used. A therapeutically
effective amount of an antibody described herein can also vary
according to factors such as the disease state, age, sex, and
weight of the individual, and the ability of the antibody (and one
or more additional active agents) to elicit a desired response in
the individual, e.g., amelioration of at least one condition
parameter, e.g., amelioration of at least one symptom of the cancer
and/or the presence of at least one of the immunomodulatory effect
biomarkers described herein. A therapeutically effective amount is
also one in which any toxic or detrimental effects of the
composition are outweighed by the therapeutically beneficial
effects.
[0343] Toxicity and therapeutic efficacy of such compositions can
be determined by known pharmaceutical procedures in cell cultures
or experimental animals (e.g., animal models of any of the
disorders described herein). These procedures can be used, e.g.,
for determining the LD.sub.50 (the dose lethal to 50% of the
population) and the ED.sub.50 (the dose therapeutically effective
in 50% of the population). The dose ratio between toxic and
therapeutic effects is the therapeutic index and it can be
expressed as the ratio LD.sub.50/ED.sub.50. An anti-CD200 antibody
that exhibits a high therapeutic index is preferred. While
compositions that exhibit toxic side effects may be used, care
should be taken to design a delivery system that targets such
compounds to the site of affected tissue and to minimize potential
damage to normal cells and, thereby, reduce side effects.
[0344] The inventors have also discovered an inverse correlation
between the peripheral tumor load (e.g., B CLL tumor cell load) and
the concentration (or number) of T cells present in cancer
patients. That is, the greater the concentration of non-cancer T
cells present in a cancer patient, the lower the tumor burden in
the patient.
[0345] While the discovery is not by any particular theory or
mechanism of action, the inventors believe that a cancer patient
may receive an enhanced benefit from an anti-CD200 antibody therapy
if the cancer patient exhibits normal or elevated levels of T cells
at the time of therapy. Similarly, the inventors have also
determined that an anti-CD200 antibody therapy is likely to have
even more efficacy and/or a stronger immunomodulatory effect in
patients with an intact immune system, e.g., an immune system that
is capable of mounting an immune response against a cancer present
in the patient. To wit, as described below, all four of the cancer
patients in the study who had not received prior chemotherapy
before samalizumab treatment had clinically stable or improved
disease after samalizumab treatment. In fact, patient 102-502, who
had not received an immunosuppressive or chemotherapeutic therapy
prior to administration of the anti-CD200 antibody, exhibited a
substantial reduction in tumor burden, which correlated with
changes in a number of the immunomodulatory biomarkers described
herein, including, a marked reduction in the concentration of
CD45.sup.+ B CLL cells, an increase in CD8.sup.+ T cells, a
decrease in regulatory T cells, an increase in activated T cells,
and an increase in the ratio of percent activated T cells to
percent regulatory T cells.
[0346] Accordingly, a cancer patient who has an intact immune
system capable of mounting an immune response to the patient's
cancer can be selected for treatment with an anti-CD200 antibody.
Selection can include, e.g., quantifying the concentration of
CD3.sup.+ cells present in a biological sample from a patient
suffering from a cancer; and administering to the patient the
anti-CD200 antibody in an amount effective to treat the cancer in
the patient if the patient has a concentration of T cells
sufficient to enhance the efficacy of the anti-CD200 antibody
therapy in the patient. The average concentration of CD3.sup.+
cells in blood from a healthy human and a human having a cancer
such as B-CLL are well known in the art. In some embodiments, a
sufficient concentration of CD3.sup.+ cells in the biological
sample is a concentration of CD3.sup.+ cells that is greater than
300 (e.g., 325, 350, 375, 400, 425, 450, 475, 500, 525, 550, 575,
600, 625, 650, 675, 700, 725, 750, 775, 800, 825, 850, 875, 900,
925, 950, 975, 1000, 1100, 1200, or 1300 or more) cells per
microliter. In some embodiments, a sufficient concentration of
CD3.sup.+ cells in the biological sample is a concentration of
CD3.sup.+/CD4.sup.+ cells that is greater than or equal to 200
(e.g., 225, 250, 275, 300, 325, 350, 375, 400, 425, 450, 475, or
500 or more) cells per microliter. In some embodiments, a
sufficient concentration of CD3.sup.+ cells in the biological
sample is a concentration of CD3.sup.+/CD8.sup.+ cells that is
greater than or equal to 150 (e.g., 175, 200, 225, 250, 275, 300,
325, 350, 375, 400, 425, 450, 475, 500, 525, 550, 575, 600, 625,
650, 675, 700, 725, 750, 775, 800, 825, 850, 875, 900, 925, 950,
975, 1000, 1100, 1200, or 1300 or more) cells per microliter.
Methods for determining the concentration of CD3.sup.+ cells are
known in the art and include, e.g., flow cytometry. Methods for
administering an anti-CD200 antibody therapeutically to a cancer
patient are known in the art, described herein, and elaborated on
in, e.g., U.S. Pat. No. 7,408,041.
[0347] In some embodiments, immune competence can be determined by
quantifying the absolute number of certain lymphocyte populations
in a biological sample (e.g., a blood sample) obtained from a
patient as measured by, e.g., flow cytometry. See, e.g., Shearer et
al. (2003) J Allergy Clin Immunol 112(5):973-980 and Paglieroni and
Holland (1994) Transfusion 34:512-516. For example, in some
embodiments, immune competence is indicated by a CD45.sup.+
lymphocyte count, by flow cytometry, of: 0.66-4.60.times.10.sup.3
cells/4 (for patients 0 to 17 years of age);
0.99-3.15.times.10.sup.3 cells/.mu.L (for patients aged 18 to 55
years); or 1.00-3.33.times.10.sup.3 cells/.mu.L (for patients older
than 55 years).
[0348] In some embodiments, immune competence can be determined by
quantifying the absolute number of CD3.sup.+ T cells, by flow
cytometry, in a biological sample obtained from a patient. For
example, in some embodiments, immune competence is indicated by a
CD3.sup.+ lymphocyte count, by, e.g., flow cytometry, of:
2,500-5,500 cells/.mu.L (for patients 0 to 2 months of age);
2,500-5,600 cells/.mu.L (for patients aged 3 to 5 months);
1,900-5,900 cells/.mu.L (for patients aged 6 to 11 months);
2,100-6,200 cells/.mu.L (for patients aged 12 to 23 months);
1,400-3,700 cells/.mu.L (for patients aged 2 to 5 years);
1,200-2,600 cells/.mu.L (for patients aged 6 to 11 years);
1,000-2,200 cells/.mu.L (for patients aged 12 to 17 years);
677-2,383 cells/.mu.L (for patients aged 18 to 55 years); or
617-2,254 cells/.mu.L (for patients older than 55 years of
age).
[0349] In some embodiments, immune competence can be determined by
quantifying the absolute number of CD19.sup.+ B cells, by, e.g.,
flow cytometry, in a biological sample obtained from a patient. For
example, in some embodiments, immune competence is indicated by a
CD19.sup.+ B cell count, by flow cytometry, of: 300-2,000
cells/.mu.L (for patients 0 to 2 months of age); 430-3,000
cells/.mu.L (for patients aged 3 to 5 months); 610-2,600
cells/.mu.L (for patients aged 6 to 11 months); 720-2,600
cells/.mu.L (for patients aged 12 to 23 months); 390-1,400
cells/.mu.L (for patients aged 2 to 5 years); 270-860 cells/.mu.L
(for patients aged 6 to 11 years); 110-570 cells/.mu.L (for
patients aged 12 to 17 years); 99-527 cells/.mu.L (for patients
aged 18 to 55 years); or 31-409 cells/.mu.L (for patients older
than 55 years of age).
[0350] In some embodiments, immune competence can be determined by
quantifying the absolute number of CD16.sup.+CD56.sup.+ Natural
Killer (NK) cells, by, e.g., flow cytometry, in a biological sample
obtained from a patient. For example, in some embodiments, immune
competence is indicated by a CD16.sup.+CD56.sup.+NK cell count, by
flow cytometry, of: 170-1,100 (for patients 0 to 2 months of age);
170-830 cells/.mu.L (for patients aged 3 to 5 months); 160-950
cells/.mu.L (for patients aged 6 to 11 months); 180-920 cells/.mu.L
(for patients aged 12 to 23 months); 130-720 cells/.mu.L (for
patients aged 2 to 5 years); 100-480 cells/.mu.L (for patients aged
6 to 11 years); 110-570 cells/.mu.L (for patients aged 12 to 17
years); 101-678 cells/.mu.L (for patients aged 18 to 55 years); or
110-657 cells/.mu.L (for patients older than 55 years of age).
[0351] In some embodiments, immune competence can be determined by
quantifying the absolute number of CD4.sup.+ Helper T cells, by,
e.g., flow cytometry, in a biological sample obtained from a
patient. For example, in some embodiments, immune competence is
indicated by a CD4.sup.+ Helper T cell count, by flow cytometry,
of: 1,600-4,000 (for patients 0 to 2 months of age); 1,800-4,000
cells/.mu.L (for patients aged 3 to 5 months); 1,400-4,300
cells/.mu.L (for patients aged 6 to 11 months); 1,300-3,400
cells/.mu.L (for patients aged 12 to 23 months); 700-2,200
cells/.mu.L (for patients aged 2 to 5 years); 650-1,500 cells/.mu.L
(for patients aged 6 to 11 years); 530-1,300 cells/.mu.L (for
patients aged 12 to 17 years); 424-1,509 cells/.mu.L (for patients
aged 18 to 55 years); or 430-1,513 cells/.mu.L (for patients older
than 55 years of age).
[0352] In some embodiments, immune competence can be determined by
quantifying the absolute number of CD8.sup.+ T cells, by, e.g.,
flow cytometry, in a biological sample obtained from a patient. For
example, in some embodiments, immune competence is indicated by a
CD8.sup.+ T cell count, by flow cytometry, of: 560-1,700 (for
patients 0 to 2 months of age); 590-1,600 cells/.mu.L (for patients
aged 3 to 5 months); 500-1,700 cells/.mu.L (for patients aged 6 to
11 months); 620-2,000 cells/.mu.L (for patients aged 12 to 23
months); 490-1,300 cells/.mu.L (for patients aged 2 to 5 years);
370-1,100 cells/.mu.L (for patients aged 6 to 11 years); 330-920
cells/.mu.L (for patients aged 12 to 17 years); 169-955
cells/.mu.l. (for patients aged 18 to 55 years); or 101-839
cells/.mu.L (for patients older than 55 years of age).
[0353] It is understood that immune cell counts that fall below
these levels, as measured in a biological sample obtained from a
patient, may indicate that the patient is immunocompromised. Immune
cell counts that fall within one or more of the ranges set forth
above may indicate that the patient is immunocompetent and likely
to receive an enhanced benefit from an anti-CD200 antibody therapy
described herein. Any of the methods described herein can include
assaying a biological sample to determine: (a) the number per
microliter of one or more of the immune cell subsets described
herein and/or (b) whether the assayed numbers fall within a
pre-determined range such as the pre-determined ranges described
above.
[0354] In some embodiments, the methods described herein can
include identifying or selecting a subject that has an intact
immune system, e.g., one competent to mount an immune response
against the cancer present in or on the subject. Methods for
determining whether an immune system is competent to mount an
immune response against a cancer are well known in the art. For
example, a medical practioner may assay for antibody (e.g., IgG,
IgM, or IgA) responses specific for cancer by testing for the
presence of antibodies that bind to cancer tissue systemically
(e.g., in serum) or, for example, at various mucosal sites (e.g.,
in saliva or gastric and bronchoalveolar lavages) using in vitro
assays familiar to those in the art, e.g., an ELISA. Practitioners
may also assess the general immunocompetence of the patient by
evaluating one or more of: (a) the ability to mount a normal
proliferative response to mitogens (e.g., PHA or LPS) or anti-CD3
antibody stimulation; (b) CD4.sup.+ cell:CD8.sup.+ cell ratios in a
predetermined normal range (e.g., >1.0); and (c) tumor-specific
immune responses such as quantitating T cells specific for tumor
antigens using, e.g., ELISPOT or tetramer or cytokine analysis.
[0355] Alternatively, or in addition, since CD4' T cell responses
are generally required for antibody responses, in vitro CD4.sup.+ T
cell responses to the cancer can be measured using methods known in
the art. Such methods include CD4.sup.+ T cell proliferation or
lymphokine (e.g., interleukin-2, interleukin-4, or
interferon-.gamma.) production assays. Part of the determination
can include a quantitative or qualitative assessment/evaluation as
to whether the patient has previously been administered a
chemotherapeutic or immunosuppressive therapy as such therapies are
known to inhibit the immune system of the patient to which the
therapies are administered.
[0356] The following examples are intended to illustrate, not
limit, the invention.
EXAMPLES
Example 1
Preliminary Results of a Dose Escalation Trial Evaluating an
Anti-CD200 Antibody in Humans
[0357] Samalizumab (Alexion Pharmaceuticals, Inc.) is a
first-in-class recombinant, humanized monoclonal antibody that is
currently being evaluated clinically for the treatment of B-CLL.
The antibody inhibits the interaction between CD200 and CD200R and
thus, in patients with cancers expressing CD200, inhibits
CD200-dependent immune suppression. Accordingly, administration to
the patient of samalizumab enables the patient's immune system to
adequately identify and eradicate the cancer.
[0358] An ongoing dose escalation trial was performed to evaluate
the safety and maximum tolerated dose (MTD) of samalizumab in
patients with relapsing or refractory B-CLL or Multiple myeloma
(MM) using a modified Fibonacci design of three patients per
cohort. Cohorts were to be expanded to six patients if
dose-limiting toxicities (DLT) occurred. Study patients received a
single intravenous dose per 28 day cycle and optional additional
intravenous doses of samalizumab at 28 day intervals. A total of
seven cohorts have been evaluated, ranging from 50 mg/m.sup.2 to
600 mg/m.sup.2 per treatment cycle. The study has assessed in the
patients, among other things, complete blood counts, computed
tomography (CT) scans, standard safety evaluations, pharmacokinetic
(PK) and pharmacodynamic (PD) measurements of the antibody; and
whether an anti-samalizumab antibody response has been generated in
the patients.
[0359] The study enrolled 26 B-CLL patients, including three
multiple myeloma (MM) patients and one patient with small
lymphocytic lymphoma (SLL), in seven (7) dose cohorts with doses
ranging from 50 mg/m.sup.2 to 600 mg/m.sup.2. Two of the MM
patients were enrolled in the 500 mg/m.sup.2 cohort and one in the
600 mg/m.sup.2 cohort. The SLL patient was enrolled in the 500
mg/m.sup.2 cohort. Four patients had received no prior chemotherapy
for their cancer, whereas the other 22 patients had received a
median of two regimens (ranging between 1 to 9 cycles per patient)
of chemotherapy prior to administration of the first dose of
samalizumab. There were 18 male patients and 8 female patients, the
patients having an age range of 41-87 years (the median age being
67). Twenty patients received optional dosing; three of these (one
dosed at 50 mg/m.sup.2; two dosed at 200 mg/m.sup.2) developed a
human anti-human antibody response against samalizumab. Nine of 13
patients who completed four (4) dosing cycles exhibited stable
disease (SD) based on serial assessments of peripheral blood counts
and CT scans. The protocol was amended to allow for greater than
four (4) treatment cycles for patients exhibiting SD at four
cycles. No clinically adverse cytokine reactions were observed. One
non-drug, non-malignancy related death occurred. Adverse events
were mostly mild or moderate in severity, and no maximum tolerated
dose (MTD) has been observed as of the end of the cohort six
evaluation period. Samalizumab exibits a dose-dependent linear
increase in serum area under the curve (AUC). The mean AUC of serum
drug levels (100-400 mg/m.sup.2) for the first four (4) cycles of
treatment are consistent with a linear relationship between dose
and AUC (FIG. 1).
[0360] Initial results suggest that samalizumab is generally safe
and well tolerated and exhibits a desired immunomodulatory activity
in this patient population as elaborated on below.
Example 2
Observation of Biomarkers of the Occurrence of an Immunomodulatory
Effect in Humans Treated with Samalizumab
[0361] Among patients with evaluable cell populations, antibody
treatment resulted in observable immunomodulatory effects on both
immune cells and B-CLL cancer cells in the peripheral blood. A
summary of the effects is shown in Table 1. For example, a
reduction in CD200.sup.+ T cells was observed in 19 out of 20
patients (95%) following administration of samalizumab (Table
1).
TABLE-US-00001 TABLE 1 Dosing and Pharmacodynamic (PD) Parameters
by Cohort N/N evaluable Antibody N (patients) Antibody- CD200.sup.+
CD200.sup.+ CD200R.sup.+ Dose .gtoreq.4 Th1 bound loss on T cell T
cell (mg/m.sup.2) Total cycles HAHA cytokines.sup.a CLL CLL
reduction increase 50 4.sup.b .sup. 1.sup.c .sup. 1.sup.c 3/4 1/4
1/4 2/2 0/2 100 5.sup.b 5 0 5/5 2/5 5/5 5/5 2/5 200 3.sup. .sup.
1.sup.d 2 3/3 2/2 2/2 3/3 2/3 300 3.sup. 2 0 3/3 1/2 1/2 2/2 2/2
400 3.sup. 2 0 2/3 2/3 3/3 2/2 0/2 500 7.sup.e 2 0 5/6.sup.f 2/5
2/5 4/5 2/4 600 1.sup.g 0 0 1/1 NA NA 1/1 0/1 "Antibody" refers to
samalizumab. "N" is the number of patients. "HAHA" refers to the
occurrence of a human anti-human antibody response against
samalizumab in the patient. .sup.aTh1 cytokines detected at any
time point during the treatment of the patient. .sup.bGreater than
three patients enrolled to evaluate multiple-dose safety.
.sup.cRefers to the same patient. .sup.dNo HAHA response detected.
.sup.eTwo of seven patients were Multiple Myeloma (MM) patients
.sup.fTh1 cytokine information was not available for one patient in
this cohort. .sup.gPatient afflicted with multiple myeloma. "NA"
refers to Not Applicable
[0362] FIG. 2 provides a representative analysis of a treated
patient showing a reduction in CD200.sup.+ T cells. As shown in
FIG. 3, the reduction of CD200.sup.+ T cells in patients was
transient, with CD200.sup.+ T cells beginning to recover to
pre-treatment levels around day 14. However, administration of a
second dose of samalizumab to these patients again resulted in a
transient reduction in the concentration of CD200.sup.+ T cells
(FIG. 3). The transient nature of the effect was observed more
frequently at lower doses of samalizumab (e.g., 50 to 200
mg/m.sup.2), compared to a sustained effect at higher doses (300 to
500 mg/m.sup.2) of the antibody. This indicated that the
immunomodulatory effect of the anti-CD200 antibody in the patients
was dose-dependent and that modification of a dosing schedule to
maintain the immunomodulatory effect in the patients could be
achieved by one or both of an increase in the dose of samalizumab
and/or more frequent administration of samalizumab.
[0363] The reduction or recovery in CD200.sup.+ T cells was not
associated with an overall change in the total concentration of
CD3.sup.+ T cells in the patients suggesting that the CD200.sup.+ T
cells are either downregulating CD200 expression and/or are being
mobilized out of the periphery, rather than being deleted.
(Samalizumab does not crossblock the binding of the antibody used
to detect CD200 expression by the leukocytes in the patient blood
samples, and thus does not substantially affect the ability to
detect CD200 expression using these assays.)
[0364] In addition, an elevated level of CD200R expression on
leukocyte subsets (e.g., CD200R.sup.+/CD4.sup.+ leukocyte subsets)
by day seven (7) following the administration of samalizumab was
also observed in eight of 19 patients (see, e.g., FIG. 4).
Reductions in CD200.sup.+ cells and increases in CD200R expression
by leukocytes were predominately observed in the CD4.sup.+ T cell
populations (FIG. 4). As described above, increases in CD200R
expression by leukocyte subsets may be the result of compensation
by the cells to the reduction in CD200 expression.
[0365] Ten out of 25 patients (40%) exhibited modest first-dose Th1
cytokine responses, whereas twenty-two of twenty-five (88%)
patients had detectable Th1 cytokines at one or more time points
during the study. This is also consistent with immunomodulatory
activity of samalizumab in the patients.
[0366] A loss of regulatory T cells (Tregs) was also observed in
patients administered samalizumab. Particularly four out of nine
(44.4%) patients with clinically stable or improved disease
exhibited a reduction in Tregs, whereas only five out of sixteen
(31.2%) patients whose disease clinically progressed exhibited a
similar loss of Tregs (FIG. 5).
[0367] A reduction of CD200 protein expression by B-CLL tumor cells
in the peripheral blood was also observed in 14 of 21 patients
(67%) following administration of samalizumab. The reduction was
transient at lower doses of samalizumab (e.g., 50 to 200
mg/m.sup.2), with CD200 expression by B-CLL cells beginning to
recover to (or nearly to) pre-treatment levels around day 14.
However, administration of a second dose of samalizumab to these
patients again resulted in a transient reduction in the expression
of CD200 protein by the cells (FIG. 6). A sustained loss of CD200
on B-CLL tumor cells was observed at higher doses (300 to 500
mg/m.sup.2) of the antibody. As noted above, this result further
indicated that the immunomodulatory effect of the anti-CD200
antibody in the patients was dose-dependent and that modification
of a dosing strategy to maintain the immunomodulatory effect in the
patients could be achieved by one or both of an increase in the
dose of samalizumab and/or more frequent administration of
samalizumab. Such an anti-CD200 antibody dosing strategy will
likely provide improved clinical benefit to treated patients.
[0368] Changes in expression of CD200R and/or CD200 on other
leukocyte subsets, or a change in the concentration of other
CD200.sup.+ or CD200R.sup.+ leukocytes was not observed due to a
lack of sufficient quantity of cells to make such an
observation.
[0369] Of the nine patients exhibiting stable disease, one was from
cohort 1, three were from cohort 2, one in each of cohorts 3, 4 and
5, and two in the higher dose cohort (500 mg/m.sup.2). Exemplary
anti-CD200 antibody-associated immunomodulatory effects observed in
the patients are as follows: [0370] 1. CD4.sup.+/CD200.sup.+ T
cells: All patients with stable disease in cohorts 1, 2 and 3
showed a transient reduction in CD200.sup.+/CD4.sup.+ T cells after
the 1.sup.st and subsequent samalizumab doses. Patient in cohorts
4, 5 and 6 with stable disease exhibited a sustained reduction in
CD200.sup.+/CD4.sup.+ T cells. [0371] 2. CD200R.sup.+/CD4.sup.+ T
cell: One of three of the patients in cohort 2 with stable disease,
the patient in cohort 3, and the patient in cohort 4 (all with
stable disease) exhibited an increase in CD200R.sup.+/CD4.sup.+ T
cells after the first dose. These patients with stable disease had
varying numbers of T cells at baseline (2%, 2%, 14%, 23% and 39% of
the CD45.sup.+ leukocytes) and percent CD4.sup.+/CD200.sup.+ T
cells varied from 10-35% of total CD3.sup.+ T cells at baseline in
these patients. In addition, in all patients with stable disease,
the expression of CD200 on the B CLL cells was reduced.
[0372] These results indicate that the anti-CD200 antibody is
capable of producing an immunomodulatory effect in patients to whom
the antibody was administered. As nine patients treated with
samalizumab exhibited stable disease at four treatment cycles, the
biomarkers may also indicate that the dose of samalizumab, by
virtue of its observed immunomodulatory effect in the human, is
sufficient to achieve a clinically-meaningful effect on the
disease.
Example 3
Biomarkers, Immunomodulatory Effect, and Efficacy of Samalizumab
Treatment in Patients Who Had not Previously Received
Chemotherapy
[0373] As described above, four of the patients enrolled in the
study had not received chemotherapy prior to the samalizumab
therapy. All four of these patients received samalizumab therapy
and exhibited clinically stable or improved disease--four of the
nine responders. One of the four patients, patient 102-502, is a 66
year old male who presented with advanced CLL (RAI stage 4 at study
entry with no prior treatment), including a large abdominal mass
and fatigue at the point of enrollment. Prior to beginning the
anti-CD200 antibody treatment regimen, patient 102-502 had not
received any chemotherapeutic treatments or other immunosuppressive
therapies for CLL. Within weeks after receiving the first 400
mg/m.sup.2 dose of samalizumab, the patient's abdominal mass had
been reduced by 57.6% as determined by CT scan. The patient's
treatment with samalizumab continued for an additional four cycles
(4 doses) at 400 mg/m.sup.2. After the fourth cycle, the patient's
abdominal mass had been further reduced--a total of a 71% reduction
since the time of enrollment. The patient's fatigue had also been
eliminated. The patient has received 13 doses of samalizumab
administered once per month and has achieved a partial response
(PR).
[0374] Concomitantly with the reduction in tumor burden (FIG. 7A),
a change in a number of anti-CD200 antibody-associated
immunomodulatory biomarkers was observed in this patient. For
example, like other evaluated patients, the concentration of
CD200.sup.+ lymphocytes (e.g., CD200.sup.+CD4.sup.+ T cells) also
decreased in this patient over the course of treatment (FIG. 8A).
In addition, the concentration of B CLL cells and the expression
level of CD200 by the remaining B CLL cells were also dramatically
reduced in this patient (FIG. 8B).
[0375] Also observed in the patient was an increase in CD8.sup.+ T
cells as well as CD4.sup.+ T cells (FIG. 7B). In contrast, there
was a loss of Tregs in patient 102-502 over the course of the
treatment. The ratio of percent activated T cells to percent
regulatory T cells increased over the course of treatment from
approximately 2:1, to 3:1, to 4:1, to 5:1, and eventually to over
6:1. See FIG. 9.
[0376] These results further indicated that the anti-CD200 antibody
is even more capable of producing an immunomodulatory effect in
patients and that changes in the biomarkers described herein
correlate with a clinically-meaningful effect on the disease. That
four of the nine responders had not, prior to administration of the
anti-CD200 antibody, received any chemotherapeutic treatment for
CLL so as to immunosuppress the patients indicates that
administration of an anti-CD200 antibody to a patient with an
intact immune system (or one that has not been compromised by
immunosuppressive agents) may likely receive an even greater
therapeutic benefit from an anti-CD200 antibody therapy described
herein.
Example 4
Efficacy of an Anti-CD200 Antibody in a Mouse Model of Autoimmune
Hemolytic Disease
[0377] Study 0 (Prevention Model). Therapeutic anti-CD200
antibodies were tested for their ability to prevent, delay, or
lessen the severity of, the production of autoantibodies associated
with autoimmune hemolytic disease using a mouse model of the
disease. See, e.g., Playfair and Marshall-Clarke (1973) Nat New
Biol 243:213-214; Naysmith et al. (1981) Immunol Rev 55:55-87.
[0378] To elicit in mice the production of autoantibodies that bind
to mouse red blood cells (RBCs), 2.times.10.sup.8 rat RBCs were
administered intraperitoneally (i.p.) to female C57BL/6 mice once
on study day 0 and then once per week thereafter for the remainder
of the study. Production of anti-rat RBC alloantibodies by the
immunized mice was observed by the second week of the study and
production by the mice of anti-mouse RBC autoantibodies was
observed by week three.
[0379] The rat RBC-immunized mice were divided into six
experimental groups designated: Group 1 (six mice), Group 2 (6
mice), Group 3 (8 mice), Group 4 (7 mice), Group 5 (9 mice), and
Group 6 (9 mice). One additional group--Group 7 (6 mice)--was also
evaluated as a control. The Group 7 mice were neither immunized
with rat RBCs nor did they receive any of the additional treatments
described below.
[0380] Starting at day 0 (that is the day of the first
administration of the rat RBCs), the mice of each of Groups 2 to 6
were administered a therapeutic agent or vehicle under the
following schedule: for each week of the study, five doses of agent
or vehicle administered as one dose per day for five consecutive
days. Group 1 mice were treated with only
vehicle--phosphate-buffered saline (PBS). Group 2 mice were treated
under the above treatment schedule using 5 mg/kg of a Control
antibody that does not bind to CD200, but possesses effector
function (IgG2a). Group 3 mice were treated under the
aforementioned treatment schedule with Antibody 1--an anti-CD200
antibody (IgG2a) having effector function--each dose being 5 mg/kg.
Group 4 mice were treated with cyclosporine at a dose of 15 mg/kg.
Group 5 mice were treated with the Control Antibody at 5 mg/kg and
cyclosporine at 15 mg/kg. Group 6 mice were treated with Antibody 1
at a dose of 5 mg/kg and cyclosporine at a dose of 15 mg/kg. The
antibody treatments were administered i.p. Cyclosporine was
administered to the mice subcutaneously (s.c.). The Group design
and treatment schedules for each group are summarized in Table
2.
TABLE-US-00002 TABLE 2 Group Design and Treatment Schedule for
Study 0. Groups N Therapeutic Administered Dose Group 1 6 Vehicle
N/A Group 2 6 Control antibody (IgG2a) that does not bind 5 mg/kg
to CD200 but possesses effector function Group 3 8 Antibody 1
(anti-CD200 antibody IgG2a with 5 mg/kg effector function) Group 4
7 Cyclosporine 15 mg/kg Group 5 9 Control antibody (IgG2a) that
does not bind 5 mg/kg to CD200 but possesses effector function; 15
mg/kg and Cyclosporine Group 6 9 Antibody 1 (anti-CD200 antibody
IgG2a with 5 mg/kg effector function); and Cyclosporine 15 mg/kg
Group 7 6 Non-immunized, non-treated control group N/A N refers to
the number of mice in each group. N/A = not applicable.
[0381] On a weekly basis, blood was drawn from the mice of Groups 1
to 7 prior to, during, and after the above treatments to evaluate
by flow cytometry whether treatment affected the titer of
anti-mouse RBC autoantibodies and/or anti-rat RBC alloantibodies in
the mice. To determine the relative concentration of anti-mouse
autoantibodies produced in a subject mouse (e.g., a treated mouse
from Group 3), whole blood obtained from the mouse was incubated
with a preparation of fluorescently-labeled anti-mouse antibody to
thereby detect the presence of anti-mouse RBC antibodies present on
the surface of mouse RBC in the blood of the animals. The cells
were washed with PBS and then subjected to flow cytometry to
evaluate the relative amount of mouse anti-mouse RBCs bound to the
mouse RBCs as a function of the mean fluorescence intensity.
Between day 13 and 27, the concentration of anti-mouse RBC
autoantibodies in the mice of Groups 1, 2, 4, 5, and 6 increased.
In contrast, the concentration of anti-mouse RBC autoantibodies in
the mice of Group 3 was markedly reduced as compared to the
concentration of autoantibody in the other groups. In addition, the
production of autoantibody by the mice in Group 3 was markedly
delayed as compared to the mice in the other groups (FIG. 10). For
example, 50% of mice in Groups 1, 2, 4, 5, and 6 developed
autoantibodies between day 20 and 27 of the study. In contrast,
autoantibody production in at least 50% of mice in Group 3 did not
occur until between day 27 and day 34. These results indicate that
Antibody 1, an anti-CD200 antibody, at 5 mg/kg was capable of not
only reducing the concentration of anti-mouse RBC autoantibodies in
a mice model of autoimmune hemolytic disease, but was also capable
of delaying significantly the production of the autoantibodies in
the mice.
[0382] To determine the relative concentration of alloantibodies
produced in a subject mouse (e.g., a treated mouse from Group 3),
serum obtained from the mouse was incubated with a sample of
isolated rat RBCs for a time and under conditions sufficient for
any rat RBC-specific alloantibodies present in the serum to bind to
the rat RBCs. The cells were washed with PBS and then incubated
with a fluorescently-labeled antibody that binds to mouse
antibodies. Following an additional washing step, the cells were
subjected to flow cytometry to evaluate the relative amount of
mouse anti-rat RBCs bound to the rat RBCs as the mean fluorescence
intensity. Sera obtained from mice of Groups 1, 2, 4, 5, and 6
contained an increasing concentration of anti-rat RBC
alloantibodies over the course of the experiment. In contrast, sera
obtained from the mice of Group 3 contained much less detectable
anti-rat RBC autoantibodies as compared to the other Groups. These
results further indicated that Antibody 1, an anti-CD200 antibody,
at 5 mg/kg was capable of reducing the titer of RBC-specific
alloantibodies, as well as anti-RBC autoantibodies, produced in a
mouse model of autoimmune hemolytic disease.
[0383] Study 1 (Treatment Model). Therapeutic anti-CD200 antibodies
were tested for their ability to reduce the production of
autoantibodies associated with autoimmune hemolytic disease using a
mouse model of the disease. To elicit in mice the production of
autoantibodies that bind to mouse red blood cells (RBCs),
2.times.10.sup.8 rat RBCs were administered intraperitoneally
(i.p.) to female C57BL/6 mice once on study day 0 and then once per
week thereafter for the remainder of the study. Production of
anti-rat RBC alloantibodies by the immunized mice was observed by
the second week of the study and production by the mice of
anti-mouse RBC autoantibodies was observed by week three.
[0384] The rat RBC-immunized mice were divided into five groups
designated Group 1 (8 mice), Group 2 (8 mice), Group 3 (8 mice),
Group 4 (7 mice), and Group 5 (8 mice). A sixth group of mice
(designated Group 6; 6 mice) was also evaluated as a control. The
Group 6 mice were neither immunized with rat RBCs nor did they
receive any of the additional treatments described below.
[0385] Starting on day 112, the mice of each of Groups 1 to 5
received an additional treatment of 14 doses of a therapeutic agent
or vehicle control administered under the following schedule: (i)
five doses of agent or vehicle administered as one dose per day for
five consecutive days; (ii) a two day break in treatment; (iii) an
additional five doses of the agent or vehicle administered one dose
per day for five consecutive days; another two day break in
treatment; and (iv) four more doses of agent or vehicle
administered one dose per day for four consecutive days. Group 1
mice were treated with only vehicle--phosphate-buffered saline
(PBS). Group 2 mice were treated under the aforementioned treatment
schedule with Antibody 1--an anti-CD200 antibody (IgG2a) having
effector function--each dose being 5 mg/kg. Group 3 mice were
treated with Antibody 1 at a dose of 1 mg/kg. Group 4 mice were
treated under the above treatment schedule with Antibody 2--an
anti-CD200 antibody that lacked effector function--each dose at 5
mg/kg. Group 5 mice were treated under the above treatment schedule
using a dose of 5 mg/kg of a Control antibody that does not bind to
CD200, but possesses effector function (IgG2a). The Group design
and treatment schedules for each group are summarized in Table
3.
TABLE-US-00003 TABLE 3 Group Design and Treatment Schedule for
Study 1. Groups N Therapeutic Administered Dose Group 1 8 Vehicle
N/A Group 2 8 Antibody 1 (anti-CD200 antibody IgG2a with 5 mg/kg
effector function) Group 3 8 Antibody 1 (anti-CD200 antibody IgG2a
with 1 mg/kg effector function) Group 4 7 Antibody 2 (anti-CD200
antibody that does not 5 mg/kg possess effector function) Group 5 8
Control antibody (IgG2a) that does not bind to 5 mg/kg CD200 but
possesses effector function Group 6 6 Non-immunized, non-treated
control group N/A N refers to the number of mice in each group. N/A
= not applicable.
[0386] On a weekly basis, blood was drawn from the mice of Groups 1
to 6 prior to, during, and after the above treatments to evaluate
by flow cytometry whether treatment affected the titer of
anti-mouse RBC autoantibodies and/or anti-rat RBC alloantibodies in
the mice. Between day 133 and 137 of the study, the mice were
sacrificed and their spleens harvested. To determine the relative
concentration of alloantibodies produced in a subject mouse (e.g.,
a treated mouse from Group 2), serum obtained from the mouse (e.g.,
at day 133) was contacted to a sample of isolated rat RBCs for a
time and under conditions sufficient for any rat RBC-specific
alloantibodies present in the serum to bind to the rat RBCs. The
cells were washed with PBS and then incubated with a
fluorescently-labeled antibody that binds to mouse antibodies.
Following an additional washing step, the cells were subjected to
flow cytometry to evaluate the relative amount of mouse anti-rat
RBCs bound to the rat RBCs as the mean fluorescence intensity. The
inventors observed that the post-treatment sera obtained from mice
of Groups 1, 3, 4, and 5 contained an increased concentration of
anti-rat RBC alloantibodies as compared to the corresponding sera
obtained from the mice prior to treatment. In contrast, sera
obtained from the mice of Group 2 post-treatment contained less
detectable anti-rat RBC alloantibodies as compared to the
corresponding sera obtained from the mice prior to treatment. These
results indicated that Antibody 1, an anti-CD200 antibody, at 5
mg/kg was capable of reducing the production of RBC-specific
antibodies in a mouse model of autoimmune hemolytic disease.
[0387] The inventors subsequently observed that Antibody 2 had a
significantly shorter half-life in the treated mice as compared to
the half-life of Antibody 1. Thus the results observed with
Antibody 2 in Study 1 and in other studies described herein may not
fully reflect the true efficacy of the Antibody 2 in the autoimmune
hemolytic disease model nor the immunodulatory effect of the
antibody in animals.
[0388] Study 2 (Prevention model). Therapeutic anti-CD200
antibodies were tested for their ability to prevent, delay, or
lessen the severity of, the production of autoantibodies associated
with autoimmune hemolytic disease using the above-described mouse
model of the disease.
[0389] To elicit in mice the production of autoantibodies that bind
to mouse red blood cells (RBCs), rat RBCs were administered
intraperitoneally (i.p.) to female BALB/c mice once on study day 0
and then once per week thereafter for the remainder of the study.
As described above, production of anti-rat RBC alloantibodies by
the immunized mice was observed by the second week of the study and
production by the mice of anti-mouse RBC autoantibodies was
observed by week three.
[0390] The rat RBC-immunized mice were divided into five groups
designated Group 1 (8 mice), Group 2 (8 mice), Group 3 (8 mice),
Group 4 (8 mice), and Group 5 (8 mice). A sixth group of mice
(designated Group 6; 6 mice) was also evaluated as a control. The
Group 6 mice were neither immunized with rat RBCs nor did they
receive any of the additional treatments described below.
[0391] Starting at day 0 (that is the day of the first
administration of the rat RBCs), the mice of each of Groups 1 to 5
were administered a therapeutic agent or vehicle under the
following schedule: for each week of the study, five doses of agent
or vehicle administered as one dose per day for five consecutive
days. Group 1 mice were treated with only
vehicle--phosphate-buffered saline (PBS). Group 2 mice were treated
under the aforementioned treatment schedule with Antibody 1--an
anti-CD200 antibody (IgG2a) having effector function--each dose
being 5 mg/kg. Group 3 mice were treated with Antibody 1 at a dose
of 1 mg/kg. Group 4 mice were treated under the above treatment
schedule with Antibody 2--an anti-CD200 antibody that lacked
effector function--each dose at 5 mg/kg. Group 5 mice were treated
under the above treatment schedule using 5 mg/kg of a Control
antibody that does not bind to CD200, but possesses effector
function (IgG2a). The Group design and treatment schedules for each
group are summarized in Table 4.
TABLE-US-00004 TABLE 4 Group Design and Treatment Schedule for
Study 2. Groups N Therapeutic Administered Dose Group 1 8 Vehicle
N/A Group 2 8 Antibody 1 (anti-CD200 antibody IgG2a with 5 mg/kg
effector function) Group 3 8 Antibody 1 (anti-CD200 antibody IgG2a
with 1 mg/kg effector function) Group 4 8 Antibody 2 (anti-CD200
antibody that does not 5 mg/kg possess effector function) Group 5 8
Control antibody (IgG2a) that does not bind to 5 mg/kg CD200 but
possesses effector function Group 6 6 Non-immunized, non-treated
control group N/A N refers to the number of mice in each group. N/A
= not applicable.
[0392] On a weekly basis, blood was drawn from the mice of Groups 1
to 6 prior to, during, and after the above treatments to evaluate
by flow cytometry whether treatment affected the titer of
anti-mouse RBC autoantibodies and/or anti-rat RBC alloantibodies in
the mice. On day 64 or 65 of the study, the mice were sacrificed
and their spleens harvested. (Four mice in each group were
sacrificed on day 64 and the other four mice in each group were
sacrificed on day 65.) To determine the relative concentration of
alloantibodies produced in a subject mouse (e.g., a treated mouse
from Group 3), serum obtained from the mouse was contacted to a
sample of isolated rat RBCs for a time and under conditions
sufficient for any rat RBC-specific alloantibodies present in the
serum to bind to the rat RBCs. The cells were washed with PBS and
then incubated with a fluorescently-labeled antibody that binds to
mouse antibodies. Following an additional washing step, the cells
were subjected to flow cytometry to evaluate the relative amount of
mouse anti-rat RBCs bound to the rat RBCs as the mean fluorescence
intensity. As shown in FIG. 11, sera obtained from mice of Groups
1, 3, 4, and 5 contained an increasing concentration of anti-rat
RBC alloantibodies over the course of the experiment. In contrast,
sera obtained from the mice of Group 2 post-treatment contained
much less detectable anti-rat RBC alloantibodies as compared to the
other Groups. These results further indicated that Antibody 1, an
anti-CD200 antibody, at 5 mg/kg was capable of reducing the titer
of RBC-specific alloantibodies produced in a mouse model of
autoimmune hemolytic disease.
[0393] Study 3 (Treatment Model). Therapeutic anti-CD200 antibodies
were tested for their ability to treat autoimmune hemolytic disease
using a mouse model of the disease. To elicit in mice the
production of autoantibodies that bind to mouse red blood cells
(RBCs), rat RBCs were administered intraperitoneally (i.p.) to
female C57BL/6 mice once on study day 0 and then once per week
thereafter for the remainder of the study. As described above,
production of anti-rat RBC alloantibodies by the immunized mice was
observed by the second week of the study and production by the mice
of anti-mouse RBC autoantibodies was observed by week three. The
rat RBC-immunized mice were divided into three groups designated
Group 1 (6 mice), Group 2 (3 mice), and Group 3 (5 mice).
[0394] Starting on day 86, the mice of each of Groups 1 to 3
received an additional treatment of 10 doses of a therapeutic agent
or vehicle control administered under the following schedule: (i)
five doses of agent or vehicle administered as one dose per day for
five consecutive days; (ii) a two day break in treatment; and (iii)
an additional five doses of the agent or vehicle administered one
dose per day for five consecutive days. Group 1 mice were treated
under the aforementioned treatment schedule with Antibody 1--an
anti-CD200 antibody (IgG2a) having effector function--each dose
being 5 mg/kg. Group 2 mice were treated with Antibody 1 at a dose
of 1 mg/kg. Group 3 mice were treated under the above treatment
schedule with Antibody 2--an anti-CD200 antibody that lacked
effector function--each dose at 5 mg/kg. The Group design and
treatment schedules for each group are summarized in Table 5.
TABLE-US-00005 TABLE 5 Group Design and Treatment Schedule for
Study 3. Groups N Therapeutic Administered Dose Group 1 6 Antibody
1 (anti-CD200 antibody IgG2a with 5 mg/kg effector function) Group
2 3 Antibody 1 (anti-CD200 antibody IgG2a with 1 mg/kg effector
function) Group 3 5 Antibody 2 (anti-CD200 antibody that does not 5
mg/kg possess effector function) Group 4 3 Non-immunized,
non-treated control group N/A N refers to the number of mice in
each group. N/A = not applicable.
[0395] At the conclusion of the study, the mice were sacrificed and
their spleens harvested. To determine whether administration of
Antibody 1 to the mice affected activation of splenocytes by RBC,
in addition to affecting the production of anti-RBC antibodies in
the mice, splenocyte activation in the presence of RBCs was
evaluated using an in vitro proliferation assay. Briefly, isolated
splenocytes were cultured with one of three different
antigens--mouse RBCs, rat RBCs, or bovine serum albumin
(control)--or with media alone. Following contact of the
splenocytes with the antigens, .sup.3H-thymidine was added to the
splenocyte culture medium for approximately 16 hours. The medium
was removed and the cells harvested. The relative activation of the
splenocytes by the antigens was then measured as a function of the
amount of .sup.3H-thymidine incorporated into the DNA of the
splenocytes.
[0396] As shown in FIG. 12, splenocytes from Group 2 and 3 mice
exhibited a robust proliferative response following contact with
rat RBCs. In contrast, splenocytes from Group 1 mice proliferated
very little in the presence of rat RBCs indicating that
administration of an anti-CD200 antibody at 5 mg/kg was capable of
inhibiting the activation of splenocytes by rat RBCs in a mouse
model of autoimmune hemolytic disease.
[0397] Study 4 (Treatment Model). As described above, to elicit in
mice the production of autoantibodies that bind to mouse red blood
cells (RBCs), rat RBCs were administered intraperitoneally (i.p.)
to female C57BL/6 mice once on study day 0 and then once per week
thereafter for the remainder of the study.
[0398] The rat RBC-immunized mice were divided into seven (7)
groups designated Group 1, Group 2, Group 3, Group 4, Group 5,
Group 6, and Group 7. An eighth group of mice (designated Group 8)
was also evaluated as a control. The Group 8 mice were neither
immunized with rat RBCs nor did they receive any of the additional
treatments described below. Ten mice were in each group.
[0399] Starting on day 21, the mice of each of Groups 1 to 7
received an additional treatment of one or more therapeutic agents
or vehicle control administered under the following schedule: for
each week of the study, five doses of one or more agents or vehicle
administered as one dose per day for five consecutive days. Group 1
mice were treated with only vehicle--phosphate-buffered saline
(PBS). Group 2 mice were treated under the above treatment schedule
using a dose of 5 mg/kg of a Control antibody that does not bind to
CD200, but possesses effector function (IgG2a). Group 3 mice were
treated under the aforementioned treatment schedule with Antibody
1--an anti-CD200 antibody (IgG2a) having effector function--each
dose being 5 mg/kg. Group 4 mice were treated under the above
schedule with 15 mg/kg cyclosporine. Group 5 mice were treated
under the above dosing schedule with both the Control antibody (at
5 mg/kg) and cyclosporine (at 15 mg/kg). Group 6 mice were treated
under the above dosing schedule with both Antibody 1 (at 5 mg/kg)
and cyclosporine (at 15 mg/kg). Group 7 mice were treated under the
above dosing schedule with both Antibody 1 (at 1 mg/kg) and
cyclosporine (at 15 mg/kg). The Group design and treatment
schedules for each group are summarized in Table 6.
TABLE-US-00006 TABLE 6 Group Design and Treatment Schedule for
Study 4. Groups N Therapeutic Administered Dose Group 1 10 Vehicle
N/A Group 2 10 Control antibody (IgG2a) that does not bind 5 mg/kg
to CD200 but possesses effector function Group 3 10 Antibody 1
(anti-CD200 antibody IgG2a 5 mg/kg with effector function) Group 4
10 Cyclosporine 15 mg/kg Group 5 10 Control antibody; and
cyclosporine 5 mg/kg 15 mg/kg Group 6 10 Antibody 1; and
cyclosporine 5 mg/kg 15 mg/kg Group 7 10 Antibody 1; and
cyclosporine 1 mg/kg 15 mg/kg Group 8 10 Non-immunized, non-treated
control group N/A N refers to the number of mice in each group. N/A
= not applicable.
[0400] On a weekly basis, blood was drawn from the mice of Groups 1
to 8 prior to, during, and after the above treatments to evaluate
by flow cytometry whether treatment affected the titer of
anti-mouse RBC autoantibodies and/or anti-rat RBC alloantibodies in
the mice. On day 37 of the study, the mice were sacrificed and
their spleens harvested. Bone marrow cells were also obtained from
the two mouse femur and tibia bones. The spleen and bone marrow
cells were subjected to flow cytometry as described below (Example
5).
[0401] A reduced concentration of anti-rat RBC alloantibodies was
present in post-treatment sera obtained from mice of Groups 3 and 4
as compared to the corresponding pre-treatment sera. The
post-treatment reduction in anti-rat RBC alloantibodies was even
greater in the mice of Groups 6 and 7, indicating that cyclosporine
and Antibody 1 have a synergistic effect on reducing alloantibody
production in the mice. These results even further indicated that
an anti-CD200 antibody was capable of reducing the titer of
RBC-specific antibodies produced in a mouse model of autoimmune
hemolytic disease and that an anti-CD200 antibody is useful for
treating the disease.
Example 5
Administration of an Anti-CD200 Antibody to Mice Affects the
Concentration of Splenocyte and Bone Marrow Cell Populations in the
Mice
[0402] Splenocytes obtained from the mice of Study 1 were evaluated
to determine the percentage of cells that express CD200. Cells were
harvested from the spleens of the mice and incubated with a
composition of biotin-labeled anti-CD200 antibodies (polyclonal)
for an amount of time and under conditions sufficient to allow for
binding of the antibodies to CD200, if present on the cells. The
polyclonal antibody preparation was used to prevent or lessen any
masking effect due to the presence of residual therapeutic
anti-CD200 antibody (e.g., Antibody 1 or Antibody 2) on the cells.
The cells were washed and incubated with a fluorescently-labeled
streptavidin moiety. Following an additional washing step, the
cells were then subjected to flow cytometry. As shown in FIG. 13,
there was a marked reduction in the concentration of CD200.sup.+
splenocytes in mice treated with fourteen 5 mg/kg doses of Antibody
1 as compared to the concentration of CD200' splenocytes in mice
treated with vehicle, the Control antibody, or Antibody 2.
[0403] Splenocytes harvested from the spleens of the mice of Study
2 were also subjected to staining and flow cytometry analysis as
described above. There was a marked reduction in the concentration
of CD200.sup.+ splenocytes in mice chronically treated with 5 mg/kg
of Antibody 1, as compared to the concentration of CD200.sup.+
splenocytes in mice treated with vehicle or the Control antibody.
There was also no change in the concentration of CD200.sup.+
splenocytes in the Group 3 mice treated with 1 mg/kg dose of
Antibody 1 and Group 4 mice trated with 5 mg/kg Antibody 2.
[0404] Splenocytes harvested from the spleens of the mice of Study
4 were also subjected to staining and flow cytometry analysis as
described above. There was a marked reduction in the concentration
of CD200.sup.+ splenocytes in mice treated with 5 mg/kg of Antibody
1 with or without cyclosporine, as compared to the concentration of
CD200.sup.+ splenocytes in mice treated with vehicle, the Control
antibody, cyclosporine alone, or a combination of the Control
antibody and cyclosporine. There was also no change in the
concentration of CD200.sup.+ splenocytes in the Group 7 mice
treated with a combination schedule of cyclosporine and a 1 mg/kg
dose of Antibody 1. An analysis of the mean fluorescence intensity
(MFI) of the CD200.sup.+ splenocytes from each mouse (which is a
measure of the relative expression level of CD200 by each
CD200.sup.+ splenocyte) was also performed. The MFI of CD200.sup.+
splenocytes from Groups 3 and 6 was markedly reduced as compared to
the MFI of CD200.sup.+ splenocytes in the remaining Groups (save
Group 8). This indicated that not only does administration of
Antibody 1 to the mice reduce the total number of CD200.sup.+
splenocytes, but the remaining cells that do express CD200.sup.+ in
Antibody 1-treated mice express CD200 at much lower levels.
[0405] Taken together, these results confirm that administration of
an anti-CD200 antibody to an animal reduces the concentration of
CD200.sup.+ splenocytes in the animal. The results also indicate
that the anti-CD200 antibody-mediated reduction in CD200.sup.+
splenocytes is neither positively nor negatively affected by
cyclosporine.
[0406] The inventors also further examined the effect of anti-CD200
antibodies on: (i) the concentration of particular CD200.sup.+
lymphocyte subsets of splenocytes from the mice of Study 4 and (ii)
the concentration of particular CD200.sup.+ subsets of bone
marrow-derived cells from the mice of Study 4.
[0407] Concentration of Splenic Lymphocyte Subsets in the Mice of
Study 4
[0408] CD3.sup.+/CD200.sup.+ Lymphocyte Subset. A sample of
splenocytes from each of the mice of Study 4 was incubated with the
polyclonal anti-CD200 antibody preparation and a detectably-labeled
antibody that binds to CD3 to thereby identify the proportion of
CD3.sup.+/CD200.sup.+ cells in the spleens of mice from Groups 1 to
8. The CD3.sup.+ population of cells includes T cells such as
CD4.sup.+ and CD8.sup.+ T cells. The labeled cells were subjected
to flow cytometry. There was a marked reduction in the
concentration of CD3.sup.+/CD200.sup.+ splenocytes in mice
chronically treated with 5 mg/kg of Antibody 1 with or without
cyclosporine, as compared to the concentration of
CD3.sup.+/CD200.sup.+ splenocytes in mice treated with vehicle, the
Control antibody, cyclosporine alone, or a combination of the
Control antibody and cyclosporine. There was also no significant
change in the concentration of CD3.sup.+/CD200.sup.+ splenocytes in
the Group 7 mice treated with a combination schedule of
cyclosporine and a 1 mg/kg dose of Antibody 1.
[0409] CD5.sup.+/CD200.sup.+ Lymphocyte Subset. A sample of
splenocytes from each of the mice of Study 4 was incubated with the
polyclonal anti-CD200 antibody preparation and a detectably-labeled
antibody that binds to CD5 to thereby identify the proportion of
CD5.sup.+/CD200.sup.+ cells in the spleens of mice from Groups 1 to
8. The CD5.sup.+ population of cells includes T cells as well as B
cells (the B1 cell population). The labeled cells were subjected to
flow cytometry. There was a marked reduction in the concentration
of CD5.sup.+/CD200.sup.+ splenocytes in mice chronically treated
with 5 mg/kg of Antibody 1 with or without cyclosporine, as
compared to the concentration of CD5.sup.+/CD200.sup.+ splenocytes
in mice treated with vehicle, the Control antibody, cyclosporine
alone, or a combination of the Control antibody and cyclosporine.
There was also no significant change in the concentration of
CD5.sup.+/CD200.sup.+ splenocytes in the Group 7 mice treated with
a combination schedule of cyclosporine and a 1 mg/kg dose of
Antibody 1.
[0410] CD19.sup.+/CD200.sup.+ Lymphocyte Subset. A sample of
splenocytes from each of the mice of Study 4 was incubated with the
polyclonal anti-CD200 antibody preparation and a detectably-labeled
antibody that binds to CD19 to thereby identify the proportion of
CD19.sup.+/CD200.sup.+ cells in the spleens of mice from Groups 1
to 8. The CD19.sup.+ population of cells includes B cells. The
labeled cells were subjected to flow cytometry. Like
CD5.sup.+/CD200.sup.+ cells and CD3.sup.+/CD200.sup.+ cells, there
was also a marked reduction in the concentration of
CD19.sup.+/CD200.sup.+ splenocytes in mice chronically treated with
5 mg/kg of Antibody 1 with or without cyclosporine, as compared to
the concentration of CD19.sup.+/CD200.sup.+ splenocytes in mice
treated with vehicle, the Control antibody, cyclosporine alone, or
a combination of the Control antibody and cyclosporine. There was
also no significant change in the concentration of
CD19.sup.+/CD200.sup.+ splenocytes in the Group 7 mice treated with
a combination schedule of cyclosporine and a 1 mg/kg dose of
Antibody 1.
[0411] CD138.sup.+/CD200.sup.+ Lymphocyte Subset. A sample of
splenocytes from each of the mice of Study 4 was incubated with the
polyclonal anti-CD200 antibody preparation and a detectably-labeled
antibody that binds to CD138 to thereby identify the proportion of
CD138.sup.+/CD200.sup.+ cells in the spleens of mice from Groups 1
to 8. The CD138.sup.+ population of cells includes plasma cells.
The labeled cells were subjected to flow cytometry. There was a
marked reduction in the concentration of CD138.sup.+/CD200.sup.+
splenocytes in mice chronically treated with 5 mg/kg of Antibody 1
with or without cyclosporine, as compared to the concentration of
CD138.sup.+/CD200.sup.+ splenocytes in mice treated with vehicle,
the Control antibody, cyclosporine alone, or a combination of the
Control antibody and cyclosporine. There was also no significant
change in the concentration of CD138.sup.+/CD200.sup.+ splenocytes
in the Group 7 mice treated with a combination schedule of
cyclosporine and a 1 mg/kg dose of Antibody 1.
[0412] F4/80.sup.+ Lymphocyte Subset. F4/80 is 125 kDa
transmembrane protein present on the cell-surface of mature mouse
macrophages. To determine whether administration of an anti-CD200
antibody affects the concentration of resident macrophages in
spleen, a sample of splenocytes from each mouse of Study 4 was
incubated with a detectably-labeled antibody that binds to F4/80.
The labeled cells were subjected to flow cytometry to thereby
identify the proportion of F4/80.sup.+ cells in the spleens of mice
from Groups 1 to 8. The concentration of F4/80.sup.+ splenocytes
increased in mice treated with 5 mg/kg of Antibody 1 (10 doses)
with or without cyclosporine, as compared to the concentration of
F4/80.sup.+ splenocytes in mice treated with vehicle, the Control
antibody, cyclosporine alone, or a combination of the Control
antibody and cyclosporine. There was also no significant change in
the concentration of F4/80.sup.+ splenocytes in the Group 7 mice
treated with a combination schedule of cyclosporine and a 1 mg/kg
dose of Antibody 1.
[0413] Taken together, these results indicate that administration
of an anti-CD200 antibody reduces a variety of CD200.sup.+
splenocyte subsets, but increases certain macrophage subsets, in
the treated animals.
[0414] Concentration of Bone Marrow Cell Subsets in the Mice of
Study 4
[0415] CD34.sup.+/CD200.sup.+ Bone Marrow Cell Subset. A sample of
bone marrow cells from each of the mice was incubated with the
polyclonal anti-CD200 antibody preparation and a detectably-labeled
antibody that binds to CD34 to thereby identify the proportion of
CD34.sup.+/CD200.sup.+ cells in the bone marrow of mice from Groups
1 to 8. The CD34.sup.+ cells include a population of hematopoietic
stem cells (HSCs). The labeled cells were subjected to flow
cytometry also selecting for those cells that are lineage low
(Lin.sup.-/Low). There was a marked reduction in the concentration
of CD34.sup.+/CD200.sup.+ bone marrow cells in mice treated with 5
mg/kg of Antibody 1 (10 doses) with or without cyclosporine, as
compared to the concentration of CD34.sup.+/CD200.sup.+ bone marrow
cells in mice treated with vehicle, the Control antibody,
cyclosporine alone, or a combination of the Control antibody and
cyclosporine. There was also no significant change in the
concentration of CD34.sup.+/CD200.sup.+ bone marrow cells in the
Group 7 mice treated with a combination schedule of cyclosporine
and a 1 mg/kg dose of Antibody 1.
[0416] Sca-1.sup.+/CD200.sup.+ Bone Marrow Cell Subsets. A sample
of bone marrow cells from each of the mice was incubated with the
polyclonal anti-CD200 antibody preparation and a detectably-labeled
antibody that binds to Sca-1 to thereby identify the proportion of
Sca-1.sup.+/CD200.sup.+ cells in the bone marrow of mice from
Groups 1 to 8. The Sca-1.sup.+ cells include a population of HSCs
and mesenchymal stem cells (MSCs). The labeled cells were subjected
to flow cytometry also selecting for those cells that are lineage
low (Lin.sup.-/Low). There was a marked reduction in the
concentration of Sca-1.sup.+/CD200.sup.+ bone marrow cells in mice
treated with 5 mg/kg of Antibody 1 (10 doses) with or without
cyclosporine, as compared to the concentration of
Sca-1.sup.+/CD200.sup.+ bone marrow cells in mice treated with
vehicle, the Control antibody, cyclosporine alone, or a combination
of the Control antibody and cyclosporine. There was also no
significant change in the concentration of Sca-1.sup.+/CD200.sup.+
bone marrow cells in the Group 7 mice treated with a combination
schedule of cyclosporine and a 1 mg/kg dose of Antibody 1.
[0417] Sca-1.sup.+/CD34.sup.+ Bone Marrow Cell Subsets. A sample of
bone marrow cells from each of the mice was incubated with a first
detectably-labeled antibody that binds to CD34 and a second
detectably-labeled antibody that binds to Sca-1 to thereby identify
the proportion of Sca-1.sup.+/CD34.sup.+ cells in the bone marrow
of mice from Groups 1 to 8. The labeled cells were subjected to
flow cytometry also selecting for those cells that are lineage low
(Lin.sup.-/Low). The Sca-1.sup.+/CD34.sup.+/Lin.sup.- cells include
a population of MSCs. There was a marked reduction in the
concentration of Sca-1.sup.+/CD34.sup.+ bone marrow cells in mice
treated with 5 mg/kg of Antibody 1 (10 doses) with or without
cyclosporine, as compared to the concentration of
Sca-1.sup.+/CD34.sup.+ bone marrow cells in mice treated with
vehicle, the Control antibody, cyclosporine alone, or a combination
of the Control antibody and cyclosporine. There was also no
significant change in the concentration of Sca-1.sup.+/CD34.sup.+
bone marrow cells in the Group 7 mice treated with a combination
schedule of cyclosporine and a 1 mg/kg dose of Antibody 1.
[0418] c-kit.sup.+/CD200.sup.+ Bone Marrow Cell Subsets. A sample
of bone marrow cells from each of the mice was incubated with the
polyclonal anti-CD200 antibody preparation and a detectably-labeled
antibody that binds to c-kit to thereby identify the proportion of
c-kit.sup.+/CD200.sup.+ cells in the bone marrow of mice from
Groups 1 to 8. The c-kit.sup.+ cells include a population of HSCs
and MSCs. The labeled cells were subjected to flow cytometry also
selecting for those cells that are lineage low (Lin.sup.-/Low).
There was a marked reduction in the concentration of
c-kit.sup.+/CD200.sup.+ bone marrow cells in mice chronically
treated with 5 mg/kg of Antibody 1 with or without cyclosporine, as
compared to the concentration of c-kit.sup.+/CD200.sup.+ bone
marrow cells in mice treated with vehicle, the Control antibody,
cyclosporine alone, or a combination of the Control antibody and
cyclosporine. There was also no significant change in the
concentration of c-kit.sup.+/CD200.sup.+ bone marrow cells in the
Group 7 mice treated with a combination schedule of cyclosporine
and a 1 mg/kg dose of Antibody 1.
[0419] CD200.sup.+/CD200R.sup.+ Bone Marrow Cell Subset. A sample
of bone marrow cells from each of the mice was incubated with the
polyclonal anti-CD200 antibody preparation and a detectably-labeled
antibody that binds to CD200R to thereby identify the proportion of
CD200.sup.+/CD200R.sup.+ cells in the bone marrow of mice from
Groups 1 to 8. The labeled cells were subjected to flow cytometry.
There was a marked reduction in the concentration of
CD200.sup.+/CD200R.sup.+ bone marrow cells in mice chronically
treated with 5 mg/kg of Antibody 1 with or without cyclosporine, as
compared to the concentration of CD200.sup.+/CD200R.sup.+ bone
marrow cells in mice treated with vehicle, the Control antibody,
cyclosporine alone, or a combination of the Control antibody and
cyclosporine. There was also no significant change in the
concentration of CD200.sup.+/CD200R.sup.+ bone marrow cells in the
Group 7 mice treated with a combination schedule of cyclosporine
and a 1 mg/kg dose of Antibody 1.
Example 6
Recovery of Bone Marrow Cell and CD200.sup.+ Splenocyte Subsets
after Withdrawal of Anti-CD200 Therapy
[0420] Study 5 (Treatment Model). The therapeutic anti-CD200
antibodies were again tested for their ability to modulate the
concentration of specific subset populations of splenocytes and
bone marrow cells. The antibodies were administered to the mice in
the context of a mouse model of autoimmune hemolytic disease. As
described above, to elicit in mice the production of autoantibodies
that bind to mouse red blood cells (RBCs), 2.times.10.sup.8 rat
RBCs were administered intraperitoneally (i.p.) to female BALB/c
mice once on study day 0 and then once per week thereafter for the
remainder of the study. Production of anti-rat RBC alloantibodies
by the immunized mice was observed by the second week of the study
and production by the mice of anti-mouse RBC autoantibodies was
observed by week three.
[0421] The rat RBC-immunized mice were divided into five groups
designated Group 2 (20 mice), Group 3 (20 mice), Group 4 (20 mice),
Group 5 (15 mice), and Group 6 (15 mice). A sixth group of mice
(designated Group 1; 20 mice) was also evaluated as a control. The
Group 1 mice were neither immunized with rat RBCs nor did they
receive any of the additional treatments described below.
[0422] Starting on day 21, the mice of each of Groups 2 to 6
received an additional treatment of 10 doses of a therapeutic agent
or vehicle control administered under the following schedule: (i)
five doses of agent or vehicle administered as one dose per day for
five consecutive days; (ii) a two day break in treatment; and (iii)
an additional five doses of the agent or vehicle administered one
dose per day for five consecutive days. Group 6 mice were treated
with only vehicle--phosphate-buffered saline (PBS). Group 2 mice
were treated under the aforementioned treatment schedule with
Antibody 1--an anti-CD200 antibody (IgG2a) having effector
function--each dose being 5 mg/kg. Group 3 mice were treated under
the above treatment schedule with Antibody 2--an anti-CD200
antibody that lacked effector function--each dose at 5 mg/kg. Group
4 mice were treated under the above treatment schedule using a dose
of 5 mg/kg of a Control antibody that does not bind to CD200, but
possesses effector function (IgG2a). Group 5 mice were treated
under the above treatment schedule using a dose of 5 mg/kg of a
Control antibody that does not bind to CD200 and does not possess
effector function. The Group design and treatment schedules for
each group are summarized in Table 7.
TABLE-US-00007 TABLE 7 Group Design and Treatment Schedule for
Study 5. Groups N Therapeutic Administered Dose Group 1 20
Non-immunized, non-treated control group N/A Group 2 20 Antibody 1
(anti-CD200 antibody IgG2a with 5 mg/kg effector function) Group 3
20 Antibody 2 (anti-CD200 antibody that does not 5 mg/kg possess
effector function) Group 4 20 Control antibody (IgG2a) that does
not bind to 5 mg/kg CD200 but possesses effector function Group 5
15 Control antibody (IgG2a) that does not bind to 5 mg/kg CD200 and
does not possess effector function Group 6 15 Vehicle N/A N refers
to the number of mice in each group. N/A = not applicable.
[0423] On a weekly basis, blood was drawn from the mice of Groups 1
to 6 prior to, during, and after the above treatments to evaluate
by flow cytometry whether treatment affected the titer of
anti-mouse RBC autoantibodies and/or anti-rat RBC alloantibodies in
the mice. On day 35 of the study, three of the mice in each group
were sacrificed and their spleens harvested. Bone marrow was also
isolated from the femurs and tibias of each mouse. As described
above, the cells were labeled with detectably-labeled antibodies
(e.g., the polyclonal anti-CD200 antibody preparation and an
additional fluorescently-labeled antibody) and subjected to flow
cytometry. A summary of the results are shown below in Table 8.
TABLE-US-00008 TABLE 8 Effect of Anti-CD200 Antibodies on
Splenocvte and Bone Marrow Cell Subsets at day 35 Reduction (R) or
Reduction (R) or Tissue Cell Subset/Expression Increase (I) in
Increase (I) in Type Profile Group 2 Mice** Group 3 Mice ** Spleen
CD200.sup.+ R R Spleen CD3.sup.+/CD200.sup.+ R -- Spleen
CD5.sup.+/CD200.sup.+ R -- Spleen CD19.sup.+/CD200.sup.+ R --
Spleen CD45R.sup.+/CD200.sup.+ R -- Spleen CD138.sup.+/CD200.sup.+
(Gated R R on CD45R.sup.+ cells) Spleen CD200.sup.+ (Gated on R R*
CD45R.sup.+ cells) Bone CD200.sup.+ R -- Marrow Bone
CDIgk.sup.+/CD200.sup.+ R -- Marrow Bone CD200+ (Gated on R --
Marrow CD45R.sup.+ cells) Bone CD200.sup.+ (Gated on R -- Marrow
CD138.sup.+/CD45R.sup.- cells) Bone c-kit.sup.+/CD200.sup.- (Gated
R R Marrow on Lin.sup.- cells) *indicates that the reduction in
concentration of a particular cell subset in mice treated with
Antibody 2 is not as profound as the reduction observed in the same
cell subset in mice treated with Antibody 1. **indicates that the
reduction or increase in the concentration of a particular cell
subset is relative the concentration of the particular subset in
vehicle treated mice (Group 6) and the corresponding isotype
control. Thus, the reduction of CD200.sup.+ splenocytes observed in
mice of Group 2 mice is relative to the concentration of
CD200.sup.+ splenocytes in Group 6 mice and Group 4 mice.
--indicates that no difference in the levels was observed between
Antibody 2 and its corresponding Control antibody.
[0424] From day 35 to day 91, the remaining mice in each group
received additional RBC immunizations but no treatments with the
antibodies, the purpose being to determine if the populations of
splenocytes and bone marrow cells would recover over time. Three
mice in each group were sacrificed at day 91 and their spleens and
bone marrow harvested as described above. Flow cytometry analysis
was performed on the isolated cells to determine whether particular
population subsets of splenocytes and bone marrow cells, which were
reduced at day 35, recovered by day 91. Each of the cell
populations recovered fully by day 91, indicating that the
immunomodulatory effects of the anti-CD200 antibody on the
concentration of bone marrow cell and splenocyte subsets is
reversible upon withdrawal of the antibody.
[0425] While the present disclosure has been described with
reference to the specific embodiments thereof, it should be
understood by those skilled in the art that various changes may be
made and equivalents may be substituted without departing from the
true spirit and scope of the disclosure. In addition, many
modifications may be made to adapt a particular situation,
material, composition of matter, process, process step or steps, to
the objective, spirit and scope of the present disclosure. All such
modifications are intended to be within the scope of the
disclosure.
Sequence CWU 1
1
391269PRTHomo sapiens 1Met Glu Arg Leu Val Ile Arg Met Pro Phe Ser
His Leu Ser Thr Tyr 1 5 10 15 Ser Leu Val Trp Val Met Ala Ala Val
Val Leu Cys Thr Ala Gln Val 20 25 30 Gln Val Val Thr Gln Asp Glu
Arg Glu Gln Leu Tyr Thr Pro Ala Ser 35 40 45 Leu Lys Cys Ser Leu
Gln Asn Ala Gln Glu Ala Leu Ile Val Thr Trp 50 55 60 Gln Lys Lys
Lys Ala Val Ser Pro Glu Asn Met Val Thr Phe Ser Glu 65 70 75 80 Asn
His Gly Val Val Ile Gln Pro Ala Tyr Lys Asp Lys Ile Asn Ile 85 90
95 Thr Gln Leu Gly Leu Gln Asn Ser Thr Ile Thr Phe Trp Asn Ile Thr
100 105 110 Leu Glu Asp Glu Gly Cys Tyr Met Cys Leu Phe Asn Thr Phe
Gly Phe 115 120 125 Gly Lys Ile Ser Gly Thr Ala Cys Leu Thr Val Tyr
Val Gln Pro Ile 130 135 140 Val Ser Leu His Tyr Lys Phe Ser Glu Asp
His Leu Asn Ile Thr Cys 145 150 155 160 Ser Ala Thr Ala Arg Pro Ala
Pro Met Val Phe Trp Lys Val Pro Arg 165 170 175 Ser Gly Ile Glu Asn
Ser Thr Val Thr Leu Ser His Pro Asn Gly Thr 180 185 190 Thr Ser Val
Thr Ser Ile Leu His Ile Lys Asp Pro Lys Asn Gln Val 195 200 205 Gly
Lys Glu Val Ile Cys Gln Val Leu His Leu Gly Thr Val Thr Asp 210 215
220 Phe Lys Gln Thr Val Asn Lys Gly Tyr Trp Phe Ser Val Pro Leu Leu
225 230 235 240 Leu Ser Ile Val Ser Leu Val Ile Leu Leu Val Leu Ile
Ser Ile Leu 245 250 255 Leu Tyr Trp Lys Arg His Arg Asn Gln Asp Arg
Glu Pro 260 265 2294PRTHomo sapiens 2Met Glu Arg Leu Thr Leu Thr
Arg Thr Ile Gly Gly Pro Leu Leu Thr 1 5 10 15 Ala Thr Leu Leu Gly
Lys Thr Thr Ile Asn Asp Tyr Gln Val Ile Arg 20 25 30 Met Pro Phe
Ser His Leu Ser Thr Tyr Ser Leu Val Trp Val Met Ala 35 40 45 Ala
Val Val Leu Cys Thr Ala Gln Val Gln Val Val Thr Gln Asp Glu 50 55
60 Arg Glu Gln Leu Tyr Thr Pro Ala Ser Leu Lys Cys Ser Leu Gln Asn
65 70 75 80 Ala Gln Glu Ala Leu Ile Val Thr Trp Gln Lys Lys Lys Ala
Val Ser 85 90 95 Pro Glu Asn Met Val Thr Phe Ser Glu Asn His Gly
Val Val Ile Gln 100 105 110 Pro Ala Tyr Lys Asp Lys Ile Asn Ile Thr
Gln Leu Gly Leu Gln Asn 115 120 125 Ser Thr Ile Thr Phe Trp Asn Ile
Thr Leu Glu Asp Glu Gly Cys Tyr 130 135 140 Met Cys Leu Phe Asn Thr
Phe Gly Phe Gly Lys Ile Ser Gly Thr Ala 145 150 155 160 Cys Leu Thr
Val Tyr Val Gln Pro Ile Val Ser Leu His Tyr Lys Phe 165 170 175 Ser
Glu Asp His Leu Asn Ile Thr Cys Ser Ala Thr Ala Arg Pro Ala 180 185
190 Pro Met Val Phe Trp Lys Val Pro Arg Ser Gly Ile Glu Asn Ser Thr
195 200 205 Val Thr Leu Ser His Pro Asn Gly Thr Thr Ser Val Thr Ser
Ile Leu 210 215 220 His Ile Lys Asp Pro Lys Asn Gln Val Gly Lys Glu
Val Ile Cys Gln 225 230 235 240 Val Leu His Leu Gly Thr Val Thr Asp
Phe Lys Gln Thr Val Asn Lys 245 250 255 Gly Tyr Trp Phe Ser Val Pro
Leu Leu Leu Ser Ile Val Ser Leu Val 260 265 270 Ile Leu Leu Val Leu
Ile Ser Ile Leu Leu Tyr Trp Lys Arg His Arg 275 280 285 Asn Gln Asp
Arg Glu Pro 290 3274PRTHomo sapiens 3Val Ile Arg Met Pro Phe Ser
His Leu Ser Thr Tyr Ser Leu Val Trp 1 5 10 15 Val Met Ala Ala Val
Val Leu Cys Thr Ala Gln Val Gln Val Val Thr 20 25 30 Gln Asp Glu
Arg Glu Gln Leu Tyr Thr Thr Ala Ser Leu Lys Cys Ser 35 40 45 Leu
Gln Asn Ala Gln Glu Ala Leu Ile Val Thr Trp Gln Lys Lys Lys 50 55
60 Ala Val Ser Pro Glu Asn Met Val Thr Phe Ser Glu Asn His Gly Val
65 70 75 80 Val Ile Gln Pro Ala Tyr Lys Asp Lys Ile Asn Ile Thr Gln
Leu Gly 85 90 95 Leu Gln Asn Ser Thr Ile Thr Phe Trp Asn Ile Thr
Leu Glu Asp Glu 100 105 110 Gly Cys Tyr Met Cys Leu Phe Asn Thr Phe
Gly Phe Gly Lys Ile Ser 115 120 125 Gly Thr Ala Cys Leu Thr Val Tyr
Val Gln Pro Ile Val Ser Leu His 130 135 140 Tyr Lys Phe Ser Glu Asp
His Leu Asn Ile Thr Cys Ser Ala Thr Ala 145 150 155 160 Arg Pro Ala
Pro Met Val Phe Trp Lys Val Pro Arg Ser Gly Ile Glu 165 170 175 Asn
Ser Thr Val Thr Leu Ser His Pro Asn Gly Thr Thr Ser Val Thr 180 185
190 Ser Ile Leu His Ile Lys Asp Pro Lys Asn Gln Val Gly Lys Glu Val
195 200 205 Ile Cys Gln Val Leu His Leu Gly Thr Val Thr Asp Phe Lys
Gln Thr 210 215 220 Val Asn Lys Gly Tyr Trp Phe Ser Val Pro Leu Leu
Leu Ser Ile Val 225 230 235 240 Ser Leu Val Ile Leu Leu Val Leu Ile
Ser Ile Leu Leu Tyr Trp Lys 245 250 255 Arg His Arg Asn Gln Asp Arg
Gly Glu Leu Ser Gln Gly Val Gln Lys 260 265 270 Met Thr
410PRTArtificial SequenceDescription of Artificial Sequence
Synthetic peptide 4Gly Phe Thr Phe Ser Gly Phe Ala Met Ser 1 5 10
516PRTArtificial SequenceDescription of Artificial Sequence
Synthetic peptide 5Ser Ile Ser Ser Gly Gly Thr Thr Tyr Tyr Leu Asp
Ser Val Lys Gly 1 5 10 15 611PRTArtificial SequenceDescription of
Artificial Sequence Synthetic peptide 6Gly Asn Tyr Tyr Ser Gly Thr
Ser Tyr Asp Tyr 1 5 10 715PRTArtificial SequenceDescription of
Artificial Sequence Synthetic peptide 7Arg Ala Ser Glu Ser Val Asp
Ser Tyr Gly Asn Ser Phe Met His 1 5 10 15 87PRTArtificial
SequenceDescription of Artificial Sequence Synthetic peptide 8Arg
Ala Ser Asn Leu Glu Ser 1 5 99PRTArtificial SequenceDescription of
Artificial Sequence Synthetic peptide 9Gln Gln Ser Asn Glu Asp Pro
Arg Thr 1 5 1010PRTArtificial SequenceDescription of Artificial
Sequence Synthetic peptide 10Gly Phe Asn Ile Lys Asp Tyr Tyr Met
His 1 5 10 1117PRTArtificial SequenceDescription of Artificial
Sequence Synthetic peptide 11Trp Ile Asp Pro Glu Asn Gly Asp Thr
Lys Tyr Ala Pro Lys Phe Gln 1 5 10 15 Gly 1213PRTArtificial
SequenceDescription of Artificial Sequence Synthetic peptide 12Lys
Asn Tyr Tyr Val Ser Asn Tyr Asn Phe Phe Asp Val 1 5 10
1310PRTArtificial SequenceDescription of Artificial Sequence
Synthetic peptide 13Ser Ala Ser Ser Ser Val Arg Tyr Met Tyr 1 5 10
147PRTArtificial SequenceDescription of Artificial Sequence
Synthetic peptide 14Asp Thr Ser Lys Leu Ala Ser 1 5
159PRTArtificial SequenceDescription of Artificial Sequence
Synthetic peptide 15Phe Gln Gly Ser Gly Tyr Pro Leu Thr 1 5
1610PRTArtificial SequenceDescription of Artificial Sequence
Synthetic peptide 16Gly Phe Asn Ile Lys Asp Tyr Tyr Ile His 1 5 10
1717PRTArtificial SequenceDescription of Artificial Sequence
Synthetic peptide 17Trp Ile Asp Pro Glu Ile Gly Ala Thr Lys Tyr Val
Pro Lys Phe Gln 1 5 10 15 Gly 1813PRTArtificial SequenceDescription
of Artificial Sequence Synthetic peptide 18Leu Tyr Gly Asn Tyr Asp
Arg Tyr Tyr Ala Met Asp Tyr 1 5 10 1911PRTArtificial
SequenceDescription of Artificial Sequence Synthetic peptide 19Lys
Ala Ser Gln Asn Val Arg Thr Ala Val Ala 1 5 10 207PRTArtificial
SequenceDescription of Artificial Sequence Synthetic peptide 20Leu
Ala Ser Asn Arg His Thr 1 5 219PRTArtificial SequenceDescription of
Artificial Sequence Synthetic peptide 21Leu Gln His Trp Asn Tyr Pro
Leu Thr 1 5 2210PRTArtificial SequenceDescription of Artificial
Sequence Synthetic peptide 22Gly Tyr Ser Phe Thr Asp Tyr Ile Ile
Leu 1 5 10 2317PRTArtificial SequenceDescription of Artificial
Sequence Synthetic peptide 23His Ile Asp Pro Tyr Tyr Gly Ser Ser
Asn Tyr Asn Leu Lys Phe Lys 1 5 10 15 Gly 248PRTArtificial
SequenceDescription of Artificial Sequence Synthetic peptide 24Ser
Lys Arg Asp Tyr Phe Asp Tyr 1 5 2511PRTArtificial
SequenceDescription of Artificial Sequence Synthetic peptide 25Lys
Ala Ser Gln Asp Ile Asn Ser Tyr Leu Ser 1 5 10 267PRTArtificial
SequenceDescription of Artificial Sequence Synthetic peptide 26Arg
Ala Asn Arg Leu Val Asp 1 5 279PRTArtificial SequenceDescription of
Artificial Sequence Synthetic peptide 27Leu Gln Tyr Asp Glu Phe Pro
Tyr Thr 1 5 2810PRTArtificial SequenceDescription of Artificial
Sequence Synthetic peptide 28Gly Tyr Thr Phe Thr Glu Tyr Thr Met
His 1 5 10 2917PRTArtificial SequenceDescription of Artificial
Sequence Synthetic peptide 29Gly Val Asn Pro Asn Asn Gly Gly Ala
Leu Tyr Asn Gln Lys Phe Lys 1 5 10 15 Gly 3012PRTArtificial
SequenceDescription of Artificial Sequence Synthetic peptide 30Arg
Ser Asn Tyr Arg Tyr Asp Asp Ala Met Asp Tyr 1 5 10
3116PRTArtificial SequenceDescription of Artificial Sequence
Synthetic peptide 31Lys Ser Ser Gln Ser Leu Leu Asp Ile Asp Glu Lys
Thr Tyr Leu Asn 1 5 10 15 327PRTArtificial SequenceDescription of
Artificial Sequence Synthetic peptide 32Leu Val Ser Lys Leu Asp Ser
1 5 339PRTArtificial SequenceDescription of Artificial Sequence
Synthetic peptide 33Trp Gln Gly Thr His Phe Pro Gln Thr 1 5
3410PRTArtificial SequenceDescription of Artificial Sequence
Synthetic peptide 34Ala Phe Asn Ile Lys Asp His Tyr Met His 1 5 10
3517PRTArtificial SequenceDescription of Artificial Sequence
Synthetic peptide 35Trp Ile Asp Pro Glu Ser Gly Asp Thr Glu Tyr Ala
Pro Lys Phe Gln 1 5 10 15 Gly 369PRTArtificial SequenceDescription
of Artificial Sequence Synthetic peptide 36Phe Asn Gly Tyr Gln Ala
Leu Asp Gln 1 5 3712PRTArtificial SequenceDescription of Artificial
Sequence Synthetic peptide 37Thr Ala Ser Ser Ser Val Ser Ser Ser
Tyr Leu His 1 5 10 387PRTArtificial SequenceDescription of
Artificial Sequence Synthetic peptide 38Ser Thr Ser Asn Leu Ala Ser
1 5 3911PRTArtificial SequenceDescription of Artificial Sequence
Synthetic peptide 39Arg Gln Tyr His Arg Ser Pro Pro Ile Phe Thr 1 5
10
* * * * *