U.S. patent application number 14/630262 was filed with the patent office on 2015-12-24 for polypeptides and antibodies derived from chronic lymphocytic leukemia cells and uses thereof.
The applicant listed for this patent is ALEXION PHARMACEUTICALS, INC.. Invention is credited to Katherine S. BOWDISH, Anke KRETZ-ROMMEL.
Application Number | 20150368341 14/630262 |
Document ID | / |
Family ID | 35907987 |
Filed Date | 2015-12-24 |
United States Patent
Application |
20150368341 |
Kind Code |
A1 |
BOWDISH; Katherine S. ; et
al. |
December 24, 2015 |
POLYPEPTIDES AND ANTIBODIES DERIVED FROM CHRONIC LYMPHOCYTIC
LEUKEMIA CELLS AND USES THEREOF
Abstract
Small animal models for assessing immunomodulatory effects of
compounds are provided.
Inventors: |
BOWDISH; Katherine S.; (Del
Mar, CA) ; KRETZ-ROMMEL; Anke; (San Diego,
CA) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
ALEXION PHARMACEUTICALS, INC. |
Cheshire |
CT |
US |
|
|
Family ID: |
35907987 |
Appl. No.: |
14/630262 |
Filed: |
February 24, 2015 |
Related U.S. Patent Documents
|
|
|
|
|
|
Application
Number |
Filing Date |
Patent Number |
|
|
13072470 |
Mar 25, 2011 |
8999328 |
|
|
14630262 |
|
|
|
|
11985322 |
Nov 13, 2007 |
7915000 |
|
|
13072470 |
|
|
|
|
11171567 |
Jun 30, 2005 |
|
|
|
11985322 |
|
|
|
|
10996316 |
Nov 23, 2004 |
7408041 |
|
|
11171567 |
|
|
|
|
10894672 |
Jul 20, 2004 |
|
|
|
10996316 |
|
|
|
|
10736188 |
Dec 15, 2003 |
|
|
|
10894672 |
|
|
|
|
10379151 |
Mar 4, 2003 |
7435412 |
|
|
10736188 |
|
|
|
|
PCT/US01/47931 |
Dec 10, 2001 |
|
|
|
10379151 |
|
|
|
|
60254113 |
Dec 8, 2000 |
|
|
|
Current U.S.
Class: |
424/135.1 ;
424/133.1; 424/143.1; 424/173.1 |
Current CPC
Class: |
A61K 2039/505 20130101;
A61P 43/00 20180101; A61P 35/00 20180101; A61K 47/6849 20170801;
C07K 16/2803 20130101; C07K 2317/92 20130101; A61P 35/02 20180101;
G01N 2333/70596 20130101; C07K 2317/732 20130101; C07K 2317/76
20130101; G01N 2800/52 20130101; A61P 37/04 20180101; G01N 33/57426
20130101; C07K 2317/24 20130101; C07K 16/3061 20130101; C07K
2317/622 20130101 |
International
Class: |
C07K 16/28 20060101
C07K016/28; C07K 16/30 20060101 C07K016/30 |
Claims
1. A method for treating CLL comprising administering to a patient
suffering from CLL an antibody, or an antigen-binding fragment
thereof, that binds to OX-2/CD200 and comprises: (a) a light chain
CDR1 having the sequence set forth in residues 26-36 of SEQ ID
NO:208, a light chain CDR2 having the sequence set forth in
residues 52-58 of SEQ ID NO:208, a light chain CDR3 having the
sequence set forth in residues 91-99 of SEQ ID NO:208, a heavy
chain CDR1 having the sequence set forth in SEQ ID NO:114, a heavy
chain CDR2 having the sequence set forth in SEQ ID NO:165, and a
heavy chain CDR3 having the sequence set forth in SEQ ID NO:189;
(d1B5) (b) a light chain CDR1 having the sequence set forth in
residues 26-40 of SEQ ID NO:206; a light chain CDR2 having the
sequence set forth in residues 56-62 of SEQ ID NO:206; a light
chain CDR3 having the sequence set forth in residues 95-103 of SEQ
ID NO:206; a heavy chain CDR1 having the sequence set forth in SEQ
ID NO:141; a heavy chain CDR2 having the sequence set forth in
residues 51-66 of SEQ ID NO:200; and a heavy chain CDR3 having the
sequence set forth in SEQ ID NO:188; (d1B10) (c) a light chain CDR1
having the sequence set forth in residues 26-35 of SEQ ID NO:207; a
light chain CDR2 having the sequence set forth in residues 51-57 of
SEQ ID NO:207; a light chain CDR3 having the sequence set forth in
residues 90-98 of SEQ ID NO:207; a heavy chain CDR1 having the
sequence set forth in SEQ ID NO:111; a heavy chain CDR2 having the
sequence set forth in SEQ ID NO:118; and a heavy chain CDR3 having
the sequence set forth in residues 100-112 of SEQ ID NO:201; (d1A5)
(d) a light chain polypeptide comprising: a light chain CDR1 having
the sequence set forth in residues 26-41 of SEQ ID NO:210; a light
chain CDR2 having the sequence set forth in residues 57-63 of SEQ
ID NO:210; a light chain CDR3 having the sequence set forth in
residues 96-104 of SEQ ID NO:210; a heavy chain CDR1 having the
sequence set forth in SEQ ID NO:140; a heavy chain CDR2 having the
sequence set forth in SEQ ID NO:162; and a heavy chain CDR3 having
the sequence set forth in SEQ ID NO:187; (c1A10) or (e) a light
chain CDR1 having the sequence set forth in residues 26-37 of SEQ
ID NO:211; a light chain CDR2 having the sequence set forth in
residues 53-58 of SEQ ID NO:211; a light chain CDR3 having the
sequence set forth in residues 92-102 of SEQ ID NO:211; a heavy
chain CDR1 having the sequence set forth in SEQ ID NO:136; a heavy
chain CDR2 having the sequence set forth in SEQ ID NO:159; and a
heavy chain CDR3 having the sequence set forth in SEQ ID NO:181,
(c2aA10) wherein the patient's CLL has been identified as
overexpressing OX-2/CD200 three to six-fold as compared to normal
peripheral blood mononuclear cells.
2. The method of claim 1, wherein the antibody, or antigen-binding
fragment of the antibody, comprises a light chain CDR1 having the
sequence set forth in residues 26-36 of SEQ ID NO:208, a light
chain CDR2 having the sequence set forth in residues 52-58 of SEQ
ID NO:208, a light chain CDR3 having the sequence set forth in
residues 91-99 of SEQ ID NO:208, a heavy chain CDR1 having the
sequence set forth in SEQ ID NO:114, a heavy chain CDR2 having the
sequence set forth in SEQ ID NO:165, and a heavy chain CDR3 having
the sequence set forth in SEQ ID NO:189.
3. The method of claim 1, wherein the antibody, or antigen-binding
fragment of the antibody, comprises a light chain CDR1 having the
sequence set forth in residues 26-40 of SEQ ID NO:206; a light
chain CDR2 having the sequence set forth in residues 56-62 of SEQ
ID NO:206; a light chain CDR3 having the sequence set forth in
residues 95-103 of SEQ ID NO:206; a heavy chain CDR1 having the
sequence set forth in SEQ ID NO:141; a heavy chain CDR2 having the
sequence set forth in residues 51-66 of SEQ ID NO:200; and a heavy
chain CDR3 having the sequence set forth in SEQ ID NO:188.
4. The method of claim 1, wherein the antibody, or antigen-binding
fragment of the antibody, comprises a light chain CDR1 having the
sequence set forth in residues 26-35 of SEQ ID NO:207; a light
chain CDR2 having the sequence set forth in residues 51-57 of SEQ
ID NO:207; a light chain CDR3 having the sequence set forth in
residues 90-98 of SEQ ID NO:207; a heavy chain CDR1 having the
sequence set forth in SEQ ID NO:111; a heavy chain CDR2 having the
sequence set forth in SEQ ID NO:118; and a heavy chain CDR3 having
the sequence set forth in residues 100-112 of SEQ ID NO:201.
5. The method of claim 1, wherein the antibody, or antigen-binding
fragment of the antibody, comprises a light chain polypeptide
comprising: a light chain CDR1 having the sequence set forth in
residues 26-41 of SEQ ID NO:210; a light chain CDR2 having the
sequence set forth in residues 57-63 of SEQ ID NO:210; a light
chain CDR3 having the sequence set forth in residues 96-104 of SEQ
ID NO:210; a heavy chain CDR1 having the sequence set forth in SEQ
ID NO:140; a heavy chain CDR2 having the sequence set forth in SEQ
ID NO:162; and a heavy chain CDR3 having the sequence set forth in
SEQ ID NO:187.
6. The method of claim 1, wherein the antibody, or antigen-binding
fragment of the antibody, comprises a light chain CDR1 having the
sequence set forth in residues 26-37 of SEQ ID NO:211; a light
chain CDR2 having the sequence set forth in residues 53-58 of SEQ
ID NO:211; a light chain CDR3 having the sequence set forth in
residues 92-102 of SEQ ID NO:211; a heavy chain CDR1 having the
sequence set forth in SEQ ID NO:136; a heavy chain CDR2 having the
sequence set forth in SEQ ID NO:159; and a heavy chain CDR3 having
the sequence set forth in SEQ ID NO:181.
7. The method of claim 1, wherein said antibody, or antigen-binding
fragment thereof, is selected from the group consisting of a
monoclonal antibody, a chimeric antibody, an Fv, an scFv, a Fab',
and an F(ab').sub.2.
8. The method of claim 1, wherein the antibody, or antigen-binding
fragment thereof, is humanized.
Description
RELATED APPLICATIONS
[0001] This application is divisional of U.S. application Ser. No.
13/072,470, filed Mar. 25, 2011, which is a continuation of U.S.
application Ser. No. 11/985,322, filed Nov. 13, 2007 (now U.S. Pat.
No. 7,915,000), which is a continuation of U.S. application Ser.
No. 11/171,567, filed Jun. 30, 2005 (now abandoned), which is a
continuation-in-part of U.S. application Ser. No. 10/996,316, filed
Nov. 23, 2004 (now U.S. Pat. No. 7,408,041), which is a
continuation-in-part of U.S. application Ser. No. 10/894,672, filed
Jul. 20, 2004, which is a continuation-in-part of U.S. application
Ser. No. 10/736,188, filed Dec. 15, 2003 (now abandoned), which is
a continuation-in-part of U.S. application Ser. No. 10/379,151,
filed on Mar. 4, 2003 (now U.S. Pat. No. 7,435,412), which, in
turn, is a continuation-in-part of PCT/US01/47931, filed on Dec.
10, 2001, which is an international application that claims
priority to U.S. Provisional Application 60/254,113, filed Dec. 8,
2000. The entire disclosures of the aforementioned U.S.,
international and provisional applications are incorporated herein
by reference.
SEQUENCE LISTING
[0002] The instant application contains a Sequence Listing which
has been submitted via EFS-Web and is hereby incorporated by
reference in its entirety. Said ASCII copy, created on Mar. 25,
2011, is named ALEXP13060Sequence.txt, and is 48,421 bytes in
size.
TECHNICAL FIELD
[0003] Cancer treatments using a therapy that provides a
combination of two mechanisms are disclosed. More specifically,
this disclosure relates to treating cancer using a therapy that: 1)
interferes with the interaction between CD200 and its receptor to
block immune suppression thereby promoting eradication of the
cancer cells; and 2) directly kills the cancer cells either by a)
complement-mediated or antibody-dependent cellular cytotoxicity or
b) by targeting cells using a fusion molecule that includes a
CD200-targeting portion.
BACKGROUND
[0004] Chronic Lymphocytic Leukemia (CLL) is a disease of the white
blood cells and is the most common form of leukemia in the Western
Hemisphere. CLL represents a diverse group of diseases relating to
the growth of malignant lymphocytes that grow slowly but have an
extended life span. CLL is classified in various categories that
include, for example, B-cell chronic lymphocytic leukemia (B-CLL)
of classical and mixed types, B-cell and T-cell prolymphocytic
leukemia hairy cell leukemia, and large granular lymphocytic
leukemia.
[0005] Of all the different types of CLL, B-CLL accounts for
approximately 30 percent of all leukemias. Although it occurs more
frequently in individuals over 50 years of age, it is increasingly
seen in younger people. B-CLL is characterized by accumulation of
B-lymphocytes that are morphologically normal but biologically
immature, leading to a loss of function. Lymphocytes normally
function to fight infection. In B-CLL, however, lymphocytes
accumulate in the blood and bone marrow and cause swelling of the
lymph nodes. The production of normal bone marrow and blood cells
is reduced and patients often experience severe anemia as well as
low platelet counts. This can pose the risk of life-threatening
bleeding and the development of serious infections because of
reduced numbers of white blood cells.
[0006] To further understand diseases such as leukemia it is
important to have suitable cell lines that can be used as tools for
research on their etiology, pathogenesis and biology. Examples of
malignant human B-lymphoid cell lines include pre-B acute
lymphoblasticleukemia (Reh), diffuse large cell lymphoma
(WSU-DLCL2), and Waldenstrom's macroglobulinemia (WSU-WM).
Unfortunately, many of the existing cell lines do not represent the
clinically most common types of leukemia and lymphoma.
[0007] The use of Epstein Barr Virus (EBV) infection in vitro has
resulted in some CLL derived cell lines, in particular B-CLL cells
lines, that are representative of the malignant cells. The
phenotype of these cell lines is different than that of the in vivo
tumors and instead the features of B-CLL lines tend to be similar
to those of lymphoblastoid cell lines. Attempts to immortalize
B-CLL cells with the aid of EBV infection have had little success.
The reasons for this are unclear but it is known that it is not due
to a lack of EBV receptor expression, binding or uptake. Wells et
al. found that B-CLL cells were arrested in the G1/S phase of the
cell cycle and that transformation associated EBV DNA was not
expressed. This suggests that the interaction of EBV with B-CLL
cells is different from that with normal B cells. EBV-transformed
CLL cell lines moreover appear to differentiate, possessing a
morphology more similar to lymphoblastoid cell lines (LCL)
immortalized by EBV.
[0008] An EBV-negative CLL cell line, WSU-CLL, has been established
previously (Mohammad et al., (1996) Leukemia 10(1):130-7). However,
no other such cell lines are known.
[0009] Various mechanisms play a role in the body's response to a
disease state, including cancer and CLL. For example, CD4.sup.+ T
helper cells play a crucial role in an effective immune response
against various malignancies by providing stimulatory factors to
effector cells. Cytotoxic T cells are believed to be the most
effective cells to eliminate cancer cells, and T helper cells prime
cytotoxic T cells by secreting Th1 cytokines such as IL-2 and
IFN-.gamma.. In various malignancies, T helper cells have been
shown to have an altered phenotype compared to cells found in
healthy individuals. One of the prominent altered features is
decreased Th1 cytokine production and a shift to the production of
Th2 cytokines. (See, e.g., Kiani, et al., Haematologica 88:754-761
(2003); Maggio, et al., Ann Oncol 13 Suppl 1:52-56 (2002); Ito, et
al., Cancer 85:2359-2367 (1999); Podhorecka, et al., Leuk Res
26:657-660 (2002); Tatsumi, et al., J Exp Med 196:619-628 (2002);
Agarwal, et al., Immunol Invest 32:17-30 (2003); Smyth, et al., Ann
Surg Oncol 10:455-462 (2003); Contasta, et al., Cancer Biother
Radiopharm 18:549-557 (2003); Lauerova, et al., Neoplasma
49:159-166(2002).) Reversing that cytokine shift to a Th1 profile
has been demonstrated to augment anti-tumor effects of T cells.
(See Winter, et al., Immunology 108:409-419 (2003); Inagawa, et
al., Anticancer Res 18:3957-3964 (1998).)
[0010] Mechanisms underlying the capacity of tumor cells to drive
the cytokine expression of T helper cells from Th1 to Th2 include
the secretion of cytokines such as IL-10 or TGF-.beta. as well as
the expression of surface molecules interacting with cells of the
immune system. OX-2/CD200, a molecule expressed on the surface of
dendritic cells which possesses a high degree of homology to
molecules of the immunoglobulin gene family, has been implicated in
immune suppression (Gorczynski et al., Transplantation 65:1106-1114
(1998)) and evidence that OX-2/CD200-expressing cells can inhibit
the stimulation of Th1 cytokine production has been provided.
Gorczynski et al. demonstrated in a mouse model that infusion of
OX-2/CD200 Fc suppresses the rejection of tumor cells in an animal
model using leukaemic tumor cells (Clin Exp Immunol 126:220-229
(2001)).
[0011] Improved methods for treating individuals suffering from
cancer or CLL are desirable, especially to the extent they can
enhance the activity of T cells.
SUMMARY
[0012] In one embodiment a CLL cell line of malignant origin is
provided that is not established by immortalisation with EBV. The
cell line was derived from primary CLL cells and is deposited under
ATCC accession no. PTA-3920. In a preferred embodiment, the cell
line is CLL-AAT. CLL-AAT is a B-CLL cell line, derived from a B-CLL
primary cell.
[0013] In a further aspect, the CLL-AAT cell line is used to
generate monoclonal antibodies useful in the diagnosis and/or
treatment of CLL. Antibodies may be generated by using the cells as
disclosed herein as immunogens, thus raising an immune response in
animals from which monoclonal antibodies may be isolated. The
sequence of such antibodies may be determined and the antibodies or
variants thereof produced by recombinant techniques. In this
aspect, "variants" includes chimeric, CDR-grafted, humanized and
fully human antibodies based on the sequence of the monoclonal
antibodies.
[0014] Moreover, antibodies derived from recombinant libraries
("phage antibodies") may be selected using the cells described
herein, or polypeptides derived therefrom, as bait to isolate the
antibodies on the basis of target specificity.
[0015] In a still further aspect, antibodies may be generated by
panning antibody libraries using primary CLL cells, or antigens
derived therefrom, and further screened and/or characterized using
a CLL cell line, such as, for example, the CLL cell line described
herein. Accordingly, a method for characterizing an antibody
specific for CLL is provided, which includes assessing the binding
of the antibody to a CLL cell line.
[0016] In a further aspect, there is provided a method for
identifying proteins uniquely expressed in CLL cells employing the
CLL-AAT cell line, by methods well known to those skilled in the
art, such as by immunoprecipitation followed by mass spectroscopy
analyses. Such proteins may be uniquely expressed in the CLL-AAT
cell line, or in primary cells derived from CLL patients.
[0017] Small molecule libraries (many available commercially) may
be screened using the CLL-AAT cell line in a cell-based assay to
identify agents capable of modulating the growth characteristics of
the cells. For example, the agents may be identified which modulate
apoptosis in the CLL-AAT cell line, or which inhibit growth and/or
proliferation thereof. Such agents are candidates for the
development of therapeutic compounds.
[0018] Nucleic acids isolated from CLL-AAT cell lines may be used
in subtractive hybridization experiments to identify CLL-specific
genes or in micro array analyses (e.g., gene chip experiments).
Genes whose transcription is modulated in CLL cells may be
identified. Polypeptide or nucleic acid gene products identified in
this manner are useful as leads for the development of antibody or
small molecule therapies for CLL.
[0019] In a preferred aspect, the CLL-AAT cell line may be used to
identify internalizing antibodies, which bind to cell surface
components which are internalized by the cell. Such antibodies are
candidates for therapeutic use. In particular, single-chain
antibodies, which remain stable in the cytoplasm and which retain
intracellular binding activity, may be screened in this manner.
[0020] In yet another aspect, a therapeutic treatment is described
in which a patient is screened for the presence of a polypeptide
that is upregulated by a malignant cancer cell and an antibody that
interferes with the metabolic pathway of the upregulated
polypeptide is administered to the patient.
[0021] The present disclosure further is directed to methods
wherein a determination is made as to whether OX-2/CD200 is
upregulated in a subject and, if so, administering to the subject a
therapy that enhances immune response. Upregulation of OX2/CD200
can be determined by measuring OX2/CD200 levels directly, or by
monitoring the level of any marker that correlates with OX2/CD200.
Suitable immunomodulatory therapies include the administration of
agents that block negative regulation of T cells or antigen
presenting cells, administration of agents that enhance positive
co-stimulation of T cells, cancer vaccines, general adjuvants
stimulating the immune system or treatment with cytokines such as
IL-2, GM-CSF and IFN-gamma. In particularly useful embodiments, the
therapy that enhances immune response includes the administration
of a polypeptide that binds to OX-2/CD200, optionally in
combination with one or more other immunomodulatory therapies. In
another embodiment, the polypeptide binds to an OX-2/CD200
receptor.
[0022] In another aspect, methods in accordance with this
disclosure are used to treat a disease state in which OX-2/CD200 is
upregulated in a subject by administering a polypeptide that binds
to OX-2/CD200 or an OX-2/CD200 receptor to the subject afflicted
with the disease state. In one embodiment, the disease state
treated by these methods includes cancer, specifically, in other
embodiments, CLL.
[0023] In a particularly useful embodiment, a cancer therapy in
accordance with this disclosure includes i) administering an
antibody that interferes with the interaction between CD200 and its
receptor to block immune suppression, thereby promoting eradication
of the cancer cells; and ii) administering a fusion molecule that
includes a CD200-targeting portion to directly kill cancer cells.
Alternatively, the antibody directly kills the cancer cells through
complement-mediated or antibody-dependent cellular
cytotoxicity.
[0024] In another embodiment in accordance with the present
disclosure, methods are provided for monitoring the progress of a
therapeutic treatment. The method involves administering an
immunomodulatory therapy and determining OX-2/CD200 levels in a
subject at least twice to determine the effectiveness of the
therapy.
[0025] In another aspect, the present disclosure provides methods
for assessing the immunomodulatory effect of molecules expressed by
cancer cells. In these methods a molecule that is expressed or
upregulated by a cancer cell is identified (e.g., experimentally or
from a database). Cancer cells, lymphocytes and the molecule that
is expressed or upregulated by a cancer cell are administered to a
subject and the rate of growth of the cancer cells is monitored.
The number of lymphocytes administered is predetermined to be
either a) sufficient to slow the growth of the cancer cells or b)
insufficient to slow the growth of cancer cells. The molecule that
is expressed or upregulated by a cancer cell can be administered as
the molecule or the active portion of the molecule itself, or by
administering cells that produce or express the molecule or
portions thereof naturally, or by administering cells that have
been engineered to produce or express the molecule or portions
thereof. If any change in the growth rate of the cancer cells is
observed compared to the rate of growth when the tumor cells and
donor lymphocytes are administered alone, the molecule is deemed to
have an immunomodulatory effect. For example, if the number of
lymphocytes administered is sufficient to slow the growth of the
cancer cells and the rate of growth of the tumor cells observed is
higher compared to the rate of growth when the tumor cells and
donor lymphocytes are administered alone, the molecule that is
expressed or upregulated by a cancer cell is considered
immunosuppressive. As another example, if the number of lymphocytes
administered is insufficient to slow the growth of the cancer cells
and the rate of growth of the tumor cells observed is lowered
compared to the rate of growth when the tumor cells and donor
lymphocytes are administered alone, the compound is considered
immune enhancing. Once the immunomodulatory effect of the molecule
is established, compounds that either enhance or inhibit the
activity of the molecule can be identified in accordance with
embodiments described herein. The enhancing or inhibiting effect
can be the result of direct interaction with the molecule expressed
or upregulated by the cancer cell or may be the result of an
interaction with other molecules in the metabolic pathway of the
compound expressed or upregulated by the cancer cell.
[0026] In another aspect, the present disclosure provides methods
for assessing the immunomodulatory effect of a compound. In these
methods cancer cells, lymphocytes and the compound to be assessed
are administered to a subject and the rate of growth of the cancer
cells is monitored. The number of lymphocytes administered is
predetermined to be either a) sufficient to slow the growth of the
cancer cells or b) insufficient to slow the growth of cancer cells.
The compound to be assessed can be administered as the compound
itself, or by administering cells that produce the compound
naturally, or by administering cells that have been engineered to
produce the compound. If any change in the growth rate of the
cancer cells is observed compared to the rate of growth when the
tumor cells and donor lymphocytes are administered alone, the
compound is deemed to have an immunomodulatory effect. For example,
if the number of lymphocytes administered is sufficient to slow the
growth of the cancer cells and the rate of growth of the tumor
cells observed is higher compared to the rate of growth when the
tumor cells and donor lymphocytes are administered alone, the
compound is considered immunosuppressive. As another example, if
the number of lymphocytes administered is insufficient to slow the
growth of the cancer cells and the rate of growth of the tumor
cells observed is lowered compared to the rate of growth when the
tumor cells and donor lymphocytes are administered alone, the
compound is considered immune enhancing.
BRIEF DESCRIPTION OF THE FIGURES
[0027] FIG. 1 schematically illustrates typical steps involved in
cell surface panning of antibody libraries by
magnetically-activated cell sorting (MACS).
[0028] FIG. 2 is a graph showing the results of whole cell ELISA
demonstrating binding of selected scFv clones to primary B-CLL
cells and absence of binding to normal human PBMC. The designation
2.degree.+3.degree. in this and other figures refers to negative
control wells stained with Mouse Anti-HA and detecting antimouse
antibodies alone. The designation RSC-S Library in this and other
figures refers to soluble antibodies prepared from original rabbit
scFv unpanned library. The designation R3/RSC-S Pool in this and
other figures refers to soluble antibodies prepared from the entire
pool of scFv antibodies from round 3 of panning. Anti-CD5 antibody
was used as a positive control to verify that equal numbers of
B-CLL and PBMC cells were plated in each well.
[0029] FIGS. 3a and 3b show the results of whole cell ELISA
comparing binding of selected scFv antibodies to primary B-CLL
cells and normal primary human B cells. Anti-CD 19 antibody was
used as a positive control to verify that equal numbers of B-CLL
and normal B cells were plated in each well. Other controls were as
described in the legend to FIG. 2.
[0030] FIGS. 4a and 4b show the results of whole cell ELISA used to
determine if scFv clones bind to patient-specific (i.e. idiotype)
or blood type-specific (i.e. HLA) antigens. Each clone was tested
for binding to PBMC isolated from 3 different B-CLL patients.
Clones that bound to only one patient sample were considered to be
patient or blood type-specific.
[0031] FIGS. 5a and 5b show the results of whole cell ELISA
comparing binding of scFv clones to primary B-CLL cells and three
human leukemic cell lines. Ramos is a mature B cell line derived
from a Burkitt's lymphoma. RL is a mature B cell line derived from
a non-Hodgkin's lymphoma. TF-1 is an erythroblastoid cell line
derived from an erythroleukemia.
[0032] FIGS. 6a, 6b and 6c show the results of whole cell ELISA
comparing binding of scFv clones to primary B-CLL cells and
CLL-AAT, a cell line derived from a B-CLL patient. TF-1 cells were
included as a negative control.
[0033] FIG. 7 shows the binding specificity of scFv antibodies in
accordance with this disclosure as analyzed by 3-color flow
cytometry. In normal peripheral blood mononuclear cells, the
antigen recognized by scFv-9 is moderately expressed on B
lymphocytes and weakly expressed on a subpopulation of T
lymphocytes. PBMC from a normal donor were analyzed by 3-color flow
cytometry using anti-CD5-FITC, anti-CD19-PerCP, and
scFv-9/Anti-HA-biotin/streptavidin-PE.
[0034] FIGS. 8a, 8b and 8c show the expression levels of antigens
recognized by scFv antibodies in accordance with this disclosure.
The antigens recognized by scFv-3 and scFv-9 are overexpressed on
the primary CLL tumor from which the CLL-AAT cell line was derived.
Primary PBMC from the CLL patient used to establish the CLL-AAT
cell line or PBMC from a normal donor were stained with scFv
antibody and analyzed by flow cytometry. ScFv-3 and scFv-9 stain
the CLL cells more brightly than the normal PBMC as measured by the
mean fluorescent intensities.
[0035] FIGS. 9A, 9B and 9C provide a summary of CDR sequences and
binding specificities of selected scFv antibodies.
[0036] FIG. 10 is Table 2 which shows a summary of flow cytometry
results comparing expression levels of scFv antigens on primary CLL
cells vs. normal PBMC as described in FIGS. 8a-8c.
[0037] FIG. 11 is a Table showing a summary of flow cytometry
results comparing expression levels of scFv-9 antigen with the
percentage of CD38.sup.+ cells in peripheral blood mononuclear
cells isolated from ten CLL patients.
[0038] FIG. 12 shows the identification of scFv antigens by
immunoprecipitation and mass spectrometry. CLL-AAT cells were
labeled with a solution of 0.5 mg/ml sulfo-NHS-LC-biotin (Pierce)
in PBS, pH 8.0 for 30'. After extensive washing with PBS to remove
unreacted biotin, the cells were disrupted by nitrogen cavitation
and the microsomal fraction was isolated by differential
centrifugation. The microsomal fraction was resuspended in NP40
Lysis Buffer and extensively precleared with normal rabbit serum
and Protein A SEPHAROSE.RTM.. Antigens were immunoprecipitated with
HA-tagged scFv antibodies coupled to Rat Anti-HA agarose beads
(Roche). Following immunoprecipitation, antigens were separated by
SDS-PAGE and detected by Western blot using streptavidin-alkaline
phosphatase (AP) or by Coomassie G-250 staining. ScFv-7, an
antibody which doesn't bind to CLL-AAT cells, was used as a
negative control. Antigen bands were excised from the
Coomassie-stained gel and identified by mass spectrometry (MS).
MALDI-MS was performed at the Proteomics Core Facility of The
Scripps Research Institute (La Jolla, Calif.). .mu.LC/MS/MS was
performed at the Harvard Microchemistry Facility (Cambridge,
Mass.).
[0039] FIG. 13 shows that three scFv antibodies bind specifically
to 293-EBNA cells transiently transfected with a human OX-2/CD200
cDNA clone. An OX-2/CD200 cDNA was cloned from CLL cells by RT-PCR
and inserted into the mammalian expression vector pCEP4
(Invitrogen). PCEP4-CD200 plasmid or the corresponding empty vector
pCEP4 was transfected into 293-EBNA cells using Polyfect reagent
(QIAGEN). Two days after transfection, the cells were analyzed for
binding to scFv antibodies by flow cytometry.
[0040] FIG. 14 shows that the presence of OX-2/CD200 transfected
cells resulted in down-regulation of Th1 cytokines such as IL-2 and
IFN-.gamma.. Addition of the anti-OX-2/CD200 antibody at 30
.mu.g/ml fully restored the Th1 response.
[0041] FIG. 15 shows that the presence of CLL cells in a mixed
lymphocyte reaction resulted in down-regulation of the Th1 response
for IL-2.
[0042] FIG. 16 shows that the presence of CLL cells in a mixed
lymphocyte reaction resulted in down-regulation of the Th1 response
for IFN-.gamma..
[0043] FIGS. 17A and B show the mean+/-SD of tumor volumes for all
groups of NOD/SCID mice were injected subcutaneously with
4.times.10.sup.6-RAJI cells either in the presence or absence of
human PBL cells.
[0044] FIG. 18 shows the results of statistical analyses performed
using 2 parametric tests (Student's t-test and Welch's test) and
one non-parametric test (the Wilcox test).
[0045] FIG. 19A shows ELISA results of representative IgG1 kappa
clones after round 3 panning on CD200-Fc captured on goat
anti-mouse IgG Fc antibody.
[0046] FIG. 19B shows ELISA results of representative IgG2a kappa
clones after round 3 panning on CD200-Fc captured on goat
anti-mouse IgG Fc antibody.
[0047] FIG. 19C shows ELISA results of representative IgG1 kappa
clones after round 3 panning on CD200-Fc directly coated on
microtiter wells.
[0048] FIG. 19D shows ELISA results of representative IgG2a kappa
clones after round 3 panning on CD200-Fc directly coated on
microtiter wells.
[0049] FIG. 20 A shows flow cytometry results of representative
IgG1 clones selected on CD200-Fc captured with goat anti-mouse IgG
Fc.
[0050] FIG. 20B shows flow cytometry results of representative
IgG2a clones selected on CD200-Fc captured with goat anti-mouse IgG
Fc.
[0051] FIG. 20C shows flow cytometry results of representative IgG1
clones selected on directly coated CD200-Fc.
[0052] FIG. 20D shows flow cytometry results of representative
IgG2a clones selected on directly coated CD200-Fc.
[0053] FIG. 21A shows deduced amino acid sequence of heavy chain
complementarity regions of CD200-specific clones.
[0054] FIG. 21B shows deduced amino acid sequence of heavy chain
complementarity regions of CD200-specific clones.
[0055] FIG. 22 shows ability of selected clones to block the
interaction of CD200 with its receptor (CD200R) in a fluorescent
bead assay.
[0056] FIG. 23 shows deduced amino acid sequences of selected CD200
Fabs for chimerization.
[0057] FIG. 24 shows ELISA results of chimeric IgG obtained from
the culture supernatant of a small-scale transient
transfection.
[0058] FIG. 25 shows bead inhibition assay results on purified IgG
showing that all antibodies directed against CD200 blocked the
receptor ligand interaction very well.
[0059] FIGS. 26A and 26B show that the presence of CLL cells
completely abrogated IFN-gamma and most of IL-2 production observed
in the mixed lymphocyte reaction but that the presence of any of
the antibodies allowed for production of these Th1 cytokines.
[0060] FIG. 26C shows that IL-10 production was downregulated in
the presence of the antibodies.
[0061] FIG. 27 shows the ability to kill CD200 expressing tumor
cells in an antibody-dependent cell-mediated cytotoxicity assay
(ADCC). All of the mouse chimeric CD200 antibodies produced similar
levels of lysis when cultured with CD200 positive cells.
[0062] FIG. 28 shows a representative example of 10 experiments
using different PBL donors compared to a group that received tumor
cells only, analyzed by a 2-tailed unpaired Student's t-test.
Significant differences were observed in the groups that received 5
or 10 million PBLs, but not in the group that received 1 million
PBLs from Day 32 on.
[0063] FIG. 29 shows a representative example of 10 experiments
using different PBL donors compared to a group that received tumor
cells only, analyzed by 2-tailed unpaired Student's t-test.
Significant differences were observed in the groups that received
10 million PBLs for both donors, but not in the group that received
2 million PBLs from Day 8 on.
[0064] FIG. 30(a) shows RAJI cells infected with CD200 expressing
tumor cells transduced from a lentivirus vector appeared to grow
somewhat more slowly than its parental RAJI cells. The growth
difference between the transduced and parental cells did not reach
statistical significance.
[0065] FIG. 30(b) shows the presence of PBLs reduced tumor growth
in the RAJI cells transduced with the reversed CD200 (non
functional CD200) by up to 84% when 5 or 10.times.10.sup.6 PBLs
were injected indicating that this particular donor rejects RAJI
tumor cells very strongly.
[0066] FIG. 30(c) shows results indicating that CD200 expression on
tumor cells does indeed prevent the immune system from slowing
tumor growth. Also this study demonstrates the usefulness of the
RAJI/PBL model to assess immunosuppressive compounds or
molecules.
[0067] FIGS. 31 (a)-(d) show whether the effects seen in the
RAJI/PBL model can also be observed with other tumor cell
models.
[0068] FIG. 31(a) shows Namalwa tumor cells resulted in rapid tumor
growth with no significant difference between transduced and
parental cells.
[0069] FIG. 31(b) shows the presence of PBLs slowed tumor growth by
about 50%. The 2-tailed Student's t-test results showed that the
differences between the PBL treated group versus groups that
received only tumor cells were statistically significant.
[0070] FIGS. 31 (c) and (d) show tumor growth in the groups that
received CD200 expressing Namalwa cells and PBLs was similar to the
tumor growth in the group that received Namalwa cells. These data
confirm that CD200 expression on tumor cells prevents slowing of
tumor growth by the human immune system.
[0071] FIG. 32 shows results demonstrating that the RAJI PBL model
is an efficient way to assess efficacy of immune-enhancing
compounds.
[0072] FIG. 33 shows that CD200 expression on the tumor cells
prevented the immune cells from reducing tumor growth.
[0073] FIG. 34 shows that CD200 expression on the tumor cells
prevented the immune cells from reducing tumor growth.
DETAILED DESCRIPTION OF PREFERRED EMBODIMENTS
[0074] In accordance with the present disclosure, methods are
provided for determining whether OX-2/CD200 is upregulated in a
subject and, if so, administering to the subject a therapy that
enhances immune response. Illustrative examples of suitable
immunomodulatory therapies include the administration of agents
that block negative regulation of T cells or antigen presenting
cells (e.g., anti-CTLA4 antibodies, anti-PD-L1 antibodies,
anti-PDL-2 antibodies, anti-PD-1 antibodies and the like) or the
administration of agents that enhance positive co-stimulation of T
cells (e.g., anti-CD40 antibodies or anti 4-1BB antibodies) or
administration of agents that increase NK cell number or T-cell
activity (e.g., anti-CD200 antibodies alone or in combination with
inhibitors such as IMiDs, thalidomide, or thalidomide analogs).
Furthermore, immunomodulatory therapy could be cancer vaccines such
as dendritic cells loaded with tumor cells, tumor RNA or tumor DNA,
tumor protein or tumor peptides, patient derived heat-shocked
proteins (hsp's) or general adjuvants stimulating the immune system
at various levels such as CpG, Luivac, Biostim, Ribominyl, Imudon,
Bronchovaxom or any other compound activating receptors of the
innate immune system (e.g., toll like receptors). Also,
immunomodulatory therapy could include treatment with cytokines
such as IL-2, GM-CSF and IFN-gamma.
[0075] In particularly useful embodiments, the therapy that
enhances immune response is the administration of a polypeptide
that binds to OX-2/CD200, alone or in combination with one of the
previously mentioned immunomodulatory therapies. In general, the
polypeptides utilized in the present disclosure can be constructed
using different techniques which are known to those skilled in the
art. In one embodiment, the polypeptides are obtained by chemical
synthesis. In other embodiments, the polypeptides are antibodies or
constructed from a fragment or several fragments of one or more
antibodies.
[0076] Preferably, the polypeptides utilized in the methods of the
present disclosure are obtained from a CLL cell line. "CLL", as
used herein, refers to chronic lymphocytic leukemia involving any
lymphocyte including, but not limited to, various developmental
stages of B cells and T cells including, but not limited to, B cell
CLL ("B-CLL"). B-CLL, as used herein, refers to leukemia with a
mature B cell phenotype which is CD5.sup.+, CD23.sup.+,
CD20.sup.dim+, sIg.sup.dim+ and arrested in G0/G1 of the cell
cycle. In a further aspect, the CLL cell line is used to generate
polypeptides, including antibodies, useful in the diagnosis and/or
treatment of a disease state in which OX-2/CD200 is upregulated,
including cancer and CLL.
[0077] As used herein, the term "antibodies" refers to complete
antibodies or antibody fragments capable of binding to a selected
target. Included are Fab, Fv, scFv, Fab' and F(ab')2, monoclonal
and polyclonal antibodies, engineered antibodies (including
chimeric, CDR-grafted and humanized, fully human antibodies, and
artificially selected antibodies), and synthetic or semi-synthetic
antibodies produced using phage display or alternative techniques.
Small fragments, such as Fv and scFv, possess advantageous
properties for diagnostic and therapeutic applications on account
of their small size and consequent superior tissue
distribution.
[0078] Antibodies may be generated by using the cells as disclosed
herein as immunogens, thus raising an immune response in animals
from which monoclonal antibodies may be isolated. The sequence of
such antibodies may be determined and the antibodies or variants
thereof produced by recombinant techniques. In this aspect,
"variants" includes chimeric, CDR-grafted, humanized and fully
human antibodies based on the sequence of the monoclonal
antibodies, as well as polypeptides capable of binding to
OX-2/CD200.
[0079] Moreover, antibodies derived from recombinant libraries
("phage antibodies") may be selected using the cells described
herein, or polypeptides derived therefrom, as bait to isolate the
antibodies or polypeptides on the basis of target specificity.
[0080] In a still further aspect, antibodies or polypeptides may be
generated by panning antibody libraries using primary CLL cells, or
antigens derived therefrom, and further screened and/or
characterized using a CLL cell line, such as, for example, the CLL
cell line described herein. Accordingly, a method for
characterizing an antibody or polypeptide specific for CLL is
provided, which includes assessing the binding of the antibody or
polypeptide to a CLL cell line.
Preparation of Cell Lines
[0081] Cell lines may be produced according to established
methodologies known to those skilled in the art. In general, cell
lines are produced by culturing primary cells derived from a
patient until immortalized cells are spontaneously generated in
culture. These cells are then isolated and further cultured to
produce clonal cell populations or cells exhibiting resistance to
apoptosis.
[0082] For example, CLL cells may be isolated from peripheral blood
drawn from a patient suffering from CLL. The cells may be washed,
and optionally immunotyped in order to determine the type(s) of
cells present. Subsequently, the cells may be cultured in a medium,
such as a medium containing IL-4. Advantageously, all or part of
the medium is replaced one or more times during the culture
process. Cell lines may be isolated thereby, and will be identified
by increased growth in culture.
[0083] In one embodiment a CLL cell line of malignant origin is
provided that is not established by immortalization with EBV.
"Malignant origin" refers to the derivation of the cell line from
malignant CLL primary cells, as opposed to non-proliferating cells
which are transformed, for example, with EBV. Cell lines useful
according to this disclosure may be themselves malignant in
phenotype, or not. A CLL cell having a "malignant" phenotype
encompasses cell growth unattached from substrate media
characterized by repeated cycles of cell growth and exhibits
resistance to apoptosis. The cell line, which was derived from
primary CLL cells, is deposited under ATCC accession no. PTA-3920.
In a preferred embodiment, the cell line is CLL-AAT. CLL-AAT is a
B-CLL cell line, derived from a B-CLL primary cell.
[0084] In one embodiment, proteins uniquely expressed in CLL cells
are identified employing the CLL-AAT cell line by methods well
known to those skilled in the art, such as by immunoprecipitation
followed by mass spectroscopy analyses. Such proteins may be
uniquely expressed in the CLL-AAT cell line, or in primary cells
derived from CLL patients.
[0085] Small molecule libraries (many available commercially) may
be screened using the CLL-AAT cell line in a cell-based assay to
identify agents capable of modulating the growth characteristics of
the cells. For example, the agents may be identified which modulate
apoptosis in the CLL-AAT cell line, or which inhibit growth and/or
proliferation thereof. Such agents are candidates for the
development of therapeutic compounds.
[0086] Nucleic acids isolated from CLL-AAT cell lines may be used
in subtractive hybridization experiments to identify CLL-specific
genes or in micro array analyses (e.g., gene chip experiments).
Genes whose transcription is modulated in CLL cells may be
identified. Polypeptide or nucleic acid gene products identified in
this manner are useful as leads for the development of antibody or
small molecule therapies for CLL.
[0087] In one embodiment, the CLL-AAT cell line may be used to
identify internalizing antibodies, which bind to cell surface
components and are then internalized by the cell. Such antibodies
are candidates for therapeutic use. In particular, single-chain
antibodies, which remain stable in the cytoplasm and which retain
intracellular binding activity, may be screened in this manner.
Preparation of Monoclonal Antibodies
[0088] Recombinant DNA technology may be used to improve the
antibodies produced in accordance with this disclosure. Thus,
chimeric antibodies may be constructed in order to decrease the
immunogenicity thereof in diagnostic or therapeutic applications.
Moreover, immunogenicity may be minimized by humanizing the
antibodies by CDR grafting and, optionally, framework modification.
See, U.S. Pat. No. 5,225,539, the contents of which are
incorporated herein by reference.
[0089] Antibodies may be obtained from animal serum, or, in the
case of monoclonal antibodies or fragments thereof produced in cell
culture. Recombinant DNA technology may be used to produce the
antibodies according to established procedure, in bacterial or
preferably mammalian cell culture. The selected cell culture system
preferably secretes the antibody product.
[0090] 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 polycistronic,
for example bicistronic, DNA sequences encoding antibody proteins
each individually operably linked to a signal peptide in the proper
reading frame.
[0091] 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.
[0092] In vitro production provides relatively pure antibody
preparations and allows scale-up 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 fibres, microcapsules, on agarose
microbeads or ceramic cartridges).
[0093] 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 pristine. After one to two weeks,
ascitic fluid is taken from the animals.
[0094] 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, for example WO97/08320; U.S. Pat. No.
5,427,908; U.S. Pat. No. 5,508,717; Smith, 1985, Science, Vol. 225,
pp 1315-1317; Parmley and Smith, 1988, Gene 73, pp 305-318; De La
Cruz et al., 1988, Journal of Biological Chemistry, 263 pp
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; Taylor, et al., Nucleic Acids
Research 20 (1992): 6287-6295; Tomizuka et al., Proc. Natl. Academy
of Sciences USA 97(2) (2000): 722-727. The contents of all these
references are incorporated herein by reference.
[0095] The cell culture supernatants are screened for the desired
antibodies, preferentially by immunofluorescent staining of CLL
cells, by immunoblotting, by an enzyme immunoassay, e.g. a sandwich
assay or a dot-assay, or a radioimmunoassay.
[0096] 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 DEAE-cellulose and/or (immuno-)
affinity chromatography, e.g. affinity chromatography with one or
more surface polypeptides derived from a CLL cell line according to
this disclosure, or with Protein-A or G.
[0097] Another embodiment provides a process for the preparation of
a bacterial cell line secreting antibodies directed against the
cell line characterized in that a suitable mammal, for example a
rabbit, is immunized with pooled CLL patient samples. 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, for example, the methods disclosed
in the various references incorporated herein by reference).
[0098] Hybridoma cells secreting the monoclonal antibodies are also
contemplated. The preferred hybridoma cells are genetically stable,
secrete monoclonal antibodies described herein of the desired
specificity and can be activated from deep-frozen cultures by
thawing and recloning.
[0099] In another embodiment, a process is provided for the
preparation of a hybridoma cell line secreting monoclonal
antibodies directed to the CLL cell line is described herein. In
that process, a suitable mammal, for example a Balb/c mouse, is
immunized with one or more polypeptides or antigenic fragments
thereof derived from a cell described in this disclosure, the cell
line 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
the present cell line are fused with cells of the myeloma cell line
PAI or the myeloma cell line Sp2/0-Ag 14, the obtained hybrid cells
are screened for secretion of the desired antibodies, and positive
hybridoma cells are cloned.
[0100] Preferred is a process for the preparation of a hybridoma
cell line, characterized in that Balb/c mice are immunized by
injecting subcutaneously and/or intraperitoneally between 10.sup.6
and 10.sup.7 cells of a cell line in accordance with this
disclosure 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 hereinbefore, 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.
[0101] In a further embodiment, recombinant DNA comprising an
insert coding for a heavy chain variable domain and/or for a light
chain variable domain of antibodies directed to the cell line
described hereinbefore are 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.
[0102] Furthermore, DNA encoding a heavy chain variable domain
and/or a light chain variable domain of antibodies directed to the
cell line disclosed herein can be enzymatically or chemically
synthesized DNA having 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 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.
[0103] 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.
[0104] 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.
[0105] Recombinant DNAs including an insert coding for a heavy
chain murine variable domain of an antibody directed to the cell
line disclosed herein fused to a human constant domain g, for
example .gamma.1, .gamma.2, .gamma.3 or .gamma.4, preferably
.gamma.1 or .gamma.4 are also provided. Recombinant DNAs including
an insert coding for a light chain murine variable domain of an
antibody directed to the cell line disclosed herein fused to a
human constant domain .kappa. or .lamda. preferably .kappa. are
also provided
[0106] 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 coding for
a peptide facilitating the purification of the antibody and/or a
cleavage site and/or a peptide spacer and/or an effector
molecule.
[0107] The DNA coding for an effector molecule is intended to be a
DNA coding for the effector molecules useful in diagnostic or
therapeutic applications. Thus, effector molecules which are toxins
or enzymes, especially enzymes capable of catalyzing the activation
of prodrugs, are particularly indicated. The DNA encoding such an
effector molecule 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.
Uses of the Present Antibodies/Polypeptides
[0108] The polypeptides and/or antibodies utilized herein are
especially indicated for diagnostic and therapeutic
applications.
[0109] The present antibodies can be administered as a therapeutic
to cancer patients, especially, but not limited to CLL patients. In
some embodiments, the antibodies are capable of interfering with
the interaction of CD200 and its receptors. This interference can
block the immune suppressing effect of CD200. By improving the
immune response in this manner, such antibodies can promote the
eradication of cancer cells.
[0110] The anti-CD200 antibody can also be administered in
combination with other immunomodulatory compounds, vaccines or
chemotherapy. For example, elimination of existing regulatory T
cells with reagents such as anti-CD25 or cyclophosphamide is
achieved in one particularly useful embodiment before starting
anti-CD200 treatment. Also, 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 cells or proteins, peptides or RNA derived
from such cells, patient-derived heat-shocked proteins (hsp's),
tumor peptides or protein. In other embodiments, an anti-CD200
antibody is 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 yet other
embodiments, efficacy of anti-CD200 treatment is improved by
blocking of immunosuppressive mechanisms such as anti-PDL1 and/or 2
antibodies, anti-IL-10 antibodies, anti-IL-6 antibodies, and the
like. In yet other embodiments, efficacy of anti-CD200 treatment is
improved by administration of agents that increase NK cell number
or T-cell such as the small molecule inhibitor IMiDs, thalidomide,
or thalidomide analogs).
[0111] Anti-CD200 antibodies in accordance with the present
disclosure can also be used as a diagnostic tool. For example,
using blood obtained from patients with hematopoietic cancers,
expression of CD200 can be evaluated on cancer cells by FACS
analysis using anti-CD200 antibodies in combination with the
appropriate cancer cell markers such as, e.g., CD38 and CD19 on CLL
cells. Patients with CD200 levels at least 1.4-fold above the
levels found on normal B cells can be selected for treatment with
anti-CD200 antibodies.
[0112] In another example of using the present anti-CD200
antibodies as a diagnostic tool, biopsies from patients with
malignancies are obtained and expression of CD200 is determined by
FACS analysis using anti-CD200 antibodies. If tumor cells express
CD200 at levels that are at least 1.4-fold higher compared to
corresponding normal tissue, cancer patients are selected for
immunomodulatory therapy. Immunomodulatory therapy can be
anti-CD200 therapy, but can also be any other therapy affecting the
patient's immune system. Examples of suitable immunomodulatory
therapies include the administration of agents that block negative
regulation of T cells or antigen presenting cells (e.g.,
anti-CTLA4, anti-PD-L1, anti-PDL-2, anti-PD-1) or the
administration of agents that enhance positive co-stimulation of T
cells (e.g., anti-CD40 or anti 4-1BB). Furthermore,
immunomodulatory therapy could be the administration of agents that
increase NK cell number or T-cell activity (e.g., anti-CD200
antibodies alone or in combination with inhibitors such as IMiDs,
thalidomide, or thalidomide analogs) or the administration of
agents that deplete regulatory T cells (e.g. anti-CD200 antibodies
alone or in combination with ONTAK). Furthermore, immunomodulatory
therapy could be cancer vaccines such as dendritic cells loaded
with tumor cells, tumor RNA or tumor DNA, tumor protein or tumor
peptides, patient derived heat-shocked proteins (hsp's) or general
adjuvants stimulating the immune system at various levels such as
CpG, Luivac, Biostim, Ribominyl, Imudon, Bronchovaxom or any other
compound activating receptors of the innate immune system (e.g.,
toll like receptors). Also, therapy could include treatment with
cytokines such as IL-2, GM-CSF and IFN-gamma.
[0113] In another embodiment in accordance with the present
disclosure, methods are provided for monitoring the progress and/or
effectiveness of a therapeutic treatment. The method involves
administering an immunomodulatory therapy and determining
OX-2/CD200 levels in a subject at least twice to determine the
effectiveness of the therapy. For example, pre-treatment levels of
OX-2/CD200 can be ascertained and, after at least one
administration of the therapy, levels of OX-2/CD200 can again be
determined. A decrease in OX-2/CD200 levels is indicative of an
effective treatment. Measurement of OX-2/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
OX-2/CD200 levels can be directly monitored or, alternatively, any
marker that correlates with OX-2/CD200 can be monitored.
[0114] The present antibodies also may be utilized to detect
cancerous cells in vivo. This is achieved by labeling the antibody,
administering the labeled antibody to a subject, and then imaging
the subject. Examples of labels useful for diagnostic imaging in
accordance with the present disclosure are radiolabels such as
.sup.131I, .sup.111In, .sup.123I, .sup.99mTC, .sup.32P, .sup.125I,
.sup.3H, .sup.14C, and .sup.188Rh, fluorescent labels such as
fluorescein and rhodamine, nuclear magnetic resonance active
labels, positron emitting isotopes detectable by a positron
emission tomography ("PET") scanner, chemiluminescers such as
luciferin, and enzymatic markers such as peroxidase or phosphatase.
Short-range radiation emitters, such as isotopes detectable by
short-range detector probes, such as a transrectal probe, can also
be employed. The antibody can be labeled with such reagents using
techniques known in the art. For example, see Wensel and Meares,
Radioimmunoimaging and Radioimmunotherapy, Elsevier, N.Y. (1983),
which is hereby incorporated by reference, for techniques relating
to the radiolabeling of antibodies. See also, D. Colcher et al.,
"Use of Monoclonal Antibodies as Radiopharmaceuticals for the
Localization of Human Carcinoma Xenografts in Athymic Mice", Meth.
Enzymol. 121: 802-816 (1986), which is hereby incorporated by
reference.
[0115] A radiolabeled antibody in accordance with this disclosure
can be used for in vitro diagnostic tests. The specific activity of
an antibody, binding portion thereof, probe, or ligand, depends
upon the half-life, the isotopic purity of the radioactive label,
and how the label is incorporated into the biological agent. In
immunoassay tests, the higher the specific activity, in general,
the better the sensitivity. Procedures for labeling antibodies with
the radioactive isotopes are generally known in the art.
[0116] The radiolabeled antibody can be administered to a patient
where it is localized to cancer cells bearing the antigen with
which the antibody reacts, and is detected or "imaged" in vivo
using known techniques such as radionuclear scanning using e.g., a
gamma camera or emission tomography. See e.g., A. R. Bradwell et
al., "Developments in Antibody Imaging", Monoclonal Antibodies for
Cancer Detection and Therapy, R. W. Baldwin et al., (eds.), pp.
65-85 (Academic Press 1985), which is hereby incorporated by
reference. Alternatively, a positron emission transaxial tomography
scanner, such as designated Pet VI located at Brookhaven National
Laboratory, can be used where the radiolabel emits positrons (e.g.,
.sup.11C, .sup.18F, .sup.15O, and .sup.13N).
[0117] Fluorophore and chromophore labeled biological agents can be
prepared from standard moieties known in the art. Since antibodies
and other proteins absorb light having wavelengths up to about 310
nm, the fluorescent moieties should be selected to have substantial
absorption at wavelengths above 310 nm and preferably above 400 nm.
A variety of suitable fluorescers and chromophores are described by
Stryer, Science, 162:526 (1968) and Brand, L. et al., Annual Review
of Biochemistry, 41:843-868 (1972), which are hereby incorporated
by reference. The antibodies can be labeled with fluorescent
chromophore groups by conventional procedures such as those
disclosed in U.S. Pat. Nos. 3,940,475, 4,289,747, and 4,376,110,
which are hereby incorporated by reference.
[0118] In other embodiments, 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 cancer cell,
the other one is for any other antigen, and preferably for a
cell-surface protein or receptor or receptor subunit.
[0119] 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,
Nature, 305:537-539 (1983)). 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, CH2, and CH3 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, for example,
Suresh et al., Methods in Enzymology, 121:210 (1986); WO 96/27011;
Brennan et al., Science 229:81 (1985); Shalaby et al., J. Exp. Med.
175:217-225 (1992); Kostelny et al., J. Immunol. 148(5):1547-1553
(1992); Hollinger et al., Proc. Natl. Acad. Sci. USA 90:6444-6448
(1993); and Gruber et al., J. Immunol. 152:5368 (1994); and Tutt et
al., J. Immunol. 147:60 (1991).
[0120] The present antibodies can also be utilized to directly kill
or ablate cancerous cells in vivo. This involves administering the
antibodies (which are optionally fused to a cytotoxic drug) to a
subject requiring such treatment. Since the antibodies recognize
CD200 on cancer cells, any such cells to which the antibodies bind
are destroyed.
[0121] Where the antibodies are used alone to kill or ablate cancer
cells, such killing or ablation can be effected by initiating
endogenous host immune functions, such as complement-mediated or
antibody-dependent cellular cytotoxicity. Assays for determining
whether an antibody kills cells in this manner are within the
purview of those skilled in the art.
[0122] The antibodies of the present disclosure may be used to
deliver a variety of cytotoxic compounds. Any cytotoxic compound
can be fused to the present antibodies. The fusion can be achieved
chemically or genetically (e.g., via expression as a single, fused
molecule). The cytotoxic compound can be a biological, such as a
polypeptide, or a small molecule. As those skilled in the art will
appreciate, for small molecules, chemical fusion is used, while for
biological compounds, either chemical or genetic fusion can be
employed.
[0123] Non-limiting examples of cytotoxic compounds include
therapeutic drugs, a compound emitting radiation, molecules of
plants, fungal, or bacterial origin, biological proteins, and
mixtures thereof. The cytotoxic drugs can be intracellularly acting
cytotoxic drugs, such as short-range radiation emitters, including,
for example, short-range, high-energy .alpha.-emitters.
Enzymatically active toxins and fragments thereof are exemplified
by diphtheria toxin A fragment, nonbinding active fragments of
diphtheria toxin, exotoxin A (from Pseudomonas aeruginosa), ricin A
chain, abrin A chain, modeccin A chain, .alpha.-sacrin, certain
Aleurites fordii proteins, certain Dianthin proteins, Phytolacca
americana proteins (PAP, PAPII and PAP-S), Morodica charantia
inhibitor, curcin, crotin, Saponaria officinalis inhibitor,
gelonin, mitogillin, restrictocin, phenomycin, and enomycin, for
example. Procedures for preparing enzymatically active polypeptides
of the immunotoxins are described in WO84/03508 and WO85/03508,
which are hereby incorporated by reference. Certain cytotoxic
moieties are derived from adriamycin, chlorambucil, daunomycin,
methotrexate, neocarzinostatin, and platinum, for example.
[0124] Procedures for conjugating the antibodies with the cytotoxic
agents have been previously described and are within the purview of
one skilled in the art.
[0125] Alternatively, the antibody can be coupled to high energy
radiation emitters, for example, a radioisotope, such as .sup.131I,
a .gamma.-emitter, which, when localized at the tumor site, results
in a killing of several cell diameters. See, e.g., S. E. Order,
"Analysis, Results, and Future Prospective of the Therapeutic Use
of Radiolabeled Antibody in Cancer Therapy", Monoclonal Antibodies
for Cancer Detection and Therapy, R. W. Baldwin et al. (eds.), pp
303-316 (Academic Press 1985), which is hereby incorporated by
reference. Other suitable radioisotopes include .alpha.-emitters,
such as .sup.212Bi, .sup.213Bi, and .sup.211At, and
.beta.-emitters, such as .sup.186Re and .sup.90Y.
[0126] The route of antibody administration of the present
antibodies (whether the pure antibody, a labeled antibody, an
antibody fused to a toxin, etc.) is in accord with known methods,
e.g., injection or infusion by intravenous, intraperitoneal,
intracerebral, intramuscular, subcutaneous, intraocular,
intraarterial, intrathecal, inhalation or intralesional routes, or
by sustained release systems. The antibody is preferably
administered continuously by infusion or by bolus injection. One
may administer the antibodies in a local or systemic manner.
[0127] The present antibodies may be prepared in a mixture with a
pharmaceutically acceptable carrier. Techniques for formulation and
administration of the compounds of the instant application may be
found in "Remington's Pharmaceutical Sciences," Mack Publishing
Co., Easton, Pa., latest edition. This therapeutic composition can
be administered intravenously or through the nose or lung,
preferably as a liquid or powder aerosol (lyophilized). The
composition may also be administered parenterally or subcutaneously
as desired. When administered systemically, the therapeutic
composition should be sterile, pyrogen-free and in a parenterally
acceptable solution having due regard for pH, isotonicity, and
stability. These conditions are known to those skilled in the
art.
[0128] Pharmaceutical compositions suitable for use include
compositions wherein one or more of the present antibodies are
contained in an amount effective to achieve their intended purpose.
More specifically, a therapeutically effective amount means an
amount of antibody effective to prevent, alleviate or ameliorate
symptoms of disease or prolong the survival of the subject being
treated. Determination of a therapeutically effective amount is
well within the capability of those skilled in the art, especially
in light of the detailed disclosure provided herein.
Therapeutically effective dosages may be determined by using in
vitro and in vivo methods.
[0129] In some embodiments, present CD200 binding antibodies
provide the benefit of blocking immune suppression in CLL by
targeting the leukemic cells directly through CD200. Specifically,
stimulating the immune system can allow the eradication of CLL
cells from the spleen and lymph nodes. Applicants are unaware of
any successful eradication of CLL cells from these
microenvironments having been achieved with agents that simply
target B cells (such as alemtuzumab). In contrast, CLL reactive T
cells can have better access to these organs than antibodies. In
other embodiments, direct cell killing is achieved by tagging the
CLL cells with anti-CD200 Abs.
[0130] In particularly useful embodiments, the combination of
direct cell killing and driving the immune response towards a Th1
profile provides a particularly powerful approach to 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
antibody-dependent cellular cytotoxicity (ADCC) or complement
dependent cytotoxicity (CDC); fusion with a toxin; fusion with a
radiolabel; fusion with a biological agent involved in cell
killing, such as granzyme B or perforin; fusion with a cytotoxic
virus; 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 an anti-CD200 antibody in combination
with inhibitors such as IMiDs, thalidomide, or thalidomide
analogs.
[0131] 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, IP-10, TAC, CCL21, CCL5 or 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.
[0132] While the above disclosure has been directed to antibodies,
in some embodiments polypeptides derived from such antibodies can
be utilized in accordance with the present disclosure.
Uses of the CLL Cell Line
[0133] There are many advantages to the development of a CLL cell
line, as it provides an important tool for the development of
diagnostics and treatments for CLL, cancer, and other disease
states characterized by upregulated levels of OX-2/CD200, e.g.,
melanoma.
[0134] A cell line according to this disclosure may be used for in
vitro studies on the etiology, pathogenesis and biology of CLL and
other disease states characterized by upregulated levels of
OX-2/CD200. This assists in the identification of suitable agents
that are useful in the therapy of these diseases.
[0135] The cell line may also be used to produce polypeptides
and/or monoclonal antibodies for in vitro and in vivo diagnosis of
CLL, cancer, and other disease states characterized by upregulated
levels of OX-2/CD200 (e.g., melanoma), as referred to above, and
for the screening and/or characterization of antibodies produced by
other methods, such as by panning antibody libraries with primary
cells and/or antigens derived from CLL patients.
[0136] The cell line may be used as such, or antigens may be
derived therefrom. Advantageously, such antigens are cell-surface
antigens specific for CLL. They may be isolated directly from cell
lines according to this disclosure. Alternatively, a cDNA
expression library made from a cell line described herein may be
used to express CLL-specific antigens, useful for the selection and
characterization of anti-CLL antibodies and the identification of
novel CLL-specific antigens.
[0137] Treatment of CLL using monoclonal antibody therapy has been
proposed in the art. Recently, Hainsworth (Oncologist 5 (5) (2000)
376-384) has described the current therapies derived from
monoclonal antibodies. Lymphocytic leukemia in particular is
considered to be a good candidate for this therapeutic approach due
to the presence of multiple lymphocyte-specific antigens on
lymphocyte tumors.
[0138] Existing antibody therapies (such as Rituximab.TM., directed
against the CD20-antigen, which is expressed on the surface of
B-lymphocytes) have been used successfully against certain
lymphocytic disease. However, a lower density CD20 antigen is
expressed on the surface of B-lymphocytes in CLL (Almasri et al.,
Am. J. Hematol., 40 (4) (1992) 259-263).
[0139] The CLL cell line described herein thus permits the
development of novel anti-CLL antibodies and polypeptides having
specificity for one or more antigenic determinants of the present
CLL cell line, and their use in the therapy and diagnosis of CLL,
cancer, and other disease states characterized by upregulated
levels of OX-2/CD200.
[0140] The antibody or polypeptide may bind to a receptor with
which OX-2/CD200 normally interacts, thereby preventing or
inhibiting OX-2/CD200 from binding to the receptor. As yet another
alternative, the antibody can bind to an antigen that modulates
expression of OX-2/CD200, thereby preventing or inhibiting normal
or increased expression of OX-2/CD200. Because the presence of
OX-2/CD200 has been associated with reduced immune response, it
would be desirable to interfere with the metabolic pathway of
OX-2/CD200 so that the patient's immune system can defend against
the disease state, such as cancer or CLL, more effectively.
[0141] In a particularly useful embodiment, the polypeptide binds
to OX-2/CD200. In one embodiment, the polypeptide can be an
antibody which binds to OX-2/CD200 and prevents or inhibits
OX-2/CD200 from interacting with other molecules or receptors. As
CLL cells and other cells overexpressing OX-2/CD200 greatly
diminish the production of Th1 cytokines, the administration of
anti-CD200 antibody or a polypeptide which binds to OX-2/CD200 to a
subject having upregulated levels of OX-2/CD200 restores the Th1
cytokine profile. Thus, these polypeptides and/or antibodies can be
useful therapeutic agents in the treatment of CLL and other cancers
or diseases overexpressing OX-2/CD200.
[0142] Thus, in another embodiment, the method of the present
disclosure includes the steps of screening a subject for the
presence OX-2/CD200 and administering a polypeptide that binds to
OX-2/CD200. It should of course be understood that the presence of
OX-2/CD200 can be directly monitored or, alternatively, any marker
that correlates with OX-2/CD200 can be detected. In a particularly
useful embodiment, a CLL patient is screened for overexpression of
OX-2/CD200 and an antibody that binds to OX-2/CD200 is administered
to the patient. One such antibody is the commercially available
anti-CD200 antibody from Serotec Inc. (3200 Atlantic Ave, Suite
105, Raleigh, N.C. 27604). As described in detail below, another
such antibody is scFv-9 (see FIG. 9B) which binds to
OX-2/CD200.
[0143] In another aspect, the present disclosure provides methods
for assessing the immunomodulatory effect of molecules expressed by
cancer cells. In these methods a molecule that is expressed or
upregulated by a cancer cell is first identified. The molecule can
be identified from a database or experimentally. Databases that
identify molecules that are expressed or upregulated by cancer
cells are known and include, for example, the NCI60 cancer
microarray project (Ross et al., Nature Genetics 24: 227-34, 2000),
the Carcinoma classification (Andrew I. Su et al., "Molecular
Classification of Human Carcinomas by Use of Gene Expression
Signatures." Cancer Research 61:7388-7393, 2001), and the
Lymphoma/Leukemia molecular profiling project (Alizadeh et al.,
Nature 403: 503-11, 2000).
[0144] Experimental methods useful for identifying molecules that
are expressed or upregulated by cancer cells are also known and
include, for example microarray experiments, quantitative PCR,
FACS, and Northern analysis.
[0145] Cancer cells, lymphocytes and the previously identified
molecule that is expressed by a cancer cell are administered to a
subject and the rate of growth of the cancer cells is monitored.
Any type of cancer cells can be employed in the present methods. In
some embodiments, the cancer cells express an immunosuppressive
compound. In particularly useful embodiments, the cancer cells
express or even overexpress CD200. Suitable cancer cells include,
but are not limited to lymphoma cell lines such as the RAJI or
Namalwa cell lines. The amount of cancer cells administered may
range from about 1.times.10.sup.6 to about 20.times.10.sup.6.
[0146] Any type of lymphocyte may be employed in the present
process. Suitable lymphocytes include, for example, PBLs, T cells,
cytotoxic T cells, dendritic cells or NK cells. In particularly
useful embodiments, the lymphocytes are human lymphocytes,
specifically human PBLs. The number of lymphocytes administered is
predetermined to be either a) sufficient to slow the growth of the
cancer cells or b) insufficient to slow the growth of cancer cells.
The amount of lymphocytes administered may be greater than or equal
to the number of cancer cells administered when the number of
lymphocytes administered is intended to be sufficient to slow the
growth of the cancer cells. In embodiments, the amount of
lymphocytes administered may be from about 5.times.10.sup.6 to
about 10.times.10.sup.6 when the number of lymphocytes administered
is intended to be sufficient to slow the growth of the cancer
cells. The amount of lymphocytes administered may be less than the
number of cancer cells administered when the number of lymphocytes
administered is intended to be insufficient to slow the growth of
the cancer cells. In embodiments, the amount of lymphocytes
administered may range from about 1.times.10.sup.6 to about
4.times.10.sup.6 when the number of lymphocytes administered is
intended to be insufficient to slow the growth of the cancer cells.
The foregoing amounts are illustrative and other suitable amounts
may be experimentally determined and used in the present
methods.
[0147] The molecule that is expressed or upregulated by a cancer
cell can be administered as the molecule or the active portion
thereof itself (either isolated or recombinantly generated), or by
administering cells that produce the molecule naturally, or by
administering cells that have been engineered to produce the
molecule or portions thereof. Methods for engineering cells to
express a desired molecule are known to those skilled in the art.
The amount of molecule administered can be any amount above the
amount found in a healthy individual. For example, the amount of
the molecule administered can be from about 1.4-fold above what is
found in a healthy individual in the same type of cell to about
10,000-fold above what is found in a healthy individual in the same
type of cell. It should be understood that the molecule being
assessed may or may not be known to have some degree of
immunomodulatory activity. Thus, the present methods may be used to
confirm the immunomodulatory effect of a molecule as well as to
determine such activity ab initio.
[0148] Any small animal may be chosen as the subject to which the
cancer cells, lymphocytes and the molecule are administered. The
subject may advantageously be immunocompromised. Suitable small
animals include, for example, immunodeficient mice, irradiated
rats, irradiated guinea pigs and the like.
[0149] The rate of cancer cell growth can be monitored using
conventional techniques. For example, tumor growth can be monitored
by measuring length and width with a caliper. Tumor volume can be
calculated, for example, based on multiplying the length of the
tumor by the width of the tumor and then multiplying by one-half
the width of the tumor. The rate of cancer cell growth can be
measured periodically, such as, for example, three times a week.
The rate of growth of cancer cells when cancer cells and
lymphocytes alone have been administered may be determined by
administering cancer cells and lymphocytes alone to a control
subject and periodically measuring tumor size. Alternatively,
cancer cells and lymphocytes alone can be administered initially,
and once a baseline growth rate is established, the molecule that
is expressed or upregulated by a cancer cell can be subsequently
administered to the subject and the rate of growth of the cancer
cells after the second administration can be measured.
Alternatively, with systemic models the rate of cancer cell growth
can be monitored using FACS, survival or other conventional
techniques.
[0150] If any change in the growth rate of the cancer cells is
observed compared to the rate of growth when the tumor cells and
donor lymphocytes are administered alone, the molecule is deemed to
have an immunomodulatory effect. If the number of lymphocytes
administered is sufficient to slow the growth of the cancer cells
and the rate of growth of the tumor cells observed is higher
compared to the rate of growth when the tumor cells and donor
lymphocytes are administered alone, the molecule that is expressed
or upregulated by a cancer cell is considered immunosuppressive. If
the number of lymphocytes administered is insufficient to slow the
growth of the cancer cells and the rate of growth of the tumor
cells observed is lowered compared to the rate of growth when the
tumor cells and donor lymphocytes are administered alone, the
molecule is considered immune enhancing. Typically, the rate of
cancer cell growth observed may be about 20 to about 1,000% higher
than the rate of growth when the tumor cells and donor lymphocytes
are administered alone for the immunosuppressing or immune
enhancing effect to be statistically significant.
[0151] Once the immunomodulatory effect of the molecule is
established, compounds that either enhance or inhibit the activity
of the molecule can be identified in accordance with embodiments
described herein. The compound that either enhances or inhibits the
activity of the molecule previously found to have immunomodulatory
effect can be any compound that alters the protein/protein
interaction that provides the immunomodulatory effect. The
enhancing or inhibiting effect can be the result of direct
interaction with the compound expressed or upregulated by the
cancer cell or may be the result of an interaction with other
compounds in the metabolic pathway of the compound expressed or
upregulated by the cancer cell.
[0152] For example, antibodies or functional antibody fragments can
be identified that interact with the molecule previously found to
have an immunomodulatory effect, the receptor with which the
molecule interacts, or some other molecule in the metabolic pathway
of the molecule responsible for the immunomodulatory effect.
Techniques for making antibodies (including antibody libraries) and
screening them for an inhibitory or enhancing effect will be
apparent to those skilled in the art. As another example, small
molecules may be screened for an inhibitory or enhancing effect.
Techniques for screening small molecule libraries for an inhibitory
or enhancing effect will be apparent to those skilled in the
art.
[0153] In another aspect, methods for assessing the
immunomodulatory effect of a compound are contemplated by the
present disclosure. Demonstrating immunomodulatory properties of
compounds or molecules acting on the human immune system is very
challenging in small animal models. Often, the compounds do not act
on the immune system of small animals, requiring reconstitution of
the human immune system in mice. Reconstitution can be accomplished
by grafting of various fetal immune organs into mice or by
injection of human lymphocytes, but none of the models described to
date has proven to be useful for demonstrating immunomodulatory
properties of compounds or molecules. The immune system is believed
to play an important role in eradicating cancer cells. Cancer cells
have found ways to evade the immune system by upregulation of
immunosuppressive receptors.
[0154] The present methods of assessing the immunomodulatory effect
of a compound is accomplished by a model that mimics the graft
versus leukemia effect observed in patients with leukemia that are
infused with donor lymphocytes (e.g., PBLs) resulting in remission
in up to 80% of patients. The method involves administering cancer
cells, lymphocytes and the compound to be assessed to a subject and
the rate of growth of the cancer cells is monitored.
[0155] Any type of cancer cells can be employed in the present
methods. In some embodiments, the cancer cells express an
immunosuppressive compound. In particularly useful embodiments, the
cancer cells express or even overexpress CD200. Suitable cancer
cells include, but are not limited to lymphoma cell lines such as
the RAJI or Namalwa cell lines. The amount of cancer cells
administered may range from about 2.times.10.sup.6 to about
20.times.10.sup.6.
[0156] Any type of lymphocyte may be employed in the present
process. Suitable lymphocytes include, for example, PBLs, dendritic
cells, T cells, cytotoxic T cells, NK cells. In particularly useful
embodiments, the lymphocytes are human lymphocytes, specifically
human PBLs. The number of lymphocytes administered is predetermined
to be either a) sufficient to slow the growth of the cancer cells
or b) insufficient to slow the growth of cancer cells. The amount
of lymphocytes administered may be greater than or equal to the
number of cancer cells administered when the number of lymphocytes
administered is intended to be sufficient to slow the growth of the
cancer cells. In embodiments, the amount of lymphocytes
administered may be from about 5.times.10.sup.6 to about
10.times.10.sup.6 when the number of lymphocytes administered is
intended to be sufficient to slow the growth of the cancer cells.
The amount of lymphocytes administered may be less than the number
of cancer cells administered when the number of lymphocytes
administered is intended to be insufficient to slow the growth of
the cancer cells. In embodiments, the amount of lymphocytes
administered may range from about 1.times.10.sup.6 to about
4.times.10.sup.6 when the number of lymphocytes administered is
intended to be insufficient to slow the growth of the cancer cells.
The foregoing amounts are illustrative and other suitable amounts
may be experimentally determined and used in the present
methods.
[0157] The compound being assessed can be any compound whose
immunomodulatory effect is sought to be determined. Illustrative
examples of compounds include the antibodies and peptides described
herein above. The amount of the compound being assessed
administered may range from about 1 mg/kg to about 200 mg/kg. The
compound to be assessed can be administered as the compound itself,
or by administering cells that produce the compound naturally, or
by administering cells that have been engineered to produce the
compound. It should be understood that the compound being assessed
may or may not be known to have some degree of immunomodulatory
activity. Thus, the present methods may be used to confirm the
immunomodulatory effect of a compound as well as to determine such
activity ab initio.
[0158] Any small animal may be chosen as the subject to which the
cancer cells, lymphocytes and the compound are administered. The
subject may advantageously be immunocompromised. Suitable small
animals include, for example, immunodeficient mice, irradiated
rats, irradiated guinea pigs and the like.
[0159] The rate of cancer cell growth can be monitored using
conventional techniques. For example, tumor growth can be monitored
by measuring length and width with a caliper. Tumor volume can be
calculated, for example, based on multiplying the length of the
tumor by the width of the tumor and then multiplying by one-half
the width of the tumor. The rate of cancer cell growth can be
measured periodically, such as, for example, three times a week.
The rate of growth of cancer cells when cancer cells and
lymphocytes alone have been administered may be determined by
administering cancer cells and lymphocytes alone to a control
subject and periodically measuring tumor size. Alternatively,
cancer cells and lymphocytes alone can be administered initially,
and once a baseline growth rate is established, the compound to be
assessed can be subsequently administered to the subject and the
rate of growth of the cancer cells after the second administration
can be measured.
[0160] When injecting cancer cells such as the lymphoma cell lines
RAJI or Namalwa into immune-deficient mice, administration of five
to 10 million PBLs results in significantly slower tumor growth. In
contrast, low PBL numbers (1-2 million depending on donor) do not
slow tumor growth.
[0161] If any change in the growth rate of the cancer cells is
observed compared to the rate of growth when the tumor cells and
donor lymphocytes are administered alone, the molecule is deemed to
have an immunomodulatory effect. If the number of lymphocytes
administered is sufficient to slow the growth of the cancer cells
and the rate of growth of the tumor cells observed is higher
compared to the rate of growth when the tumor cells and donor
lymphocytes are administered alone, the compound is considered
immunosuppressive. If the number of lymphocytes administered is
insufficient to slow the growth of the cancer cells and the rate of
growth of the tumor cells observed is lowered compared to the rate
of growth when the tumor cells and donor lymphocytes are
administered alone, the compound is considered immune
enhancing.
[0162] In order that those skilled in the art may be better able to
practice the compositions and methods described herein, the
following examples are given for illustration purposes.
Example 1
Isolation of Cell Line CLL-AAT Establishment of the Cell Line
[0163] Peripheral blood from a patient diagnosed with CLL was
obtained. The WBC count was 1.6.times.10.sup.8/ml. Mononuclear
cells were isolated by HISTOPAQUE.RTM.-1077 density gradient
centrifugation (Sigma Diagnostics, St. Louis, Mo.). Cells were
washed twice with Iscove's Modified Dulbecco's Medium (IMDM)
supplemented with 10% heat-inactivated fetal bovine serum (FBS),
and resuspended in 5 ml of ice-cold IMDM/10% FBS. Viable cells were
counted by staining with trypan blue. Cells were mixed with an
equal volume of 85% FBS/15% DMSO and frozen in 1 ml aliquots for
storage in liquid nitrogen.
[0164] Immunophenotyping showed that >90% of the CD45.sup.+
lymphocyte population expressed IgD, kappa light chain, CD5, CD19,
and CD23. This population also expressed low levels of IgM and
CD20. Approximately 50% of the cells expressed high levels of CD38.
The cells were negative for lambda light chain, CD10 and CD138
[0165] An aliquot of the cells was thawed, washed, and resuspended
at a density of 10.sup.7/mL in IMDM supplemented with 20%
heat-inactivated FBS, 2 mM L-glutamine, 100 units/ml penicillin,
100 .mu.g/ml streptomycin, 50 .mu.M 2-mercaptoethanol, and 5 ng/ml
recombinant human IL-4 (R & D Systems, Minneapolis, Minn.). The
cells were cultured at 37.degree. C. in a humidified 5% CO2
atmosphere. The medium was partially replaced every 4 days until
steady growth was observed. After 5 weeks, the number of cells in
the culture began to double approximately every 4 days. This cell
line was designated CLL-AAT.
Characterization of the Cell Line
[0166] Immunophenotyping of the cell line by flow cytometry showed
high expression of IgM, kappa light chain, CD23, CD38, and CD138,
moderate expression of CD19 and CD20, and weak expression of IgD
and CD5. The cell line was negative for lambda light chain, CD4,
CD8, and CD10.
[0167] Immunophenotyping of the cell line was also done by whole
cell ELISA using a panel of rabbit scFv antibodies that had been
selected for specific binding to primary B-CLL cells. All of these
CLL-specific scFv antibodies also recognized the CLL-AAT cell line.
In contrast, the majority of the scFvs did not bind to two cell
lines derived from B cell lymphomas: Ramos, a Burkitt's lymphoma
cell line, and RL, a non-Hodgkin's lymphoma cell line.
Example 2
Selection of scFv Antibodies for B-CLL-Specific Cell Surface
Antigens Using Antibody Phage Display and Cell Surface Panning
[0168] Immunizations and scFv Antibody Library Construction
[0169] Peripheral blood mononuclear cells (PBMC) were isolated from
blood drawn from CLL patients at the Scripps Clinic (La Jolla,
Calif.). Two rabbits were immunized with 2.times.10.sup.7 PBMC
pooled from 10 different donors with CLL. Three immunizations, two
subcutaneous injections followed by one intravenous injection, were
done at three week intervals. Serum titers were checked by
measuring binding of serum IgG to primary CLL cells using flow
cytometry. Five days after the final immunization, spleen, bone
marrow, and PBMC were harvested from the animals. Total RNA was
isolated from these tissues using Tri-Reagent (Molecular Research
Center, Inc). Single-chain Fv (scFv) antibody phage display
libraries were constructed as previously described (Barbas et al.,
(2001) Phage Display: A Laboratory Manual, Cold Spring Harbor
Laboratory Press, Cold Spring Harbor, N.Y.). For cell surface
panning, phagemid particles from the reamplified library were
precipitated with polyethylene glycol (PEG), resuspended in
phosphate-buffered saline (PBS) containing 1% bovine serum albumin
(BSA), and dialysed overnight against PBS.
Antibody Selection by Cell Surface Panning
[0170] The libraries were enriched for CLL cell surface-specific
antibodies by positive-negative selection with a
magnetically-activated cell sorter (MACS) as described by Siegel et
al. (1997, J. Immunol. Methods 206:73-85). Briefly, phagemid
particles from the scFv antibody library were preincubated in MPBS
(2% nonfat dry milk, 0.02% sodium azide in PBS, pH 7.4) for 1 hour
at 25.degree. C. to block nonspecific binding sites. Approximately
10.sup.7 primary CLL cells were labeled with mouse anti-CD5 IgG and
mouse anti-CD19 IgG conjugated to paramagnetic microbeads (Miltenyi
Biotec, Sunnyvale, Calif.). Unbound microbeads were removed by
washing. The labeled CLL cells ("target cells") were mixed with an
excess of "antigen-negative absorber cells", pelleted, and
resuspended in 50 .mu.l (10.sup.10-10.sup.11 cfu) of phage
particles. The absorber cells serve to soak up phage that stick
non-specifically to cell surfaces as well as phage specific for
"common" antigens present on both the target and absorber cells.
The absorber cells used were either TF-1 cells (a human
erythroleukemia cell line) or normal human B cells isolated from
peripheral blood by immunomagnetic negative selection (StemSep
system, StemCell Technologies, Vancouver, Canada). The ratio of
absorber cells to target cells was approximately 10 fold by volume.
After a 30 minute incubation at 25.degree. C., the cell/phage
mixture was transferred to a MiniMACS MS+ separation column. The
column was washed twice with 0.5 ml of MPBS, and once with 0.5 ml
of PBS to remove the unbound phage and absorber cells. The target
cells were eluted from the column in 1 ml of PBS and pelleted in a
microcentrifuge at maximum speed for 15 seconds. The captured phage
particles were eluted by resuspending the target cells in 200 .mu.l
of acid elution buffer (0.1 N HCl, pH adjusted to 2.2 with glycine,
plus 1 mg/ml BSA). After a 10 minute incubation at 25.degree. C.,
the buffer was neutralized with 12 .mu.L of 2M Tris base, pH10.5,
and the eluted phage were amplified in E. coli for the next round
of panning. For each round of panning, the input and output phage
titers were determined. The input titer is the number of
reamplified phage particles added to the target cell/absorber cell
mixture and the output titer is the number of captured phage eluted
from the target cells. An enrichment factor (E) is calculated using
the formula E=(R.sub.n output/R.sub.n input)/(R.sub.1
output/R.sub.1 input), where R.sub.1=round 1 and R.sub.n=round 2,
3, or 4. In most cases, an enrichment factor of 10.sup.2-10.sup.3
fold should be attained by the third or fourth round.
Analysis of Enriched Antibody Pools Following Panning
[0171] After 3-5 rounds of panning, the pools of captured phage
were assayed for binding to CLL cells by flow cytometry and/or
whole cell ELISA: [0172] 1. To produce an entire pool in the form
of HA-tagged soluble antibodies, 2 ml of a non-suppressor strain of
E. coli (e.g. TOP10F') was infected with 1 .mu.l
(10.sup.9-10.sup.10 cfu) of phagemid particles. The original,
unpanned library was used as a negative control. Carbenicillin was
added to a final concentration of 10 .mu.M and the culture was
incubated at 37.degree. C. with shaking at 250 rpm for 1 hour.
Eight ml of SB medium containing 50 .mu.g/ml carbenicillin was
added and the culture was grown to an OD 600 of .about.0.8. IPTG
was added to a final concentration of 1 mM to induce scFv
expression from the Lac promoter and shaking at 37.degree. C. was
continued for 4 hours. The culture was centrifuged at 3000.times.g
for 15'. The supernatant containing the soluble antibodies was
filtered and stored in 1 ml aliquots at -20.degree. C. [0173] 2.
Binding of the scFv antibody pools to target cells vs. absorber
cells was determined by flow cytometry using high-affinity Rat
Anti-HA (clone 3F10, Roche Molecular Biochemicals) as secondary
antibody and PE-conjugated Donkey Anti-Rat as tertiary antibody.
[0174] 3. Binding of the antibody pools to target cells vs.
absorber cells was also determined by whole-cell ELISA as described
below. Screening Individual scFv Clones Following Panning
[0175] To screen individual scFv clones following panning, TOP10F'
cells were infected with phage pools as described above, spread
onto LB plates containing carbenicillin and tetracycline, and
incubated overnight at 37.degree. C. Individual colonies were
inoculated into deep 96-well plates containing 0.6-1.0 ml of
SB-carbenicillin medium per well. The cultures were grown for 6-8
hours in a HiGro.RTM. shaking incubator (GeneMachines, San Carlos,
Calif.) at 520 rpm and 37.degree. C. At this point, a 90 .mu.l
aliquot from each well was transferred to a deep 96-well plate
containing 10 .mu.l of DMSO. This replica plate was stored at
-80.degree. C. IPTG was added to the original plate to a final
concentration of 1 mM and shaking was continued for 3 hours. The
plates were centrifuged at 3000.times.g for 15 minutes. The
supernatants containing soluble scFv antibodies were transferred to
another deep 96-well plate and stored at -20.degree. C.
[0176] A sensitive whole-cell ELISA method for screening HA-tagged
scFv antibodies was developed: [0177] 1. An ELISA plate is coated
with concanavalin A (10 mg/ml in 0.1 M NaHCO.sub.3, pH8.6, 0.1 mM
CaCl.sub.2). [0178] 2. After washing the plate with PBS,
0.5-1.times.10.sup.5 target cells or absorber cells in 50 .mu.l of
PBS are added to each well, and the plate is centrifuged at
250.times.g for 10 minutes. [0179] 3. 50 .mu.l of 0.02%
glutaraldehyde in PBS are added and the cells are fixed overnight
at 4.degree. C. [0180] 4. After washing with PBS, non-specific
binding sites are blocked with PBS containing 4% non-fat dry milk
for 3 hours at room temperature. [0181] 5. The cells are incubated
with 50 .mu.l of soluble, HA-tagged scFv or Fab antibody (TOP10F'
supernatant) for 2 hours at room temperature, then washed six times
with PBS. [0182] 6. Bound antibodies are detected using a Mouse
Anti-HA secondary antibody (clone 12CA5) and an alkaline
phosphatase (AP)-conjugated Anti-Mouse IgG tertiary antibody. An
about 10-fold amplification of the signal is obtained by using
AMDEX AP-conjugated Sheep Anti-Mouse IgG as the tertiary antibody
(Amersham Pharmacia Biotech). The AMDEX antibody is conjugated to
multiple AP molecules via a dextran backbone. Color is developed
with the alkaline phosphatase substrate PNPP and measured at 405 nm
using a microplate reader.
[0183] Primary screening of the scFv clones was done by ELISA on
primary CLL cells versus normal human PBMC. Clones which were
positive on CLL cells and negative on normal PBMC were rescreened
by ELISA on normal human B cells, human B cell lines, TF-1 cells,
and the CLL-AAT cell line. The clones were also rescreened by ELISA
on CLL cells isolated from three different patients to eliminate
clones that recognized patient-specific or blood type-specific
antigens. Results from representative ELISAs are shown in FIGS. 2-6
and summarized in FIGS. 9A-9C.
[0184] The number of unique scFv antibody clones obtained was
determined by DNA fingerprinting and sequencing. The scFv DNA
inserts were amplified from the plasmids by PCR and digested with
the restriction enzyme BstNI. The resulting fragments were
separated on a 4% agarose gel and stained with ethidium bromide.
Clones with different restriction fragment patterns must have
different amino acid sequences. Clones with identical patterns
probably have similar or identical sequences. Clones with unique
BstNI fingerprints were further analyzed by DNA sequencing.
Twenty-five different sequences were found, which could be
clustered into 16 groups of antibodies with closely related
complementarity determining regions (FIGS. 9A-9C).
Characterization of scFv Antibodies by Flow Cytometry
[0185] The binding specificities of several scFv antibodies were
analyzed by 3-color flow cytometry (FIG. 7). PBMC isolated from
normal donors were stained with FITC-conjugated anti-CD5 and
PerCP-conjugated anti-CD19. Staining with scFv antibody was done
using biotin-conjugated anti-HA as secondary antibody and
PE-conjugated streptavidin. Three antibodies, scFv-2, scFv-3, and
scFv-6, were found to specifically recognize the CD19.sup.+ B
lymphocyte population (data not shown). The fourth antibody,
scFv-9, recognized two distinct cell populations: the CD19.sup.+ B
lymphocytes and a subset of CD5.sup.+ T lymphocytes (FIG. 7).
Further characterization of the T cell subset showed that it was a
subpopulation of the CD4.sup.+CD8.sup.- TH cells (data not
shown).
[0186] To determine if the antigens recognized by the scFv
antibodies were overexpressed on primary CLL cells, PBMC from five
CLL patients and five normal donors were stained with scFv and
compared by flow cytometry (FIG. 8 and Table 2). By comparing the
mean fluorescent intensities of the positive cell populations, the
relative expression level of an antigen on CLL cells vs. normal
cells could be determined. One antibody, scFv-2, consistently
stained CLL cells less intensely than normal PBMC, whereas scFv-3
and scFv-6 both consistently stained CLL cells more brightly than
normal PBMC. The fourth antibody, scFv-9, stained two of the five
CLL samples much more intensely than normal PBMC, but gave only
moderately brighter staining for the other three CLL samples (FIG.
8 and Table 2). This indicates that the antigens for scFv-3 and
scFv-6 are overexpressed approximately 1.4 fold on most if not all
CLL tumors, whereas scFv-9 is overexpressed 3 to 6-fold on a subset
of CLL tumors.
[0187] CLL patients can be divided into two roughly equal groups:
those with a poor prognosis (median survival time of 8 years) and
those with a favorable prognosis (median survival time of 26
years). Several unfavorable prognostic indicators have been
identified for CLL, most notably the presence of VH genes lacking
somatic mutations and the presence of a high percentage of
CD38.sup.+ B cells. Since scFv-9 recognizes an antigen
overexpressed in only a subset of CLL patients, it was sought to
determine if scFv-9 antigen overexpression correlated with the
percentage of CD38.sup.+ cells in blood samples from ten CLL
patients (FIG. 11). The results indicate that scFv-9 antigen
overexpression and percent CD38.sup.+ cells are completely
independent of one another. Identification of antigens recognized
by scFv antibodies by immunoprecipitation (IP) and mass
spectrometry (MS)
[0188] To identify the antigens for these antibodies, scFvs were
used to immunoprecipitate the antigens from lysates prepared from
the microsomal fraction of cell-surface biotinylated CLL-AAT cells
(FIG. 12). The immunoprecipitated antigens were purified by
SDS-PAGE and identified by matrix assisted laser desorption
ionization mass spectrometry (MALDI-MS) or microcapillary
reverse-phase HPLC nano-electrospray tandem mass spectrometry
(.mu.LC/MS/MS) (data not shown). ScFv-2 immunoprecipitated a 110 kd
antigen from both RL and CLL-AAT cells (FIG. 12). This antigen was
identified by MALDI-MS as the B cell-specific marker CD 19. ScFv-3
and scFv-6 both immunoprecipitated a 45 kd antigen from CLL-AAT
cells (not shown). This antigen was identified by MALDI-MS as CD23,
which is a known marker for CLL and activated B cells. ScFv-9
immunoprecipitated a 50 kd antigen from CLL-AAT cells (FIG. 12).
This antigen was identified by .mu.LC/MS/MS as OX-2/CD200, a known
marker for B cells, activated CD4.sup.+ T cells, and thymocytes.
OX-2/CD200 is also expressed on some non-lymphoid cells such as
neurons and endothelial cells.
Example 3
[0189] The capability of cells overexpressing OX-2/CD200 to shift
the cytokine response from a Th1 response (IL-2, IFN-.gamma.) to a
Th2 response (IL-4, IL-10) was assessed in a mixed lymphocyte
reaction using monocyte-derived macrophages/dendritic cells from
one donor and blood-derived T cells from a different donor. As a
source of OX-2/CD200-expressing cells, either OX-2/CD200
transfected EBNA cells as described below or CLL patient samples
were used.
Transfection of 293-EBNA Cells
[0190] 293-EBNA cells (Invitrogen) were seeded at
2.5.times.10.sup.6 per 100 mm dish. 24 hours later the cells were
transiently transfected using Polyfect reagent (QIAGEN) according
to the manufacturer's instructions. Cells were cotransfected with
7.2 .mu.g of OX-2/CD200 cDNA in vector pCEP4 (Invitrogen) and 0.8
.mu.g of pAdVAntage vector (Promega). As a negative control, cells
were cotransfected with empty pCEP4 vector plus pAdVAntage. 48
hours after transfection, approximately 90% of the cells expressed
OX-2/CD200 on their surface as determined by flow cytometry with
the scFv-9 antibody.
Maturation of Dendritic Cells/Macrophages from Blood Monocytes
[0191] Buffy coats were obtained from the San Diego Blood Bank and
primary blood lymphocytes (PBL) were isolated using Ficoll. Cells
were adhered for 1 hour in Eagles Minimal Essential Medium (EMEM)
containing 2% human serum followed by vigorous washing with PBS.
Cells were cultured for 5 days either in the presence of GM-CSF,
IL-4 and IFN-.gamma. or M-CSF with or without the addition of
lipopolysaccharide (LPS) after 3 days. Matured cells were harvested
and irradiated at 2000 RAD using a .gamma.-irradiator (Shepherd
Mark I Model 30 irradiator (Cs137)).
Mixed Lymphocyte Reaction
[0192] Mixed lymphocyte reactions were set up in 24 well plates
using 500,000 dendritic cells/macrophages and 1.times.10.sup.6
responder cells. Responder cells were T cell enriched lymphocytes
purified from peripheral blood using Ficoll. T cells were enriched
by incubating the cells for 1 hour in tissue culture flasks and
taking the non-adherent cell fraction. 500,000 OX-2/CD200
transfected EBNA cells or CLL cells were added to the
macrophages/dendritic cells in the presence or absence of 30
.mu.g/ml anti-CD200 antibody (scFv-9 converted to full IgG) 2-4
hours before the lymphocyte addition. Supernatants were collected
after 48 and 68 hours and analyzed for the presence of
cytokines.
Conversion of scFv-9 to Full IgG
[0193] Light chain and heavy chain V genes of scFv-9 were amplified
by overlap PCR with primers that connect the variable region of
each gene with human lambda light chain constant region gene, and
human IgG1 heavy chain constant region CHI gene, respectively.
Variable regions of light chain gene and heavy chain gene of scFv-9
were amplified with specific primers and the human lambda light
chain constant region gene and the IgG1 heavy chain constant region
CH1 gene were separately amplified with specific primers as
follows:
TABLE-US-00001 R9VL-F1 QP: (SEQ ID NO: 103) 5' GGC CTC TAG ACA GCC
TGT GCT GAC TCA GTC GCC CTC 3'; R9VL/hCL2-rev: (SEQ ID NO: 104) 5'
CGA GGG GGC AGC CTT GGG CTG ACC TGT GAC GGT CAG CTG GGT C 3';
R9VL/hCL2-F: (SEQ ID NO: 105) 5' GAC CCA GCT GAC CGT CAC AGG TCA
GCC CAA GGC TGC CCC CTC G 3'; R9VH-F1: (SEQ ID NO: 106) 5' TCT AAT
CTC GAG CAG CAG CAG CTG ATG GAG TCC G 3'; R9VH/hCG-rev: (SEQ ID NO:
107) 5' GAC CGA TGG GCC CTT GGT GGA GGC TGA GGA GAC GGT GAC CAG GGT
GC 3'; R9VH/hCG-F: (SEQ ID NO: 108) 5' GCA CCC TGG TCA CCG TCT CCT
CAG CCT CCA CCA AGG GCC CAT CGG TC 3'; hCL2-rev: (SEQ ID NO: 109)
5' CCA CTG TCA GAG CTC CCG GGT AGA AGT C 3'; hCG-rev: (SEQ ID NO:
110) 5' GTC ACC GGT TCG GGG AAG TAG TC 3'.
Amplified Products were Purified and Overlap PCR was Performed.
[0194] Final products were digested with Xba I/Sac I (light chain)
and Xho I/Pin AI (heavy chain) and cloned into a human Fab
expression vector, PAX243hGL (see published International
Application WO 2004/078937, the disclosure of which is incorporated
herein by this reference). DNA clones were analyzed for PCR errors
by DNA sequencing. The hCMV IE promoter gene was inserted at Not
I/Xho I site (in front of the heavy chain). The vector was digested
with Xba I/Pin AI/EcoR I/Nhe I and a 3472 by fragment containing
the light chain plus the hCMV IE promoter and the heavy chain gene
was transferred to an IgG1 expression vector at the Xba I/Pin AI
site.
Cytokine Analysis
[0195] The effect of the scFv-9 converted to full IgG on the
cytokine profile in the mixed lymphocyte reaction was
determined.
[0196] Cytokines such as IL-2, IFN-.gamma., IL-4, IL-10 and IL-6
found in the tissue culture supernatant were quantified using
ELISA. Matched capture and detection antibody pairs for each
cytokine were obtained from R+D Systems (Minneapolis, Minn.), and a
standard curve for each cytokine was produced using recombinant
human cytokine. Anti-cytokine capture antibody was coated on the
plate in PBS at the optimum concentration. After overnight
incubation, the plates were washed and blocked for 1 hour with PBS
containing 1% BSA and 5% sucrose. After 3 washes with PBS
containing 0.05% Tween, supernatants were added at dilutions of
two-fold or ten-fold in PBS containing 1% BSA. Captured cytokines
were detected with the appropriate biotinylated anti-cytokine
antibody followed by the addition of alkaline phosphatase
conjugated streptavidin and SigmaS substrate. Color development was
assessed with an ELISA plate reader (Molecular Devices).
[0197] As shown in FIG. 14, the presence of OX-2/CD200 transfected
but not untransfected cells resulted in down-regulation of Th1
cytokines such as IL-2 and IFN-.gamma.. Addition of the anti-CD200
antibody at 30 .mu.g/ml fully restored the Th1 response, indicating
that the antibody blocked interaction of OX-2/CD200 with its
receptor.
[0198] As set forth in FIGS. 15 and 16, the presence of CLL cells
in a mixed lymphocyte reaction resulted in down-regulation of the
Th1 response. (FIG. 15 shows the results for IL-2; FIG. 16 shows
the results for IFN-.gamma.). This was not only the case for cells
over-expressing OX-2/CD200 (IB, EM, HS, BH), but also for CLL cells
that did not overexpress OX-2/CD200 (JR, JG and GB) (the expression
levels for these cells are set forth in FIG. 11). However, the
anti-CD200 antibody only restored the Th1 response in cells
over-expressing OX-2/CD200, indicating that for patients
overexpressing OX-2/CD200, abrogating OX-2/CD200 interaction with
its receptor on macrophages was sufficient to restore a Th1
response. In patients that did not overexpress OX-2/CD200, other
mechanisms appeared to be involved in down-regulating the Th1
response.
Animal Models to Test an Effect of Anti-CD200 on Tumor
Rejection
[0199] A model was established in which RAJI lymphoma tumor growth
is prevented by the simultaneous injection of PBLs. NOD/SCID mice
were injected subcutaneously with 4.times.10.sup.6 RAJI cells
either in the presence or absence of human PBLs from different
donors at 1.times.10.sup.6, 5.times.10.sup.6 or 10.times.10.sup.6
cells. Tumor length and width as well as body weight was determined
3 times a week. Mean+/-SD of tumor volumes for all groups is shown
in FIGS. 17 A and B. Statistical analysis was performed using 2
parametric tests (Student's t-test and Welch's test) and one
non-parametric test (the Wilcox test). Results of the statistical
analysis are found in FIG. 18. RAJI cells form subcutaneous tumors
with acceptable variation. Rejection is dependent on the specific
donor and the PBL cell number. 1.times.10.sup.6 PBLs were
insufficient to prevent tumor growth. Donor 2 at 5.times.10.sup.6
PBLs from day 22-43 and donor 3 at 5.times.10.sup.6 or
1.times.10.sup.7 PBLs starting at day 36 significantly reduced
tumor growth. Donor 4 is very close to being significant after day
48.
[0200] To test for an effect of anti-CD200, RAJI cells are stably
transfected with CD200. Animals are injected as described in the
previous paragraph. In the presence of CD200-transfected cells,
tumors grow even in the presence of human PBLs. Anti-CD200 antibody
is administered to evaluate tumor rejection in this model.
[0201] Also, a liquid tumor model is established. RAJI cells are
injected intraperitoneally into NOD/SCID mice. Cells disseminate to
bone marrow, spleen, lymph node and other organs resulting in
paralysis. Concurrent injection of human PBLs prevents or slows
tumor growth. Tumor growth is monitored by assessing the mice for
signs of movement impairment and paralysis. Once these signs are
observed, mice are sacrificed and the number of tumor cells is
assessed in various organs including bone marrow, spleen, lymph
nodes and blood by FACS analysis and PCR.
[0202] Similar to the subcutaneous model, CD200 transfected cells
are injected intraperitoneally. They grow even in the presence of
human PBLs. Treatment with anti-CD200 results in tumor rejection or
slower tumor growth.
Example 4
Library Construction
[0203] A mouse was immunized alternately with baculovirus expressed
recombinant CD200 extracellular domain fused to mouse IgG Fc
(CD200-Fc) (Orbigen Inc., San Diego, Calif.) and 293-EBNA cells
transiently transfected with a vector containing full length CD200.
Total RNA was prepared from mouse spleen using TRI reagent
(Molecular Research Center, Inc., Cincinnati, Ohio) according to
the manufacturer's protocol. Messenger RNA (mRNA) was purified
using Oligotex (QIAGEN Inc., Valencia, Calif.) according to the
manufacturer's manual. First strand cDNA was synthesized using
SuperScript II RTase (Invitrogen Life Technologies, Carlsbad,
Calif.) according to the manufacturer's protocol. First strand cDNA
was digested with restriction endonuclease and second strand cDNA
was synthesized according to the method fully described in
published PCT application WO03/025202A2, published Mar. 27, 2003.
Second strand cDNA was cleaned up with PCR purification kit
(QIAGEN) and single primer amplification was performed according to
the method described in published PCT application WO03/025202A2,
published Mar. 27, 2003. Amplified products were pooled and
purified with PCR purification kit. Kappa light chain was digested
with Xba I and BspE I, and IgG1 and IgG2a heavy chains were
digested with Xho I and Bln I. Digested fragments were purified
from the agarose gel using Gel extraction kit (QIAGEN) and cloned
into PAX313m/hG vector as described in published PCT application
WO/04078937A2 published Sep. 16, 2004.
Library Panning
[0204] The libraries (IgG1 kappa and IgG2a kappa) were panned on
CD200-Fc either directly coated on the microtiter wells (Costar
Group, Bethesda, Md.) or captured with goat anti-mouse IgG Fc
specific antibody (Sigma-Aldrich Corp., St. Louis, Mo.). For the
preparation of library phage, electrocompetent XL1-Blue cells
(Stratagene, La Jolla, Calif.) were electroporated with library DNA
and grown in SOC medium for 1 hour and in SB medium for 2 hours
with carbenicillin. Phage production was induced with the addition
of VCS M13 helper phage (Amersham Biosciences Corp., Piscataway,
N.J.) and 1 mM IPTG at 30.degree. C. overnight. The culture was
spun down and phage were precipitated with 4% polyethylene glycol
and 3% NaCl. The phage were spun down and resuspended in 1% BSA/PBS
containing unrelated antigen, FLJ32028 that is also baculovirus
expressed extracellular domain fused to mouse IgG Fc (FLJ32028-Fc)
(Orbigen, San Diego), as a soluble competitor. For the panning on
directly coated CD200-Fc, four wells were coated with 100 .mu.l of
CD200-Fc (5 .mu.g/ml in 0.1 M NaHCO.sub.3 pH8.6) at 4.degree. C.
overnight. The wells were washed 5 times with phosphate buffered
saline (PBS) pH7.0 and blocked with 1% bovine serum albumin
(BSA)/PBS at 37.degree. C. for 1 hr. For the panning on CD200-Fc
captured on goat anti-mouse IgG Fc, four microtiter wells were
coated with 100 .mu.l goat anti-mouse IgG Fc (20 .mu.g/ml in PBS)
at 4.degree. C. overnight. The wells were washed 5 times with PBS
and incubated with 100 .mu.l CD200-Fc (20 .mu.g/ml in PBS) for 1
hour at 37.degree. C. The wells were washed 5 times with PBS and
blocked with 1% BSA/PBS at 37.degree. C. for 1 hour. For both
directly-coated and captured methods of panning, the blocker was
replaced with the mixture of soluble Fabs obtained from the panning
of another library (the library described in Example 3 of PCT
application serial No. PCT/US04/17118 filed Jun. 2, 2004 (not yet
published), the entire disclosure of which is incorporated herein
by this reference) on FLJ32028 to mask epitopes on mouse IgG Fc and
the wells were incubated for 30 min at 37.degree. C. These masking
Fabs were shown to also bind to CD200-Fc. Library phage were added
on top of the masking Fabs and the wells were incubated for
approximately 1.5 hours at 37.degree. C. The unbound phage were
washed with PBS with increasing stringency (3 times in the first
round, 5 times in the 2.sup.nd round and 10 times in the 3.sup.rd
and the 4.sup.th rounds) with 5 minute incubation and pipetting up
and down 5 times for each wash. The bound phage were eluted twice
with 100 .mu.l 0.1 M HCl with 1 mg/ml BSA, pH2.2 and neutralized
with 2 M Tris Base pH 11.5. The freshly grown ER2738 cells were
infected with eluted phage and titrated onto LB agarose plates
containing carbenicillin and glucose. The remaining phage were
propagated overnight at 30-37.degree. C. with the addition of VCS
M13 helper phage and 1 mM IPTG for the next round of panning.
Library Screening
[0205] Ninety five colonies from round 3 and 4 titration plates
were grown in 1 ml SB containing 12.5 .mu.g/ml tetracycline and 50
.mu.g/ml carbenicillin for approximately 6 hours at 37.degree. C.
VCS M13 helper phage were added and the culture was incubated for 2
hours at 37.degree. C. 1 mM IPTG and 70 .mu.g/ml kanamycin were
added and Fab-phage production was induced at 30.degree. C.
overnight. Microtiter wells were coated with 50 .mu.l of rabbit
anti-mouse IgG F(ab').sub.2 (4 .mu.g/ml in PBS), CD200-Fc (4
.mu.g/ml in 0.1 M NaHCO.sub.3 pH8.6), or FLJ32028-Fc (4 .mu.g/ml in
0.1 M NaHCO.sub.3 pH8.6) at 4.degree. C. overnight. The wells were
washed 3 times with PBS and blocked with 100 .mu.l 1% BSA/PBS for 1
hour at 37.degree. C. The culture was spun down. The blocker was
replaced with the culture supernatant containing Fab-phage and the
wells were incubated for 1.5-2 hours at 37.degree. C. The remaining
Fab-phage was stored at -80.degree. C. for flow cytometry. The
plates were washed 3 times with PBS and the binding was detected
with 50 .mu.l alkaline phosphatase (AP)-conjugated goat anti-mouse
IgG F(ab').sub.2 antibody (Pierce)(1:500 in 1% BSA/PBS) for 1 hr at
37.degree. C. The plates were washed 3 times with PBS and developed
with AP substrate (Sigma-Aldrich) in pNPP buffer. Almost all of the
clones from round 3 were already specifically positive to CD200
(FIGS. 19A-D). Clones were also screened by high throughput flow
cytometry analysis. One hundred microliters of 293 cells
transiently transfected with CD200 (1.times.10.sup.5 cells) were
aliquoted into a 96 well plate (Costar). Fifty microliters
Fab-phage was added to the cells and mixed by pipetting and
incubated on ice for 30 minutes. The cells were washed twice with
1% BSA/PBS containing 0.01% N.sub.aN.sub.3. The cells were
resuspended in 100 .mu.l PE-conjugated goat anti-mouse IgG antibody
(Sigma-Aldrich) in 1% BSA/PBS containing 0.01% NaN.sub.3 and
incubated on ice for 30 minutes. The cells were washed twice with
1% BSA/PBS containing 0.01% N.sub.aN.sub.3 and resuspended in 200
.mu.l 1% paraformaldehyde in PBS. Representative clones showing
positive binding to CD200 expressing cells are shown in FIGS.
20A-D.
[0206] DNA sequences were analyzed and deduced amino acid sequences
of the heavy chain were grouped according to the complementarity
determining region 3 (CDR3) (FIGS. 21 A, B). They were divided into
17 groups.
Fluorescent Bead Assay
[0207] 23 clones were selected for further analysis. They were
cG2aR3B5, dG1R3A5, cG2aR3A2, dG2aR3B2, dG1R3A1, cG2aR3A1, cG2aR3B,
dG1R3B, cG1R3A, cG1R3A, cG1R3A1, dG1R3B, dG1R3B, cG1R3C, dG2aR3C,
dG2aR3A1, cG2aR3B, cG2aR3B, dG1R3B, cG2aR3B, cG2aR3C, dG1R3H, and
dG2aR3A6. DNA of selected Fabs was digested with Spe I/Nhe I for
gene III removal and soluble Fab expression and purification. The
purified Fabs were evaluated for their ability to block the
interaction of CD200 with its receptor (CD200R) in a fluorescent
bead assay. TransFluoSpheres carboxylate-modified microspheres
(488/645) (Molecular Probes Invitrogen Detection Technologies,
Eugene, Oreg.) were coated with streptavidin followed by a
biotin-labeled anti-human Fc antibody and baculovirus-produced
CD200-Fc protein. 293 cells were transiently transfected with
CD200R. Cell surface expression was confirmed by FACS analysis. 1
million CD200-coated beads were pre-incubated with various amounts
of anti-CD200 Fabs or chimeric IgG for 10 minutes before the
addition of 50,000 CD200R transfected cells. After a 30 minute
incubation at 37.degree. C., the cells were washed in Tris buffer
containing 1% BSA and analyzed using a FACS Calibur. Fabs c1A10,
c2aB7, and d1A5 showed the best blocking of CD200 and CD200R
interaction at 6.7 .mu.g/ml of Fab (FIG. 22). These clones are
referred to as cG1R3A10, cG2aR3B7 and dG1R3A5, respectively in
FIGS. 21A and/or B.
Chimerization and IgG Conversion
[0208] Six antibodies were selected for chimerization and IgG
conversion. (See FIG. 23.) They were c1A10 (cG1R3A10), c2aA10
(cG2aR3A10), c2aB7 (cG2aR3B7), d1A5 (dG1R3A5), d1B5 (dG1R3B5), and
d1B10 (dG1R3B10). For the chimerization, overlap PCR was performed
to connect mouse kappa chain variable region and human kappa chain
constant region. Mouse heavy chain variable region was amplified
with a 3' primer that contains a partial human IgG1 constant region
and Apa I site for cloning. Amplified kappa chain fragments and
heavy chain fragments were cloned into PAX243hGK vector (see
published International Application WO 2004/078937) that contains
human IgG1 constant region at Xba I/Not I for kappa light chain and
Xho I/Apa. I for heavy chain fragment. Binding of chimeric Fab to
CD200 was confirmed by ELISA and flow cytometry. These chimeric
Fabs were converted into IgG by insertion of human cytomegalovirus
immediate early promoter (hCMV IE Pro) sequence for the heavy chain
expression at Not I/Xho I, then the transfer of the light chain and
heavy chain into a human IgG1 expression vector at Xba I/Pin AI
sites. This vector has an additional hCMV IE Pro sequence upstream
Xba I site for the light chain expression in mammalian cells. The
DNA sequences were confirmed and maxi prep DNA was prepared using
HiSpeed Maxi prep columns (QIAGEN) for mammalian cell transfection.
Transient transfection was performed in 293-EBNA cells using
Effectene (QIAGEN) according to the manufacturer's protocol with
the addition of pAdVAntage vector (Promega US, Madison, Wis.).
Stable cell line transfection was performed in NS0 cells using
Effectene according to the manufacturer's protocol. After a small
scale transient transfection, culture supernatant for each antibody
was tested by ELISA (FIG. 24). After a large scale transient
transfection, each IgG was purified from the culture supernatant by
anti-human IgG F(ab')2 affinity column using FPLC (Amersham
Biosciences).
[0209] The purified IgG were tested in a bead inhibition assay as
described for the Fabs. All antibodies directed against CD200
blocked the receptor ligand interaction very well as shown below in
FIG. 25.
Mixed Lymphocyte Reaction
[0210] Whether blocking of CD200 interaction with its receptor also
prevents the cytokine shift from Th1 to Th2 observed in mixed
lymphocytes reactions in the presence of CD200 was evaluated. Buffy
coats were obtained from the San Diego Blood Bank and primary blood
lymphocytes (PBL) were isolated using HISTOPAQUE.RTM.
(Sigma-Aldrich). Cells were adhered for 1 h in EMEM containing 2%
human serum followed by vigorous washing with PBS. Cells were
cultured for 5 days in the presence of GM-CSF, IL-4 and
IFN-.gamma.. Matured cells were harvested and irradiated at 2000
RAD using a 7-irradiator (University of California San Diego).
Mixed lymphocyte reactions were set up in 24 well plates using
500,000 dendritic cells and 1.times.10.sup.6 responder cells.
Responder cells were T cell enriched lymphocytes purified from
peripheral blood using HISTOPAQUE.RTM.. T cells were enriched by
incubating the cells for 1 hour in tissue culture flasks and taking
the non-adherent cell fraction. Five hundred thousand CD200
expressing primary irradiated CLL cells were added to the dendritic
cells in the presence or absence of various amounts of anti-CD200
antibodies 2-4 hours before the lymphocyte addition. Supernatants
were collected after 48 and 68 hours and cytokines such as IL-2,
IFN-.gamma., IL-4, IL-10 and IL-6 were quantified using ELISA.
Matched capture and detection antibody pairs for each cytokine were
obtained from R+D Systems (Minneapolis), and a standard curve for
each cytokine was produced using recombinant human cytokine.
Anti-cytokine capture antibody was coated on the plate in PBS at
the optimum concentration. After overnight incubation, the plates
were washed and blocked for 1 h with PBS containing 1% BSA and 5%
sucrose. After 3 washes with PBS containing 0.05% TWEEN.RTM.,
supernatants were added at the indicated dilutions in PBS
containing 1% BSA. Captured cytokines were detected with the
appropriate biotinylated anti-cytokine antibody followed by the
addition of alkaline phosphatase conjugated streptavidin and SigmaS
substrate. Color development was assessed with an ELISA plate
reader (Molecular Devices Corp., Sunnyvale, Calif.). As shown in
FIGS. 26A and B, the presence of CLL cells completely abrogated
IFN-gamma and most of IL-2 production observed in the mixed
lymphocyte reaction. Presence of any of the antibodies allowed for
production of these Th1 cytokines (FIGS. 26A and B). In contrast,
IL-10 production was downregulated in the presence of the
antibodies. (See FIG. 26C.)
Antibody-Dependent Cell-Mediated Cytotoxicity Assay
[0211] Furthermore, the six chimeric mouse anti-CD200 antibodies
were evaluated for their ability to kill CD200 expressing tumor
cells in an antibody-dependent cell-mediated cytotoxicity assay
(ADCC). 293-EBNA cells transfected with CD200 were labeled with 100
.mu.Ci/million cells in 0.5 ml medium for 1 hr at 37.degree. C.
After 3 washes, cells were counted, resuspended in medium (RPMI
supplemented with 10% human AB serum) at 0.2 million/ml and 50
.mu.l (10,000 cells/well) was dispensed in triplicate into a 96
well round bottom plate. 20 .mu.l of anti-CD200 antibodies were
dispensed into each well so as to achieve a final concentration of
20 .mu.g/ml. Peripheral blood mononuclear cells (effector cells)
were isolated on a Ficoll gradient, red blood cells were lysed with
ammonium chloride, washed and resuspended in culture medium and 50
.mu.l of cells were dispensed into each well. The assay plates were
spun (1,500 rpm/5 minutes/low brake) and transferred to the cell
culture incubator. After 4 hours, assay plates were spun as before.
36 .mu.l of the supernatants were transferred to pico plates and
mixed with 250 .mu.l microscint-20 cocktail, and placed on the
orbital shaker for 2 minutes and read on a Top count. As
illustrated in the FIG. 27, all of the mouse chimeric CD200
antibodies produced similar levels of lysis when cultured with
CD200 positive cells. No lysis was observed with CD200 negative
cells. In addition, the extent of lysis was statistically
significant (p<0.05) when compared to isotype control antibody,
d2A6 (anti-FLJ32028 antibody).
Example 5
Raji/PBL Model
[0212] NOD.CB17-Prkdc<scid> mice (Jackson Laboratory) were
injected with 200 .mu.l RPMI containing 4.times.10.sup.6 RAJI cells
(ATCC) s.c. along with 0, 1, 5 or 10 million PBLs. Nine or ten mice
were included per group. PBLs were isolated from 250 ml whole
ammonium chloride. Tumor growth was monitored three times a week by
measuring length and width with a caliper. Tumor volume was
calculated based on length.times.width.times.width/2.
[0213] Differences between the groups that were injected with PBLs
compared to the group that received tumor cells only were analyzed
by 2-tailed unpaired Student's t-test. Significant differences were
observed in the groups that received 5 or 10 million PBLs, but not
in the group that received 1 million PBLs from Day 32 on. The data
shown in FIG. 28 are a representative example of 10 experiments
using different PBL donors.
Namalwa PBL Model
[0214] NOD.CB17-Prkdc<scid> mice (Jackson Laboratory, Bar
Harbor, Me.) were injected with 200 .mu.l RPMI containing
4.times.10.sup.6 Namalwa cells (ATCC) s.c. along with 0, 2 or 10
million PBLs. 9-10 mice were included per group. PBLs were isolated
from 250 ml whole blood on a histopaque gradient followed by red
blood cell lysis using 0.9% ammonium chloride. Tumor growth was
monitored three times a week by measuring length and width with a
caliper. Tumor volume was calculated based on
length.times.width.times.width/2.
[0215] FIG. 29 shows differences between the groups that were
injected with PBLs compared to the group that received tumor cells
only analyzed by 2-tailed unpaired Student's t-test. Significant
differences were observed in the groups that received 10 million
PBLs for both donors, but not in the group that received 2 million
PBLs from day 8 on.
Creation of Stable CD200-Expressing Cell Lines
[0216] Stable CD200-expressing Raji and Namalwa cell lines were
generated using the Virapower Lentiviral Expression System
(Invitrogen, Carlsbad, Calif.). A CD200 cDNA was isolated from
primary CLL cells by RT-PCR using forward primer
5'-GACAAGCTTGCAAGGATGGAGAGGCTGGTGA-3' (SEQ ID NO: 212) and reverse
primer 5'-GACGGATCCGCCCCTTTTCCTCCTGCTTTTCTC-3' (SEQ ID NO: 213).
The PCR product was cloned into the Gateway entry vector
pCR8/GW/TOPO-TA and individual clones were sequenced. Clones with
the correct sequence were recombined in both the sense and
antisense orientations into the lentiviral vectors pLenti6/V5/DEST
and pLenti6/UbC/V5/DEST using Gateway technology (Invitrogen,
Carlsbad, Calif.). The primary difference between these two vectors
is the promoter used to drive CD200 expression: pLenti6/V5/DEST
contains the human CMV immediate early promoter, whereas
pLenti6/UbC/V5/DEST contains the human ubiquitin C promoter.
[0217] High-titer, VSV-G pseudotyped lentiviral stocks were
produced by transient cotransfection of 293-FT cells as recommended
by the manufacturer. Raji or Namalwa cells were transduced by
resuspending 10.sup.6 cells in 1 ml of growth medium containing 12
.mu.g/ml Polybrene and adding 1 ml of lentiviral stock. After
incubating the cells overnight at 37.degree. C., the medium
containing virus was removed and replaced with 4 ml of fresh
medium. Two days later, the infected cells were analyzed for CD200
expression by flow cytometry. In all experiments, .gtoreq.70% of
the cells were CD200.sup.+, whereas CD200 was undetectable in the
parental cell lines and in cells transduced with the negative
control (antisense CD200) viruses.
[0218] To isolate clonal cell lines that overexpress CD200, the
infected cells were selected with blasticidin for 13 days. The
concentrations of blasticidin used were 6 .mu.g/ml for Raji cells
or 2 .mu.g/ml for Namalwa cells. Stable clones were then isolated
by limiting dilution of the blasticidin-resistant cells into
96-well plates. Clones were screened in 96-well format by flow
cytometry using PE-conjugated Mouse Anti-Human CD200 (clone MRC
OX104, Serotec) and a BD FACSCalibur equipped with a High
Throughput Sampler. After screening a total of 2000 Raji and 2000
Namalwa clones, those clones with the highest CD200 expression were
expanded for further characterization using conventional
techniques.
Immunosuppressive Effect of CD200 in the RAJI/PBL Model
[0219] In accordance with the methods disclosed, it has been
demonstrated that CD200 is upregulated on CLL cells. Upregulation
of this molecule might potentially be immunosuppressive. To test
whether cancer cells expressing CD200 prevent the immune system
from eradicating the cancer cells, RAJI cells, that normally do not
express CD200, were infected with a lentivirus vector system
encoding for CD200 as described above. RAJI clones stably
expressing CD200 were selected. As a control to ensure that there
was no effect of vector infection, clones expressing a reversed,
nonfunctional form of CD200 (CD200rev) were also selected. RAJI
cells expressing CD200, CD200REV or the parental RAJI cells were
injected subcutaneously into NOD.CB17-Prkdc<scid> mice. The
following groups were included in the study: [0220] group 1:
4.times.10.sup.6 RAJI s.c.; 9 mice [0221] This group was needed to
ensure that the lentivirus transduced cells show similar growth as
the parent cells. [0222] group 2: 4.times.10.sup.6 RAJICD200 s.c.;
9 mice [0223] This group was needed to ensure that the CD200
transduced cells showed similar growth as the parent cells. Also,
this group will give the maximum tumor growth. Group 3 and group 4
were compared to this group. [0224] group 3: 4.times.10.sup.6
RAJICD200+5.times.10.sup.6 PBL s.c.; 9 mice [0225] This PBL number
has been shown to reduce tumor growth in some mice in previous
experiments. Rejection is not as strong as with 10 million cells,
but in order to determine whether CD200 can affect only a certain
number of cells, and 5.times.10.sup.6 is the minimum amount of PBLs
which can be used to get rejection; rejection should be prevented
by the presence of CD200. [0226] group 4: 4.times.10.sup.6
RAJICD200+10.times.10.sup.6 PBL s.c.; 8 mice [0227] This is the
optimum number of PBLs to see rejection in the RAJI/PBL model. The
design was that CD200 expression would prevent this rejection.
[0228] group 5: 4.times.10.sup.6 RAJICD200rev s.c.; 9 mice [0229]
This group was needed to ensure that the lentivirus transduced
cells show similar growth as the parent cells. [0230] group 6:
4.times.10.sup.6 RAJICD200rev+2.times.10.sup.6 PBL s.c.; 9 mice
[0231] This number of PBLs should not result in strong rejection or
reduction of tumor growth. This is the positive control for group 3
and group 4 (maximum expected tumor growth). If there is no
rejection in this group, then the donor PBLs were hyperactivated to
start with which could explain lack of an effect by CD200. group 7:
4.times.10.sup.6 RAJICD200rev+5.times.10.sup.6 PBL s.c.; 9 mice
Controls that any observed effects in the CD200 group 3 are really
related to CD200 and not to lentivirus transduction. [0232] group
8: 4.times.10.sup.6 RAJICD200rev+10.times.10.sup.6 PBL s.c.; 8 mice
[0233] Controls that any observed effects in the CD200 group 4 are
really related to CD200 and not to lentivirus transduction.
[0234] Animals were sacrificed at day 38 based on tumors reaching a
size above acceptable limits. Tumors from 4 animals/group were
removed. Two tumors/group were frozen in OCT, the other 2 were used
to isolate cells and analyze by FACS for CD200 expression. FIGS.
30(a-c) demonstrate the results for this study.
[0235] Although RAJICD200 cells appeared to grow somewhat more
slowly, the growth difference between transduced and parental cells
did not reach statistical significance as shown in FIG. 30(a).
[0236] The presence of PBLs slowed tumor growth by up to 84% when 5
or 10.times.10.sup.6 PBLs were injected, although generally
10.times.10.sup.6 PBL resulted in a stronger reduction over time
compared to 5.times.10.sup.6 PBL. The reduction in growth compared
to the parent tumor cells was significant from day 20 on.
2.times.10.sup.6 PBL resulted in a significant tumor growth
reduction from d22-d29, but that reduction was overcome at later
timepoints (FIG. 30(b)). This study indicated that this particular
donor rejects RAJI tumor cells very strongly.
[0237] Tumor growth in the groups that received CD200 expressing
RAJI cells and PBLs was not significantly different from the tumor
growth in the group that only received RAJI cells although mice
that received 10.times.10.sup.6 PBL showed a trend of reduced tumor
growth, but the difference reached no statistical significance at
any time point after tumors reach 100 mm.sup.3 Every mouse in the
group that received RAJI cells and 5.times.10.sup.6 PBL developed a
second tumor, some mice as early as d7, while this was not observed
in any other group. For analysis, the second tumor was added to the
first tumor and the combined size is shown in FIG. 30(c).
[0238] These results indicate that CD200 expression on tumor cells
does indeed prevent the immune system from slowing tumor growth.
Also, this study demonstrates the usefulness of the RAJI/PBL model
to assess immunosuppressive compounds or molecules.
Immunosuppressive Effect of CD200 in the Namalwa/PBL Model
[0239] To evaluate whether the effects seen in the RAJI/PBL model
can also be observed in other tumor models, Namalwa tumor cells
were also infected with the lentivirus CD200 system and stable
clones selected. As a control to ensure that there is no effect of
vector infection, clones expressing a reversed, nonfunctional form
of CD200 (CD200rev) were also selected. NOD.CB17-Prkdc<scid>
mice were injected according to the following scheme as shown in
FIGS. 31(a)-(d): [0240] group 1: 4.times.10.sup.6 Namalwa s.c.; 9
mice [0241] This group was needed to ensure that the lentivirus
transduced cells show similar growth as the parent cells. [0242]
group 2: 4.times.10.sup.6 Namalwa CD200 (1D12Ub) s.c; 9 mice [0243]
This group was needed to ensure that the CD200 transduced cells
showed similar growth as the parent cells. Also, this group will
give the maximum tumor growth. Group 3 and group 4 were compared to
this group. [0244] group 3: 4.times.10.sup.6 Namalwa CD200
(1D12Ub)+5.times.10.sup.6 PBL s.c.; 9 mice [0245] This PBL number
has been shown to reduce tumor growth in some mice in previous
experiments. Rejection is not as strong as with 10 million cells,
but if CD200 can affect only a certain number of cells, and this is
the minimum PBL we can use to get rejection; rejection will be
prevented by the presence of CD200. [0246] group 4:
4.times.10.sup.6 Namalwa CD200 (1D12Ub)+10.times.10.sup.6 PBL s.c.;
8 mice [0247] This is the optimum number of PBLs to see rejection
in the Namalwa/PBL model. This was done to show that CD200
expression prevents this rejection. [0248] group 5:
4.times.10.sup.6 Namalwa CD200rev (C5Ubrev) s.c.; 9 mice [0249]
This group was needed to ensure that the lentivirus transduced
cells showed similar growth as the parent cells. [0250] group 6:
4.times.10.sup.6 Namalwa CD200rev+2.times.10.sup.6 PBL s.c.; 9 mice
[0251] This number of PBLs should not result in strong rejection of
reduction of tumor growth. This is the positive control for group 3
and group 4 (maximum expected tumor growth). If there is no
rejection in this group, then the donor PBLs were hyperactivated to
start with which would explain lack of an effect by CD200. [0252]
group 7: 4.times.10.sup.6 Namalwa CD200rev+5.times.10.sup.6 PBL
s.c.; 9 mice [0253] This group was needed as a control to detect
any effects in the CD200 group 3 that were related to CD200 and not
to lentivirus transduction. [0254] group 8: 4.times.10.sup.6
Namalwa CD200rev+10.times.10.sup.6 PBL s.c.; 8 mice [0255] This
group was needed as a control to detect any effects in the CD200
group 4 that were related to CD200 and not to lentivirus
transduction.
[0256] Tumor length and width were assessed three times/week.
[0257] All tumor cells resulted in rapid tumor growth. There was no
significant growth difference between transduced and parental
cells. The tumor grows more aggressively than previously observed
as shown in FIG. 31(a).
[0258] FIG. 31(b) shows the presence of PBLs slows tumor growth by
about 50%. This trend was observed from d12 on. The differences of
the PBL treated group versus groups that received only tumor cells
are statistically significant (as determined by 2-tailed Student's
t-test) at d17 and d19 when 2 million or 10 million PBLs were
injected. Injection of 5 million PBLs resulted in tumor growth
reduction, but did not reach significance.
[0259] Tumor growth in the groups that received CD200 expressing
Namalwa cells and PBLs was similar to the tumor growth in the group
that only received Namalwa cells (FIG. 31(c)).
[0260] These data confirm that CD200 expression on tumor cells
prevents slowing of tumor growth by the human immune system.
Blockage of the Immunosuppressive Effect of CD200 in the RAJI/PBL
Model by Anti-CD200 Antibodies
[0261] To evaluate whether anti-CD200 antibodies can block the
immunosuppressive effect of CD200 expressed on tumor cells, RAJI
cells transduced with CD200 were injected s.c. into
NOD.CB17-Prkdc<scid> mice, and the ability of PBLs to reduce
tumor growth in the presence or absence of chimeric anti-CD200
antibodies d1B5 and c2aB7 or a control antibody that does not bind
tumor cells (alxn4100) was assessed. Antibodies were administered
initially at 10 mg/kg or 2.5 mg/kg with the tumor cells, and then
at concentrations indicated below twice/week i.v. The following
groups were set up: [0262] 1. 4.times.10.sup.6 RAJICD200; 10 mice
[0263] 2. 4.times.10.sup.6 RAJICD200+5.times.10.sup.6 PBL; 10 mice
[0264] 3. 4.times.10.sup.6 RAJICD200+5.times.10.sup.6 PBL+100 mg/kg
d1B5; 12 mice [0265] 4. 4.times.10.sup.6 RAJICD200+5.times.10.sup.6
PBL+20 mg/kg d1B5; 9 mice [0266] 5. 4.times.10.sup.6
RAJICD200+5.times.10.sup.6 PBL+100 mg/kg c2aB7; 11 mice [0267] 6.
4.times.10.sup.6 RAJICD200+5.times.10.sup.6 PBL+20 mg/kg c2aB7; 9
mice [0268] 7. 4.times.10.sup.6 RAJICD200+5.times.10.sup.6 PBL+100
mg/kg alxn4100; 9 mice
[0269] Tumor length and width was measured 3 times a week, and the
tumor volume was calculated by tumor length*width*width/2. FIG. 33
shows that as expected, CD200 expression on the tumor cells
prevented the immune cells from reducing tumor growth. However,
addition of anti-CD200 antibodies reduced the tumor volume by
50-75%. The reduction in growth by the antibodies was statistically
significant as determined by Student's t-test and Mann Whitney test
from day 18 on through the end of the study. In contrast, treatment
with the control antibody did not reduce the tumor growth. These
data demonstrate the usefulness of anti-CD200 in anti-cancer
treatment. Also, this study demonstrates the usefulness of the
RAJI/PBL model to assess immunomodulatory therapeutics.
Blockage of the Immunosuppressive Effect of CD200 in the
Namalwa/PBL Model by Anti-CD200 Antibodies
[0270] To evaluate whether the effect seen with the anti-CD200
antibodies in the RAJI/PBL model can also be observed in other
tumor models, RAJI cells transduced with CD200 were injected s.c.
into NOD.CB17-Prkdc<scid> mice, and the ability of PBLs to
reduce tumor growth in the presence or absence of chimeric
anti-CD200 antibodies d1B5 and c2aB7 or a control antibody that
does not bind tumor cells (alxn4100) was assessed. Antibodies at
concentrations indicated below were administered initially with the
tumor cells, and then twice/week s.c. within 0.5 cm of the tumor.
The following groups were set up (10 mice/group unless indicated
otherwise): [0271] 1. 4.times.10.sup.6 NamalwaCD200 [0272] 2.
4.times.10.sup.6 NamalwaCD200+5.times.10.sup.6 PBL; 12 mice [0273]
3. 4.times.10.sup.6 NamalwaCD200+5.times.10.sup.6 PBL+10 mg/kg
d1B5; 12 mice [0274] 4. 4.times.10.sup.6
NamalwaCD200+5.times.10.sup.6 PBL+2.5 mg/kg dl B5 [0275] 5.
4.times.10.sup.6 NamalwaCD200+5.times.10.sup.6 PBL+10 mg/kg c2aB7;
12 mice [0276] 6. 4.times.10.sup.6 NamalwaCD200+5.times.10.sup.6
PBL+2.5 mg/kg c2aB7 [0277] 7. 4.times.10.sup.6
NamalwaCD200+5.times.10.sup.6 PBL+10 mg/kg alxn4100
[0278] Tumor length and width was measured 3 times a week, and the
tumor volume was calculated by tumor length*width*width/2. FIG. 34
shows that as expected, CD200 expression on the tumor cells
prevented the immune cells from reducing tumor growth. However,
addition of anti-CD200 antibodies reduced the tumor volume by up to
97%. The reduction in growth by the antibodies was statistically
significant as determined by Student's t-test and Mann Whitney test
from day 12 on through the end of the study. In contrast, treatment
with the control antibody did not reduce the tumor growth. These
data confirm the usefulness of anti-CD200 in anti-cancer treatment,
and the use of the tumor/PBL models in assessing immunomodulatory
therapeutics.
Detection of a Potential Immune-Enhancing Effect of Compounds in
the RAJI/PBL Model
[0279] T cells isolated from PBLs using CD3 columns (Miltenyi) were
incubated in vitro with an ascites preparation of AZND1
(anti-DC-SIGN antibody) or an ascites preparation of a control
antibody (BB5). NOD.CB17-Prkdc<scid> mice were injected
according to the following scheme:
Group 6: 8 mice, 4.times.10.sup.6 RAJI+0.8.times.10.sup.6
AZND1-potentiated T cells+2.times.10.sup.6 PBL s.c. Group 5: 10
mice, 4.times.10.sup.6 RAJI s.c. Group 4: 8 mice, 4.times.10.sup.6
RAJI+8.times.10.sup.6 PBL s.c. Group 3: 8 mice, 4.times.10.sup.6
RAJI+0.8.times.10.sup.6 fresh T cells s.c. Group 2: 8 mice,
4.times.10.sup.6 RAJI+0.8.times.10.sup.6 AZND1-potentiated T cells
s.c. Group 1: 8 mice, 4.times.10.sup.6
RAJI+0.8.times.10.sup.6BB5.1-potentiated T cells s.c. Tumor Length
and Width were Measured 3 Times/Week.
[0280] While T cells incubated with the negative control BB5 did
not reduce tumor growth, AZND1 treated T cells did reduce tumor
growth significantly (See, FIG. 32).
[0281] These results demonstrate that the RAJI PBL model can be
used to assess efficacy of immune-enhancing compounds.
Example 6
Determination of CD200 Upregulation in CLL
Patients
[0282] Lymphocytes from 15 CLL patients were stained with
FITC-conjugated anti-CD5 (e-bioscience), APC-conjugated anti-CD 19
(e-bioscience) and PE-conjugated anti-CD200 (Serotec). Lymphocytes
from healthy donors were stained accordingly. CD200 expression on
CD5+CD19+ cells was determined. As shown in the table below,
although the level of CD200 expression varied among CLL patient
samples, all CLL samples showed elevated levels (1.6-4-fold range)
higher CD200 expression compared to CD200 expression on normal B
cells. The CLL patients showing elevated levels of CD200 expression
are selected for anti-CD200 treatment in accordance with the
methods described herein.
TABLE-US-00002 FACS analysis of CD200 expression on B-CLL cells in
comparison to normal B cells CLL sample Healthy donor B-CLL CD200
Normal B Ratio Donor ID (GMFI) CD200 (GMFI) (CLL/normal B) RC011731
93 58 1.6 RF020934 659 185 3.6 JA073031 334 64 5.2 GR011846 156 64
2.4 BB101735 420 95 4.4 DM6988172 290 97 2.9 MR8074020 403 97 4.2
CB8267677 300 97 3.1 GB1325248 178 77(7) 2.3 VN7029373 154 77(7)
2.0 DG8942820 146 77(7) 1.9 MM8451869 237 77(7) 3.1 JR4539931 215
77(7) 2.8 HS6787771 305 77(7) 4.0 VB040439 123 41 3.0 MEAN = 3.1
STDEV = 1.0
REFERENCES
[0283] The following references are incorporated herein by
reference to more fully describe the state of the art to which the
present invention pertains. Any inconsistency between these
publications below or those incorporated by reference above and the
present disclosure shall be resolved in favor of the present
disclosure. [0284] 1) Agarwal, et al., (2003). Disregulated
expression of the Th2 cytokine gene in patients with intraoral
squamous cell carcinoma. Immunol Invest 32:17-30. [0285] 2)
Almasri, N M et al. (1992). Am J Hemato 140 259-263. [0286] 3)
Contasta, et al., (2003). Passage from normal mucosa to adenoma and
colon cancer: alteration of normal sCD30 mechanisms regulating
TH1/TH2 cell functions. Cancer Biother Radiopharm 18:549-557.
[0287] 4) Gorczynski, et al., (1998). Increased expression of the
novel molecule OX-2 is involved in prolongation of murine renal
allograft survival. Transplantation 65:1106-1114. [0288] 5)
Gorczynski, et al., (2001). Evidence of a role for CD200 in
regulation of immune rejection of leukaemic tumour cells in C57BL/6
mice. Clin Exp Immunol 126:220-229. [0289] 6) Hainsworth, J D
(2000). Oncologist 2000; 5(5):376-84. [0290] 7) Inagawa, et al.,
(1998). Mechanisms by which chemotherapeutic agents augment the
antitumor effects of tumor necrosis factor: involvement of the
pattern shift of cytokines from Th2 to Th1 in tumor lesions.
Anticancer Res 18:3957-3964. [0291] 8) Ito, et al., (1999). Lung
carcinoma: analysis of T helper type 1 and 2 cells and T cytotoxic
type 1 and 2 cells by intracellular cytokine detection with flow
cytometry. Cancer 85:2359-2367. [0292] 9) Kiani, et al., (2003).
Normal intrinsic Th1/Th2 balance in patients with chronic phase
chronic myeloid leukemia not treated with interferon-alpha or
imatinib. Haematologica 88:754-761. [0293] 10) Lauerova, et al.,
(2002). Malignant melanoma associates with Th1/Th2 imbalance that
coincides with disease progression and immunotherapy response.
Neoplasma 49:159-166. [0294] 11) Maggio, et al., (2002).
Chemokines, cytokines and their receptors in Hodgkin's lymphoma
cell lines and tissues. Ann Oncol 13 Suppl 1:52-56. [0295] 12)
Nilsson, K (1992). Burn Cell. 5(1):25-41. [0296] 13) Podhorecka, et
al., (2002). T type 1/type 2 subsets balance in B-cell chronic
lymphocytic leukemia--the three-color flow cytometry analysis. Leuk
Res 26:657-660. [0297] 14) Pu, Q Q and Bezwoda, W (2000).
Anticancer Res. 20(4):2569-78. [0298] 15) Smyth, et al., (2003).
Renal cell carcinoma induces prostaglandin E2 and T-helper type 2
cytokine production in peripheral blood mononuclear cells. Ann Surg
Oncol 10:455-462. [0299] 16) Tatsumi, et al., (2002).
Disease-associated bias in T helper type 1 (Th1)/Th2 CD4(+) T cell
responses against MAGE-6 in HLA-DRB10401(+) patients with renal
cell carcinoma or melanoma. J Exp Med 196:619-628. [0300] 17) Walls
A V et al. (1989). Int. J Cancer 44846-853. [0301] 18) Winter, et
al., (2003). Tumour-induced polarization of tumour vaccine-draining
lymph node T cells to a type 1 cytokine profile predicts inherent
strong immunogenicity of the tumour and correlates with therapeutic
efficacy in adoptive transfer studies. Immunology 108:409-419.
[0302] It will be understood that various modifications may be made
to the embodiments disclosed herein. For example, as those skilled
in the art will appreciate, the specific sequences described herein
can be altered slightly without necessarily adversely affecting the
functionality of the polypeptide, antibody or antibody fragment
used in binding OX-2/CD200. For instance, substitutions of single
or multiple amino acids in the antibody sequence can frequently be
made without destroying the functionality of the antibody or
fragment. Thus, it should be understood that polypeptides or
antibodies having a degree of identity greater than 70% to the
specific antibodies described herein are within the scope of this
disclosure. In particularly useful embodiments, antibodies having
an identity greater than about 80% to the specific antibodies
described herein are contemplated. In other useful embodiments,
antibodies having an identity greater than about 90% to the
specific antibodies described herein are contemplated. Therefore,
the above description should not be construed as limiting, but
merely as exemplifications of preferred embodiments. Those skilled
in the art will envision other modifications within the scope and
spirit of this disclosure.
Sequence CWU 1
1
213114PRTrabbit 1Thr Leu Ser Thr Gly Tyr Ser Val Gly Ser Tyr Val
Ile Ala 1 5 10 211PRTrabbit 2Gln Ala Ser Glu Ser Ile Arg Asn Tyr
Leu Ala 1 5 10 311PRTrabbit 3Gln Ala Ser Glu Ser Ile Ser Asn Trp
Leu Ala 1 5 10 411PRTrabbit 4Gln Ala Ser Glu Ser Ile Ser Asn Tyr
Leu Ala 1 5 10 511PRTrabbit 5Gln Ala Ser Gln Asn Ile Tyr Ser Asn
Leu Ala 1 5 10 611PRTrabbit 6Gln Ala Ser Gln Ser Val Asn Asn Leu
Leu Ala 1 5 10 711PRTrabbit 7Gln Ala Ser Glu Ser Ile Asn Asn Tyr
Leu Ala 1 5 10 811PRTrabbit 8Leu Ala Ser Glu Asn Val Tyr Ser Ala
Val Ala 1 5 10 911PRTrabbit 9Leu Ala Ser Glu Asn Val Tyr Gly Ala
Val Ala 1 5 10 1011PRTrabbit 10Gln Ala Ser Gln Ser Ile Ser Asn Leu
Leu Ala 1 5 10 1111PRTrabbit 11Leu Ala Ser Glu Asn Val Ala Ser Thr
Val Ser 1 5 10 1214PRTrabbit 12Thr Leu Ser Thr Gly Tyr Ser Val Gly
Glu Tyr Pro Val Val 1 5 10 1314PRTrabbit 13Thr Leu Arg Thr Gly Tyr
Ser Val Gly Glu Tyr Pro Leu Val 1 5 10 1411PRTrabbit 14Leu Ala Ser
Glu Asp Ile Tyr Ser Gly Leu Ser 1 5 10 1511PRTrabbit 15Gln Ala Ser
Gln Ser Val Ser Asn Leu Leu Ala 1 5 10 1611PRTrabbit 16Gln Ala Ser
Glu Asp Ile Glu Ser Tyr Leu Ala 1 5 10 1712PRTrabbit 17Gln Ser Ser
Gln Ser Ile Ala Gly Ala Tyr Leu Ser 1 5 10 1810PRTrabbit 18His Ser
Glu Glu Ala Lys His Gln Gly Ser 1 5 10 197PRTrabbit 19Gly Ala Ser
Asn Leu Glu Ser 1 5 207PRTrabbit 20Arg Ala Ser Thr Leu Ala Ser 1 5
217PRTrabbit 21Leu Ala Phe Thr Leu Ala Ser 1 5 227PRTrabbit 22Gly
Ala Ser Asp Leu Glu Ser 1 5 2310PRTrabbit 23His Thr Asp Asp Ile Lys
His Gln Gly Ser 1 5 10 247PRTrabbit 24Leu Ala Ser Lys Leu Ala Ser 1
5 2513PRTrabbit 25Ala Thr Ala His Gly Ser Gly Ser Ser Phe His Val
Val 1 5 10 2610PRTrabbit 26Gln Ser Gly Asp Tyr Ser Ala Gly Leu Thr
1 5 10 2710PRTrabbit 27Gln Ser Gly Tyr Tyr Ser Ala Gly Leu Thr 1 5
10 2810PRTrabbit 28Gln Ser Gly Tyr Tyr Ser Ala Gly Val Thr 1 5 10
2914PRTrabbit 29Gln Gly Gly Asp Tyr Ser Ser Ser Ser Ser Tyr Gly Tyr
Gly 1 5 10 3010PRTrabbit 30Gln Ser Gly Tyr Tyr Ser Pro Gly Val Thr
1 5 10 3110PRTrabbit 31Gln Ser Gly Tyr Tyr Ser Gly Gly Ala Thr 1 5
10 329PRTrabbit 32Gln Gly Tyr Ser Ser Tyr Pro Pro Thr 1 5
338PRTrabbit 33Gln Gly Tyr Ser Ser Tyr Pro Thr 1 5 3412PRTrabbit
34Ala Gly Tyr Lys Ser Ser Ser Thr Asp Gly Ile Ala 1 5 10
3511PRTrabbit 35Gln Ser Gly Tyr Tyr Ser Ala Gly His Leu Thr 1 5 10
3612PRTrabbit 36Leu Gly Gly Phe Gly Tyr Ser Thr Thr Gly Leu Thr 1 5
10 3713PRTrabbit 37Ala Ile Ala His Gly Thr Glu Ser Ser Phe His Val
Val 1 5 10 3813PRTrabbit 38Ala Thr Gly His Gly Ser Gly Ser Ser Ala
Gly Val Val 1 5 10 3912PRTrabbit 39Leu Gly Gly Tyr Pro Tyr Ser Ser
Thr Gly Thr Ala 1 5 10 4011PRTrabbit 40Gln Ser Gly Trp Tyr Ser Ala
Gly Ala Leu Thr 1 5 10 4111PRTrabbit 41Gln Ser Gly Tyr Tyr Arg Ala
Gly Asp Leu Thr 1 5 10 4211PRTrabbit 42Gln Ser Gly Tyr Tyr Ser Ala
Gly Ala Leu Thr 1 5 10 4310PRTrabbit 43Gln Ser Asn Ala Trp Ser Val
Gly Met Thr 1 5 10 4410PRTrabbit 44Ala Ala Gln Tyr Ser Gly Asn Ile
Tyr Thr 1 5 10 455PRTrabbit 45Asn Tyr Ala Met Thr 1 5 465PRTrabbit
46Ser Tyr Gly Leu Ser 1 5 475PRTrabbit 47Thr Tyr Gly Val Ser 1 5
485PRTrabbit 48Ser Asn Ala Met Gly 1 5 495PRTrabbit 49Thr Asn Ala
Met Gly 1 5 506PRTrabbit 50Ser Ser Asp Trp Ile Cys 1 5 515PRTrabbit
51Ser Asp Val Ile Ser 1 5 525PRTrabbit 52Thr Tyr Ala Met Gly 1 5
535PRTrabbit 53Ser Asn Ala Met Thr 1 5 545PRTrabbit 54Asp Phe Ala
Met Ser 1 5 555PRTrabbit 55Ser Tyr Gly Met Asn 1 5 565PRTrabbit
56Ser Asn Ala Met Ser 1 5 575PRTrabbit 57Thr Asn Ala Ile Ser 1 5
585PRTrabbit 58Ser Tyr Tyr Met Ser 1 5 595PRTrabbit 59Ser Tyr Thr
Met Ser 1 5 605PRTrabbit 60Ser Asn Ala Ile Ser 1 5 615PRTrabbit
61Thr Asn Ala Met Ser 1 5 626PRTrabbit 62Ser Ser Tyr Trp Ile Cys 1
5 635PRTrabbit 63Asn Tyr Gly Val Asn 1 5 6415PRTrabbit 64Ile Ile
Ser Ser Asn Gly Gly Ala Asp Tyr Ala Ser Trp Ala Lys 1 5 10 15
6516PRTrabbit 65Tyr Phe Asp Pro Val Phe Gly Asn Ile Tyr Tyr Ala Thr
Trp Val Asp 1 5 10 15 6616PRTrabbit 66Tyr Asn Asp Pro Ile Phe Gly
Asn Thr Tyr Tyr Ala Thr Trp Val Asn 1 5 10 15 6715PRTrabbit 67Ile
Ile Ser Ser Ser Gly Gly Thr Tyr Tyr Ala Ser Trp Ala Lys 1 5 10 15
6815PRTrabbit 68Ile Ile Ser Ser Ser Gly Ser Thr Tyr Tyr Ala Ser Trp
Ala Lys 1 5 10 15 6917PRTrabbit 69Cys Ile Tyr Thr Gly Ser Ser Ser
Ser Thr Trp Tyr Ala Ser Trp Ala 1 5 10 15 Lys 7016PRTrabbit 70Tyr
Ile Tyr Thr Gly Asp Gly Ser Thr Asp Tyr Ala Ser Trp Val Asn 1 5 10
15 7115PRTrabbit 71Ser Ile Tyr Ala Ser Arg Ser Pro Tyr Tyr Ala Ser
Trp Ala Lys 1 5 10 15 7215PRTrabbit 72Thr Ile Ile Tyr Gly Asp Asn
Thr Tyr Tyr Ala Ser Trp Ala Lys 1 5 10 15 7317PRTrabbit 73Val Val
Tyr Ala Gly Thr Arg Gly Asp Thr Tyr Tyr Ala Asn Trp Ala 1 5 10 15
Lys 7416PRTrabbit 74Tyr Ile Asp Pro Asp Tyr Gly Ser Thr Tyr Tyr Ala
Ser Trp Val Asn 1 5 10 15 7515PRTrabbit 75Ile Thr Tyr Pro Ser Gly
Asn Val Tyr Tyr Ala Ser Trp Ala Lys 1 5 10 15 7615PRTrabbit 76Tyr
Ser Ser Tyr Gly Asn Asn Ala His Tyr Thr Asn Trp Ala Lys 1 5 10 15
7715PRTrabbit 77Ile Ile Ile Gly Ser Gly Thr Thr Tyr Tyr Ala Asn Trp
Ala Lys 1 5 10 15 7815PRTrabbit 78Ile Ile Ser Ser Ser Gly Thr Ser
Tyr Tyr Ala Thr Trp Ala Lys 1 5 10 15 7915PRTrabbit 79Ile Ile Ser
Ser Ser Gly Ser Ala Tyr Tyr Ala Thr Trp Ala Lys 1 5 10 15
8015PRTrabbit 80Ile Ile Val Gly Ser Gly Thr Thr Tyr Tyr Ala Asp Trp
Ala Lys 1 5 10 15 8115PRTrabbit 81Thr Ile Thr Tyr Gly Thr Asn Ala
Tyr Tyr Ala Ser Trp Ala Lys 1 5 10 15 8217PRTrabbit 82Cys Ile Tyr
Thr Gly Ser Asn Gly Ser Thr Tyr Tyr Ala Ser Trp Ala 1 5 10 15 Lys
8316PRTrabbit 83Tyr Ile Asp Pro Val Phe Gly Ser Thr Tyr Tyr Ala Ser
Trp Val Asn 1 5 10 15 8417PRTrabbit 84Asp Asp Glu Gly Tyr Asp Asp
Tyr Gly Asp Tyr Met Gly Tyr Phe Thr 1 5 10 15 Leu 8514PRTrabbit
85Asp Arg Ile Tyr Val Ser Ser Val Gly Tyr Ala Phe Asn Leu 1 5 10
8614PRTrabbitMISC_FEATURE(11)..(14)Xaa = is an unknown amino acid
86Asp Arg Ala Tyr Ala Ser Ser Ser Gly Tyr Xaa Xaa Xaa Xaa 1 5 10
8713PRTrabbit 87Asp Trp Ile Ala Ala Gly Lys Ser Tyr Gly Leu Asp Leu
1 5 10 889PRTrabbit 88Arg Tyr Thr Gly Asp Asn Gly Asn Leu 1 5
8913PRTrabbit 89Asp Ala Ala Tyr Ala Gly Tyr Gly Trp Ile Phe Asn Leu
1 5 10 9011PRTrabbit 90Gly Asp Ala Gly Ser Ile Pro Tyr Phe Lys Leu
1 5 10 917PRTrabbit 91Gly Asn Val Phe Ser Asp Leu 1 5 927PRTrabbit
92Gly Leu Thr Tyr Tyr Pro Leu 1 5 9312PRTrabbit 93Gly Ala Tyr Ser
Gly Tyr Pro Ser Tyr Phe Asn Leu 1 5 10 945PRTrabbit 94Gly Phe Phe
Asn Leu 1 5 957PRTrabbit 95Gly Asn Ala Tyr Ser Asp Leu 1 5
9622PRTrabbit 96Asp Gln Pro Ile Ile Tyr Gly Ala Tyr Gly Asp Tyr Gly
Leu Ala Thr 1 5 10 15 Gly Thr Arg Leu Asp Leu 20 9722PRTrabbit
97Asp Gln Pro Ile Ile Asp Ala Ala Tyr Gly Asp Tyr Gly Ile Ala Thr 1
5 10 15 Gly Thr Arg Leu Asp Leu 20 9822PRTrabbit 98Asp Gln Pro Ile
Ile Thr Thr Asp Tyr Gly Gly Tyr Gly Ile Ala Thr 1 5 10 15 Gly Thr
Arg Leu Asp Leu 20 9919PRTrabbit 99Asp Gln Pro Ile Thr Tyr Ala Gly
Tyr Gly Tyr Ala Thr Gly Thr Arg 1 5 10 15 Leu Asp Leu 1007PRTrabbit
100Gly Asn Thr Tyr Ser Asp Leu 1 5 10113PRTrabbit 101Ala Tyr Ile
Tyr Tyr Gly Gly Tyr Gly Phe Phe Asp Leu 1 5 10 10210PRTrabbit
102Glu Ala Ser Phe Tyr Tyr Gly Met Asp Leu 1 5 10
10336DNAartificial sequenceprimer 103ggcctctaga cagcctgtgc
tgactcagtc gccctc 3610443DNAartificial sequenceprimer 104cgagggggca
gccttgggct gacctgtgac ggtcagctgg gtc 4310543DNAartificial
sequenceprimer 105gacccagctg accgtcacag gtcagcccaa ggctgccccc tcg
4310634DNAartificial sequenceprimer 106tctaatctcg agcagcagca
gctgatggag tccg 3410747DNAartificial sequenceprimer 107gaccgatggg
cccttggtgg aggctgagga gacggtgacc agggtgc 4710847DNAartificial
sequenceprimer 108gcaccctggt caccgtctcc tcagcctcca ccaagggccc
atcggtc 4710928DNAartificial sequenceprimer 109ccactgtcag
agctcccggg tagaagtc 2811023DNAartificial sequenceprimer
110gtcaccggtt cggggaagta gtc 2311110PRTmurine 111Gly Phe Asn Ile
Lys Asp Tyr Tyr Met His 1 5 10
11210PRTmurineMISC_FEATURE(9)..(9)Xaa = any amino acid 112Asp Phe
Asn Ile Lys Asp Tyr Tyr Xaa His 1 5 10 11310PRTmurine 113Gly Leu
Asn Ile Lys Asp Tyr Tyr Met His 1 5 10 11410PRTmurine 114Gly Phe
Asn Ile Lys Asp Tyr Tyr Ile His 1 5 10 11510PRTmurine 115Gly Phe
Asn Ile Lys Asp Tyr Tyr Leu His 1 5 10 11610PRTmurine 116Gly Tyr
Thr Phe Thr Ser Tyr Val Met His 1 5 10
11717PRTmurineMISC_FEATURE(4)..(4)Xaa = any amino acid 117Trp Ile
Asp Xaa Glu Asn Gly Asp Thr Lys Tyr Ala Pro Lys Phe Gln 1 5 10 15
Gly 11817PRTmurine 118Trp Ile Asp Pro Glu Asn Gly Asp Thr Lys Tyr
Ala Pro Lys Phe Gln 1 5 10 15 Gly
11917PRTmurineMISC_FEATURE(6)..(6)Xaa = any amino acid 119Trp Ile
Asp Pro Glu Xaa Asp Asp Thr Lys Tyr Ala Pro Lys Phe Gln 1 5 10 15
Gly 12017PRTmurine 120Trp Ile Asp Pro Glu Asn Gly Asn Thr Lys Tyr
Ala Pro Lys Phe Gln 1 5 10 15 Gly 12117PRTmurine 121Trp Ile Asp Pro
Asp Asn Gly Asp Thr Lys Tyr Ala Pro Lys Phe Arg 1 5 10 15 Gly
12217PRTmurine 122Trp Ile Asp Pro Asp Asn Gly Asp Thr Lys Tyr Ala
Pro Lys Phe Arg 1 5 10 15 Asp 12317PRTmurine 123Tyr Ile Asn Pro Tyr
Asn Asp Val Thr Lys Asn Asn Glu Lys Phe Arg 1 5 10 15 Gly
12417PRTmurine 124Tyr Ile Asn Pro Tyr Asn Asp Ile Thr Asn Tyr Asn
Glu Lys Phe Lys 1 5 10 15 Gly 12517PRTmurine 125Tyr Ile Asn Pro Tyr
Asn Asp Gly Ser Lys Tyr Asn Glu Lys Phe Lys 1 5 10 15 Gly
12617PRTmurine 126Tyr Ile Asn Pro Tyr Asn Asp Gly Thr Lys Tyr Asn
Glu Lys Phe Lys 1 5 10 15 Gly 12717PRTmurine 127Tyr Ile Asn Pro Tyr
Asn Asp Gly Thr Arg Tyr Asn Glu Lys Phe Lys 1 5 10 15 Gly
12817PRTmurine 128Tyr Ile Asn Pro Tyr Asn Asp Val Thr Asn Tyr Asn
Glu Lys Phe Lys 1 5 10 15 Gly 12917PRTmurine 129Tyr Ile Asn Pro Tyr
Asn Asp Gly Thr Asn Tyr Asn Glu Lys Phe Lys 1 5 10 15 Gly
13017PRTmurine 130Tyr Ile Asn Pro Tyr Asn Asp Val Thr Lys Tyr Asn
Glu Lys Phe Arg 1 5 10 15 Gly 13113PRTmurine 131Lys Asn Tyr Tyr Val
Ser Asn Tyr Asn Tyr Phe Asp Val 1 5 10 13213PRTmurine 132Lys Asn
Tyr Tyr Val Ser Asp Tyr Asn Tyr Phe Asp Val 1 5 10 13313PRTmurine
133Lys Asn Tyr Tyr Val Ser Asn Tyr Asn Phe Phe Asp Val 1 5 10
13412PRTmurine 134Lys Arg Gly Gly Tyr Asp Gly Ala Trp Phe Ala Tyr 1
5 10 13510PRTmurine 135Gly Phe Asn Ile Lys Asp His Tyr Met His 1 5
10 13610PRTmurine 136Ala Phe Asn Ile Lys Asp His Tyr Met His 1 5 10
13710PRTmurine 137Gly Phe Asn Leu Lys Asp Tyr Tyr Met His 1 5 10
13810PRTmurine 138Gly Tyr Thr Phe Thr Asp Tyr Trp Leu His 1 5 10
13910PRTmurine 139Gly Phe Thr Phe Ser Ala Ala Trp Met Asp 1 5 10
14010PRTmurine 140Gly Tyr Thr Phe Thr Glu Tyr Thr Met His 1 5 10
14110PRTmurine 141Gly Phe Thr Phe Ser Gly Phe Ala Met Ser 1 5 10
14210PRTmurine 142Gly Phe Thr Phe Thr Gly Tyr Ala Met Ser 1 5 10
14310PRTmurine 143Gly Phe Thr Phe Ser Ser His Ala Met Ser 1 5 10
14410PRTmurine 144Gly Tyr Thr Phe Thr Glu Phe Thr Met His 1 5 10
14510PRTmurine 145Gly Tyr Ile Phe Thr Ser Phe Tyr Ile His 1 5 10
14610PRTmurine 146Gly Tyr Thr Phe Thr Ser Phe Tyr Ile His 1 5 10
14710PRTmurine 147Gly Tyr Thr Phe Thr Asp Tyr Trp Met His 1 5 10
14810PRTmurine 148Gly Tyr Thr Phe Thr Asp Asn Trp Ile His 1 5 10
14910PRTmurine 149Gly Tyr Ser Phe Thr Asp Tyr Ile Ile Leu 1 5 10
15010PRTmurine 150Gly Phe Asn Ile Lys Asp Ser Tyr Ile His 1 5 10
15110PRTmurineMISC_FEATURE(6)..(6)Xaa = any amino acid 151Gly Phe
Asn Ile Lys Xaa Ser Tyr Ile His 1 5 10 15210PRTmurine 152Gly Tyr
Thr Phe Thr Ser Tyr Thr Ile His 1 5 10 15310PRTmurine 153Gly Tyr
Thr Phe Thr Glu Tyr Ile Met His 1 5 10 15416PRTmurine 154Trp Ile
Pro Glu Asn Gly Asp Thr Glu Tyr Ala Pro Lys Phe Gln Gly 1 5 10 15
15516PRTmurine 155Trp Ile Pro Glu Ser Gly Asp Thr Glu Tyr Ala Pro
Lys Phe Gln Gly 1 5 10 15 15616PRTmurine 156Trp Ile Pro Glu Asn Gly
Asn Thr Glu Tyr Ala Pro Lys Phe Gln Gly 1 5 10 15
15716PRTmurineMISC_FEATURE(16)..(16)Xaa = any amino acid 157Trp Ile
Pro Glu Asn Gly Asn Thr Glu Tyr Ala Pro Lys Phe Gln Xaa 1 5 10 15
15817PRTmurine 158Trp Ile Asp Pro Glu Asn Gly Asn Thr Glu Tyr Ala
Pro Lys Phe Gln 1 5 10 15 Gly 15917PRTmurine 159Trp Ile Asp Pro Glu
Ser Gly Asp Thr Glu Tyr Ala Pro Lys Phe Gln 1 5 10 15 Gly
16017PRTmurine 160Thr Ile Asp Thr Ser Thr Gly Tyr Thr Gly Tyr Asn
Gln Lys Phe Lys 1 5 10 15 Gly 16119PRTmurine 161Glu Ile Arg Ser Lys
Ala Asn Asn His Ala Thr Tyr Tyr Ala Glu Ser 1 5 10 15 Val Lys Gly
16217PRTmurine 162Gly Val Asn Pro Asn Asn Gly Gly Ala Leu Tyr Asn
Gln Lys Phe Lys 1 5 10 15
Gly 16316PRTmurine 163Ser Ile Ser Ser Gly Gly Thr Thr Tyr Tyr Leu
Asp Ser Val Lys Gly 1 5 10 15 16416PRTmurine 164Ser Ile Ser Ser Gly
Gly Ser Ala Tyr Tyr Pro Asp Ser Val Lys Gly 1 5 10 15
16517PRTmurine 165Trp Ile Asp Pro Glu Ile Gly Ala Thr Lys Tyr Val
Pro Lys Phe Gln 1 5 10 15 Gly 16617PRTmurine 166Ser Ile Ser Ser Gly
Gly Gly Thr Tyr Tyr Pro Asn Ser Val Lys Gly 1 5 10 15 Arg
16718PRTmurine 167Gly Ile Asn Pro Glu Asn Asn Gly Gly Tyr Ser Tyr
Asn Gln Lys Phe 1 5 10 15 Lys Gly 16818PRTmurine 168Gly Ile Asn Pro
Glu Asn Thr Gly Gly Tyr Ser Tyr Asn Gln Lys Phe 1 5 10 15 Lys Gly
16918PRTmurineMISC_FEATURE(8)..(8)Xaa = any amino acid 169Gly Ile
Asn Pro Glu Asn Thr Xaa Gly Xaa Ala Tyr Asn Gln Lys Phe 1 5 10 15
Lys Gly 17017PRTmurine 170Ala Ile Asp Thr Phe Asp Ser Asn Thr Lys
Tyr Asn Gln Lys Phe Lys 1 5 10 15 Gly 17117PRTmurine 171Ala Ile Asp
Thr Phe Asp Ser Asn Thr Arg Tyr Asn Gln Lys Phe Lys 1 5 10 15 Gly
17217PRTmurine 172Ala Ile Asp Thr Phe Asp Ser Asn Thr Arg Tyr Asn
Pro Lys Phe Lys 1 5 10 15 Gly 17317PRTmurine 173Thr Ile Asp Ala Ser
Asp Arg Tyr Ile Ser Tyr Asn Gln Lys Phe Arg 1 5 10 15 Gly
17417PRTmurine 174His Ile Asp Pro Tyr Tyr Gly Ser Ser Asn Tyr Asn
Leu Lys Phe Lys 1 5 10 15 Gly 17517PRTmurine 175Trp Ile Asp Pro Glu
Asn Gly Gly Thr Glu Tyr Ala Pro Lys Phe Gln 1 5 10 15 Gly
17617PRTmurine 176Tyr Ile Asn Pro Ser Ser Gly Tyr Thr Asn Tyr Asn
Gln Lys Phe Lys 1 5 10 15 Asp 17717PRTmurine 177Gly Ile Asn Pro Asn
Thr Gly Ala Tyr Asn Tyr Asn Gln Lys Phe Lys 1 5 10 15 Gly
1789PRTmurine 178Phe Asn Gly Tyr Tyr Ala Met Asp Tyr 1 5
1799PRTmurine 179Phe Asn Gly Tyr Leu Ala Leu Asp Tyr 1 5
1809PRTmurineMISC_FEATURE(9)..(9)Xaa = any amino acid 180Phe Asn
Gly Tyr Gln Ala Leu Asp Xaa 1 5 1819PRTmurine 181Phe Asn Gly Tyr
Gln Ala Leu Asp Gln 1 5 1829PRTmurine 182Phe Asn Gly Tyr Leu Ala
Leu Asp Gln 1 5 1839PRTmurine 183Arg Asn Glu Tyr Tyr Thr Met Asp
Tyr 1 5 1849PRTmurine 184Arg Asn Glu Tyr Tyr Ile Met Asp Tyr 1 5
18510PRTmurine 185Gly Gly Asp Asn Tyr Val Trp Phe Ala Tyr 1 5 10
18611PRTmurine 186Asn Gly Tyr Asp Asp Gly Val Pro Phe Asp Tyr 1 5
10 18712PRTmurine 187Arg Ser Asn Tyr Arg Tyr Asp Asp Ala Met Asp
Tyr 1 5 10 18811PRTmurine 188Gly Asn Tyr Tyr Ser Gly Thr Ser Tyr
Asp Tyr 1 5 10 18913PRTmurine 189Leu Tyr Gly Asn Tyr Asp Arg Tyr
Tyr Ala Met Asp Tyr 1 5 10 19013PRTmurine 190Arg Gly Asp Tyr Tyr
Arg Tyr Pro Tyr Ala Met Asp Tyr 1 5 10 19112PRTmurine 191Met Ile
Thr Thr Gly Tyr His Tyr Ala Met Asp Tyr 1 5 10 19212PRTmurine
192Lys Ala Arg Gly Asp Ser Gly Ala Trp Phe Ala Tyr 1 5 10
1934PRTmurine 193Gly Val Asp Tyr 1 19410PRTmurine 194Leu Glu Gly
Ser Gly Tyr Gly Phe Ala Tyr 1 5 10 1958PRTmurine 195Ser Lys Arg Asp
Tyr Phe Asp Tyr 1 5 19611PRTmurine 196Cys Asn Phe Tyr Gly Asn Pro
Tyr Phe Asp Tyr 1 5 10 19711PRTmurine 197Cys Asn Phe Tyr Ala Asn
Pro Tyr Phe Asp Tyr 1 5 10 19812PRTmurine 198Arg Pro Met Ile Thr
Ala Gly Ala Trp Phe Ala Tyr 1 5 10 19913PRTmurine 199Ile Thr Thr
Val Val Gly Tyr Tyr Tyr Ala Met Asp Tyr 1 5 10 200120PRTmurine
200Leu Glu Val Lys Leu Val Glu Ser Gly Gly Gly Leu Val Lys Pro Gly
1 5 10 15 Gly Ser Leu Lys Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe
Ser Gly 20 25 30 Phe Ala Met Ser Trp Val Arg Gln Thr Pro Glu Lys
Arg Leu Glu Trp 35 40 45 Val Ala Ser Ile Ser Ser Gly Gly Thr Thr
Tyr Tyr Leu Asp Ser Val 50 55 60 Lys Gly Arg Phe Thr Ile Ser Arg
Asp Ile Ala Arg Asn Ile Leu Tyr 65 70 75 80 Leu Gln Met Ser Ser Leu
Arg Ser Glu Asp Thr Ala Met Tyr Tyr Cys 85 90 95 Ala Arg Gly Asn
Tyr Tyr Ser Gly Thr Ser Tyr Asp Tyr Trp Gly Gln 100 105 110 Gly Thr
Thr Leu Thr Val Ser Ser 115 120 201123PRTmurine 201Leu Glu Val Gln
Leu Gln Gln Ser Gly Ala Glu Leu Val Arg Ser Gly 1 5 10 15 Ala Ser
Val Lys Leu Ser Cys Thr Ala Ser Gly Phe Asn Ile Lys Asp 20 25 30
Tyr Tyr Met His Trp Val Lys Gln Arg Pro Glu Gln Gly Leu Glu Trp 35
40 45 Ile Gly Trp Ile Asp Pro Glu Asn Gly Asp Thr Lys Tyr Ala Pro
Lys 50 55 60 Phe Gln Gly Lys Ala Thr Met Thr Ala Asp Thr Ser Ser
Asn Thr Ala 65 70 75 80 Tyr Leu Gln Leu Ser Ser Leu Thr Ser Glu Asp
Thr Ala Val Tyr Tyr 85 90 95 Cys Asn Ala Lys Asn Tyr Tyr Val Ser
Asn Tyr Asn Phe Phe Asp Val 100 105 110 Trp Gly Ala Gly Thr Thr Val
Thr Val Ser Ser 115 120 202123PRTmurine 202Leu Glu Val Gln Leu Gln
Gln Pro Gly Ala Glu Leu Val Arg Ser Gly 1 5 10 15 Ala Ser Val Lys
Leu Ser Cys Lys Ala Ser Gly Phe Asn Ile Lys Asp 20 25 30 Tyr Tyr
Ile His Trp Val Lys Gln Arg Pro Glu Gln Gly Leu Glu Trp 35 40 45
Ile Gly Trp Ile Asp Pro Glu Ile Gly Ala Thr Lys Tyr Val Pro Lys 50
55 60 Phe Gln Gly Lys Ala Thr Met Thr Thr Asp Thr Ser Ser Asn Thr
Ala 65 70 75 80 Tyr Leu Gln Leu Ser Ser Leu Thr Ser Glu Asp Thr Ala
Val Tyr Tyr 85 90 95 Cys Asn Ala Leu Tyr Gly Asn Tyr Asp Arg Tyr
Tyr Ala Met Asp Tyr 100 105 110 Trp Gly Gln Gly Thr Ser Val Thr Val
Ser Ser 115 120 203118PRTmurine 203Leu Glu Val Gln Leu Gln Gln Ser
Gly Pro Glu Leu Val Lys Pro Gly 1 5 10 15 Ala Ser Leu Lys Met Ser
Cys Lys Ala Ser Gly Tyr Ser Phe Thr Asp 20 25 30 Tyr Ile Ile Leu
Trp Val Lys Gln Asn His Gly Lys Ser Leu Glu Trp 35 40 45 Ile Gly
His Ile Asp Pro Tyr Tyr Gly Ser Ser Asn Tyr Asn Leu Lys 50 55 60
Phe Lys Gly Lys Ala Thr Leu Thr Val Asp Lys Ser Ser Ser Thr Ala 65
70 75 80 Tyr Met Gln Leu Asn Ser Leu Thr Ser Glu Asp Ser Ala Val
Tyr Tyr 85 90 95 Cys Gly Arg Ser Lys Arg Asp Tyr Phe Asp Tyr Trp
Gly Gln Gly Thr 100 105 110 Thr Leu Thr Val Ser Ser 115
204122PRTmurine 204Leu Glu Val Gln Leu Gln Gln Ser Gly Pro Glu Leu
Val Lys Pro Gly 1 5 10 15 Ala Ser Val Lys Ile Ser Cys Lys Thr Ser
Gly Tyr Thr Phe Thr Glu 20 25 30 Tyr Thr Met His Trp Val Lys Gln
Ser His Gly Lys Ser Leu Glu Trp 35 40 45 Ile Gly Gly Val Asn Pro
Asn Asn Gly Gly Ala Leu Tyr Asn Gln Lys 50 55 60 Phe Lys Gly Lys
Ala Thr Leu Thr Val Asp Lys Ser Ser Ser Thr Ala 65 70 75 80 Tyr Met
Glu Leu Arg Ser Leu Ala Ser Glu Asp Ser Ala Val Tyr Tyr 85 90 95
Cys Ala Arg Arg Ser Asn Tyr Arg Tyr Asp Asp Ala Met Asp Tyr Trp 100
105 110 Gly Gln Gly Thr Ser Val Thr Val Ser Ser 115 120
205119PRTmurine 205Leu Glu Val Gln Leu Gln Gln Ser Gly Ala Glu Leu
Val Arg Ser Gly 1 5 10 15 Ala Ser Val Lys Leu Ser Cys Thr Ala Ser
Ala Phe Asn Ile Lys Asp 20 25 30 His Tyr Met His Trp Val Lys Gln
Arg Pro Glu Gln Gly Leu Glu Trp 35 40 45 Ile Gly Trp Ile Asp Pro
Glu Ser Gly Asp Thr Glu Tyr Ala Pro Lys 50 55 60 Phe Gln Gly Lys
Ala Thr Met Thr Ala Asp Ile Ser Ser Asn Thr Ala 65 70 75 80 Tyr Leu
Gln Leu Asn Ser Leu Thr Ser Glu Asp Thr Ala Val Tyr Tyr 85 90 95
Cys Asn Ala Phe Asn Gly Tyr Gln Ala Leu Asp Gln Trp Gly Gln Gly 100
105 110 Thr Ser Val Thr Val Ser Ser 115 206114PRTmurine 206Ser Arg
Asp Ile Val Leu Thr Gln Ser Pro Ala Ser Leu Ala Val Ser 1 5 10 15
Leu Gly Gln Arg Ala Thr Ile Ser Cys Arg Ala Ser Glu Ser Val Asp 20
25 30 Ser Tyr Gly Asn Ser Phe Met His Trp Tyr Gln Gln Lys Pro Gly
Gln 35 40 45 Pro Pro Lys Leu Leu Ile Tyr Arg Ala Ser Asn Leu Glu
Ser Gly Ile 50 55 60 Pro Ala Arg Phe Ser Gly Ser Gly Ser Arg Thr
Asp Phe Thr Leu Thr 65 70 75 80 Ile Asn Pro Val Glu Ala Asp Asp Val
Ala Thr Tyr Tyr Cys Gln Gln 85 90 95 Ser Asn Glu Asp Pro Arg Thr
Phe Gly Gly Gly Thr Lys Leu Glu Ile 100 105 110 Lys Arg
207109PRTmurine 207Ser Arg Glu Ile Val Leu Thr Gln Ser Pro Ala Thr
Met Ser Ala Ser 1 5 10 15 Pro Gly Glu Lys Val Thr Met Thr Cys Ser
Ala Ser Ser Ser Val Arg 20 25 30 Tyr Met Tyr Trp Tyr Gln Gln Lys
Ser Ser Thr Ser Pro Lys Leu Trp 35 40 45 Ile Tyr Asp Thr Ser Lys
Leu Ala Ser Gly Val Pro Gly Arg Phe Ser 50 55 60 Gly Ser Gly Ser
Gly Asn Ser Tyr Ser Leu Thr Ile Ser Ser Met Glu 65 70 75 80 Ala Glu
Asp Val Ala Thr Tyr Tyr Cys Phe Gln Gly Ser Gly Tyr Pro 85 90 95
Leu Thr Phe Gly Ser Gly Thr Lys Leu Glu Ile Lys Arg 100 105
208110PRTmurine 208Ser Arg Asp Ile Val Met Thr Gln Ser Gln Lys Phe
Met Ser Thr Ser 1 5 10 15 Val Gly Asp Arg Val Ser Ile Thr Cys Lys
Ala Ser Gln Asn Val Arg 20 25 30 Thr Ala Val Ala Trp Tyr Gln Gln
Lys Pro Gly Gln Ser Pro Lys Ala 35 40 45 Leu Ile Tyr Leu Ala Ser
Asn Arg His Thr Gly Val Pro Asp Arg Phe 50 55 60 Thr Gly Ser Gly
Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Asn Val 65 70 75 80 Gln Ser
Glu Asp Leu Ala Asp Tyr Phe Cys Leu Gln His Trp Asn Tyr 85 90 95
Pro Leu Thr Phe Gly Ala Gly Thr Lys Leu Glu Leu Lys Arg 100 105 110
209110PRTmurine 209Ser Arg Asp Ile Gln Met Thr Gln Ser Pro Ser Ser
Met Tyr Ala Ser 1 5 10 15 Leu Gly Glu Arg Val Thr Ile Thr Cys Lys
Ala Ser Gln Asp Ile Asn 20 25 30 Ser Tyr Leu Ser Trp Phe Gln Gln
Lys Pro Gly Lys Ser Pro Lys Thr 35 40 45 Leu Ile Tyr Arg Ala Asn
Arg Leu Val Asp Gly Val Pro Ser Arg Phe 50 55 60 Ser Gly Ser Gly
Ser Gly Gln Asp Tyr Ser Leu Thr Ile Ser Ser Leu 65 70 75 80 Glu Tyr
Glu Asp Met Gly Ile Tyr Tyr Cys Leu Gln Tyr Asp Glu Phe 85 90 95
Pro Tyr Thr Phe Gly Gly Gly Thr Lys Leu Glu Ile Lys Arg 100 105 110
210115PRTmurine 210Ser Arg Asp Val Val Met Thr Gln Thr Pro Leu Thr
Leu Ser Val Thr 1 5 10 15 Ile Gly Gln Pro Ala Ser Ile Ser Cys Lys
Ser Ser Gln Ser Leu Leu 20 25 30 Asp Ile Asp Glu Lys Thr Tyr Leu
Asn Trp Phe Leu Gln Arg Pro Gly 35 40 45 Gln Ser Pro Lys Arg Leu
Ile Tyr Leu Val Ser Lys Leu Asp Ser Gly 50 55 60 Val Pro Asp Arg
Phe Thr Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu 65 70 75 80 Lys Ile
Ser Arg Val Glu Ala Glu Asp Leu Gly Val Tyr Tyr Cys Trp 85 90 95
Gln Gly Thr His Phe Pro Gln Thr Phe Gly Gly Gly Thr Lys Leu Glu 100
105 110 Ile Lys Arg 115 211113PRTmurine 211Ser Arg Glu Ile Val Leu
Thr Gln Ser Pro Ala Ile Met Ser Ala Ser 1 5 10 15 Leu Gly Glu Arg
Val Thr Met Thr Cys Thr Ala Ser Ser Ser Val Ser 20 25 30 Ser Ser
Tyr Leu His Trp Tyr Gln Gln Lys Pro Gly Ser Ser Pro Lys 35 40 45
Leu Trp Ile Tyr Ser Thr Ser Asn Leu Ala Ser Gly Val Pro Ala Arg 50
55 60 Phe Ser Gly Ser Gly Ser Gly Thr Ser Tyr Ser Leu Thr Ile Ser
Ser 65 70 75 80 Met Glu Ala Glu Asp Ala Ala Thr Tyr Tyr Cys Arg Gln
Tyr His Arg 85 90 95 Ser Pro Pro Ile Phe Thr Phe Gly Ser Gly Thr
Lys Leu Glu Ile Lys 100 105 110 Arg 21231DNAArtificial
Sequenceprimer 212gacaagcttg caaggatgga gaggctggtg a
3121334DNAArtificial Sequenceprimer 213gacggatccg cccccttttc
ctcctgcttt tctc 34
* * * * *