Note: Descriptions are shown in the official language in which they were submitted.
CA 02787818 2012-08-21
a,
POLYPEPTIDES AND,ANTIBODIES DERIVED FROM
CHRONIC LYM#HOCYTIC LEUKEMIA CELLS AND USES THEREOF
10 This is a division of Canadian Serial No. 2,517,968 filed March 4,
2004.
TECHNICAL FIELD
Cell lines derived from chronic iymphocytic leukemia (CLL) cells and the uses
thereof in the study and treatment of CLL and other diseases are disclosed. In
particular, this disclosure relates to a CLL cell line designated "CLL-AAT',
deposited
on December 11, 2001 with the American Type Culture Collection (Manassas,
Virginia, USA) in accordance with the terms of the Budapest Treaty under ATCC
accession no. PTA-3920.
BACKGROUND
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.
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
yearsof
CA 02787818 2012-08-21
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.
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
(VVSU-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.
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.
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.
2
CA 02787818 2012-08-21
Various mechanisms play a role in the body's response to a disease state,
including cancer and CLL. For example, CD4+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 Thl
cytokines such as IL-2 and IFN-y. 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 Thl 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
at.,
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 at., 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 Thl profile has been
demonstrated to augment anti-tumor effects of T cells. (See Winter, et al.,
Immunology 108:409-419 (2003); lnagawa, et al., Anticancer Res 18:3957-3964
998).)
Mechanisms underlying the capacity of tumor cells to drive the cytokine
expression of T helper cells from Thl to Th2 include the secretion of
cytokines such
as 11-10 or TGF-p as well as the expression of surface molecules.; interacting
with
cells of the immune system. 0K-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 at., Transplantation 65:1106-1114 (1998)) and evidence that OX-
2/CD200-expressing cells can inhibit the stimulation of Thl cytokine
production has
been provided. Gorczinski 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)).
Improved methods for treating individuals suffering from cancer or CLL are
desirable, especially to the extent they can enhance the activity of T cells.
3
CA 02787818 2012-08-21
SUMMARY
In one embodiment an CLL cell line of malignant origin is provided that is not
established by immortalisation with EBV. The cell line, which 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 B-CLL cell line,
derived
from a B-CLL primary cell.
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.
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.
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 On cell line, such as, for .ecample, 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.
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 with 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.
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
4
CA 02787818 2012-08-21
,odulating 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.
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.
In a preferred aspect, the CLL-AAT cell line may be used to identify
internalizing antibodies, which bine' 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.
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.
The present disclosure further is directed to methods wherein a determination
is made as to whether OX-2/CD200 is upregulated in El subject and, if so,
administering to the subject a polypeptide that binds to OX-2/CD200. in
another
embodiment, the polypeptide binds to an OX-2/CD200 receptor.
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.
5
CA 02787818 2012-08-21
BRIEF DESCRIPTION OF THE FIGURES
Fig. 1 schematically illustrates typical steps involved in cell surface
panning of
antibody libraries by magnetically-activated cell sorting (MACS).
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 +3 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
'Orepared from 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.
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-CD19 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.
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 (1 patient sample were considered to be patient
or
blood type-specific.
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-I is an erythroblastoid cell line
derived
from a erythroleukemia.
6
CA 02787818 2012-08-21
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-I cells were included as a negative control.
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.
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.
FIGS. 9A ¨ 9C provide a summary of CDR sequences and binding specificities of
selected scFv antibodies. As shown in Figure 9B, the clone numbers listed in
the left column are
also referred to herein by scFv numbers as follows: A2c (scFv-1), G12.1c (scFv-
2), B4.2a (scFv-
17), El c (scFv-3), F2d (scFv-18), E5e (scFv-4), H6.2b (scFv-5), G10.1 (scFv-
19), D11.1c (scFv-
6), A5.2c (scFv-20), Fld (scFv-7), Fle (scFv-8), E4.2 (scFv-21J, E2c (scFv-9),
A9c (scFv-9),
El le (scFv-10), A1.1 (scFv-11), F5.2 (scFv-12), FlOb (scFv-22), F7a (scFv-
23), F6b (scFv-13),
Cl2b (scFv-24), D2.1b (scFv-14), D1.1 (scFv-25), D2.2a (scFv-15), and D2.2b
(scFv-16).
7
CA 02787818 2012-08-21
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.
Fig 11 is a Table showing a summary of flow cytometry results comparing
expression levels of scFv-9 antigen with the percentage of CD38+ cells in
peripheral
blood mononuclear cells isolated from ten CLL patients.
Fig 12 shows the identification of scFv antigens by immunoprecipitation and
mass spectrometry. CLL-AAT cells were labeled with a solution of 0.5mg/m1
sulfo-
NHS-LC-biotin (Pierce) in PBS, pH8.O 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
7a
CA 02787818 2012-08-21
normal rabbit serum and protein A sepharose. Antigens were immunoprecipitatec'
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). MALD1-MS was
performed at the Proteomics Core Facility of The Scripps Research Institute
(La
Jolla, CA). pLC/MS/MS was performed at the Harvard Microchemistry Facility
(Cambridge, MA).
Fig 13 shOws that three scFv antibodies bind specifically to 293-EBNA cells
transiently transfected with a human OX-2/CD200 cDNA clone. A 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
(01AGEN). Two days after transfection, the cells were analyzed for binding to
scFv
antibodies by flow cytometry.
Fig 14 shows that the presence of OX-2/CD200 transfected cells resulted in
down-regulation of Thl cytokines such as IL-2 and IFN-y. Addition of the anti-
OX-
2/CD200 antibody at 30 pg/ml fully restored the Thl response.
Fig. 15 shows that the presence of CLL cells in a mixed lymphocyte reaction
resulted in down-regulation of the Thl response for IL-2.
Fig. 16 shows that the presence of CLL cells in a mixed lymphocyte reaction
resulted in down-regulation of the Thl response for IFN-y.
Figs. 17A and B show the mean +/-SD of tumor volumes for all groups of
NOD/SC1D mice were injected subcutaneously with 4x106 RAJI cells either in the
presence or absence of human.
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.
8
CA 02787818 2012-08-21
DETAILED DESCRIPTION
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 polypeptide that binds to OX-2/CD200. 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.
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+, CD23+, CD206m+, sIgdir" and arrested in GO/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.
As used herein, the term "antibodies" refers to complete antibodies or
antibody fragments capable of binding to a selected target. Included are Fv,
scFv,
Fab' and F(abe)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 pnage display or alternative techniques. Small fragments, such as FY and
scFv, possess advantageous properties for diagnostic and therapeutic
applications
on account of their small size and consequent superior tissue distribution.
The polypeptides and/or antibodies utilized herein are especially indicated
for
diagnostic and therapeutic applications. Accordingly, they may be altered with
an
effector protein such as a toxin or a label. Especially preferred are labels
which allow
the imaging of the distribution of the polypeptide or antibody in vivo. Such
labels may
9
CA 02787818 2012-08-21
be radioactive labels or radiopaque labels, such as metal particles, which are
reaf
visualisable within the body of a patient. Moreover, the labels may be
fluorescent
labels or other labels which are visualisable on tissue samples removed from
patients.
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.
1
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.
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
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.
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
CA 02787818 2012-08-21
f 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.
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 B-CLL cell line, derived from a B-CLL primary cell.
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.
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.
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.
In one embodiment, the CLL-AAT cell line may be used to identify
internalizing antibodies, which bind to cell surface components and are then
11
CA 02787818 2012-08-21
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
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. Patent No. 5,225,539.
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_
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.
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
12\
CA 02787818 2012-08-21
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 x YT, or M9 Minimal Medium. For yeast, suitable culture media include medium
YPD, YEPD, Minimal Medium, or Complete Minimal Dropout Medium.
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,
microcapsu)es, on agarose microbeads or ceramic cartridges).
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 8p2/0 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.
The foregoing, and other, techniques are discussed in, for example, Kohler
and Milstein, (1975) Nature 256:495-497; U.S. Patent No. 4,376,110; Harlow and
Lane, Antibodies: a Laboratory Manual, (1988) Cold Spring Harbor.
Techniques for the preparation of
recombinant antibody molecules is described in the above references and also
in,
for example W097/08320; U.S. Patent No. 5,427,908; U.S. Patent No. 5,508,717;
Smith, 1985, Science, Vol. 225, pp 1315-1317; Parmiey and Smith 1988, Gene 73,
13
CA 02787818 2012-08-21
pp 305-318; De La Cruz et al, 1988, Joumal of Biological Chemistry, 263 pp
4318-
4322; U.S. Patent No. 5,403,484; U.S. Patent No. 5223409; W088/06630;
W092/15679; U.S. Patent No. 5780279; U.S. Patent No. 5571698; U.S. Patent No.
6040136; Davis et al., Cancer Metastasis Rev.,1999;18(4):421-5; Taylor, et
al.,
Nucleic Acids Research 20 (1992): 6287-6295; Tomizuka et al.,Proc. Nat.
Academy
of Sciences USA 97(2) (2000): 722-727.
The cell culture supernatants are screened for the desired antibodies,
preferentially by immunofluorescent staining of CLL cells, by immunoblofting,
by an
enzyme immunoassay, e.g. a sandwich assay or a dot-assay, or a
radioimmunoassay.
For isolation of the antibodies, the imMunoglobulins in the culture
= supematants 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 (immune-) affinity chromatography, e.g. affinity
chromatography with
a one or more surface polypeptides derived from a CLL cell line according to
this
disclosure, or with Protein-A or G.
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.
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.
14
CA 02787818 2012-08-21
1 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 a one or more polypeptides or antigenic fragments thereof
derived
Preferred is a process for the preparation of a hybridoma cell line,
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
CA 02787818 2012-08-21
coding DNAs and of complementary DNAs thereto, or these complementary (sin&
stranded) DNAs themselves.
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 humanizati\on 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 degenerated sequence. Degenerated sequences
are degenerated 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 degenerated 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. coil, to obtain an
optimal
expression of the heavy chain murine variable domain and/or a light chain
murine
variable domain.
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.
ror 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.
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
16
CA 02787818 2012-08-21
iluman constant domain g, for example y1 , y2, 73 or y4, preferably
or y4 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 K or A, preferably K are also provided
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.
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.
Antibodies and antibody fragments disclosed herein are useful in diagnosis
and therapy. Accordingly, a composition for therapy or diagnosis comprising an
antibody disclosed herein is provided.
In the case of a diagnostic composition, the antibody is preferably provided
together with means for detecting the antibody, which may be enzymatic,
fluorescent, radioisotopic or other means. The antibody and the detection
means
may be provided for simultaneous, separate or sequential use, in a diagnostic
kit
intended for diagnosis.
, 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
17
CA 02787818 2012-08-21
There are many advantages to the development of a CLL cell line, as it
t
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.
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.
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.
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.
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.
Existing antibody therapies (such as RituximabTM, 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).
18
CA 02787818 2012-08-21
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.
= 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.
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
20- of OX-2/CD200 restores the Thl cytokine profile. Thus, these
polypeptides and/or
antibodies can be useful therapeutic agents in the treatment of CU_ and other
cancers or diseases over-expressing OX-2/CD200.
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. 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. As described in detail below, one
such
antibody is scFv-9 (see Fig. 9B) which binds to OX-2/CD200.
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.
19
CA 02787818 2012-08-21
EXAMPLE 1
Isolation of Cell Line CLL-AAT
Establishment of the cell line
Peripheral blood from a patient diagnosed with CLL was obtained. The WBC
count was 1.6x108/ml. Mononuclear cells were isolated by Histopaque-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 /0 DMSO and frozen in 1 ml aliquots
for
storage in liquid nitrogen.
Immunophenotyping showed that >90% of the CD45+ 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
An aliquot of the cells was thawed, washed, and resuspended at a density of
107/mL in IMDM supplemented with 20% heat-inactivated FBS, 2mM L-glutamine,
100 units/ml penicillin, 100 pg/ml streptomycin, 50 pM 2-mercaptoethanol, and
5
ng/ml recombinant human IL-4 (R & D Systems, Minneapolis, MN). The cells were
cultured at 37C 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
lmmunophenotyping 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.
CA 02787818 2012-08-21
lmmunophenotyping 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 Phaqe Display and Cell Surface Panning
Immunizations and scFv antibody library construction
Peripheral blood mononuclear cells (PBMC) were isolated from blood drawn
from CLL patients at the Scripps Clinic (La Jolla, CA). Two rabbits were
immunized
with 2x107 PBMC pooled from 10 different donors with CLL. Three immunizations,
two sub-cutaneous 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, New York). 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
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
21
CA 02787818 2012-08-21
milk, 0.02% sodium azide in PBS, pH 7.4) for 1 hour at 25 C to block
nonspecific
binding sites. Approximately 107 primary CLL cells were labeled with mouse
anti-
CD5 igG and mouse anti-CD19 IgG conjugated to paramagnetic microbeads
(Miltenyi Biotec, Sunnyvale, CA). 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 50p1(101 -1011 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 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 pl of acid
elution
buffer (0.1 N HCI, pH adjusted to 2.2 with glycine, plus1 pg/ml BSA). After a
10
minute incubation at 25 C, the buffer was neutralized with 12 pL of 2M Tris
base,
p1-110.5, 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=(Rn
output/Rr, input)/(Ri output/R1 input). In most cases, an enrichment factor of
102-103
fold should be attained by the third or fourth round.
Analysis of enriched antibody pools following panning
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:
22
CA 02787818 2012-08-21
. _
. To produce an entire pool in the form of HA-tagged soluble antibodies, 2m1
of a
non-suppressor strain of E. coli (e.g. TOP1OF') was infected with lpl (109-101
cfu) of phagemid particles. The original, unpanned library was used as a
negative control. Carbenicillin was added to a final concentration of 10pM and
the culture was incubated at 37 C with shaking at 25Orpm for 1 hour. Eight ml
of
SB medium containing 50pg/mIcarbenicillin was added and the culture was
grown to an OD 600 of-O.8. IPTG was added to a final concentration of 1mM to
induce scFv expression from the Lac promoter and shaking at 37 C was
continued for 4 hours. The culture was centrifuged at 3000xg for 15'. The
supernatant containing the soluble antibodies was filtered and stored in 1 ml
aliquots at ¨20 C.
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.
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
To screen individual scFv clones following panning, TOP1OF' cells were
infected with phage pools as described above, spread onto LB plates containing
carbenicillin and tetracycline, and incubated overnight at 37 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 shaking
incubator (GeneMachines, San Carlos, CA) at 520 rpm and 37 C. At this point, a
90
pl aliquot from each well was transferred to a deep 96-well plate containing
10 pL of
DMSO. This replica plate was stored at ¨80 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 3000xg for 15 minutes. The supernatants containing
soluble scFv antibodies were transferred to another deep 96-well plate and
stored at
¨20 C.
23
CA 02787818 2012-08-21
A sensitive whole-cell ELISA method for screening HA-tagged scFv
antibodies was developed:
1. An ELISA plate is coated with concanavalin A (10mg/m1 in 0.1 M NaHCO3,
pH8.6, 0.1 mM CaCl2).
2. After washing the plate with PBS, 0.5-1x105 target cells or absorber cells
in 50p1
of PBS are added to each well, and the plate is centrifuged at 250xg for 10
minutes.
3. 50p1 of 0.02% glutaraldehyde in PBS are added and the cells are fixed
overnight
at 4 C.
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.
5. The cells are incubated with 50p1 of soluble, HA-tagged scFv or Fab
antibody
(TOP1OF' supernatant) for 2 hours at room temperature, then washed six times
with PBS.
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 4.05nm using a microplate
reader.
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 Figures 2-6 and summarized in Figures 9A
¨9C .
The number of unique scFv antibody clones obtained was determined by
DNA fingerprinting and sequencing. The scFv DNA inserts were amplified from
the
24
CA 02787818 2012-08-21
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 (Figures 9A -9C ).
Characterization of scFv antibodies by flow cytometry
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+ B lymphocyte population (data not shown).
The
fourth antibody, scFv-9, recognized two distinct cell populations: the CD19+ B
lymphocytes and a subset of CD5+ T lymphocytes (Fig 7). Further
characterization
of the T cell subset showed that it was a subpopulation of the CD4+CD8" TH
cells
(data not shown).
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 stqined 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
CA 02787818 2012-08-21
iipproximately 2-fold on most if not all CLL tumors, whereas scFv-9 is
overexpress,'
3 to 6-fold on a subset of CLL tumors.
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+ B cells. Since scFv-9
recognizes an antigen overexpressed in only a subset of CLL patients, we
sought to
determine if scFy-9 antigen overexpression correlated with the percentage of
CD38+
cells in blood samples from ten CLL patients (Fig 11). The results indicate
that
scFv-9 antigen overexpression and percent CD384. cells are completely
independent
of one another.
Identification of antigens recognized by scFv antibodies by
immunoprecipitation (JP)
and mass spectrometry (MS)
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 speckrometry (pLC/MS/MS) (data not
shown). ScFv-2 immunoprecipitated a 110 lzd antigen from both RL and CLL-AAT
cells (Fig12). This antigen was identified by MALDI-MS as the B cell-specific
marker
CD19. 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 pLC/MS/MS
as
OX-2/CD200, a known marker for B cells, activated CD4+ T cells, and
thymocytes.
OX-2/CD200 is also expressed on some non-lymphoid cells such as neurons and
endothelial cells.
26
CA 02787818 2012-08-21
EXAMPLE 3
The capability of cells overexpressing OX-2/CD200 to shift the cytokine
response from a Th1 response (IL-2, IFN-y) to a Th2 response (IL-4, IL-10) was
. 5 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
293-EBNA cells (Invitrogen) were seeded at 2.5x106 per 100mm dish. 24
hours later the cells were transiently transfected using Polyfect reagent
(QIAGEN)
according to the manufacturer's instructions. Cells were cotransfected with
7.2pg of
OX-2/CD200 cDNA in vector pCEP4 (Invitrogen) and 0.8pg 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 cienciritic cells/macrophages from blood monocytes
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-y 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 y-irradiator (Shepherd Mark I Model 30 irradiator (Cs137)).
Mixed lymphocyte reaction
27
CA 02787818 2012-08-21
Mixed lymphocyte reactions were set up in 24 well plates using. 500,000
dendritic cells/macrophages and 1 x106 responder cells. Responder cells were T
cell enriched lymphocytes purified from peripheral blood using Flo II. 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
iAg/mlanti-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
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 CHI gene were separately
amplified with specific primers as follows:
R9VL-F1 QP: 5' GGC CTC TAG ACA GCC TGT GCT GAC TCA GTC
GCC CTC 3' (SEQ ID N0103);
R9VUhCL2-rev: 5' CGA GGG GGC AGC; CTT GGG CTG ACC TGT
GAG GOT CAG CTG GGT C 3' (SEQ ID NO 104);
R9VIACL2-F: 5' GAC CCA GCT GAC CGT CAC AGG TCA GCC
CAA GGC TGC CCC CTC G 3' (SEQ ID NO
105);
R9VH-F1: 5' TCT AAT CTC GAG CAG CAG CAG CTG ATG GAG
TCC G 3' (SEQ ID NO
106);
R9VH/hCG-rev: 5' GAC CGA TGG GCC CTT GGT GGA GGC TGA
GGA GAC GGT GAC CAG GGT GC 3' (SEQ ID NO 107);
R9VH/hCG-F: 5' GCA CCC TGG TCA CCG TCT CCT CAG CCT CCA
CCA AGG GCC CAT CGG TC 3' (SEQ ID NO 108);
28
CA 02787818 2012-08-21
hCL2-rev : 5' CCA CTG TCA GAG CTC CCG GGT AGA AGT C 3'
(SEQ ID NO 109);
hCG-rev : 5' GTC ACC GGT TCG GGG AAG TAG TC 3'
(SEQ ID NO110).
Amplified products were purified and overlap PCR was performed.
Final products were digested with Xba I/Sac I (light chain) and Xho I/Pin Al
(heavy chain) and cloned into a human Fab expression vector, PAX243hGL. DNA
clones were analyzed for PCR errors by DNA sequencing. The hCMV IE promoter
gene was inserted at Not 1/ Xho I site (in front of the heavy chain). The
vector was
digested with Xba I/Pin Al/EcoR I/Nhe I and a 3472 bp fragment containing the
light
chain plus the hCMV IE promoter and the heavy chain gene was tiansferred to an
IgG1 expression vector at the Xba I/Pin Al site.
Cytokine analysis
The effect of the scFv-9 converted to full IgG on the cytokine profile in the
mixed lymphocyte reaction was determined.
Cytokines such as IL-2, IFNI, IL-4, IL-10 and 1L-6 found in the tissue culture
supernatant were quantified using EL1SA. Matched capture and detection
antibody
pairs for each cytokine were obtained from R+D Systems (Minneapolis, MN), 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 ovemight 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 EL1SA plate reader (Molecular Devices).
As shown in Figure 14, the presence of OX-2/CD200 transfected but not
untransfected cells resulted in down-regulation of Th1 cytokines such as IL-2
and
29
CA 02787818 2012-08-21
IFNI. Addition of the anti-CD200 antibody at 30 g/m1 fully restored the Thl
response, indicating that the antibody blocked interaction of OX-2/CD200 with
its
receptor.
As set forth in Figures 15 and 16, the presence of CLL cells in a mixed
lymphocyte reaction resulted in down-regulation of the Th1 response. (Figure
15
shows the results for IL-2; Figure 16 shows the results for IFNI() 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 over-express OX-2/CD200 (JR, JG and GB) (the expression
levels
for these cells are set forth in Figure 11). However, the anti-CD200 antibody
only
restored the Th1 response in cells over-expressing OX-2/CD200, indicating that
for
patients over-expressing OX-21CD200, abrogating OX-2/CD200 interaction with
its
receptor on macrophages was sufficient to restore a Th1 response. In patients
that
did not over-express 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
A model was established in which RAJI lymphoma tumor growth is prevented
by the simultaneous injection of PBL's. NOD/SCID mice were injected
subcutaneously with 4x106 RAJI cells either in the presence or absence of
human
PBL's from different donors at 1x106, 5x106 or 10x106 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 figure 17 A and B. Statistical analysis was
performed using 2 parametric tests (Student's 'Rest and Welch's test) and one
non-
parametric test, the Wilcox test. Results of the statistical analysis are
found in figure
18. RAJI cells form subcutaneous tumors with acceptable variation. Rejection
is
dependent on the specific donor and the PBL cell number. 1x106 PBL's were
insufficient to prevent tumor growth. Donor 2 at 5x106 PBL's from day 22-43
and
donor 3 at 5x106 or 1x107 PBL's starting at day 36 significantly reduced tumor
growth. Donor 4 is very close to being significant after day 48.
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
CA 02787818 2012-08-21
PBL's. Anti-CD200 antibody is administered to evaluate tumor rejection in this
model.
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
PBL's 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.
Similar to the subcutaneous model, CD200 transfected cells are injected
intraperitoneally. They grow even in the presence of human PBL's. Treatment
with
anti-CD200 results in tumor rejection or slower tumor growth.
REFERENCES
The following references more fully describe the state of the art to which
the present invention pertains. Any inconsistency between these publications
below
and the present disclosure shall be resolved in favor of the present
disclosure.
1) Agarwal, et al., (2003). Disregulated expression of the Th2 cytokine
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Winter, et al., (2003). Tumour-induced polarization of tumour vaccine-draining
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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 homology greater
than 70% to the specific antibodies described herein are within the scope of
this
disclosure. In particularly useful embodiments, antibodies having a homology
greater than about 80% to the specific antibodies described herein are
contemplated. In other useful embodiments, antibodies having a homology
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.
34
