Title: Chronic lymphocytic
leukemia cell line
United States Patent: 7,435,412
Issued: October 14, 2008
Katherine S. (Del Mar, CA), McWhirter; John (San Diego, CA)
Pharmaceuticals, Inc. (Chesire, CT)
Appl. No.: 10/379,151
Filed: March 4, 2003
Executive MBA in Pharmaceutical Management, U. Colorado
The preparation and characterization of
antibodies that bind to antigens on CLL or other cancer cells, especially
to antigens upregulated in the cancer cells, and the identification and
characterization of antigens present on or upregulated by cancer cells are
useful in studying and treating cancer.
Description of the
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-MT 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
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.
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
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 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.
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.
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 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.
Preparation of Monoclonal Antibodies
Antibodies, as used herein, refers to complete antibodies or antibody
fragments capable of binding to a selected target. Included are 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 Fv and ScFv, possess advantageous properties for
diagnostic and therapeutic applications on account of their small size and
consequent superior tissue distribution.
The antibodies are especially indicated for diagnostic and therapeutic
applications. Accordingly, they may be altered antibodies comprising an
effector protein such as a toxin or a label. Especially preferred are labels
which allow the imaging of the distribution of the antibody in vivo. Such
labels may be radioactive labels or radiopaque labels, such as metal
particles, which are readily 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.
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.
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 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.
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
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.
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 is 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.,
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 contents of all these references are
incorporated herein by reference.
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
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 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 (such as, for example, the methods
disclosed in the various references incorporated herein by reference).
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 reckoning.
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 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.
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.
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.
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 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. coli, 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.
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.
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
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
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.
Uses of the CLL Cell Line
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
A cell line according to this disclosure may be used for in vitro studies on
the etiology, pathogenesis and biology of CLL. This assists in the
identification of suitable agents that are useful in the therapy of CLL
The cell line may also be used to produce monoclonal antibodies for in vitro
and in vivo diagnosis of CLL, 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
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 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).
The CLL cell line described herein thus permits the development of novel
anti-CLL antibodies having specificity for one or more antigenic
determinants of the present CLL cell line, and their use in the therapy and
diagnosis of CLL.
In a particularly useful embodiment, the antibody binds to or otherwise
interferes with the metabolic pathway of a polypeptide that is upregulated
by a malignant cancer cell. For instance, the antibody can bind to the
upregulated polypeptide and in this manner prevent or inhibit the
polypeptide from interacting with other molecules or receptors.
Alternatively, the antibody may bind to a receptor with which the
upregulated polypeptide normally interacts, thereby preventing or inhibiting
the polypeptide from binding to the receptor. As yet another alternative,
the antibody can bind to an antigen that modulates expression of the
polypeptide, thereby preventing or inhibiting normal or increased expression
of the polypeptide. For example, the peptide OX-2/CD200 is upregulated in a
portion of CLL patients. 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 cancer more effectively.
Thus, in another embodiment, a method for treating a cancer patient in
accordance with this disclosure includes the steps of screening a cancer
patient for the presence of a polypeptide that is upregulated by a malignant
cancer cell and administering an antibody that interferes with the metabolic
pathway of the upregulated polypeptide. In a particularly useful embodiment,
a CLL patient is screened for overexpression of OX-2/CD200 and an antibody
that interferes with the metabolic pathway of OX-2/CD200 is administered to
the patient. As described in detail below, one such antibody is scFv9 (see
FIG. 9B, see Original Patent) which binds to OX-2/CD200.
Claim 1 of 15 Claims
1. A method of treating chronic
lymphocytic leukemia (CLL) comprising administering to a patient suffering
from CLL an antibody or antigen-binding fragment thereof that specifically
binds to OX-2/CD200, wherein said antibody or antigen-binding fragment
thereof comprises a light chain CDR1 having the sequence set forth in SEQ
ID NO: 5; a light chain CDR2 having the sequence set forth in SEQ ID NO:
21; a light chain CDR3 having the sequence set forth in SEQ ID NO: 29; a
heavy chain CDR1 having the sequence set forth in SEQ ID NO: 50; a heavy
chain CDR2 having the sequence set forth in SEQ ID NO: 69; and a heavy
chain CDR3 having the sequence set forth in SEQ ID NO: 88.
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