U.S. patent application number 09/929209 was filed with the patent office on 2001-12-20 for selective apoptosis of neoplastic cells by an hla-dr specific monoconal antibody.
This patent application is currently assigned to Genelabs Technologies, Inc.. Invention is credited to Laus, Reiner, Vidovic, Damir.
Application Number | 20010053360 09/929209 |
Document ID | / |
Family ID | 22268657 |
Filed Date | 2001-12-20 |
United States Patent
Application |
20010053360 |
Kind Code |
A1 |
Vidovic, Damir ; et
al. |
December 20, 2001 |
Selective apoptosis of neoplastic cells by an HLA-DR specific
monoconal antibody
Abstract
Anti-human major histocompatibility complex (MHC) class II,
HLA-DR-specific monoclonal antibodies which can induce apoptosis of
HLA-DR positive cells are disclosed. The antibodies are used to
specifically eliminate HLA-DR antigen positive tumor cells by
cross-linking of HLA-DR. Also disclosed are methods for treating
cancer using such antibodies, and compositions containing them.
Inventors: |
Vidovic, Damir; (Bellevue,
WA) ; Laus, Reiner; (San Carlos, CA) |
Correspondence
Address: |
IOTA PI LAW GROUP
350 CAMBRIDGE AVENUE SUITE 250
P O BOX 60850
PALO ALTO
CA
94306-0850
US
|
Assignee: |
Genelabs Technologies, Inc.
|
Family ID: |
22268657 |
Appl. No.: |
09/929209 |
Filed: |
August 13, 2001 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
|
|
09929209 |
Aug 13, 2001 |
|
|
|
09383663 |
Aug 26, 1999 |
|
|
|
60098292 |
Aug 28, 1998 |
|
|
|
Current U.S.
Class: |
424/133.1 ;
530/388.8 |
Current CPC
Class: |
C07K 2317/24 20130101;
C07K 2317/56 20130101; A61K 38/00 20130101; C07K 2317/55 20130101;
C07K 2317/21 20130101; C07K 2317/54 20130101; C07K 16/2833
20130101 |
Class at
Publication: |
424/133.1 ;
530/388.8 |
International
Class: |
A61K 039/395; C07K
016/30 |
Claims
It is claimed:
1. An antibody or fragment thereof wherein said antibody binds an
epitope on the HLA-DR polypeptide and induces apoptosis in cells
expressing HLA-DR, without concomitant suppression of HLA-DR
dependent immune responses.
2. The antibody of claim 1 wherein said antibody is a monoclonal
antibody.
3. The antibody of claim 1 wherein said monoclonal antibody is
produced by the hybridoma cell line Danton.
4. An isolated antibody fragment according to claim 1, wherein said
isolated fragment is an F(ab').sub.2 fragment.
5. An isolated antibody fragment according to claim 1, wherein said
isolated fragment is bound to a solid support and is selected an
Fab fragment or an Fv fragment.
6. The antibody of claim 1 wherein said antibody binds an epitope
in the first domains of an HLA-DR polypeptide.
7. The antibody of claim 1 wherein said antibody is a humanized
antibody.
8. The antibody of claim 1 wherein said antibody is a human
antibody.
9. A hybridoma cell line capable of producing the antibody of claim
1.
10. The antibody of claim 1 wherein the HLA-DR-expressing cells are
tumor cells.
11. An isolated antibody or fragment thereof wherein said antibody
or fragment thereof binds the HLA-DR antigen and such binding
results in the same triggering effect on apoptosis of HLA-DR
expressing tumor cells as results from the binding of monoclonal
antibodies produced by hybridoma Danton to HLA-DR- expressing tumor
cells.
12. A composition comprising an amount of an antibody or fragment
thereof according to claim 1 sufficient to induce apoptosis in
HLA-DR-expressing tumor cells in a mixture with a physiological
acceptable carrier, excipient, or stabilizer.
13. The composition of claim 14 wherein the antibody is a
monoclonal antibody.
14. The composition of claim 14 wherein the antibody is a humanized
antibody.
15. The composition of claim 14 wherein the antibody is a human
antibody.
16. The composition of claim 13 for administration to a cancer
patient, further comprising a cytotoxic drug.
17. A method of triggering apoptosis, comprising administering to a
subject, a therapeutically effective amount of an anti-HLA-DR
antibody effective to stimulate apoptosis in HLA-DR-expressing
tumor cells.
18. The method of claim 17 wherein the therapeutic outcome of the
subject is improved following said administration.
19. The method of treating cancer according to claim 18 wherein
said cancer involves HLA-DR expressing cells and is selected from
the group consisting of plasmacytoma/multiple myeloma, Hodgkin's
lymphomas, non-Hodgkin's lymphomas and B cell leukemias.
20. The method of claim 18 wherein the therapeutic anti-HLA-DR
antibody is administered intravenously (IV).
21. The method of claim 18 wherein the therapeutic anti-HLA-DR
antibody is administered parenterally.
Description
[0001] This application claims the benefit of U.S. Provisional
Application No. 60/098,292, filed Aug. 28, 1998, incorporated
herein by reference in its entirety.
FIELD OF THE INVENTION
[0002] The present invention relates to monoclonal antibodies (mAb)
that specifically react with the HLA-DR antigen and induce
apoptosis in HLA-DR expressing cells. In particular, the invention
relates to the use of such monoclonal antibodies in the treatment
of cancers involving HLA-DR positive cells, and to pharmaceutical
compositions containing anti-HLA-DR antibodies.
REFERENCES
[0003] Babbitt, et al., Nature 317:359-361 (1985).
[0004] Boerner, et al., J. Immunol. 147(1):86-95 (1991).
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[0006] Brodeur, et al., MONOCLONAL ANTIBODY PRODUCTION TECHNIQUES
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[0029] Ritts, et al., Int. J. Cancer 31:133-141 (1983).
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[0032] Truman, et al., Blood 89(6): 1996-2007 (1997).
[0033] Truman, et al., Int. Immunol. 6(6):887-896 (1994).
[0034] Verhoeyen, et al., Science 239:1534-1536 (1988).
[0035] Vermes, et al., J. Immunol. Meth. 184:39-51 (1995).
[0036] Vidovic, et al., Eur. J Immunol. 25:3349-3355 (1995).
[0037] Vidovic and Toral, Cancer Lett. 128:127-135 (1998).
[0038] Vitale, et al., Histochemistry 100:223-229 (1993).
[0039] Wyllie, et al., J. Pathol. 142:67-77 (1984).
[0040] Zola, MONOCLONAL ANTIBODIES: A MANUAL OF TECHNIQUES, CRC
Press, Inc. pp. 147-158 (1987).
BACKGROUND OF THE INVENTION
[0041] Class II major histocompatibility complex (MHC) molecules,
constitutively expressed on normal antigen presenting cells (APC),
are responsible for the presentation of antigen-derived peptides to
CD4+ helper T (Th) cells. (Babbitt, et al., 1985; Truman, et al.,
1997). Signaling via these molecules initiates the generation of
second messengers leading to programmed cell death (PCD or
apoptosis) of activated B lymphocytes. Besides antigen
presentation, class II molecules transduce signals that can
modulate cell growth and certain class II MHC-specific mAb have
been shown to induce apoptosis of cancer cells (Newell, et al.,
1993). The practical utility of this observation in cancer therapy
has been hampered by the intrinsic lack of selectivity, in that the
class II MHC-specific antibodies that have been shown to induce
apoptosis of cancer cells, also interfere with normal Th cell
functions. (Vidovic, et al., 1995). More specifically, the
presently available apoptosis-inducing class II-specific mAb
recognize epitopes located on the first protein domains of the
HLA-DR heterodimer, in apparent close proximity to the
peptide-binding site, and these mAbs interfere with antigen
presentation, causing a potent in vitro and in vivo inhibition of
Th responses (Vidovic, et al., 1995).
[0042] Hence, the main problem in using the currently available
anti-HLA-DR antibodies for the treatment of cancers involving
HLA-DR positive cells is the potential for side effects such as
immunosuppression of HLA-DR mediated immune responses based on the
lack of definitive specificity of the antibodies for the
apoptogenic epitope, and as a result such anti-HLA-DR antibodies
may not find practical utility in therapeutic applications.
[0043] One approach to overcoming these problems is to administer
an anti-Class II (anti-HLA-DR) antibody specifically reactive with
tumor cells which can trigger apoptosis in such cells and which
does not have immunosuppressive properties associated with the
binding to HLA-DR-expressing cells.
SUMMARY OF THE INVENTION
[0044] Accordingly, it is an object of the invention to provide a
composition for in vivo administration, comprising a monoclonal
antibody which specifically binds to HLA-DR-expressing tumor cells
and triggers apoptosis of the tumor cells to which it binds.
[0045] The present invention is based, in part, on the discovery
that antibodies which specifically react with human major
histocompatibility complex (MHC) class II can induce apoptosis of
cells expressing HLA-DR molecules on their surface.
[0046] The antibodies of the present invention are highly specific
in that the monoclonal antibodies affect neither the viability nor
function of non-neoplastic HLA-DR positive cells.
[0047] In one aspect, the invention includes Fab fragments of
monoclonal antibodies specific for HLA-DR anchored to a solid
support.
[0048] The apoptosis-inducing effect of such monoclonal antibodies
is associated with a cross-linking of HLA-DR, and monovalent Fab
fragments cannot mediate cytotoxicity (Vidovic and Toral,
1998).
[0049] According to an important feature of the present invention,
the tumoricidal effects of anti-class II MHC mAb can be achieved
without simultaneous suppression of class II-dependent immune
responses, although both properties are associated with the mAb
recognizing an epitope in the first protein domains of HLA-DR.
[0050] As outlined herein, the anti HLA-DR antibodies of the
present invention specifically bind to the first domains of HLA-DR
molecules which are expressed by a variety of types of cancer
cells, including, but not limited to B cell cancers. As further
outlined herein, the anti-HLA-DR antibodies exert a triggering
effect on apoptosis which is specific to HLA-DR positive tumor
cells.
[0051] An important practical implication of this work is that a
mAb, designated Danton; and produced by a hybridoma cell line, also
designated Danton; may be effective for the selective
antibody-based therapy of HLA class II positive neoplasms,
including, but not limited to, blood cell neoplasms, e.g
plasmacytoma/multiple myeloma, Hodgkin's and non-Hodgkin's
lymphomas and B cell leukemias. In vitro studies indicate that the
Danton mAb does not interfere with normal Th function, therefore,
therapy with the Danton mAb should not affect the subject's normal
HLA-DR-expressing cells. Accordingly, it would be reasonable to
expect fewer side effects than with the currently available
therapeutic agents.
[0052] These and other objects and features of the invention will
become more fully apparent when the following detailed description
of the invention is read in conjunction with the accompanying
drawings.
BRIEF DESCRIPTION OF THE DRAWINGS
[0053] FIG. 1 shows a general epitope map of HLA-DR molecule
indicating: CY, cytoplasmic tail; TM, transmembrane part; .alpha.1
& .beta.1, the first (extracellular) domains of alpha and beta
chains, respectively; and .alpha.2 & .beta.2, the second
(extracellular ) domains of alpha and beta chains,
respectively.
[0054] FIGS. 2A-F show the results of FACS analysis indicating the
viability of different cell populations after 16 hours coculture in
medium alone (first column) or in the presence of the Danton mAb
(second column). FIGS. 2A and B reflect an analysis of the EBV-LCL
cell line, RPMI 1788; FIGS. 2C and D reflect an analysis of the
plasmacytoma cell line, MC/CAR; and FIGS. 2E and F reflect an
analysis of PBMC (peripheral blood mononuclear cells); with live
cells located in the lower right quadrants of the two-dimensional
dot-plots.
[0055] FIGS. 3A-F show the results of FACS analysis indicating the
viability of the EBV-LCL cell line, RPMI 1788 (first column) and
the plasmacytoma cell line, MC/CAR (second column) after 16 h
coculture with in medium alone (FIGS. 3A, D); in the presence of
the Danton mAb (FIGS. 3B, E); and in the presence of the 10F12 mAb
(FIGS. 3C, F).
[0056] FIGS. 4A and B show the viability of EBV-LCL RPMI 1788 cells
after incubation with the Danton mAb under different temperature
conditions as indicated, for 0.1 to 10 hours (FIG. 4A) and 1 to 21
days (FIG. 4B).
[0057] FIG. 5 shows the absence of immunosuppressive effects of the
Danton mAb relative to a medium control, staphylococcal enterotoxin
B (SEB) alone and SEB plus a different anti-HLA-DR mAb, L243, on
the SEB specific in vitro proliferative response of human PBMC, as
indicated by [.sup.3H]thymidine incorporation.
[0058] FIG. 6 shows the survival of scid mice injected with the
HLA-DR.sup.+ human plasmacytoma cell line, MC/CAR; and treated with
either mAb Danton (open triangles), or phosphate buffered saline
(PBS; filled squares).
DETAILED DESCRIPTION OF THE INVENTION
[0059] Definitions
[0060] The term "antibody" is used in the broadest sense and
specifically covers single anti-HLA-DR polypeptide monoclonal
antibodies and anti-HLA-DR antibody compositions with polyepitopic
specificity.
[0061] The term "monoclonal antibody" as used herein refers to an
antibody obtained from a population of substantially homogeneous
antibodies, i.e., the individual antibodies comprising the
population are identical except for possible naturally-occurring
mutations that may be present in minor amounts.
[0062] "Active" or "activity" for the purposes herein refers to
anti-HLA-DR antibodies which retain the biologic and/or immunologic
activities of the Danton anti-HLA-DR antibody described herein.
[0063] The term "epitope" as used herein refers to the specific
portion of an antigen which interacts with the complementarity
determining region (CDR) of an antibody.
[0064] The term "Fab fragment" as used herein refers to a partial
antibody molecule containing a single antigen binding region which
consists of a portion of both the heavy and light chains of the
molecule.
[0065] The term "F(ab').sub.2 fragment" as used herein refers to a
partial antibody molecule containing both antigen binding regions,
and which consists of the light chains and a portion of the heavy
chains of the molecule.
[0066] The term "Fv fragment" as used herein refers to the portion
of the antibody molecule involved in antigen recognition and
binding.
[0067] The term "complementary determining region" (CDR) as used
herein refers to the hypervariable region of an antibody molecule
which forms a surface complementary to the 3-dimensional surface of
a bound antigen.
[0068] The term "HLA-DR" as used herein refers to the "human
leukocyte antigen" (HLA) DR gene loci and their protein products,
the latter being alloantigens expressed on human leukocytes.
Alloantigens are the product of polymorphic genes which distinguish
self from foreign tissues.
[0069] The term "Class II major histocompatibility complex" or
"Class II MHC" or "Class II" antigens as used herein refers to
antigens that are expressed at various levels on different types of
cells and which play an essential role in the recognition of all
protein antigens by T cells. Class II MHC molecules typically bind
peptides of from 7 to 30 or more amino acids and form complexes
that are recognized by antigen-specific CD4+T cells. The CD4
molecule binds to the second domains of class II molecules.
[0070] "Apoptotic cell death" or "programmed cell death" or
"apoptosis" as used herein refers to any cell death that results
from the complex cascade of cellular events that occur at specific
stages of cellular differentiation and in response to specific
stimuli. Apoptotic cell death is characterized by condensation of
the cytoplasm and nucleus of dying cells.
[0071] The term "solid support" as used herein refers to e.g.,
microtiter plates, membranes and beads, etc. For example, such
solid supports may be made of glass, plastic (e.g., polystyrene),
polysaccharides, nylon, nitrocellulose, or teflon, etc. The surface
of such supports may be solid or porous and of any convenient
shape.
[0072] The term "tumor" or "cancer" or "neoplasm" as used herein
refers to a malignant growth that arises from normal tissue, but
grows abnormally with an absence of structure. Tumor or cancer
cells generally have lost contact inhibition and may be invasive
and/or have the ability to metastasize.
[0073] The term "cytotoxic drug" as used herein refers to a drug
which is used to inhibit the growth, or facilitate the death of
cancer cells. Examples of cytotoxic drugs include chemotherapeutic
agents such as ara-C, bleomycin, cisplatin, cladribine,
cyclophosphamide, doxorubicin, etoposide, and 5-fluorouracil
(5-FU).
[0074] By "therapeutically effective amount" as used herein is
meant a dose that reduces or eliminates HLA-DR expressing tumor
cells by stimulating apoptosis thereof. The exact dose will depend
on the purpose of the treatment, and will be ascertainable by one
skilled in the art using known techniques.
[0075] A "subject" for the purposes of the present invention
includes both humans and other animals, particularly mammals. Thus
the methods are applicable to both human therapy and veterinary
applications. In the preferred embodiment the subject is a mammal,
and in the most preferred embodiment the subject is human.
[0076] As used herein, the term "improved therapeutic outcome" or
"decrease in the number of tumor cells" means a 50% decrease,
preferably an 80% decrease, more preferably a 90% decrease, and
even more preferably a 100% decrease in either the tumor size, or
in the number of detectable circulating cancer cells in the blood
and/or affected tissue or organ as determined by examination of a
patient and/or samples taken from a patient prior to and following
treatment.
[0077] The terms "treating", "treatment" and "therapy" as used
herein refer to curative therapy, prophylactic therapy, and
preventative therapy.
[0078] Class II MHC and Apoptosis
[0079] HLA class II molecules are constitutively expressed on human
B lymphocytes and are induced on human T lymphocytes after
activation, through which signal transduction via HLA class II has
been extensively described. Up to 60% cell death has been observed
after stimulation of lymphocytes via HLA-DR molecules. Certain
HLA-DR-specific mAbs cause up to a 90% decrease in the cell surface
expression of class II molecules, which is also class II
isotype-specific, ie. HLA-DR-specific mAb do not affect the
expression of HLA-DP and HLA-DQ molecules. (Truman, et al.,
1994).
[0080] Previously described anti-HLA-DR mAbs which down-regulate
class II expression have been shown to recognize the first (peptide
binding) domains of class II heterodimers, and as a result strongly
inhibit the activation of class II-restricted human T cells in
vitro, in addition to being cytotoxic for B lymphoblastoid cell
lines and for a small proportion of normal activated B cells. Their
F(ab').sub.2 fragments mediate both down-regulation and
cytotoxicity, whereas their monovalent Fab fragments are not
cytotoxic, but retain the down-regulatory and T cell inhibitory
properties.
[0081] Class II molecules transduce signals that can modulate cell
growth and class II MHC-specific mAb can induce apoptosis of cancer
cells (Newell, et al., 1993). Monoclonal antibodies to the first
domains of the class II MHC molecule, HLA-DR, prepared as described
below serve as the basis for the present invention.
[0082] A human major histocompatibility complex (MHC) class II
molecule-specific mAb, designated Danton was generated and found to
induce apoptosis in tumor cells which express HLA-DR on their
surface. The anti-cancer activity of Danton is highly selective in
that it affects neither viability nor function of non-malignant
HLA-DR positive cells.
[0083] Class II MHC
[0084] Anti-HLA-DR mAb that are both "immunosuppressive and
cytotoxic" may inhibit antigen presentation by recognizing the
first domains and binding to an epitope located close to the
peptide-binding groove of HLA-DR. The anti-HLA-DR mAb of the
present invention, e.g. Danton are "cytotoxic only". Accordingly,
it is likely that the precise sequence of HLA-DR to which such a
"cytotoxic only" antibody binds, differs from that of antibodies
which are both "immunosuppressive and cytotoxic" (Vidovic, et al.,
1995).
[0085] Apoptotic Cell Death
[0086] As described above, apoptosis (programmed cell death) has
been associated with the binding of anti-MHC Class II antibodies to
Class II-expressing cells. Class II MHC-encoded molecules expressed
on the surface of APC (such as B lymphocytes, macrophages,
monocytes, dendritic cells, etc.) function as restriction elements
for the presentation of antigen to T lymphocytes, an interaction
that ultimately leads to activation and differentiation of both
cell types.
[0087] HLA-DR mediated cell death has been demonstrated to be very
rapid, independent of Fc receptors and complement, and
non-necrotic. (Truman, et al., 1994; Truman, et al., 1997).
[0088] The present discovery relates to the role of anti-HLA-DR
antibodies in triggering apoptosis of tumor cells and thus provides
methods for inducing programmed cell death in such cells. In a
preferred embodiment, the anti-HLA antibodies, and fragments
thereof which are capable of cross-linking the HLA-DR antigen, are
useful in the study or treatment of conditions which are mediated
by tumor cells which express HLA-DR, i.e. to treat or prevent
disorders associated with HLA-DR-expressing tumor cells.
Accordingly, the antibodies of the present invention are useful to
treat various diseases, including, but not limited to, any disease
characterized by cancer of B cell origin (where increased apoptosis
would be desirable), e.g., Hodgkin's and non-Hodgkin's lymphomas,
chronic lymphocytic leukemia, myeloma and plasmacytoma. (Holland,
et al., 1996).
[0089] Evaluation of the Mechanism of Cell Death
[0090] This section describes in vitro assays which are useful for
evaluating the extent of apoptotic cell death. Cell death may be
detected by staining of cells with propidium iodide (PI), or by use
of assays specific to apoptotic cell death, e.g. staining with
arnexin V (Vermes, et al., 1995). Necrotic cell death may be
distinguished from apoptotic cell death by evaluating the results
of a combination of the assays for cell viability, as described
below, together with microscopic observation of the morphology of
the relevant cells.
[0091] Assay for Necrotic Cell Death
[0092] Necrosis is a passive process in which collapse of internal
homeostasis leads to cellular dissolution involving a loss of
integrity of the plasma membrane and subsequent swelling, followed
by lysis of the cell (Schwartz, et al., 1993). Necrotic cell death
is characterized by loss of cell membrane integrity and
permeability to dyes such as propidium iodide (PI) which is known
by those in the art to bind to the DNA of cells undergoing primary
and secondary necrosis (Vitale, et al., 1993; Swat, et al., 1991).
Necrosis may be distinguished from apoptosis in that cell membranes
remain intact in the early stages of apoptosis. As a consequence
dye exclusion assays using PI may be used in parallel with an assay
for apoptosis, as described below in order to distinguish apoptotic
from necrotic cell death. Fluorescent-activated cell sorter (FACS)
based flow cytometry assays using PI allow for rapid evaluation and
quantitation of the percentage of necrotic cells.
[0093] Assay for Apoptotic Cell Death
[0094] Detection of programmed cell death or apoptosis may be
accomplished as will be appreciated by those in the art, e.g. by
staining with annexin V (Vermes, et al., 1995). The percentage of
cells undergoing apoptosis may be measured at various times after
stimulation of apoptosis with or without administration of
anti-HLA-DR antibodies. The morphology of cells undergoing
apoptotic cell death is generally characterized by a shrinking of
the cell cytoplasm and nucleus and condensation and fragmentation
of the chromatin. (Wyllie, et al., 1984).
[0095] Partial DNA degradation in apoptotic B cells has been
previously reported (Truman, et al., 1994; Cohen and Duke, 1992).
Consistent with this observation, DNA fragmentation was not
detected after incubating tumor B cells with an earlier described
apoptogenic anti-HLA-DR mAb (Vidovic and Toral, 1988), however, the
relative cell size and PI-uptake flow cytometry profiles of these
cultures are essentially the same as those previously demonstrated
for cells undergoing apoptosis (Newell, et al., 1993; Truman, et
al., 1994).
[0096] Anti-HLA-DR Antibodies
[0097] The present invention provides anti-HLA-DR antibodies.
Exemplary antibodies include polyclonal, monoclonal, and humanized
antibodies, as well as fragments thereof. The anti-HLA-DR
antibodies of the present invention specifically react with a
determinant or epitope in the first domains of the HLA-DR protein.
In most instances, antibodies made to an epitope or fragment of the
HLA-DR protein will be able to bind to the full length protein.
Preferably, the antibodies are generated to epitopes unique to the
HLA-DR protein; that is, the antibodies show little or no
cross-reactivity to other proteins. In a preferred embodiment, the
antibodies are generated to the first domains of the HLA-DR
molecule The first domains of the HLA-DR correspond to amino acids
1 to 88 and 1 to 96 of the alpha and beta HLA-DR chain,
respectively.
[0098] The anti-HLA-DR antibodies of the invention specifically
bind to HLA-DR proteins. By "specifically bind" herein is meant
that the antibodies bind to the protein with a binding constant in
the range of at least 10.sup.6-10.sup.8 M, with a preferred range
being 10.sup.7-10.sup.9 M.
[0099] Polyclonal Antibodies
[0100] The anti-HLA-DR antibodies of the present invention may be
polyclonal antibodies. Methods of preparing polyclonal antibodies
are known to the skilled artisan. Polyclonal antibodies can be
raised in a mammal, for example, by one or more injections of an
immunizing agent and, if desired, an adjuvant.
[0101] Typically, the immunizing agent and/or adjuvant will be
injected in the mammal by multiple subcutaneous or intraperitoneal
(IP) injections. The immunizing agent may include the HLA-DR
antigen or a fragment or fusion protein thereof. It may be useful
to conjugate the immunizing agent to a protein known to be
immunogenic in the mammal being immunized. Examples of such
immunogenic proteins include but are not limited to keyhole limpet
hemocyanin, serum albumin, bovine thyroglobulin, and soybean
trypsin inhibitor. Examples of adjuvants which may be employed
include Freund's complete adjuvant and MPL-TDM adjuvant
(monophosphoryl Lipid A, synthetic trehalose dicorynomycolate). The
specific immunization protocol may be selected from the numerous
protocols which are available, without undue experimentation.
[0102] Monoclonal Antibodies
[0103] Preferably, the anti-HLA-DR antibodies are monoclonal
antibodies. Monoclonal antibodies may be prepared using hybridoma
methods, such as those described by Kohler and Milstein, (1975). In
a hybridoma method, a mouse, hamster, or other appropriate host
animal, is injected with an immunizing agent to elicit lymphocytes
that produce or are capable of producing antibodies that will
specifically bind to the immunizing agent. Alternatively, the
lymphocytes may be immunized in vitro.
[0104] The immunizing agent includes the HLA-DR polypeptide,
fragments or a fusion protein thereof. Generally, either PMBC are
used if cells of human origin are desired, or spleen cells or lymph
node cells are used if non-human mammalian sources are desired. The
lymphocytes are then fused with an immortalized cell line using a
suitable fusing agent, such as polyethylene glycol, to form a
hybridoma cell (Goding, 1986). Immortalized cell lines are usually
transformed mammalian cells, particularly myeloma cells of rodent,
bovine and human origin. Usually, rat or mouse myeloma cell lines
are employed. The hybridoma cells may be cultured in a suitable
culture medium that preferably contains one or more substances that
inhibit the growth or survival of the unfused, immortalized cells.
For example, if the parental cells lack the enzyme hypoxanthine
guanine phosphoribosyl transferase (HGPRT), the culture medium for
the hybridomas typically will include hypoxanthine, aminopterin,
and thymidine ("HAT medium"), which substances prevent the growth
of HGPRT-deficient cells.
[0105] Preferred immortalized cell lines are those that fuse
efficiently, support stable high level expression of antibody by
the selected antibody-producing cells, and are sensitive to a
medium such as HAT medium. More preferred immortalized cell lines
are murine myeloma lines, which can be obtained, for instance, from
the Salk Institute Cell Distribution Center, San Diego, Calif. and
the American Type Culture Collection (ATCC), Rockville, Md. Human
myeloma and mouse-human heteromyeloma cell lines also have been
described for the production of human monoclonal antibodies
(Kozbor, 1984; Brodeur, et al., 1987).
[0106] The culture medium in which the hybridoma cells are cultured
can then be assayed for the presence of monoclonal antibodies
directed against the HLA-DR polypeptide. The binding specificity of
monoclonal antibodies produced by the hybridoma cells may be
determined by immunoprecipitation or by an in vitro binding assay,
such as radioimmunoassay (RIA) or enzyme-linked immunosorbent assay
(ELISA). Such techniques and assays are known in the art. The
binding affinity of the monoclonal antibody can, for example, be
determined by the Scatchard analysis of Munson and Pollard,
(1980).
[0107] After the desired hybridoma cells are identified, the clones
may be subcloned by limiting dilution procedures and grown by
standard methods [Goding, supra]. Alternatively, the hybridoma
cells may be grown in vivo as ascites in a mammal.
[0108] The monoclonal antibodies secreted by the subclones may be
isolated or purified from the culture medium or ascites fluid by
conventional immunoglobulin purification procedures such as, for
example, protein A-Sepharose, hydroxylapatite chromatography, gel
electrophoresis, dialysis, or affinity chromatography.
[0109] The monoclonal antibodies may also be made by recombinant
DNA methods, such as those described in U.S. Pat. No. 4,816,567.
DNA encoding the monoclonal antibodies of the invention can be
readily isolated and sequenced using conventional procedures (e.g.,
by using oligonucleotide probes that are capable of binding
specifically to genes encoding the heavy and light chains of murine
antibodies). The hybridoma cells of the invention serve as a
preferred source of such DNA. Once isolated, the DNA may be placed
into expression vectors, which are then transfected into host cells
such as simian COS cells, Chinese hamster ovary (CHO) cells, or
myeloma cells that do not otherwise produce immunoglobulin protein,
to obtain the synthesis of monoclonal antibodies in the recombinant
host cells. The DNA also may be modified, for example, by
substituting the coding sequence for human heavy and light chain
constant domains in place of the homologous murine sequences (U.S.
Pat. No. 4,816,567; Morrison, et al., supra) or by covalently
joining to the immunoglobulin coding sequence all or part of the
coding sequence for a non-immunoglobulin polypeptide. Such a
non-immunoglobulin polypeptide can be substituted for the constant
domains of an antibody of the invention, or can be substituted for
the variable domains of one antigen-combining site of an antibody
of the invention to create a chimeric bivalent antibody.
[0110] Digestion of antibodies to produce fragments thereof,
particularly, F(ab), F(ab').sub.2 and Fv fragments, can be
accomplished using routine techniques known in the art.
[0111] The methods of the present invention require that
anti-HLA-DR antibodies be bivalent, in order to facilitate
cross-linking of HLA-DR molecules and thereby stimulate apoptosis.
Accordingly one or more fragments of anti-HLA-DR antibodies may be
bound to a solid support thereby facilitating the cross-linking of
HLA-DR.
[0112] Humanized Antibodies
[0113] The anti-HLA-DR antibodies of the invention may further
comprise humanized antibodies or human antibodies. Humanized forms
of non-human (e.g., murine) antibodies are chimeric
immunoglobulins, immunoglobulin chains or fragments thereof (such
as Fv, Fab, Fab', F(ab').sub.2 or other antigen-binding regions of
antibodies) which contain minimal sequence derived from non-human
immunoglobulin. Humanized antibodies include human immunoglobulins
(recipient antibody) in which residues from a complementary
determining region (CDR) of the recipient are replaced by residues
from a CDR of a non-human species (donor antibody) such as mouse,
rat or rabbit having the desired specificity, affinity and
capacity. In some instances, Fv framework residues of the human
immunoglobulin are replaced by corresponding non-human residues.
Humanized antibodies may also comprise residues which are found
neither in the recipient antibody nor in the imported CDR or
framework sequences. In general, the humanized antibody will
comprise substantially all of at least one, and typically two,
variable domains, in which all or substantially all of the CDR
regions correspond to those of a non-human immunoglobulin and all
or substantially all of the FR regions are those of a human
immunoglobulin consensus sequence. The humanized antibody optimally
also will comprise at least a portion of an immunoglobulin constant
region (Fc), typically that of a human immunoglobulin (Jones, et
al., 1986; Riechmann, et al., 1988; and Presta, 1992).
[0114] Methods for humanizing non-human antibodies are well known
in the art. Generally, a humanized antibody has one or more amino
acid residues introduced into it from a source which is non-human.
Humanization can be essentially performed following the method of
Winter and co-workers (Jones, et al., 1986; Riechmann, et al.,
1988; Verhoeyen, et al., 1988), by substituting rodent CDRs or CDR
sequences for the corresponding sequences of a human antibody.
Accordingly, such "humanized" antibodies are chimeric antibodies
wherein substantially less than an intact human variable domains
has been substituted by the corresponding sequence from a non-human
species. In practice, humanized antibodies are typically human
antibodies in which some CDR residues and possibly some framework
residues are substituted by residues from analogous sites in rodent
antibodies.
[0115] Human antibodies can also be produced using various
techniques known in the art, including phage display libraries
(Hoogenboom and Winter, 1991; Marks, et al., 1991). Exemplary
additional techniques that are available for the preparation of
human monoclonal antibodies are described in Cole et al., (1985)
and Boemer, et al., (1991).
[0116] Uses for anti-HLA-DR Antibodies
[0117] The anti-HLA-DR antibodies of the present invention have
various utilities. For example, anti-HLA-DR antibodies may be used
in diagnostic assays for, and therapy involving, HLA-DR expressing
tumor cells, e.g., detecting expression of such cells in tissues or
serum, and serving as the basis for therapy to improve the clinical
outcome of subjects with such tumors, respectively.
[0118] Various diagnostic assay techniques known in the art may be
used, such as competitive binding assays, direct or indirect
sandwich assays and immunoprecipitation assays conducted in either
heterogeneous or homogeneous phases (Zola, 1987). The antibodies
used in the diagnostic assays can be labeled with a detectable
moiety. The detectable moiety should be capable of producing,
either directly or indirectly, a detectable signal. For example,
the detectable moiety may be a radioisotope, such as .sup.3H,
.sup.14C, .sup.32p, .sup.35S, or .sup.125I, a fluorescent or
chemiluminescent compound, such as fluorescein isothiocyanate,
rhodamine, or luciferin, or an enzyme, such as alkaline
phosphatase, beta-galactosidase or horseradish peroxidase.
Molecules which facilitate specific binding also include pairs,
such as biotin and streptavidin, digoxin and antidigoxin, etc. One
of the members of a pair of such molecules which facilitate
specific binding may be labeled such that it provides for detection
in accordance with known procedures, wherein the label can directly
or indirectly provide a detectable signal. Any method known in the
art for conjugating the antibody to the detectable moiety may be
employed, including those methods described by Hunter, et al.,
(1962); David, et al., (1974); Pain, et al., (1981); and Nygren,
(1982).
[0119] Therapy with anti-HLA-DR antibodies is further described
below.
[0120] Anti-HLA-DR antibodies also are useful for the affinity
purification of HLA-DR expressing cancer cells from cell culture or
natural sources. In this process, the antibodies against a HLA-DR
are immobilized on a suitable support, such a Sephadex resin or
filter paper, using methods well known in the art. The immobilized
antibody is then contacted with a sample containing HLA-DR
expressing cancer cells to be purified, and thereafter the support
is washed with a suitable medium that will remove substantially all
the material in the sample except the HLA-DR expressing cancer
cells, which are bound to the immobilized antibody. Finally, the
support is washed with another suitable medium that will release
the HLA-DR expressing cancer cells from the antibody.
[0121] Biological Effects of Anti-HLA-DR Specific Monoclonal
Antibodies
[0122] Class II MHC specific mAbs are described which recognize the
first domains of HLA-DR. The antibody or CDR region of the HLA-DR
specific mAbs of the invention is immunoreactive with, and capable
of inducing apoptosis in, tumor cells that express detectable
levels of the HLA-DR. The HLA-DR-specific mAb, Danton, has
demonstrated in vitro specificity for induction of apoptosis in
tumor (plasmacytoma MC/CAR) cells relative to non-neoplastic cells
both of which express HLA-DR. (Example 2 and FIGS. 2A-4B).
[0123] In addition, the lack of interference with normal T.sub.h
responses by Danton has been demonstrated in vitro. (See Example 2
and FIG. 5).
[0124] In vivo Cancer Therapy with Anti-HLA-DR Antibodies
[0125] The antibodies of the present invention are therapeutically
effective and can stimulate apoptotic cell death of tumor cells
that express the HLA-DR antigen. These findings raise the
possibility of a selective antibody-based anti-tumor therapy for
HLA-DR positive cancers, particularly those of the blood.
[0126] The tumoricidal effects of anti-class II MHC mAb can be
achieved without simultaneous suppression of class II-dependent
immune responses, although both properties are associated with mAb
recognizing the first domains of the protein.
[0127] Antibodies having the desired therapeutic effect may be
administered in a physiologically acceptable carrier to a host, and
may be administered in a variety of ways, e.g., parenterally,
subcutaneously (SC), intraperitoneally (IP), intravenously (IV),
etc. Depending upon the manner of introduction, the antibodies may
be formulated in a variety of ways. The concentration of
therapeutically active antibody in the formulation may vary from
about 1 mg/ml to 1 g/ml.
[0128] Preferably, the antibody is formulated for parenteral
administration in a suitable inert carrier, such as a sterile
physiological saline solution. For example, the concentration of
antibody in the carrier solution is typically between about 1-100
mg/ml. The dose administered will be determined by route of
administration. Preferred routes of administration include
parenteral or IV administration. A therapeutically effective dose
is a dose effective to produce a significant increase in apoptotic
cell death of HLA-DR expressing neoplastic cells. A significant
increase in apoptotic cell death of HLA-DR expressing neoplastic
cells is a 2-fold increase, more preferably a 5-fold increase, even
more preferably a 10-fold increase, and most preferably a 20-fold
or greater increase in apoptotic cell death of HLA-DR expressing
cells relative to cells which do not express a detectable amount of
HLA-DR.
[0129] According to an important feature of the invention, the
anti-HLA-DR antibody may be administered alone, or in combination
with other anti-cancer agents, such as chemotherapeutic agents, for
example, cisplatin, taxol, methotrexate, etc.; tumor necrosis
factor-alpha (TNF-.alpha.); FADD, PMA; ionomycin; staurosporine or
Rituxan.RTM..
[0130] The therapeutically effective amount of an anti-HLA-DR
antibody, e.g Danton, can be estimated by comparison with
established effective doses for known antibodies, taken together
with data obtained for Danton in in vitro models for the apoptotic
cell death of HLA-DR positive tumor cells, as described herein. As
is known in the art, adjustments in the dose may be necessary due
to antibody degeneration, systemic versus localized delivery, as
well as the age, body weight, general health, sex, diet, time of
administration, drug interactions and the severity of the
condition. Such adjustments may be made and appropriate doses
determined by one of skill in the art through routine
experimentation.
[0131] The following examples serve to more fully describe the
manner of using the above-described invention, as well as to set
forth the best modes contemplated for carrying out various aspects
of the invention. It is understood that these examples in no way
serve to limit the true scope of this invention, but rather are
presented for illustrative purposes.
[0132] All patent and literature references cited in the present
specification are hereby incorporated by reference in their
entirety.
EXAMPLE 1
Generation of Danton mAb
[0133] The mouse monoclonal antibody Danton, was prepared according
to standard techniques known in the art (Harlow and Lane, 1988).
Inbred laboratory mice of BALB/c strain (Jackson Laboratory, Bar
Harbor, Me.), hyperimmunized with an immunogen were donors of
immune B cells. BALB/c-derived mutant B lymphoma line M12.C3,
transfected with chimeric human/mouse class II gene was used as the
immunogen. The MHC class II molecule expressed by this transfectant
(designated M12.C3.25) was composed of the first extracellular
(alphal and beta 1) domains of the HLA-DR, and the second
extracellular (alpha2 and beta2), transmembrane and
intracytoplasmic domains of the corresponding mouse MHC class II
molecule H2-E. (Vidovic, et al., 1995). Mice were immunized at
monthly intervals with 5 IP injections, each consisting of 10.sup.7
(-irradiated (100 Gy) M12.C3.25 cells resuspended in 1 ml of
phosphate buffered saline (PBS). Three days after the last
injection, immune splenocytes were fused with the HAT-sensitive
Ig-negative mouse myeloma cells PAI-0 (Stocker, et al., 1982). The
supernatant fluids of single hybridoma cultures were screened for
their toxicity on EBV-LCL RPMI 1788 after a 4 hour incubation at
4EC. A colony, which was identified based on stable secretion of a
mAb having the desired bioactivity, was designated "Danton"
(DR-specific antibody for oncology), and subcloned two times by the
limiting dilution method. Using the standard isotyping kit (Zymed,
South San Francisco, Calif.), the mAb Danton was found to be of a
mouse IgG16 isotype.
EXAMPLE 2
Evaluation of in vitro Tumoricidal Effects of the Danton mAb
[0134] Human cell lines MC/CAR (plasmacytoma) (Ritts, et al.,
1983), and RPMI 1788 (Epstein-Barr virus transformed lymphoblastoid
B cell line, EBV-LCL) were purchased from ATCC (Rockville, Md.).
Cells were cultured at the density of 10.sup.5 /ml in IMDM medium
supplemented with 10% FCS, 2 mM L-glutamine, 0.1 mg/ml kanamycin
sulfate and 3.times.10.sup.-5 M 2-ME (Gibco, Grand Island, N.Y.) at
37EC in a humidified atmosphere containing 5% CO.sub.2 (tissue
culture incubator). Sterile filtered supernatant fluids of the
HLA-DR-specific mAb-secreting mouse B cell hybridoma cell lines
Danton and 10F12, cultured at 5.times.10.sup.5 cells/ml, were added
to the human cells at the final concentration of 20%. Following the
indicated coculture period, cells were washed and their viability
was determined after an additional 5 minute incubation with 1
.mu.g/ml of propidium iodide (PI, Sigma, St. Louis, Mo.) and a
subsequent analysis of cell size (forward light scatter, FSC) vs.
red PI fluorescence on a FACScan.RTM. flow cytometer using
CELLQuest 3.1f software (Becton-Dickinson, San Jose, Calif.) (Otten
and Yokoyama, 1997, Coligan, et al., 1997)]. Live cells were shown
to actively exclude PI, while dead cells took it up in a direct
proportion to the accessibility of their DNA (Swat, et al.,
1991).
[0135] The ability of the Danton mAb to induce apoptosis of
neoplastic cells was shown by coculture of 2 independent human B
cell tumor lines (EBV-LCL RPMI 1788 and plasmacytoma MC/CAR which
resulted in greater than 75% cell death (FIGS. 2A-D). The cytotoxic
effect was completely absent in normoplastic (i.e., non-neoplastic)
HLA-DR.sup.+ lymphocytes obtained from human peripheral blood
(FIGS. 2E-F). In contrast to Danton, 10F12, an anti-DR mAb specific
for a common epitope located within the second protein domains, did
not affect viability of MC/CAR (FIGS. 3A-F).
[0136] The cytotoxicity time course under two different incubation
conditions is shown in FIG. 4A. It appears that Danton-induced cell
death is temperature dependent; with the faster rate at human body
temperature (37.degree. C., the cytotoxic effect evident within 30
minutes, and retarded, yet still occurring at 4.degree. C. (the
cytotoxic effect evident after 1-2 hours). The undiminished
tumorotoxicity of Danton even after the prolonged (3 weeks)
coculture with tumor cells indicates their inability to become
resistant to this mAb (FIG. 4B).
EXAMPLE 3
Evaluation of Immunosuppressive Effects of the Danton mAb
[0137] HLA-DR dependent in vitro proliferative responses of human
T.sub.h cells against Staphylococcal enterotoxin B (SEB) were
generated as previously described (Mollick et al., 1991). Briefly,
2.times.10.sup.5 fresh human PBMC, obtained from a heparinized
blood by Ficoll separation, were cultured with 0.1 ug/ml SEB (Toxin
Technology, Sarasota, Fla.) in 0.2 ml of the IMDM medium
supplemented with 10% FCS, 2 mM L-glutamine, 0.1 mg/ml kanamycin
sulfate and 3.times.10.sup.-5 M 2-ME at 37.degree. C. in a
humidified atmosphere containing 5% CO.sub.2 (tissue culture
incubator) for 3 days. Sterile filtered supernatant fluids of the
HLA-DR-specific mAb-secreting mouse B cell hybridoma cell lines
Danton and L243 (ATCC, Rockville, Md.) (Lampson and Levy, 1980; Fu
and Karr, 1994), cultured at 5.times.10.sup.5 cells/ml, were added
at the initiation of the assay at the final concentration of 20%. T
cell proliferation of triplicate cultures was measured by
[.sup.3H]thymidine incorporation during the final 16 hours
(Bradley, 1980). While mAb L243 suppressed about 70% of
SEB-triggered T cell response, Danton had no effect (FIG. 5),
although both mAbs recognize epitopes on the first HLA-DR
domains.
EXAMPLE 4
Evaluation of in vivo Anti-Tumor Activity of the Danton mAb
[0138] The in vivo anti-tumor activity of Danton was evaluated in
severe combined immunodeficiency inbred (scid) mice injected with
the HLA-DR positive human plasmacytoma MC/CAR. Twenty 8-week-old
mice, randomized according to their body weight were injected IP
with 10.sup.7 MC/CAR cells in 0.1 ml RPMI 1640 medium per mouse
(Ritts, et al., 1983). Subsequently, half the animals received a
single IP dose of 0.625 mg Danton mAb in 0.1 ml of PBS. The
remaining mice were given corresponding IP injections of 0.1 ml
PBS. Mice were monitored daily, and their survival recorded. As
shown in FIG. 6, Danton exhibited a notable therapeutic activity,
significantly prolonging the survival of tumor-bearing mice.
[0139] Although the invention has been described with respect to
particular treatment methods and composition, it will be apparent
to those skilled that various changes and modifications can be made
without departing from the invention.
* * * * *