Monoclonal antibodies to human high molecular weight-melanoma associated
United States Patent: 7,550,568
Issued: June 23, 2009
Inventors: Ferrone; Soldano
Assignee: University of
Pittsburgh - Of the Commonwealth System of Higher Education (Pittsburgh,
Appl. No.: 12/039,282
Filed: February 28, 2008
The present invention provides monoclonal
antibodies that react against high molecular weight melanoma-associated
antigen. These antibodies may be used for diagnostic and/or therapeutic
Description of the
FIELD OF THE INVENTION
This invention relates generally to the field of antibodies and more
particularly to monoclonal antibodies to the human high molecular
weight-melanoma associated antigen.
BACKGROUND OF THE INVENTION
Melanomas are tumors of the skin, less frequently of mucous membranes,
some of which are benign. Malignant melanomas are carcinomas of
neuroectodermal origin generally derived from melanocytes
(pigment-producing cells), sometimes from mucous membranes, the chorioid
coat or the meninges. There are several types of malignant melanoma which
differ in localization, way of spreading and production of metastases.
Conventional treatment of melanoma includes surgery, radiation or
chemotherapy, and the application of biological response modifiers.
However, these methods have proven to be insufficient to prevent tumor
recurrence, and are complicated by significant side effects. Therefore, it
is desirable to develop therapeutic approaches which overcome these
drawbacks and can replace or be used in combination with conventional
Since the immune system seems to be heavily involved in the pathogenesis
of this disease, attention has now turned to active immunotherapy, for
example based on specific antigens. In melanoma, an example of suitable
targets for the production of antibodies and the development of
immunotherapeutic approaches are the melanoma associated antigens (MAA), a
number of which have been identified and characterized by their molecular
weight, for example high molecular weight-melanoma associated antigen (HMW-MAA)
with a molecular weight of >1,000,000.
Although not immunogenic in patients with melanoma, HMW-MAA is expressed
in at least 80% of melanoma lesions; has a restricted tissue distribution;
patients with melanoma possess an immune repertoire that recognizes this
antigen and is considered to play a role in the metastatic potential of
SUMMARY OF THE INVENTION
The invention provides monoclonal antibodies and/or antibody fragments to
human high molecular weight melanoma associated antigens. The antibodies
or fragments thereof may be used for diagnosis of melanoma or for
therapeutic purposes. The invention also provides hybridoma cell lines
which produce the monoclonal antibodies specific for the HMW-MAA.
DESCRIPTION OF THE INVENTION
This invention provides monoclonal antibodies or antibody fragments to
melanoma associated antigens. The antibodies or fragments thereof may be
used for diagnosis of melanoma and/or for therapeutic purposes.
The term "antibody fragments" as used herein for purposes of the
specification means a portion or fragment of the intact antibody molecule
wherein the fragment retains antigen binding function. Examples of such
fragments include F(ab').sub.2, Fab', Fab, Fv, scFv, Fd' and Fd fragments.
Method for producing various fragments are well known to those skilled in
For preparation of the antibodies, the High Molecular weight-Melanoma
Associated Antigen (HMW-MAA) bearing human melanoma cells can be used. The
cells can be incubated with IFN-.gamma.. Animals may be immunized with
whole cells, cell lysates or purified HMW-MAA. Animals suitable for
immunization include mice, rats, rabbits and goats.
Immunogenic conjugates of HMW-MAA can be prepared by standard methods
known in the art such as by adsorption of the immunizing antigen to the
carrier or by coupling using periodate, glutaraldehyde, carbodiimides e.g.
aminopropyl)-carbodiimide or the like. A commonly used conjugate is
involves conjugating the antigen to keyhole limpet haemocyanin (KLH) with
The immunogen may be mixed with adjuvants, i.e. agents which will further
increase the immune response, for the immunization procedure. Examples of
suitable adjuvants are Freund's complete adjuvant (emulsion of mineral
oil, water, and mycobacterial extracts), Freund's incomplete adjuvant
(emulsion of water and oil only), mineral gels, e.g. aluminum hydroxide
gels, surface active substances such as lysolecithin, polyanions,
peptides, BCG (Bacillus Calmette-Guerin), etc.
Immunization can be carried out by standard routes of immunization
including intradermal, subcutaneous, intramuscular, intraperitoneal,
intravascular and intracranial injections. Since high antibody titers are
desired, a series of injections is commonly given. The immunization is for
example performed by injecting the antigen two, three, four or more times
parenterally, e.g. intraperitoneally and/or subcutaneously, in regular or
irregular intervals of a few days, e.g. three to seven days, up to several
months, for example four weeks.
After immunization, the antibody producing cells may be recovered from the
immunized animal. The antibody producing cells may be spleen cells or
lymph node derived B cells. Antibody-producing cells recovered from the
immunized mice, preferably lymphoid cells such as spleen lymphocytes,
taken for example one to five days after the final injection, are fused
with the cells of a continuous cell line, i.e. a continuously replicating
cell clone which confers this replication ability to the hybrid cells
resulting from the fusion. An example for such a cell line is a tumor cell
line (myeloma) which does not itself actually produce inununoglobulins or
fragments thereof but has the potential to produce and secrete large
amounts of antibody, and which carries a genetic marker so that the hybrid
cells can be selected against non-fused parent cells. Several suitable
myeloma cell lines are known in the art. Preferred are myeloma cell lines
lacking the enzyme hypoxanthine guanine phosphoribosyl transferase (HGPRT)
or the enzyme thymidine kinase (TK), which therefore do not survive in a
selective culture medium containing hypoxanthine, aminopterin and
thymidine (HAT medium). Particularly preferred are myeloma cells and
derived cell lines that do not survive in HAT medium and do not secrete
immunoglubulins or fragments thereof, such as the cell lines
P3.times.63Ag8.653 or Sp2/0-Ag14.
The fusion is performed in the presence of a fusion promoter, for example
Sendai virus or other pararnyxo viruses, optionally in UV-inactivated
form, or chemical fusogens such as calcium ions, surface-active lipids,
e.g. lysolecithin, or polyethylene glycol (PEG), or by electrofusion.
Preferentially, the myeloma cells are fused with a three- to twentyfold
excess of spleen cells from immunized mammals in a solution containing
about 30% to about 60% of polyethylene glycol of a molecular weight
between 1000 and 4000.
After the fusion, the cells are resuspended and cultivated in a selective
medium chosen depending on the genetic selection marker, for example HAT
medium. In this medium, only hybridoma cells will survive, because they
combine the ability to grow and replicate in vitro inherited from the
parent myeloma cells and the missing HGPRT or TK genes essential for the
survival in HAT medium inherited from the antibody-producing spleen cells
of the immunized mammals.
Suitable culture media for the expansion of hybridoma cells are the
standard culture media, such as Dulbecco's Modified Eagle Medium (DMEM),
minimum essential medium, RPMI 1640 and the like, optionally replenished
by a mammalian serum, e.g. 5% to 15% fetal calf serum. The culture media
are supplemented with selective medium in order to prevent myeloma cells
from overgrowing the hybridoma cells. The supernatant of the hybridoma
cells can be screened for the monoclonal antibodies. For example,
hybridomas may be selected from the fused cells by radioimmunoassay,
enzyme-labeled immunoassay (ELISA), fluorescence labeled immunoassays or
the like using HMW-MAA.
Positive hybridoma cells are cloned, e.g. by limiting dilution or in soft
agar, preferentially twice or more. Optionally, hybridoma cells are
passaged through animals, e.g. mice, by intraperitoneal injection and
harvesting of ascites, which stabilizes hybridomas and improves growth
characteristics. The cloned cell lines may be frozen in a conventional
manner. In a preferred embodiment, hybridomas producing the monoclonal
antibodies TP109 and VF20-VT1.7 were produced. These hybridomas were
deposited at the American Type Culture Collection (ATCC) as ATCC Accession
No. PTA 9582 (murine hybridoma VF4 TP109.2) and ATCC Accession No. PTA
9583 (murine hybridoma VF20-VT.1), respectively, in accordance with the
Budapest Treaty on Nov. 4, 2008.
Also provided in this invention are the hybridoma cell lines which secrete
the monoclonal antibodies of the present invention. In particular, the
invention provides hybridoma cell lines which are hybrids of myeloma cells
and B lymphocytes of a mouse immunized with a monoclonal antibody directed
against high molecular weight-melanoma associated antigen. Preferentially,
these cell lines are hybrids of mouse myeloma cells and B lymphocytes of a
mouse, for example a Balb/c mouse, immunized with melanoma cells.
Especially preferred is the hybridoma cell line which secretes monoclonal
antibody TP109 and the hybridoma cell line which secretes the monoclonal
antibodies VF20-VT1.7. The hybridoma cell lines of the invention may be
kept in deep-frozen cultures and reactivated by thawing and optionally
The invention also provides pharmaceutical compositions comprising a
monoclonal antibody and/or a fragment thereof according to the invention.
The pharmaceutical compositions comprise, for example, the monoclonal
antibodies and/or fragments thereof in a therapeutically effective amount
together or in admixture with a pharmaceutically acceptable carrier.
The antibodies of the present invention may be used for diagnostic and
therapeutic purposes. The antibodies of the present invention can be used
for qualitative or quantitative detection of melanoma and other tumor
cells. The detection is carried out by standard immunological methods well
known in the art. The antibodies obtained according to the present
invention can thereby be used as such or may be labeled or conjugated
(such as to toxins). Thus, the monoclonal antibodies can be used for tumor
The antibodies according to the present invention can also be used for the
progress of a treatment regimen or as a prognostic indicator. After
treatment of a melanoma patient by conventional treatments such as
radiation etc., the antibodies of the present invention can be used to
determine the presence of the antigen in the body fluids. The level of the
antigen may also be determined following treatment.
The present invention also provides a process for the diagnosis or therapy
of tumors and especially of melanomas, wherein there is administered one
or a mixture of several antibodies according to the present invention,
optionally together with conventional pharmaceutical carrier, adjuvant,
filling and additive materials.
The antibodies of the present invention have the characteristics as
described in the examples given below. The following examples are
presented for illustrative purposes and are not intended to be
For this antibody, a BALB/c mouse was immunized four times at 2 week
intcrvals with intramuscular injections of 3.times.10.sup.6 cultured human
melanoma cells Colo38 which had been incubated with IFN-.gamma. (final
concentration 1000 U/ml) for 48 hours at 37.degree. C. Spleen cells were
obtained from the immunized mouse and fused with murine myeloma cells
Ag8.X.653. Hybridizations and subcloning were performed according to
standard procedures. The supernatant from the hybridomas was tested for
reaction with melanoma cells Colo 38. A positive reacting hybridoma,
labeled herein as TP109 was identified. The supernatant of this hybridoma
reacted with Colo 38 cultured human melanoma cells but not with cultured
human B lymphoid cells LG2 as determined by ELISA.
When tested with a panel of cultured human melanoma cells in a binding
assay, mAb TP109 reacted with HMW-MAA bearing human melanoma cells but did
not react with cultured human B lymphoid cells and with cultured human
carcinoma cells. The latter two types of cells do not express HMW-MAA.
Furthermore, mAb TP109 immunoprecipitated from radiolabeled cultured human
melanoma cells Colo 38 components with the characteristic electrohpretic
profile of HMW-MAA when analyzed by SDS-PAGE.
mAb VF20-VT1.7 and VT68.2
For these antibodies a BALB/c mouse was immunized five times at 3 week
intervals with intramuscular injections of approximately 200 .mu.l of a
bead suspension. The bead suspension had been preincubated with a NP40
lysate of 1.times.10.sup.7 cultured human melanoma Colo 38 cells. Spleen
cells were obtained from the immunized mouse and fused with murine myeloma
cells Ag8.X.653. Hybridizations and subcloning were performed according to
standard procedures. The supernatants from the hybridomas were tested for
reaction with melanoma cells Colo 38. The hybridomas, VF20-VT1.7 and
VT68.2 were found to react with cultured human melanoma cells Colo38, but
not with cultured human B lymphoid cells LG2 in a binding assay.
When tested with a panel of cultured human melanoma cells in a binding
assay, mAbs VF20-VT1.7 and VT68.2 reacted with HMW-MAA bearing human
melanoma cells but did not react with cultured human B lymphoid cells and
with cultured human carcinoma cells. Furthermore, mAb VF20-VT1.7 and
VT68.2 immunoprecipitated from radiolabeled cultured human melanoma cells
Colo 38 components with the characteristic electrohpretic profile of
HMW-MAA when analyzed by SDS-PAGE.
Claim 1 of 11 Claims
1. A monoclonal antibody which
specifically binds to human high molecular weight melanoma associated
antigen (HMW-MAA) bearing human melanoma cells but not to human B lymphoid
cells, wherein the monoclonal antibody is designated as TP109 ATCC
Accession No. PTA-9582.
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