Note: Descriptions are shown in the official language in which they were submitted.
~ 095121244 2 1 S ~ 5 7 2 PCT~S95/01440
Monoclonal Antibody 88BV59, Subclones and Method of Making
This application is a continuation-in-part of U.S.
Serial Number 08/065,517, filed May 21, 1993, which is
a continuation of U.S. Serial Number 07/636,179, filed
December 31, 1990, now Ah~n~oned, which is a
continuation-in-part of U.S. Serial Number 07/302,155,
filed January 25, 1989, now U.S. Patent No. 5,106,738,
which is a continuation-in-part of U.S. Serial Number
06/697,078, filed January 31, 1985, now U.S. Patent No.
4,828,991, which is a continuation-in-part of
06/575,533, filed January 31, 1984, now abandoned.
FIELD OF THE INVENTION
This invention relates to monoclonal antibodies
produced by hybridoma or transformed B-cell lines
derived from B-cells of cancer patients actively
immunized with autologous tumor antigen. These
monoclonal antibodies can be used in both diagnostic
procedures and therapy for human cancers. This
invention also relates to cell lines producing these
monoclonal antibodies, and to diagnostic procedures and
therapeutic approaches using them.
~CKGROUND OF THE lN V~N l ION
This invention relates to new human monoclonal
antibodies that react specifically with antigens
associated with particu:ar cancers and to hybridoma and
transformed B-cell lin~-~ for their production derived
from peripheral blood B-cells of actively immunized
patients. This invention also relates to diagnostic
procedures and cancer therapy using these monoclonal
antibodies.
WOgS/21244 2 is 8~7 2 PCT1595101440
Currently available treatments for cancer,
particularly radiation therapy and chemotherapy, are
based upon the rationale that cancer cells are
relatively more sensitive to these treatments than
normal cells. However, severe toxicity for normal
tissues imposes major limitations to these therapies.
In contrast, antibody molecules exhibit exquisite
specificity for their antigens. Researchers have
therefore sought to isolate antibodies specific for
cancer cells as the "long-sought 'magic bullet' for
cancer therapy" (Science, 1982, 216:283).
Antibodies are protein molecules normally
synthesized by the B-cell lymphocytes produced by bone
marrow and carried in the blood stream. For any antigen
entering the body, i.e., any foreign molecule from a
simple organic chemical to a complex protein, antibodies
are produced which recognize and attach to that
particular chemical structure. The unique chemical
structure on the antigen to which a particular antibody
can bind is referred to as an antigenic determinant or
epitope. B-cell lymphocytes in the body, referred to as
B-cells, lymphocytes, or leukocytes, exist as hundreds
of millions of different genetically programmed cells,
each producing an antibody specific for a different
determinant. An antigen, which stimulates antibody
production, can have several determinants on its
surface. On encountering an antigen, a B-cell carrying
on its surface an antibody specific for a determinant on
that antigen will replicate. This clonal expansion
results in many daughter cells that secrete that
antibody into the blood stream.
Because of the specificity of antibodies in
recognizing and binding to antigens, it was desired to
produce antibodies in quantity that are specific for a
single determinant, thus binding only to antigens or
tissues having that particular determinant.
_WO95/21244 PCT~S95/01440
~ 21S~72
B-cells do not grow in a continuous culture unless
they have been altered by hybridization with an
"immortal" cell or by being transformed with either
viral or tumor DNA. Kohler and Milstein (Nature, 1975,
256:495) demonstrated that hybrid cells could be
prepared by somatic cell fusion between lymphocytes and
myeloma cells that grow in culture and produce
antibodies specific for a single determinant. These
hybrids are referred to as "hybridoma cells." Hybridoma
cells are prepared by fusing lymphocytes that have been
activated to produce a particular antibody with myeloma
cells. When cultured, hybridomas produce antibodies
specific for a single determinant on a particular
antigen. Such an~ibodies are referred to as "monoclonal
antibodies."
Monoclonal antibodies may also be produced by B-
lymphocyte cell lines that have been transformed, either
spontaneously or intentionally, with a lymphotropic
virus such as Epstein-Barr Virus (EBV). Transformation
can also be accomplished using other transforming
agents, such as viral DNA and cellular DNA. These
cells, unlike hybridoma cells, possess a normal human
diploid number (46) of chromo~omes. This invention
permits the isolation of both hybridomas and transformed
B-cell lines that produce monoclonal antibodies. For
sake of simplicity, both cell types will be referred to
as monoclonal antibody producing cells below.
Monoclonal antibodies are synthesized in pure form
uncontaminated by other immunoglobulins. With
monoclonal antibody producing cells it is possible to
produce virtually unlimited quantities of an antibody
that is specific for one determinant on a particular
antigen.
It has been believed that if antibodies specific
for particular cancer cells were available, they could
be used in various methods of treatment and diagnosis.
WO95/21244 ~! PCT~Sg5/01440 ~
2l 5~g~7~
--4--
Such antibodies could inactivate or kill particular
tumor cells merely by attaching to the cell at the
determinant for which they are specific. Alternatively,
these antibodies may bind to the surface of effector
lymphocytes or macrophages, converting them into tumor
antigen-specific killer cells.
Monoclonal antibodies can also increase the
specificity of chemotherapeutic drugs, toxins and
radioactive isotopes, thus increasing their efficacy
while decreasing their toxicity by being conjugated to
them. In addition, antibodies conjugated with
radionuclides or metallic tracers can be used for
imaging for in vivo diagnosis and localization of
metastases, such as with proton emission (PET), nuclear
magnetic resonance (NMR), computed tomography (CT), and
planar and single photon emission computed tomography.
The antibodies can also be used for detecting the
presence of tumor antigens in blood, as a diagnostic
and/or prognostic test for cancer. Also, monoclonal
antibodies can be used to isolate tumor antigens for
potential use in a standardized vaccine.
The existence of antigens associated with animal
tumors was documented in the last century, and the
antigenic character of human cancers has been well
established, primarily through recent studies with
monoclonal antibodies. However, until the research
which resulted in this invention, few cancer antigens
have actually been characterized in molecular terms and
only one group of antigenic determinants associated with
human cancers, imml~noglobulin idiotypes of B-cell
tumors, has been described as being uniquely tumor-
specific, i.e., occurring with a high frequency on tumor
cells and not occurring to any significant degree on
normal tissues (Oldham and Smalley, J. Biol. Response
Modifiers, 1983; Stratte et al, J. Biol. Response
Modifiers, Volume 1, 1982).
PCT~Sg5/01440
WO95/21244
~ls8s72
--5--
Past attempts at deriving monoclonal antibodies
specific for human cancers have taken two routes with
respect to B -cells: 1) B-cells have been extracted
from spleens of mice that were immunized against human
tumors, U.S. Patent 4,172,124; and 2) human B-cells have
been extracted from either peripheral blood or from
lymph nodes draining tumors in cancer patients. Neither
approach has yielded satisfactory results.
Mice immunized against human tumors have too broad
a reactivity. That is, most of the mouse monoclonal
antibodies generated react with human antigens present
on normal as well as on tumor tissue. An antibody that
reacts only with tumor cells is very difficult to select
from among the large variety of antibodies produced.
For example, 20,000 hybridomas derived from mice
immunized with human small-cell lung carcinoma were
screened for reactivity with tumor cells (Science, 1982,
216:283). In contrast to a very low frequency (~0.4~)
observed by this research group, the present invention
results in up to 16~ of the hybridomas derived from
immunized colon patients producing monoclonal antibodies
that react speci~ically with tumor cells. In addition,
monoclonal antibodies derived from mouse B-cells have
limited potential for application in cancer therapy.
After repeated administration they stimulate the human
immune system to produce "anti-mouse" antibodies which,
in clinical trials, have been shown to neutralize the
activity of mouse monoclonal antibodies. The use of our
human monoclonal antibodies can circumvent these
difficulties.
Another apparent difference between human and mouse
monoclonal antibodies is their patterns of labeling.
Previous studies with mouse antibodies have demonstrated
that there is often a heterogenous labeling of cells
within tumor sections. This pattern of reactivity has
been attributed by some authors to antigenic
WO95/21244 ~ PCT~S95/01440
2~5~s~
--6--
heterogeneity of tumor cells (Hand et al., Cancer
Research, 43:728-735, 1983). In contrast, the human
monoclonal antibodies developed by our strategy were
homogeneous in terms of their reactivity with tumors to
which they did react. A plausible explanation for the
heterogenous staining of mouse monoclonal antibodies is
that it is a reflection of the murine immune recognition
of phase- or cell-cycle-specific differentiation
antigens abundant on the tumor cells rather than
putative tumor associated antigens. It is not
unreasonable to expect that when one immunizes mice with
human tumor cells there would be substantial antigenic
competition resulting in the more abundant and more
predominant tissue-type and differentiation antigens
successfully competing with relatively minor tumor
associated antigens for immune responsiveness by the
host. Thus, autologous immunization of man may result
in the elicitation of antibodies against the group of
antigens normally poorly immunogenic in mice. This
evidence suggests that humans and mice may respond to
different tumor antigens. In concert with this
hypothesis is our finding that none of the first 36
human monoclonal antibodies we produced appeared to
react with carcinoembryonic antigen (CEA), an antigen
frequently recognized by murine monoclonal antibodies
made against human tumor cells.
The majority of past attempts to develop human
monoclonal antibodies have used B-cells extracted from
either peripheral blood or lymph nodes from patients
bearing tumors. It was believed that the presence of
the antigenic tumor would cause a tumor-bearing
individual to mount an immune response against his tumor
and produce specifically immune B-cells. Thus, B-cells
were taken from lymph nodes draining tumors in cancer
patients or from circulating lymphocytes found in
peripheral blood. However, prior to the present
095/21244 PCT~S95/01440
~w 2ls8s7~
invention, there has been limited success in creating
tumor-specific monoclonal antibodies.
The major problem in creating monoclonal antibodies
specific for human tumor antigens has been the inability
to find a source of specifically immune B-cells
(Science, 1982, 216:285). In humans, the initial foci
of cancer cells tend to grow over long periods of time,
from 1~ to 10~ of the human lifespan, before there is
any palpable clinical evidence of the disease. By this
time patients are immunologically hyporesponsive to
their tumors, or possibly immunologically tolerant.
Thus, prior to the present invention, human monoclonal
antibodies reactive with tumor cells could not
reproducibly be obtained. Furthermore, of the small
number of human monoclonal antibodies obtained from
cancer patients, very few reacted with determinants
found on the surface of tumor cells, but rather with
intracellular determinants (R. J. Cote et al, PNAS,
1983, 80:2026). The present invention permits the
development of monoclonal antibodies reactive with
surface antigens, a requisite activity for tumor imaging
and therapy.
SUMMARY OF THE INVENTION
One object of the present invention was to develop
monoclonal antibodies specifically reactive with tumor-
associated antigens that induce an immune response in
patients having particular cancers. Such antibodies
provide a means for detecting and diagnosing tumors. A
second objective of this invention wa~ to obtain
monoclonal antibodies that are effective for treating
patients with particular types of cancer.
We have developed a new and more effective approach
for obtaining monoclonal antibodies by using peripheral
blood B-cells from patients i~ml~n;zed with cells from
their own tumors in specific vaccine preparations. To
WO95/21244 ~ PCT~Sg5/01440
--8--
achieve active specific immunotherapy, patients were
immunized with autochthonous tumor cells, that is, cells
from their own tumors. This approach was taken based on
our theory that tumor cells express tumor-specific
antigens.
Humans mounting an objective immune response
against tumor cells were specifically found to be a good
source of activated B-cells. We have shown that the
peripheral blood of patients who had been actively
immunized against their own tumors is an abundant source
of such activated B-cells.
We demonstrated in clinical studies that an
objective immune response is generated on treating
patients having the particular cancer by skin testing,
i.e., delayed cutaneous hypersensitivity (DCH).
Immunized patients showed delayed cutaneous hypersensi-
tivity to their own colorectal cancers. In addition,
the monoclonal antibodies developed from the immunized
patient's B-cells reacted with tumors of the same
histological type in other patients. These results
indicate that the patient's humoral immune response,
production of antibodies, is directed against colorectal
cancer generally and is not unique to the immunized
patient's own tumor. This general response is
especially important for the development of a
standardized vaccine.
The generation of B-cells that produce antibodies
having reactivity specific for epitopes on tumor cell
associated antigens, particularly cell surface antigens
as in the majority of cases, is an advantageous result
that was speculative, at best, when the immunization
studies were begun. Only the ;mmlln;zation treatment was
observed and measured during the animal studies on which
the human immunization procedures were based, not the
production of tumor specific antibodies.
~WO 95121244 ~S PCT~S95/01440
_g_
The general immune response accompanied by an
improvement in the subject's condition was indicative of
a cellular response in which macrophages and T-cells
become activated in the presence of tumor cell antigens
and destroy the tumor cells. Although an antibody
response would predictably be triggered by immunization
under most circumstances, the time course of the
antibody response and the cellular response would in
most instances be different. Moreover, the fact that
the patients were being immunized with autologous tumor
cells, i.e., the patient's own tumor cells, and the
experience of previous investigators that little or no
antibody production is triggered by a patient's own
tumor, made our discovery that B-cells that produce
tumor specific antibodies are generated after
immunization an unexpected beneficial result.
Some cellular and humoral immune responses can
occur independently of each other. For example, it is
possible to mount a humoral response in the absence of
demonstrable cellular immunity. Conversely, potent
cellular ; mml~n; ty, particularly delayed cutaneous
hypersensitivity (DCH), may develop despite a minimal
antibody response. It was surprising, therefore, for
the subjects who showed a positive response to active
immunotherapy to have been excellent sources of B-cells
producing tumor specific antibodies, particularly cell
surface antibodies.
This invention comprises the preparation of
successful vaccines for active specific immunization,
procedures for extracting ;mmlln;zed B-cells, the
production of monoclonal antibody prod~cing cell lines
and the production of monoclonal antibodies. Malignant
tumors are digested using enzyme preparations. The
cells obtained are treated to yield a non-tumorigenic
tumor cell preparation having the requisite cell
viability, which is injected as a vaccine into the
WO95/21244 ~ ~ PCT~S95/01440 ~
--10--
subject from which the tumor was obtained. Peripheral
blood B-cells are obtained from the inoculated subject
after a predetermined interval and are used to prepare
monoclonal antibody producing cells by fusing with
myeloma cells, after which the fused cells are screened
for the synthesis of immunoglobulin. Monoclonal
antibody producing cells may also be obtained by
selecting spontaneously transformed B-cells that are
able to survive in continuous culture, or by exposing B-
cells to an agent capable of transforming cells such asEpstein Barr Virus (EBV) or another lymphotropic virus.
Larger amounts of antibodies may be obtained by
fusing EBV-transformed cells with mouse myeloma cells or
human-mouse heteromyelomas. Cells that æynthesize
immunoglobulin are tested for production of antibodies
that react with antigens characteristic of the malignant
tissue. Those selected are cultured to produce
monoclonal antibodies that react with the particular
type of tumor with which the subject was afflicted.
This invention also comprises the immunodetection
of cancer with labeled monoclonal antibodies. That is,
the monoclonal antibodies can be used as
radioimmunoscintography (RIS) agents for diagnostic
purposes.
DETAILED DESCRIPTION OF THE INVENTION
This invention comprises, æpecifically, a human
diploid cell line, an immortalized human B-cell line
that we transformed by exposure to EBV, designated
CO88BV59, and subclones and derivatives thereof. One
particular derivative of this EBV transformed B-cell
line, which has desirable characteristics such as high
production of antibody, is the cell line designated
CO88BV59H21-2V67-66. This cell line was obtained by
first incubating the CO88BV59 cell line with a human-
_WO95/21244 PCT~S95/01440
'~ 05~
mouse heteromyeloma under conditions suitable for cell
fusion to produce a cell line designated CO88BV59H21-2.
CO88BV59H21-2 was then exposed to EBV under conditions
suitable for transformation to produce CO88BV59H21-2V67-
66. This method of re-exposing an EBV-transformed
lymphoblastoid cell line with EBV is different from
methods known in the art. It was found that by this
method we unexpectedly obtained a cell line
(C088BV59H21-2V67-66) that has a much higher potential
to be passaged in culture than the original cell line
(making it more useful for large scale manufacturing),
and produced an amount of antibody three to five times
higher than the original cell line.
A11 clones and derivative cell lines of CO88BV59
studied so far produce a human IgG3 kappa light chain
antibody specifically reactive wi~h an epitope on a
cytoplasmic antigen defined in copending application
Serial Number 07/343,475, filed February 28, 1989 and
referred to as "CTA-1" or as the 16.88 antigen. This
antigen was first identified using human IgM antibody
16-88, defined in our copending application Serial
Number 07/038,811, filed April 15, 1987, now U.S. Patent
4,977,762. Hereinafter, the IgG3 antibody produced by
all of these "88BV59" cell lines will be referred to as
the "88BV59" antibody. Both the 88BV59 antibody and the
16-88 antibody recognize the same ~umor associated
antigen, but react with different epitopes on that
antigen. The present invention includes not only the
antibodies produced by the aforementioned cell lines,
but an antibody produced by any cell line that functions
in the same way as the 88BV59 antibody; in order words,
any antibody that binds to the same epitope on the same
antigen as the 88BV59 antibody. Finally, the term
"antibody" is intended to include any functional
fragments of the 88BV59 antibody, such as fragments
containing the variable region(s) (heavy and/or light
WO95/21244 PCT~S95/01440
~ 12-
chain) and portions containing complimentary determining
regionts). Such fragments may be produced recombinantly
by methods known in the art. The advantage of using
fragments of the antibody, for instance as single chain
antibody fragments or Fab fragments, is that a smaller
antibody size allows for better tumor penetration and
more rapid clearance,and therefore are more efficacious
as imaging agents. The variable regions of the heavy
and light chain can also be used to recombinantly
produce an antibody in which the isotype has been
changed. For instance, the IgG3 isotype can be switched
to an IgG1 isotype; IgGl stays longer in the human body
and therefore may be advantageous as a therapeutic tool.
Another example of a recombinantly produced antibody is
one in which the CH2 region is deleted (~CH2). Methods
are known in the art for making a ~CH2 antibody, and it
is believed that modifying the antibody in this manner
will allow for more rapid in vivo pharmacokinetics due
to the absence of the Fc receptor binding region and N-
linked carbohydrate, resulting in a much more
efficacious imaging agent. Such an antibody, insofar as
it is a fragment of the original antibody, is intended
to be covered by the present invention. The variable
region(s) have been sequenced and are disclosed in
copending USSN 07/807,300, filed December 13, l99l,
which is incorporated herein by reference.
The key aspects of this invention are:
l) Criteria for successful vaccines for active
specific ; mmlln; zation:
Tumor cells, whole cells enzymatically
dissociated from tissue, cryopreserved and X-irradiated
for non-tumorigenicity.
Adjuvant, an immunomodulator that is capable
of inducing immunogenicity to the tumor cell
preparation.
PcT~Sss/01440
95/21244 -13- ~ ~S ~
Components and administration, including ratio
of adjuvant to tumor cells, optimum doses of tumor
cells, and regimen of vaccination.
Patient, regional lymph nodes draining the
vaccination site must be present during the first 21
days after vaccination.
2) Procedures and timing for the extraction of
immunized B-cells from the patients.
3) Procedures for the production of hybridomas or
transformation of lymphocytes and the production of
monoclonal antibodies.
4) Procedures for the use of the monoclonal
antibodies in diagnosis and therapy of cancer.
We have successfully digested solid human
malignancies using various enzyme preparations. The
tumor dissociations were evaluated for yield of tumor
cells per gram of tissue, cell types recovered, cell
viability, cell size, and sterility. The criteria for
successful vaccines for active specific immunotherapy
are shown in Table 1.
Tumor tissue was obtained from patients suffering
from the particular solid cancer for which monoclonal
antibodies were to be prepared. The tumor tissue was
surgically removed from the patient, separated from any
non-tumor tissue, and cut into small pieces. We found
it satisfactory to cut the tumor tissue into fragments
2-3 mm in diameter. The tumor fragments were then
digested to free individual tumor cells by incubation in
an enzyme solution.
After digestion, the freed cells were pooled and
counted, and cell viability was assessed. The trypan
- blue exclusion test was found to be an acceptable
meaæure of cell viability. The tumor cells were then
cryopreserved and stored in liquid nitrogen.
The vaccine was prepared for injection by rapidly
thawing cryopreserved cells, diluting the cells, washing
WO95/21244 PCT~s95/01440 ~
51 ~
-14-
with HBSS, resuspending, counting, and assessing
viability.
Viable tumor cells were irradiated to render them
non-tumorigenic. We found that irradiation with 4020
rads/min for a total of 20,000 rads resulted in non-
tumorigenic but viable cells. The volume of the cell
suspension in HBSS was adjusted such that 107 viable
cells remained in the tube. The cells were centrifuged,
the supernatant was removed, and 107 viable BCG were
added in a volume of O.l ml. Hank's Balanced Salt
Solution (HBSS) was added in sufficient quantity for a
final volume of 0.2 ml. A third vaccine was similarly
prepared, omitting the BCG.
Immunization of Patients
Patients afflicted with the particular solid cancer
for which antibodies were to be generated were immunized
by intradermal inoculation with the tumor cell vaccine.
107 viable tumor cells admixed with BCG were used for the
first two vaccinations and 107 tumor cells alone were
used for the third vaccination. Scheduling each
vaccination one week apart was found to be a successful
procedure for inducing antibody production by the
patient's peripheral blood lymphocytes.
Collection of Immunized B-Cells
Venous blood was collected from the ;mmlln;zed
patients one week after each vaccination. Peripheral
blood lymphocytes (PBLs) were separated from the
collected blood for use in hybridoma production or
transformation.
Separation of lymphocytes from the blood was
accomplished using two different methods. The first
comprised dilution with calcium and magnesium-free HBSS,
layering on lymphocyte separation medium, centrifuging,
and removing cells at the interface. These cells were
~WO9St21244 ~S~ PCT~Sg5/01440
-15-
diluted with HBSS and pelleted. The lymphocytes were
then resuspended in serum-free Hepes-buffered Dulbecco's
MEM (DMEM), counted, and assayed for viability (GIBC0
Biologics, Grand Island, New York).
An alternative method that was used to recover
peripheral blood lymphocytes (PBLs) enriched for B-cells
comprised the removal of T-lymphocytes by rosetting
with 2-aminoethylisothiouronium bromide hydrobromide
(AET) treated sheep erythrocytes. Treated erythrocytes
were mixed with peripheral blood lymphocytes, pelleted
by centrifugation, and the pellet incubated on ice.
After resuspension, layering over lymphocyte separation
medium (LSM, Litton Bionetics), and centrifugation of
the rosetted cells, the T-cell depleted PBLs were
collected at the interface, washed, and pelleted. The
PBLs enriched for B-cells were then used for hybridoma
generation after counting and viability determination.
Preparation of Human H~bridomas for the Production of
Anti-Tumor Monoclonal Antibodies
Peripheral blood lymphocytes and cultured myeloma
cells were mixed together, pelleted, and resuspended in
a serum-free medium. Polyethylene glycol (PEG) was
added, the cells pelleted and resuspended in HT medium
(DMEM containing 20~ fetal bovine serum, hypoxanthine
and thymidine) and distributed into microtiter wells.
Twenty-four hours later, HAT medium (HT medium
containing aminopterin) was added to each well, with
one-half of the medium being replaced every three days.
After maintenance in HAT medium for 14 days, the cells
were maintained on HT medium for an adc~tional two
weeks, after which the cells were grown on a DMEM medium
containing 20~ fetal bovine serum.
The hybridomas were pre-screened for the synthesis
of hllm~n immunoglobulin using the standard enzyme
;mmllnoassay. Hybridomas synthesizing hllm~n
WO95121244 2~5~ PCT~S95/01440
immunoglobulin in sufficient amounts were tested on
tissues. Particular tissue samples were incubated with
hybridoma supernatant fluids. Supernatants that
demonstrated reactivity with particular tumor tissues
indicated that hybridoma cells in the wells from which
the particular supernatants were drawn produced tumor-
specific antibodies. If the same supernatant failed to
show a reaction with samples of normal tissue after
extensive screenings, the hybridomas in that particular
well were considered tumor-specific. These tumor-
specific supernatants were further tested against
carcinoembryonic antigen (CEA) to be sure of their
narrow specificity.
In addition to hybridoma cells that produced tumor-
specific antibodies, transformed human B-cells (diploid
cells) that produced tumor-specific antibodies were also
prepared by these procedures. The transformed B-cells
were detected in the same way as tumor-specific
antibody-producing hybridomas. Thus, well supernatants
that tested positively for reactions with tumor tissue
and negatively for reactions with normal tissue and with
CEA contained either hybridomas or transformed B-cells.
The two types of cells were differentiated by observing
that the transformed B-cells contained 46 human
chromosomes, whereas the hybridomas contained many more
chromosomes, not all of which were of the human type.
It is apparent that spontaneously transformed B-
cells had been exposed to a transforming agent, either
in vivo or during the procedures after peripheral blood
was collected. One of such agents is Epstein Barr Virus
(EBV). We have used EBV transformation for producing
antibody producing cells that will live in continuous
culture. By this method, B-cells are incubated with EBV
for a period of time to let the virus be adsorbed, after
which the cells are separated from the EBV containing
~wogsnl~44 ~2 PCT/US9s/0l440
-17-
medium, resuspended, and screened in a similar manner to
that described above for screening hybridomas.
Use of the Monoclonal Antibodies in Diaqnosis and
TherapY of Cancer
The 88BV59 antibody is labeled by conventional methods
with radioisotopes or metallic traceræ typically used in
radiological scanning. T~ese isotopes include, but are
not limited to, iodine-131, iodine-125, indium-111 and
technetium-99m. The specific activity of the
radiolabeled antibody is not particularly limited, and
about 2 to about 4 mCi/mg. of antibody has been found
acceptable. About 15 to about 41 mCi of 99mTC-88BV59 has
been infused intravenously over a 30 minute period and
good imaging resulted, although this amount may be
varied depending on such factors as weight of the
patient and safety. Other methods of introduction of
the radiolabeled antibody into the body may be used,
such as through intralymphatic and intraperitoneal
administration. The details of immunodetection with
radiolabeled 88BV59 antibody may be fou}~d in the review
article, DeJager et al., "Current Status of Cancer
Immunodetection with Radiolabeled Human Monoclonal
Antibodies", Seminars in Nuclear Medicine, Volume XXIII,
No. 2 (April), 1993: pages 165-179, incorporated herein
by reference. The admini~tration of radiolabeled 88BV59
has been shown to be safe and well tolerated with few
side effects reported. The data collected so far
clearly indicate that antibody scanning with 99mTC-88BV59
using both planar and tomographic techniques is superior
to CT scanning for the detection of intraabdominal and
- pelvic metastases. The combination of the two
modalities appears to give optimal detection. The major
advantage 88BV59 has over murine antibodies is its lack
of immunogenicity, which makes possible repeated
administrations.
WO95/21244 ~ PCT~S95/01440
-18-
Example I: Preparation of Sensitized B-Cells
A. Patient Selection.
Patients undergoing surgical resection of colon or
rectal cancers were selected for a randomized trial of
active specific immunotherapy. Randomization was done
with stratification according to pathologic stage and
tumor was obtained from all patients who met the
clinical criteria. Candidates for the study were
colorectal cancer patients with no previous history of
cancer, who had received no prior chemotherapy or
radiation therapy, and who were in suitable medical
condition to comply with the outpatient treatment
protocol. Patients eligible for the trial were those
with tumor extending through the bowel wall (Astler-
Coller B2), positive lymph nodes (stages Cl, C2) orpatients with metastatic disease (stage D). Within
these classifications, patients were randomly selected
for participation in treatment and non-treatment groups.
Randomization cards were computer generated and
sequentially drawn from each category postoperatively.
B. Tumor Acquisition.
After surgical resection the bowel specimen was
taken immediately to the hospital pathology department
and opened under sterile conditions. Tumor tissue was
excised, placed in sterile tubes containing Hank's
Balanced Salt Solution (HBSS) containing 50 ~g
gentamicin per ml and carried immediately on ice to the
laboratory for processing and freezing.
C. Dissociation of Solid Tumor and Colon Mucosa.
The tissue dissociation procedure of Peters et al
(Cancer Research, 39:1353-1360, 1979) was employed using
sterile techniques throughout under a l~m-n~r flow hood.
Tumor tissue was rinsed three times in the centrifuge
tube with HBSS and gentamicin and transferred to a petri
dish on ice. Scalpel dissection removed extraneous
tissue and the tumor was minced into pieces
WO95/21244 PCT~S95/01440
8 S 72
--19--
approximately 2 to 3 mm in diameter. Tissue fragments
were placed in a 75 ml flask with 20-40 ml of 0.14~
(200 units/ml) Collagenase Type 1 (Sigma C - 0130) and
0.1~ (500 Kunitz units/ml) deoxyribonuclease type l
(Sigma D - 0876) (DNAase 1, Sigma D-0876) prewarmed to
37C. Flasks were placed in a 37C waterbath with
submersible magnetic stirrers at a speed which caused
tumbling, but not foaming. After a 30-minute incubation
free cells were decanted through three layers of sterile
medium-wet nylon mesh (166t: Martin Supply Co.,
Baltimore, Maryland) into a 50 ml centrifuge tube. The
cells were centrifuged at 1200 rpm (250 x g) in a
refrigerated centrifuge for 10 minutes. The supernatant
was poured off and the cells were resuspended in 5-10 ml
15 of DNAase (0.1~ in HBSS) and held at 37C for 5-10
minutes. The tube was filled with HBSS, washed by
centrifugation, resuspended to 15 ml in HBSS and held on
ice. The procedure was repeated until sufficient cells
were obtained, usually three times for tumor cells.
Cells from the different digests were then pooled,
counted, and cell viability assessed by the trypan blue
exclusion test. The cells were centrifuged for a final
wash prior to cryopreservation.
D. Cryopreservation.
Optimal cryopreservation was a primary concern.
For vaccine preparation, the dissociated tumor cells
were adjusted to 5-8 x 107/ml in HBSS and added in equal
volume to chilled 2 X freezing medium containing 15~
dimethylsulfoxide (DMSO) and 4~ human serum albumin
30 (HSA). The final suspension of 2 to 4 x 107 cells/ml
were placed in 1.2 ml Nunc freezer vials. For DCH cell
testing the procedure was the same except that no HSA
was used. In both cases, in preparation for freezing,
the Nunc vials were transferred on ice to a Cryo-Med
35 model 990 Biological Freezer with a model 700 Controller
and a model 500 Temperature Recorder for controlled-rate
WO9S/21244 2i~ PCT~S95/01440
~ -20-
freezing. Care was taken that the temperature of the
individual vials, including the monitor vial, was
uniform at the beginning of the freezing process. Vials
were cooled at a controlled rate of -1C/min to a final
temperature of -80C. The vials were transferred in
liquid nitrogen to liquid nitrogen storage.
E. Clinical Protocol.
Patients with tumors of the appropriate pathologic
stages were randomized to receive either the autologous
tumor cell-BCG vaccine or to have no further therapy.
The stage D patients all received 5-fluorouracil
chemotherapy and all patients with lesions below the
peritoneal reflection (rectal cancer) received 5040 rads
of pelvic X-irradiation two weeks after ;mml7notherapy
was completed. The vaccines were started at 4-5 weeks
after tumor resection to allow sufficient time for
recovery of immunologic suppression induced by
anesthesia and surgery. At 3-4 weeks after resection
both control and treatment patients were skin tested
with standard recall antigens as well as graded doses of
their autologous tumor cells. Recall antigens used
were: Mumps skin test antigen, USP, Eli Lilly,
Indianapolis, Indiana; Aplisol, PPD, (Tuberculin
Purified Protein Derivative), Parke-Davis, Detroit,
Michigan; Trichophyton, diluted 1:30, Center
Laboratories, Port Wa hington, New York; and Candida
albicans diluted 1:100, Center ~aboratories, Port
Washington, New York, 0.1 ml of each was placed
intradermally on the forearm and ~x~mlned for erythema
and induration at 24 and 48 hours.
Patients selected for treatment protocol received
3 weekly intradermal vaccine injections consisting of 107
irradiated, autologous tumor cells and 107 BCG in the
first 2 vaccines with 107 tumor cells alone in the final.
Fresh-frozen Tice BCG, was stored at -70C (Organon,
Inc., West Orange, N.J., previously supplied by
WO95/21244 ~ S PCT~S95/01440
-21-
University of Illinois Medical Center, Chicago, IL.).
The first vaccine was placed on the left anterior thigh
approximately 10 cm below the groin crease, the second
in a comparable location on the right thigh and the
third in the right deltoid area.
F. Preparation of Vaccine.
On the day of the first and second vaccinations,
the vial was rapidly thawed in a 37C waterbath, tumor
cells were diluted slowly to 15 ml in HBSS, washed once
by centrifugation at 1200 rpm and resuspended to 15 ml
in HBSS. Cell counts and viability determinations were
made using the trypan blue exclusion test. Viability
ranged between 70 and 90~, with a mean of 80~. The
cells were washed once by centrifugation at 1200 rpm and
resuspended to 15 ml in HBSS. The suspension of tumor
cells was placed on ice and irradiated at 4020 rads/min
for a total of 20,000 rads. The volume of the cell
suspension was adjusted such that 107 viable tumor cells
re~;ned in the tube (1.3 x 107 viable cells are included
to allow for cell loss in tubes and syringes, and for
the possibility of approximately 20~ misidentification
of lymphoid cells). The cells were centrifuged, the
supernatant removed and 107 BCG were added in a volume of
0.1 ~1. HBSS was added in sufficient quantity for a
~inal volume of 0.2 ml. The third vaccine was similarly
prepared, omitting the BCG.
The vaccine suspension was drawn up through a 20
gauge needle into a 1.0 ml tuberculin syringe. The 20
gauge needle was replaced with a 27 gauge needle for the
intradermal injection, and the syringe was placed on ice
for transport to the clinic.
The patients were observed closely after each
vaccine for erythema and induration at the site of
injections, fever, lymphadenopathy or any adverse
reactions. The first two vaccine sites ulcerated after
2-3 weeks but always healed within 10 to 12 weeks.
W095/2124~ PCT~S95tO1440
5r¦ 2
-22-
G. Results of Immunization.
ReactivitY to Standard Recall Antiqens
A11 patients were reactive initially to at least
one of the standard recall antigens. In the first group
5two of 29 were reactive to candida, 26 of 29 were
reactive to mumps, 16 of 29 were reactive to PPD and 3
of 29 reacted to trichophyton. There was no significant
change in reactivity in the follow-up period except that
all but two of the ;mmlln;zed patients converted to PPD
positivity.
H. Delayed Cutaneous HY~ersensitivitY (DCH) to Tumor
Cells
Four of 24 patients (17~) had a positive DCH to 106
tumor cells prior to the course of immunization. This
was not significantly different from the one of 11
patients (9~) testing positive in the non-;mml~n;zed
control group. Of significance (p ~ 0.1), all of the
initially four positive responders and 12 of the
negative responders in the immunization group boosted to
greater DCH reactivity following a course of
immunotherapy (67~ became positive). Seven of these
patients have been tested at one year, with three
maintaining a positive response. Only three of the 16
objectively ;mmlln;zed patients demonstrated a positive
DCH response to 105 tumor cells at 6 weeks, with none
showing a response to 104 cells.
Example II: Production of Cells Producinq Human
Monoclonal Antibodies
A. Removal and Processinq of Immunized B-Cells from
Patients
Patients were bled at the time of the second
;mml~n;zation, one week after the first immunization, and
at the time of the third vaccination, one week after the
second ;mmlln;zation~ Venous blood was collected
aseptically in the presence of preservative-free heparin
WO9S/~1244 ~ S~ S~rcT~s9slol44o
-23-
tO'Neill, Jones and Feldman, St. Louis, Missouri) at a
final concentration of 17 units/ml. The blood was
maintained at room temperature and transported to the
laboratory expeditiously, within a few hours of
collection.
The blood, diluted 1:2 with calcium and magnesium-
free HBSS, was layered (4 ml) over 3 ml of lymphocyte
separation medium (LSM, Litton Bionetics) and
centrifuged in a 15 ml centrifuge tube for 30 minutes at
400 x g. The cells at the interface were removed,
diluted with three times their volume of HBSS and
pelleted (1000 rpm for 10 minutes). The peripheral blood
lymphocytes were resuspended in 10 ml of serum-free
Hepes-buffered Dulbecco's MEM (DMEM), counted and
viability determined.
An alternative method was also used to recover
immunized B-cells. The T-lymphocytes were removed by
rosetting with AET-treated sheep erythrocytes. Sheep
erythrocytes (in Alsever's solution) were washed three
times with balanced salt solution (BSS) and incubated at
37OC for 20 minutes with four times the packed cell
volume with 0.14 M AET (Sigma). The treated cells were
then washed three times with HBSS and resuspended to a
10~ suspension. The treated erythrocytes were layered
over LSM, centrifuged at 2500 rpm and the pellet
collected. Following three washes with HBSS, the sheep
erythrocytes were resuspended to a 10~ suspension in
undiluted fetal bovine serum and used within two weeks.
The PBLs (up to 80 million cells) were mixed with 1 ml
of AET-treated sheep erythrocytes and pelleted at 1000
rpm for 10 minutes at 4C. The pellet was incubated on
ice for 45 minutes, gently resuspended with a wide bore
pipette and layered over 3 ml LSM. The rosetted cells
were centrifuged at 400 x g for 40 minutes at room
3~ temperature. The T-cell depleted PBLs were collected at
the interface, washed with three times the volume HBSS,
WO95/21244 PCT~S95/01440
-24-
and pelleted. Following counting and viability
determination, the PBLs enriched for B-cells were then
used for hybridoma generation.
B. Generation of Human Hybridomas.
Mouse myeloma cells (NS-1) were grown in the
presence of 8-azaguanine (20 ~g/ml). Three days before
fusion, the cells were pelleted and passaged in medium
free of 8-azaguanine. The cells were passaged again the
day before fusion to maintain them in log phase. The
myeloma cells were washed once with serum-free medium,
counted, and viability determined. The PBLs and myeloma
cells were mixed at a ratio of 3:1 and pelleted together
at 1000 rpm for 10 minutes. All supernatant fluid was
removed and the cell pellet resuspended in less than 100
~l of serum-free medium. One ml of polyethylene glycol
(50~ w/v) prewarmed to 37C was added dropwise to the
cell pellet over the course of one minute with constant
agitation of the tube. Twice the previous volume of
pre-warmed serum-free medium was added to the cell
suspension over the course of one minute until the 50 ml
tube was filled. The cells were pelleted at 800 rpm for
15 minutes. The cells were gently resuspended in HT
medium (DMEM containing 20~ fetal bovine serum,
hypoxanthine 13.6 ~g/ml and thymidine 3.9 ~g/ml) at a
concentration of 2.5 x 106 cells/ml (pre-fusion count)
and 100 ~l was added to each microtiter well. Twenty-
four hours later, 100 ~l of HAT medium (HT medium
containing .18 ~g/ml aminopterin) was added to each
well. Half of the medium was replaced every three days
with fresh HAT medium. After maintenance of HAT medium
for 14 days, the cells were maintained on HT medium for
an additional two weeks, at which time the cells were
grown on a DMEM medium containing 20~ fetal bovine
serum.
Alternatively, co-cultivation of PBLs with myeloma
cells may be used to generate transformed diploid B-
.
WO95121244 ~ ~ S~ PCT~S95/01440
-25-
cells. PBLs and myeloma cells were mixed (at a ratio of
3:l), pelleted at 800 rpm and selected in HAT medium, as
described above.
C. Screeninq of HYbridomas.
The hybridomas were first quantified and isotyped
by a capture enzyme-linked immunoassay (ELISA) for the
synthesis of human im~unoglobulin (IgA, IgG and IgM).
The standard Bio-Enza3ead method was utilized, which is
sensitive in the range of 10-300 ng/ml. The hybridoma
supernatant fluids were diluted l:30 with an effective
range of .3-9 ~g/ml. Only hybridomas that synthesized
human immunoglobulin at a concentration of greater than
or equal to l ~g/ml were tested by indirect
;~mllnoperoxidase on tissues after the isotype of the
antibody (IgA, IgG or IgM) was determined.
Polycarbonate-coatedmetallic beads (Bio-EnzaBead~,
Litton Bionetics) were incubated with goat antibodies to
human immunoglobulins (IgG + IgA + IgM) overnight at 4C
and then blocked (30 min at room temperature) with 2.5~
BSA to prevent non-specific binding. The beads were
then air dried and stored at 4C. The ELISA for
detection of immunoglobulin was performed as follows.
Supernatant fluid from a 96-well culture plate was
diluted, incubated with the antibody-capture bead for l
hr aL 37C, washed, and then incubated for l hr at 37C
with peroxidase-labeled affinity-purified goat antibody
to human immunoglobulins (IgG + IgA + IgM). The washed
beads were then incubated (l0 min at room temperature)
with 2,2'-Azino-di[3-ethyl-benzthiazoline-6-sulfonic
acid], and the optical density was determined at 405 nm.
The immunoglobulin concentrations were interpolated
mathematically from the linear portion of a standard
curve (30-l000 ng/ml) of human gamma globulin.
Supernatant fluids containing >l ~g/ml were then
isotyped using this ELISA with peroxidase-labeled goat
antibodies to human ~, ~, and ~ ch~~ n~ . Subsequent
WO95/21244 ~ 5 ~ æ PCT~SgS/014~0
-26-
quantitative assays used an immunoglobulin standard
appropriate for the monoclonal antibody isotype. Mouse
immunoglobulins were assayed with Bio-EnzaBeads coated
with goat antimouse IgG + IgM (H + L) and peroxidase-
conjugated goat antimouse IgG + IgM (H + L). In otherexperiments, supernatant fluids were incubated with the
anti-human Ig beads and the peroxidase-conjugated goat
antimouse IgG + IgM (H + L).
Cryostat sections of normal and tumor tissue,
stored at -30C, were post-fixed in PLP (0.5~ p-
formaldehyde, 0.075 M L-lysine, 0.01 M sodium periodate)
for 20 minutes at 4C. The sections were then washed.
Paraffin sections of 10~ formalin-fixed tissues were
deparaffinized immediately before use. The cryostat and
paraffin sections were then incubated at room
temperature in 1~ bovine serum albumin in PBS containing
0.075 M L-lysine for 20 minutes. The sections were
incubated overnight at 4C with hybridoma supernatant
fluids. Following three washes with PBS, the sections
were then incubated with the appropriate anti-human
peroxidase-labeled reagent for 60 minutes at 37C,
washed and incubated at room temperature for 15 minutes
with diaminobenzidine (0.5 mg/ml, Ph 7.6) in PBS
containing 0.1~ hydrogen peroxide. The sections were
washed with PBS, stained with hematoxylin, dehydrated,
and mounted with permount.
These methods permitted the widest spectrum of
tissue reactive antibodies to be detected (i.e.,
directed against surface or cytoplasmic antigens).
To isolate broadly reactive antibodies, the
supernatant fluids were screened against a panel of
tumor sections. Cell lines producing monoclonal
antibodies were then cloned by limiting dilution.
Twenty-two fusions were performed with peripheral blood
lymphocytes obtained from ten patients, and two fusions
were done with lymphocytes from patients before
WO95/2124~ ! ~S~S~ PCT~S95/01440
-27-
immunization. Optimal results were obtained with
lymphocytes removed one week after the second
immunization. The frequency of immunoglobulin producing
clones isolated after the second immunization was almost
twice that after the first immunization. Seven of the
36 tissue-positive monoclonal antibodies reacted with
cryostat sections but not with paraffin embedded
tissues. This finding underscores the need for broad
screening procedures. More than two-thirds of the
clones produced IgM, most probably a consequence of the
source of the lymphocytes (peripheral blood).
D. Identification of Diploid Cells.
One-third of the cell lines had morphology typical
of hybridomas and grew as dispersed cells. Karyotypic
analysis of six representative hybrids demonstrated that
they were human-mouse heterohybridomas. By contrast,
the majority of the monoclonal antibody synthesizing
cell lines (24 out of 36) were atypical in appearance.
These cells were predominantly irregular in shape and
grew in large aggregates. These cluster-forming cells
were isolated in seven fusions performed with PBLs from
seven of ten colon patients. Thus, they appear to be
quite common. Six cell lines representing five fusions
from four patients, were karyotyped and were found
to contain 46 chromosomes. G-h~n~;ng of the
chromosomes confirmed that they were of human origin.
Thus, based upon the criterion of cell morphology, it
appears that the majority of the monoclonal antibody-
synthesizing cell lines are not hybridomas but rather
are transformed hllm~n B-cells (diploid cells).
No clear differences exist between these cell types
in the isotype of secreted immunoglobulin or the type of
tissue stained. The amounts of immunoglobulin (1-60
~g/ml) secreted by both cell types were essentially
comparable, with most of the human cells producing 5-20
~g/ml. As may be expected, the diploid cells appear to
WO95/21244 ~ ~ PCT~S95/01440
-28-
be more stable with regard to immunoglobulin production.
These cells were grown in continuous culture for up to
9 months without any indication of a finite life span
for antibody production. In fact, increases in antibody
production during long-term culture were observed for
some diploid lines. The clones which subsequently
became non-producers during extensive cell passage had
growth properties typical of hybridomas. However, most
hybrids had sufficient stability to permit the
production of useful quantities of antibody. For
example, human-mouse heterohybridoma 7a2 was passaged
for more than 20 generations from a recently cloned seed
stock at 5 x 106 cells without a decrease in antibody
production. Thus, the cells theoretically could be
expanded to 7 x lOl3 cells. This hybrid produced
approximately 30 ~g/ml/l06 cells and thus 7 x lol3 cells
could conceivably produce over 2 kg of antibody.
E. EBV Transformation Procedure
As an alternative to hybridization, peripheral
blood s-cells from ;mmllnlzed patients can be
intentionally exposed to transforming agents, resulting
in continuously growing cell lines that produce
monoclonal antibodies. We have used EBV as the
transforming agent, although any effective lymphotropic
virus or other transforming agent able to transform the
B-cells to grow in continuous culture and still produce
monoclonal antibodies specific for tumor associated
antigens can be used.
By our method, heparinized blood was separated on
an LSM gradient and the mononuclear cell fraction was
collected at the interface. The mononuclear cell
fraction can either be used at this point or
cryopreserved for future transformation.
Prior to transformation, in some instances, we
depleted the mononuclear cell fraction of macrophages
and other cells that might inhibit transformation. Two
-
WO95/2124~ PCT~S95/01440
~S~7
-29-
techniques used were plastic adherence and treatment
with the methyl ester of L-leucine. In the plastic
adherence technique, the cells were suspended in cell
culture medium (RPMI 1640 medium, Gibco, Grand Island,
N.Y.) containing 20~ fetal bovine serum (2 x lO6/ml) and
incubated overnight in plastic cell culture dishes.
Non-adherent cells were removed from the plastic by
pipetting, leaving the lymphocytes. Alternatively, the
cells were incubated in methyl ester L-leucine (5mM in
serum-free cell culture medium) for 40 minutes at room
temperature and then washed.
The lymphocytes, either fresh or cryopreserved,
either unfractionated or depleted of some non-B cells,
were counted and between 2 and 5 x 106 cells were
pelleted. The pelleted cells were resuspended in 5 ml
of freshly harvested Epstein Barr Virus in the form of
undiluted B95-8 supernatant fluid harvested from a 4 -
6 day old culture of B95-8 cells, clarified by
centrifugation at 2,000 rpm for 15 minutes at 4C and
filtered through a 0.8 micron filter to insure that all
cells had been removed. The B95-8 cell line was
obtained from Dr. G. Tostado, Division of Biologics,
FDA. The cells and EBV were incubated at 37C for 90
minutes for virus adsorption. During virus adsorption,
the cells were agitated periodically.
After virus adsorption the cells were pelleted at
room temperature, resuspended in cell culture medium
containing 20~ fetal bovine serum and counted. The
cells were then diluted to about 5 x 104 cells/ml and
approximately lO0 ~l plated into each well of a g6 well
plate. An additional lO0 ~l of cell culture medium was
then added to each well. Alternatively, the cells may
be plated into wells containing irradiated feeder cells
(such as J774). The mouse macrophage line J774 (ATCC,
Rockville, Md.) were irradiated (20,000 rads) and then
cryopreserved. The cells were thawed and then plated (5
WO95/21244 7 PCT~SgS/01440
-30-
x 103 cells/well) into 96 well plates one day before the
EBV transformation were to be seeded.
The cell culture media was changed twice per week
for up to 6-8 weeks. Screening of supernatant fluid
from wells exhibiting extensive cell growth to select
those synthesizing human immunoglobulin and the
culturing of selected cell lines was performed according
to the procedures described above for selection and
culturing of monoclonal antibody producing cells.
F. Production of Monoclonal Antibodies.
Human monoclonal antibody producing cells were
grown in RPMI 1640 medium (Gibco, Grand Island, New
York) supplemented with 10~ fetal bovine serum, 3 Mm L-
glutamine and 5 ~g/ml gentamicin. The medium was in
some cases further supplemented with 25~ D-glucose
(final concentration 0.25~). The cells were at 37C
(35-38C) under a humidified atmosphere of 7.5~ CO2 in
air. The antibody was harvested from the highly
metabolized spent medium by pelletizing the medium free
of cells (e.g., by centrifuging at 500 rpm for 15
minutes).
Example III: Reactivity of Monoclonal Antibodies to
Normal and Tumor Tissue
Most of the antibodies exhibited substantially
reduced binding to normal colonic mucosa. The
antibodies reactive with paraffin sections were also
tested for reactivity with normal tissue. 88BV59 showed
negative reactivity with the following normal human
tissues: ovary, uterus, testes, vagina, adrenal glands,
prostate, thyroid, thymus, lymph nodes, spleen, bone
marrow, myocardium, cerebral cortical cells, skin,
muscle and hemopoietic cells. 88BV59 exhibited slight
reactivity with the following tissues: colon (brush
border and superficial glands), small intestine (brush
border and superficial glands), stomach (gastric pits
~WO 95121244 ~ PCT~S95/01440
--31-
and superficial glands), esophagus (glands), pancreas
(some ductal and exocrine glandular epithelium), kidney
; (50~ of collecting tubules), cervix (epithelial lining
(2/3 tissues were positive)), breast (acini and ductal
epithelium), lung (some a].veolar and bronchial cells),
brain (astrocytes (2/3 tissues were positive)), spinal
cord (neuropil), skin (50~ of glands in dermis) and
liver (bile ducts). Reactivity of 88BV59 with human
tumor cell lines is shown in Table 2. Table 3 shows the
reactivity of 88BV59 with tumor tissue specimens.
Example IV: Cancer Immunodetection With Radiolabelled
88BV59 AntibodY
Clinical trials have been performed for the
_5 detection of cancer with radiolabelled 88BV59 antibody.
A Phase I imaging trial consisted of five patients who
received 4.0 to 8.8 mg. of 88BV59 antibody labelled with
5 to 13 mCi of 99mTc (1.1 to l.7 mCi/mg.) by IV infusion
over 30 minutes; none had a severe adverse reaction.
The serum clearance was biphasic with a mean T~ ~ of 0.9
hour and T% ~ o~ 14 hours. Planar and single photon
emission computer tomography (SPECT) images were
obtained at 4 hours and 20 to 24 hours. More metastatic
lesions were observed using radioimmunoscintography
(RIS) with SPECT than by using the standard computed
tomography (CT) or magnetic resonance imaging (MRI). No
human ant;h1lm~n antibod~ response was detected in serum.
Sixty-eight patients entered phase II studies;
thirty-six of these patients underwent surgery. The
data of the surgical patients were used to evaluate the
imaging characteristics and to compare the antibody and
CT scans in terms of tumor localization. The study was
a nonrandomized, single-arm, open-phase II study
evaluating the efficacy and safety of 99mTc-88BV59 as a
RIS agent. Patients were not preselected on the basis
.
WO95/2124~ PCT~S95/01440
-32-
of immunohistochemistry or skin test reactivity. They
had at least one documented site of tumor involvement by
conventional diagnostic techniques or were suspected of
recurrent disease on the basis of an elevated CEA. In
order to block thyroid uptake and gastric secretion of
pertechnetate, fifty-one patients were administered 400
mg. of potassium prochlorate before infusion, 4 hours
post infusion, and 24 hours post infusion. Seventeen
patients received 88BV59 without potassium prochlorate.
Labelling of 88BV59 with 99mTc was performed by a direct
labelling method using stannous chloride as the
reductant. The specificity of 99mTc-88BV59 was 2 to 4
mCi/mg. of antibody and the antibody bound 99mTc greater
than 90~. Following an IV test dose of 300 ~g., sixty-
eight patients received 15 to 41 mCi of 99mTc-88BV59 over
a 30 minute period by IV infusion.
The distribution of99mTc-88BV59 in normal organs was
assessed at two imaging times: 3 to 4 hours and 16 to
24 hours after antibody administration. At the early
imaging time, a vascular blood-pool scan was observed
with concentration of the isotope in the heart and great
vessels, liver, spleen, kidneys, as well as bladder
excretion. At 16 to 24 hours, there was still
significant but reduced background activity. The
calculated bone marrow dose suggested that there was no
targeting of the antibody or accumulation of the 99mTc in
the bone marrow. Technetium-99m-88BV59 in doses of less
than or equal to 40 mCi may be administered safely for
diagnostic purposes.
Technetium-99m-88BV59 using SPECT imaging detected
75~ of known abdominal and pelvic lesions. The imaging
characteristics of 99mTc-88BV59 are best defined in the
subset of 36 surgical patients for whom histopathologic
validation of imaging is available. The smallest nodule
detected was .5 cm. in diameter. In the surgical
patients, the sensitivity of the antibody scan was
WO 9~/21244 ælS PCTfUS95/01440
--33--
greater than the sensitivity of the CT scan: 68~ vs.
40~ in detecting tumors within the abdomen and pelvis,
excluding the liver. The difference is statistically
significant (McNemar's test, P ~ .05). The antibody
scan and CT scan appeared to detect diferent subsets of
tumors within the abdomen. Optimal detection results
from the combination of the antibody scan and the CT
scan. Together they detect 80~ of surgically proven
lesions vs. 40~ for C~;- scan alone (McNemar's test, P ~
.01). In the case of hepatic metastases an analysis by
sight was conducted; the CT scan correctly identified 10
of 13 metastatic livers; the antibody scan, 9 of 13; and
the CT and antibody scans combined, ll of 13. These
differences are not statistically significant.
The antibody scan is clearly superior to the CT
scan in detecting abdominal and pelvic disease. The
antibody scan identifies twice as many lesions as the CT
scan. 84~ of primary tumors were correctly detected by
the antibody scan compared with 37~ by CT scan. For
recurrent and metastatic tumors, the antibody scan
identified 52~ of the lesions compared with 43~ by CT
scan alone. However, antibody and CT scans combined
showed a sensitivity of 81~. In order to further
evaluate these data, the isotopic dose effect on imaging
sensitivity was analyzed. In the analysis of abdominal
and pelvic lesions, a dose effect was evident, with the
optimal range being 30 to 35 mCi. At the 30 to 35 mCi
dose, the sensitivity of the antibody scan was 78~, the
specificity 67~, the positive predictive value 82~, the
negative predictive value 60~, and the accuracy 74~.
These studies are comparable to those of other RIS
studies using whole murine or rh;meric IgG's.
Techniques including the preparation of protein
extracts and the use of ;~ noadsorbent lectins for the
immunization of mice are required to produce monoclonal
antibodies against protein antigens derived from colon
WO95/21244 PCT~S95/01440
-34-
tumors. Thus, autologous immunization of man elicits
antibodies against a group of antigens normally poorly
immunogenic for mice. It is therefore possible that man
and mice may respond to different tumor-associated
antigens. In concert with this hypothesis is the
finding that none of 28 different monoclonal antibodies
prepared by this method that we ex~lned to-date reacted
with purified CEA, an antigen frequently seen by murine
monoclonal antibodies made against colon tumor cells,
(Koprowski et al, Somat. Cell Genet., 5:957-972, 1979,
and Morgan et al, supra).
In addition to providing monoclonal antibodies
reactive with tumor cell surface antigens for the i
vivo diagnosis and immunotherapy of cancer, the
invention provides monoclonal antibodies which will be
useful as probes to isolate and characterize the
antigens relevant to human cancer immunity. These
antigens may ultimately prove useful as a tumor vaccine.
In addition, the generation of antibody producing
diploid cells adds a dimension of genetic stability to
the production of human monoclonal antibodies reactive
with tumor cell surface antigens.
The foregoing describes the formation of novel
monoclonal antibodies specific for certain tumors,
monoclonal antibody producing cell lines, and methods
for their preparation. The techniques for preparing the
novel monoclonal antibodies, hybridomas, and diploid
cells have been described in detail, particularly with
reference to specific embodiments included by way of the
examples. It will be understood that the products and
techniques of the present invention are of far-reaching
significance in the field of cancer detection and
treatment. They include a wide range of monoclonal
antibodies, each specific for determinants found on an
individual strain of tumor forming cancer, as the
technique disclosed herein can be used to generate
WO95/21244 2~ S8 S PCT~S95/01440
-35-
antibodies for every such case. It will be further
understood that many variations and modifications of the
techniques disclosed herein are available to those of
ordinary skill in the relevant art and that such
variations and modifications are contemplated as being
within the scope of the invention.
The embodiments provided to illustrate this
invention relate to carcinoma tumors, particularly well-
differentiated colorectal adenocarcinomas. Clearly,
however, the invention pertains to all carcinomas, such
as lung, breast, and other malignancies in areas which
arise from the same type of embryonic tissue. Moreover,
the procedures described can be adjusted, if necessary,
by one skilled in the art to be used to apply this
invention to other types of cancer.
The cells line producing the IgG-3 human monoclonal
antibody 88BV59 were deposited with the American Type
Culture Collection, 12301 Parklawn Drive, Rockville,
Maryland 20852, USA, on December 13th, 1990, and January
31, 1994. The cell lines deposited are identified as
follows:
Identification Accession Number
Human B-Cell Derived Cell Line, CRL 10624
C088BV59-1
Human B-Cell Derived Cell Line, CRL
CO88BV59H21-2
Human B-Cell Derived Cell Line, CRL
CO88BV59H21-2V67-66
WO95/21244 PCT~S95/01440
-36-
TABLE l
CRITERIA FOR SUCCESSFUL VACCINES FOR
ACTIVE SPECIFIC IMMUNOTHERAPY
Adjuvant
(a) BCG (Phipps, Tice, Connaught); Lyophilized,
frozen (dose-dependence ~ lo6 (l07-l08)
(b) C. parvum (Wellcome Labs) (dose-dependence
7 ~g (70 ~g-700~g)
Tumor Cells
(a) Enzymatic dissociation
(l) Collagenase type I (l.5-2.0 U/ml HBSS)
(2) DNAase (450 D.U./ml HBSS)
(3) 37C with stirring
(b) Cryopreservation
(l) Controlled-rate freezing (-1C/min) (7.5
DMSO, 5~ HSA, HBSS)
(2) Viability 80
(c) X-irradiation
(l) Rendered non-tumorigenic at 12,000
20,000 R.
Components and ~m; ni strationl
(a) Ratio of adjuvant to tumor cells - l0:l - l:l
(optimum)
(b) 107 tumor cells (optimum)
(c) 2-3 i.d. vaccinations at weekly intervals.
Third vaccination contains tumor cells only.
Isoniazid chemoprophylaxis of BCG infection optional.
BCG - Bacillus Calmette Guerin
HBSS - Hanks' Balanced saline solution
DMSO - Dimethylsulfoxide
HSA - Human serum albumin
R - Rads
PBS - Phosphate buffered saline
EDTA - Ethylenediaminetetraacetic acid
~WO 95121244 ~1 S8~ PCTIUS95/01440
--37 -
TABLE 2
REACTIVITY OF HUMAN MONOCLONAL ANTIBODY 88BV59
Indirect Immunofluorescence with Acetone-filed Tumor
Cells~
Ht-29 Colon Carcinoma 3+
SKCO-1C Colon Carcinoma 3+
LS174 Colon Carcinoma 4+
WiDr Colon Carcinoma N.T. e
HCT-8 Colon Carcinoma
Bt-20b Breast Carcinoma 3+
EP~ Breast Carcinoma 2+
MCF-7 Breast Carcinoma 4+
SKBR-III Breast Carcinoma
CaLu-1C Lung Adenocarcinoma 4+
A2780 Ovarian Carcinoma
Ovcar3C Ovarian Carcinoma 4+(30~) d
WI-38 Normal Fibroblasts
a) Florescence Intensity: 4+strong, 3+moderate, 2+weak
to moderate, 1+ weak, -negative. Concentration of
88BV59-1 was 10 ~g/ml. St~;n-ng with a control
hnm~n IgG at 10 ~g/ml was negative on all cells.
b~ Staining prefer~ntially on cells in mitosis.
c) Staining show~ a filamentous cytoskeletal staining
pattern.
d) Percentage of cells showing the indicated
fluorescence intensity was 100~ unless otherwise
noted.
e) NT = not tested.
-
PCT~S95/01440
WO95/212~4
-38-
TABLE 3
REACTIVITY OF 88BV59 WITH VARIOUS TUMOR TYPES
Colon 17 23 74
Breast l9 l9 l00
Ovarian 13 17 76
Pancreatic 3 9 33
Lung 3 4 75
Prostate 4 6 67
