U.S. patent number 5,484,596 [Application Number 08/122,257] was granted by the patent office on 1996-01-16 for active specific immunotherapy.
This patent grant is currently assigned to Akzo N.V.. Invention is credited to Michael G. Hanna, Jr., Herbert C. Hoover, Jr., Leona C. Peters.
United States Patent |
5,484,596 |
Hanna, Jr. , et al. |
January 16, 1996 |
Active specific immunotherapy
Abstract
This invention relates to a method of cancer therapy for
treating human patients with resectable solid tumors to inhibit
recurrence and formation of metastases, comprising surgically
removing tumor tissue from a human cancer patient, treating the
tumor tissue to obtain tumor cells, irradiating the tumor cells to
be viable but non-tumorigenic, preparing a vaccine comprising about
10.sup.7 viable but non-tumorigenic tumor cells per dose and
injecting the vaccine intradermally into the human patient after
the patient's immune system has recovered from surgery.
Inventors: |
Hanna, Jr.; Michael G.
(Frederick, MD), Hoover, Jr.; Herbert C. (Hingham, MA),
Peters; Leona C. (Frederick, MD) |
Assignee: |
Akzo N.V. (Arnhem,
NL)
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Family
ID: |
27076722 |
Appl.
No.: |
08/122,257 |
Filed: |
November 1, 1993 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
Issue Date |
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937460 |
Aug 28, 1992 |
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639406 |
Jan 10, 1991 |
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426479 |
Oct 23, 1989 |
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930597 |
Nov 12, 1986 |
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697078 |
Jan 31, 1985 |
4828991 |
May 9, 1989 |
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575533 |
Jan 31, 1984 |
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Current U.S.
Class: |
424/277.1;
424/138.1; 424/93.1; 424/93.7 |
Current CPC
Class: |
C07K
16/30 (20130101); C07K 16/3046 (20130101); A61K
38/00 (20130101); Y10S 436/813 (20130101); Y10S
530/865 (20130101) |
Current International
Class: |
C07K
16/18 (20060101); C07K 16/30 (20060101); A61K
38/00 (20060101); A61K 039/00 (); A61K
039/395 () |
Field of
Search: |
;424/85.8,88,277.1,93.7,93.1,138.1,277.1 ;530/388.8
;435/814,188 |
References Cited
[Referenced By]
U.S. Patent Documents
Foreign Patent Documents
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|
Primary Examiner: Lacey; David L.
Assistant Examiner: Loring; Susan A.
Attorney, Agent or Firm: Blackstone; William M.
Parent Case Text
This application is a continuation of U.S. patent application Ser.
No. 07/937,460, filed Aug. 28, 1992, now abandoned; which is a
continuation of U.S. patent application Ser. No. 07/639,406, filed
Jan. 10, 1991, now abandoned; which is a continuation of U.S.
patent application Ser. No. 07/426,479, filed Oct. 23, 1989, now
abandoned; which is a continuation of U.S. patent application Ser.
No. 06/930,597, filed Nov. 12, 1986, now abandoned; which is a
division of U.S. patent application Ser. No. 06/697,078, filed Jan.
31, 1985 now U.S. Pat. No. 4,828,991, issued May 9, 1989; which is
a continuation-in-part of U.S. patent application Ser. No.
06/575,533, filed Jan. 31, 1984, now abandoned.
Claims
We claim:
1. A method of cancer therapy for treating a human patient with a
resectable, solid carcinoma tumor to inhibit recurrence or
formation of metastases, comprising surgically removing solid tumor
tissue from a human cancer patient and preparing a viable
autologous tumor cell vaccine by treating the tumor tissue to
obtain viable tumor cells by the method of digesting tumor tissue
with collagenase to free tumor cells, whereby said cells remain
viable and intact, irradiating the tumor cells to be viable but
non-tumorigenic, preparing a vaccine comprising about 10.sup.7
viable but non-tumorigenic tumor cells per dose and injecting at
least three doses of the vaccine intradermally into the human
patient from whom the tumor tissue had been removed after the
patient's immune system has recovered from surgery, wherein at
least the first two of the at least three doses comprise about
10.sup.7 BCG cells as adjuvant and the last dose is free of
adjuvant, and wherein at the time of injection cell viability is at
least 70% when measured by trypan blue exclusion testing.
2. The therapeutic method of claim 1, wherein the cancer patient is
inoculated with said vaccine once each week for at least three
weeks.
3. The therapeutic method of claim 1, wherein the patient is
injected once each week for two weeks with said vaccine comprising
about 10.sup.7 BCG cells, and a third vaccination is performed with
said vaccine comprising about 10.sup.7 deactivated tumor cells but
no BCG cells.
4. The therapeutic method of claim 1, wherein the tumor cells are
deactivated by irridation with a total of about 20,000 rads.
5. The therapeutic method of claim 1, wherein the viability of the
deactivated tumor cells is in the range of about 70 to 90% when
measured by trypan blue exclusion testing.
6. The therapeutic method of claim 1, wherein the tumor cells are
obtained from tumor tissue by the method of disecting the tumor
tissue to remove extraneous tissue, mincing the tumor tissue into
tissue fragments and digesting the tumor tissue fragments.
7. The therapeutic method of claim 1, wherein the tumor cells are
cryopreserved after digestion until time for injecting said vaccine
into the patient, at which time the cryopreserved tumor cells are
rapidly thawed and deactivated by irridation, and wherein the cells
are cryopreserved by controlled rate freezing at about -1.degree.
C./min. to a temperature of about -80.degree. C.
8. The therapeutic method of claim 1, wherein recovery of the
patient's immune system after surgery is tested by skin
testing.
9. The therapeutic method of claim 1, wherein the patient is
injected with said vaccine beginning 3 to 5 weeks after surgical
removal of the tumor.
Description
DESCRIPTION OF THE INVENTION
This invention relates to a method of cancer therapy for treating
human patients with resectable solid tumors to inhibit recurrence
and formation of metastases, comprising surgically removing tumor
tissue from a human cancer patient, treating the tumor tissue to
obtain tumor cells, irradiating the tumor cells to be viable but
non-tumorigenic, preparing a vaccine comprising about 10.sup.7
viable but non-tumorigenic tumor cells per dose and injecting the
vaccine intradermally into the human patient after the patient's
immune system has recovered from surgery.
BACKGROUND OF THE INVENTION
This invention relates to active specific immunotherapy and to new
human monoclonal antibodies which react specifically with antigens
associated with particular cancers and to hybridoma and transformed
B-cell lines for their production derived from peripheral blood
B-cells of actively immunized patients. This invention also relates
to methods having general applicability to all solid cancers for
preparing hybridomas and monoclonal antibodies and to diagnostic
procedures and cancer therapy using these monoclonal
antibodies.
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 which 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 which
are specific for a single determinant, thus binding only to
antigens or tissues having that particular determinant.
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 which grow in culture and produce an antibody specific for a
single determinant. These hybrids are referred to as "hybridoma
cells." Hybridoma cells are prepared by fusing lymphocytes, which
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 antibodies are
referred to as "monoclonal antibodies."
Monoclonal antibodies may also be produced by B-lymphocyte cell
lines that have been spontaneously transformed, either prior to or
subsequent to being placed in culture. These cells, in distinction
to hybridoma cells, possess a normal human diploid number (46) of
chromosomes. 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 by a monoclonal
antibody producing cell cultures uncontaminated by other
immunoglobulins. With such a cell culture, 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. 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. A
monoclonal antibody can be conjugated with a toxin, radionuclide or
chemotherapeutic drug; this conjugated antibody may be
simplistically viewed as a guided missile with the antibody as the
guidance system and the drug as the warhead. In addition,
antibodies conjugated with radionuclides or metallic tracers can be
used for proton emission (PET) and nuclear magnetic resonance (NMR)
imaging for in vivo diagnosis and localization of metastases. 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 the
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,
immunoglobulin 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).
DESCRIPTION OF THE PRIOR ART
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. Pat. No. 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 specifically
with tumor cells. In addition, monoclonal antibodies derived from
mouse B-cells have limited potential for application in cancer
therapy. After repeated administration they tend to 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
heterogeneous labeling of cells within tumor sections. This pattern
of reactivity has been attributed by some authors to antigenic
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 to 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 36 human monoclonal antibodies we produced appear to react
with carcino-embryonic 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 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 loci 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 reactive with tumor-specific antigens that induce-an
immune response in patients having particular cancers. A valid in
vivo assay for the immunogenicity of tumor-specific antigens in
tumor immunized patients is by delayed cutaneous hypersensitivity.
Such antibodies provide a means for detecting and diagnosing
tumors. A second objective of this invention was to obtain
monoclonal antibodies which would be effective in 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 immunized with cells from their own tumors in a specific
vaccine preparations. To 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.
Animal model studies have supported the concept that antigens not
found in normal adult tissues are frequently found in tumors, and
that the immunogenicity of these tumor cells can be expressed, and
even enhanced, in both normal and tumor-bearing hosts. These
experimental results validated the rationale of active specific
immunotherapy in human neoplasia.
Humans mounting an objective immune response against tumor cells
were specifically found to be a good source of activated B-cells.
The peripheral blood of patients who had been actively immunized
against their own tumors was shown in clinical trials to be an
abundant source of such activated B-cells.
It was 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 hypersensitivity
to their own colorectal cancers. In addition, the monoclonal
antibodies developed from the immunized patients' 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 treatment also proved to be highly beneficial. Forty-two months
after the immunization of the first patients there has been an
objective and significant improvement in the patients with respect
to duration of the disease-free period following surgery, and the
survival data are encouraging. Only 3 of 20 treated patients had
recurrences and none have died. Comparatively, 9 of 20 patients in
a control group had recurrences and four have died.
The generation of B-cells which produce antibodies having
reactivity specific for tumor cell 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 immunization treatment was observed and
measured during the animal studies on which the human immunization
procedures were based, not the production of tumor specific
antibodies.
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, 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 which 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 immunity, 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.
A third objective of this invention was to prepare a standardized
vaccine for use in detecting and treating specific cancers in the
general population which did not require the custom preparation of
a new immunogen suitable for each individual patient. Without a
standardized vaccine, only a vaccine prepared for each individual
patient from his own tumor tissue could be used for therapy, and
only known cancers could have been treated on a limited scale in
large institutions would not have been possible to make individual
preparations for treating the approximately 139,000 cases of
colorectal cancer that are discovered in the United States every
year.
This invention comprises the preparation of successful vaccines for
active specific immunization, procedures for extracting immunized
B-cells, the production of monoclonal antibody producing cells 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
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. Cells that
synthesize immunoglobulin are tested for production of antibodies
which 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.
Mouse myeloma cells grown in culture were used to prepare
hybridomas in the research which led to this invention. However, as
the problems with developing easy-to-grow human myeloma cell lines
that do not produce antibodies of their own are solved, human
myelomas will be preferred for preparing the hybridomas of this
invention.
DETAILED DESCRIPTION OF THE INVENTION
The key aspects of this invention are:
1) Criteria for successful vaccines for active specific
immunization:
Tumor cells, whole cells enzymatically dissociated from tissue,
cryopreserved and X-irradiated for non-tumorigenicity
Adjuvant, an immunomodulator that is capable of inducing
immunogenecity to the tumor cell preparation.
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 and transformed
lymphocytes and production of monoclonal antibodies.
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 therapy 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 measure 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 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 10.sup.7 viable
cells remained in the tube. The cells were centrifuged, the
supernatant was removed, and 10.sup.7 viable BCG were added in a
volume of 0.1 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. 10.sup.7 viable tumor
cells admixed with BCG were used for the first two vaccinations and
10.sup.7 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
patients' peripheral blood lymphocytes.
Collection of Immunized B-Cells
Venous blood was collected from the immunized patients one week
after each vaccination. Peripheral blood lymphocytes (PBL) were
separated from the collected blood for use in hybridoma
production.
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
diluted with HBSS and pelleted. The lymphocytes were then
resuspended in serum-free Hepes-buffered Dulbecco's MEM (DMEM),
counted, and assayed for viability (GIBCO Biologics, Grand Island,
N.Y.).
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 Hybridomas for the Production of Anti-Tumor
Monoclonal Antibodies
Peripheral blood lymphocytes (PBLs) 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
additional 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 human
immunoglobulin using the standard enzyme immunoassay. Hybridomas
synthesizing human immunoglobulin in sufficient amounts were tested
on tissues. Particular tissue samples were incubated with hybridoma
supernatant fluids. Supernatants which 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 supernatants 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 which produced tumor-specific
antibodies, transformed human B-cells (diploid cells) were also
prepared by these procedures which also produced tumor-specific
antibodies. The transformed B-cells were detected in the same way
as tumor-specific antibody-producing hybridomas. Thus, well
supernatants which 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.
The mechanism by which B-cells become transformed during the above
described procedures has not been precisely determined.
BRIEF DESCRIPTION OF THE FIGURES
FIG. 1A
Chromosome spread of a cell with growth characteristics typical of
hybridomas (X1600). LiCo 21B27 was incubated with colcemid (0.05
.mu.g/ml ) for two hours and treated with hypertonic (0.075M) KCl
for three minutes. The cells were fixed with methanol-acetic acid
(3:1), dropped onto microscope slides, air-dried and stained with
Giemsa. Both human and mouse chromosomes are present.
FIG. 1B
Phase photomicrograph of a clusterforming monoclonal antibody (LiCo
18-15) producing cell line (X270). Note the aggregation and
irregular shape of the cells.
FIG. 1C
G-banded chromosome spread of the cell line shown in FIG. 1D
(X1360). Note the absence of mouse Chromosomes. The cells were
incubated with colcemid (0.01 .mu.g/ml ) overnight. The chromosome
spreads were prepared as described above. The unstained slide was
aged for 10 days. The chromosomes were treated with trypsin (0.19%
for 30 seconds at room temperature), dehydrated with ethanol and
stained with Giemsa.
FIG. 1D
Formalin-fixed (10%) paraffin-embedded section of a colon carcinoma
reacted with LiCo 16-88 (4 .mu.g/ml IgM.times.380). Both
surface-like and cytoplasmic labeling are seen. The deparaffinized
section was blocked (20 min. at room temperature ) with
phosphate-buffered saline (PBS) (pH 7.3) containing 0.75M L-lysine
and 1% bovine serum albumin and then incubated with LiCo 16-88
overnight at 4.degree. C. After washing with PBS the section was
incubated (60 min. at 37.degree. C.) with affinity-purified
peroxidas-labeled goat antibody to human immunoglobulins
(IgG+IgA+IgM), washed and then reacted (15 min. at room
temperature) with diaminobenzidine (0.5 mg/ml) in PBS (pH 7.6)
containing 0.1% H.sub.2 O.sub.2. After counterstaining with
hematoxylin, the section was dehydrated, cleared and mounted with
permount.
FIG. 1E
Colon tumor as in FIG. 1D, reacted with normal human IgM (4
.mu.g/ml) (x380). No staining is observed.
FIG. 1F
Cryostat section of a colon tumor stained by LiCo 16-88 (x640).
Note the intense label of the periphery of the tumor cells
(arrows). The section was air dried and stored at -30.degree. C.
This section was post-fixed (20 min. at 4.degree. C.) with PLP in
PBS and processed as described in FIG. 1D, except that
peroxidase-labeled goat antibody specific to human .mu. chains was
used.
FIG. 1G
Cryostat sections of the colon tumor seen in FIG. 1F reacted with
normal human immunoglobulin (.times.640). No labeling of the tumor
cells is seen.
FIG. 1H
Cytospin preparation of air-dried unfixed SW1463 cells stained by
LiCo 16-88 (4 .mu.g/ml) (x280). The colon tumor cell line was
harvested with ethylenediaminetetraacetic acid (EDTA) (0.02%),
washed and suspended in medium containing 1% bovine serum albumin.
Cells (2.times.10.sup.4 in 0.1 ml) were pelleted onto the glass
slides in a cytocentrifuge, air dried and stored at -30.degree. C.
Cells were incubated with monoclonal antibody (1 hr. at room
temperature and then overnight at 4.degree. C.), washed and then
processed as described above.
FIG. 2
Distribution of antigens in paraffin sections of colorectal tumors.
Shaded area indicates positive indirect immunoperoxidase staining
of 15 tumors by 10 human monoclonal antibodies.
FIG. 3
Two monoclonal antibodies react with most colorectal tumors. The
reactivity of two monoclonal antibodies to paraffin sections of 15
colorectal tumors and air-dried cytospin preparations of
dissociated tumors from 9 patients are compared. Shaded area
indicates positive indirect immunoperoxidase staining.
FIG. 4
Follow-up of all control and immunized patients in active specific
immunotherapy clinical trials according to site and pathologic
stage.
FIG. 5A
Disease-free status of all patients.
FIG. 5B
Survival status of all patients.
FIG. 6A
Disease-free status of patients with positive regional lymph nodes
(Astler-Coller C)
FIG. 6B
Survival status of patients with positive regional lymph nodes
(Astler-Coller C).
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 C1, C2) or patients with metastatic disease
(stage D). Within these classifications, patients were randomly
selected for participation in treatment and nontreatment 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 .mu.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 laminar 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
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 1 (Sigma D-0876) (DNAase 1, Sigma D-0876)
prewarmed to 37.degree. C. Flasks were placed in a 37.degree. C.
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, Md.) into a 50 ml
centrifuge tube. The cells were centrifuged at 1200 rpm
(250.times.g) in a refrigerated centrifuge for 10 minutes. The
supernatant was poured off and the cells were resuspended in 5-10
ml of DNAase (0.1% in HBSS) and held at 37.degree. C. 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.times.10.sup.7 /ml in HBSS and added in equal volume to chilled
2X freezing medium containing 15% dimethylsulfoxide (DMSO) and 4%
human serum albumin (HSA). The final suspension of 2 to
4.times.10.sup.7 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 model 990 Biological
Freezer with a model 700 Controller and a model 500 Temperature
Recorder for controlled-rate 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 -1.degree. C./min to a final
temperature of -80.degree. C. 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 immunotherapy 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, Ind.: Aplisol, PPD, (Tuberculin Purified Protein
Derivative), ParkeDavis, Detroit, Mich.: Trichophyton, diluted
1:30, Center Laboratories, Port Washington, N.Y.: and Candida
albicans diluted 1:100, Center Laboratories, Port Washington, N.Y.,
0.1 ml of each was placed intradermally on the forearm and examined
for erythema and induration at 24 and 48 hours.
Patents selected for treatment protocol received 3 weekly
intradermal vaccine injections consisting of 10.sup.7 irradiated,
autologous tumor cells and 10.sup.7 BCG in the first 2 vaccines
with 10.sup.7 tumor cells alone in the final. Fresh-frozen Tice
BCG, supplied by Dr. Ray Crispen, University of Illinois Medical
Center, Chicago, Ill., was stored at -70.degree. C. 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 37.degree. C. 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
10.sup.7 viable tumor cells remained in the tube
(1.3.times.10.sup.7 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 10.sup.7 BCG were
added in a volume of 0.1 ml. HBSS was added in sufficient quantity
for a final 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 erytherma
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.
G. Results of Immunization
Reactivity to Standard Recall Antigens
All patients were reactive initially to at least one of the
standard recall antigens. Two of the 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 followup period except that all but two of the
immunized patients converted to PPD positivity.
H. Delayed Cutaneous Hypersensitivity (DCH) to Tumor Cells
The delayed cutaneous hypersensitivity reaction to 10.sup.6
autologous tumor cells in 24 immunized and 11 nonimmunized control
patients is shown in Table 2. A 48-hour induration measurement of
greater than 5 mm was considered positive. Four of 24 patients
(17%) had a positive DCH to 10.sup.6 tumor cells prior to the
course of immunization. This was not significantly different from
the one of 11 patients (9%) testing positive in the nonimmunized
control group. Of significance (p<0.01) 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 immunized
patients demonstrated a positive DCH response to 10.sup.5 tumor
cells at 6 weeks, with none showing a response to 10.sup.4
cells.
EXAMPLE II: Production of Hybridomas for Human Monoclonal
Antibodies
A. Removal and Processing of Immunized B-Cells from Patients
Patients were bled at the time of the second immunization, one week
after the first immunization, and at the time of the third
vaccination, one week after the second immunization. Venous blood
was collected asceptically in the presence of preservative-free
heparin (O'Neill, Jones and Feldman, St. Louis, Mo.) 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.times.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 (PBL) 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 time with balanced salt solution (BSS) and incubated
at 37.degree. C. for 20 minutes with four times the packed cell
volume with 0.14M AET (Sigma). The treated cells were then washed
three tires 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 PBL (up
to 80 million cells) were mixed with 1 ml of AET-treated sheep
erythrocytes and pelleted at 1000 rpm for 10 minutes at 4.degree.
C. 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.times.g for 40 minutes at
room temperature. The T-cell depleted PBLs were collected at the
interface, washed with three times the volume HBSS, 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 .mu.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 PBL 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 .mu.l of serum-free medium. One
ml of polyethylene glycol (50% w/v) prewarmed to 37.degree. C. 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 .mu.g/ml and thymidine 3.9 .mu.g/ml) at a
concentration of 25.times.10.sup.6 cells/ml (pre-fusion count) and
100 .mu.l was added to each microtiter well. Twenty-four hours
later, 100 .mu.l of MAT medium (HT medium containing 0.18 .mu.g/ml
aminopterin) was added to each well. Half of the medium was
replaced every three days with fresh HAT medium. After maintenance
in 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 PBL with myeloma cells may be used
to generate transformed diploid B-cells. PBL and myeloma cells were
mixed (at a ratio of 3:1), pelleted at 800 rpm and selected in HAT
medium, as described above.
C. Screening of Hybridomas
The hybridomas were first quantified and iso-typed by a capture
enzyme-linked immunoassay (ELISA) for the synthesis of human
immunoglobulin (IgA, IgG and IgM). The standard Bio-EnzaBead method
was utilized, which is sensitive in the range of 10-300 ng/ml. The
hybridoma supernatant fluids were diluted 1:30 with an effective
range of 0.3-9 .mu.g/ml. Only hybridomas that synthesized human
immunoglobulin at a concentration of greater than or equal to 1
.mu.g/ml were tested by indirect immunoperoxidase on tissues after
the isotype of the antibody (IgA, IgG or IgM) was determined.
Polycarbonate-coated metallic beads (Bio-EnzaBead.TM., Litton
Bionetics ) were incubated with goat antibodies to human
immunoglobulins (IgG+IgA+IgM) overnight at 4.degree. C. 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
4.degree. C. 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 1
hr at 37.degree. C., washed, and then incubated for 1 hr at
37.degree. C. with peroxidase-labeled affinity-purified goat
antibody to human immunoglobulins (IgG+IgA+IgM). The washed beads
were then incubated (10 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 (30-1000 ng/ml) of human gamma
globulin. Supernatant fluids containing >1 .mu.g/ml were then
isotyped using this ELISA with peroxidase-labeled goat antibodies
to human .gamma., .alpha., and .mu. chains. Subsequent 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 other
experiments, supernatant fluids were incubated with the antihuman
Ig beads and the peroxidase-conjugated goat antimouse IgG+IgM
(H+L).
Cryostat sections of normal and tumor tissue, stored at -30.degree.
C., were post-fixed in PLP (0.5% p-formaldehyde, 0.075M L-lysine,
0.01M sodium periodate) for 20 minutes at 4.degree. C. 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 0.075M L-lysine for 20
minutes. The sections were incubated overnight at 4.degree. C. 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 37.degree. C., 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
hematoxyline, 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 immunization. Optimal results were
obtained with lymphocytes removed one week after the second
immunzation (Table 8). 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).
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
hetero-hybridomas (FIG. 1A). By contrast, the majority of the
monoclonal antibody synthesizing cell lines (24 out of 36) were
atypical in appearance (FIG. 1B). These cells were predominantly
irregular in shape and grew in large aggregates. These
cluster-forming cells were isolated in seven fusions performed with
PBL 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-banding of the chromosomes confirmed that they were of human
origin (FIG. 1C). 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 human B-cells (diploid cells). The mechanism of this
spontaneous transformation is not known but may be related to the
immunization procedure.
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 .mu.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 be more stable with regard to immunoglobulin production. These
cells were grown An 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 of 5.times.10.sup.6 cells without a decrease in antibody
production. Thus, the cells theoretically could be expanded to
7.times.10.sup.13 cells. This hybrid produced approximately 30
.mu.g/ml/10.sup. 6 cells and thus 7.times.10.sup.13 cells could
conceivably produce over 2 kg of antibody.
D. Production of Monoclonal Antibodies
Human monoclonal antibody producing cells were grown in RPMI 1640
medium (Gibco, Grand Island, N.Y.) supplemented with 10% fetal
bovine serum, 3 mM L-glutamine and 5 .mu.g/ml gentamicin. The
medium was in some cases further supplemented with 25% D-glucose
(final concentration 0.25%). The cells were at 37.degree. C.
(35.degree.-38.degree. C.) under a humidified atmosphere of 7.5%
CO.sub.2 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 breast, lung,
gall bladder and liver and were found to be negative.
The pattern of reactivity of 10 of the human monoclonal antibodies
(MCA) to histological sections of colorectal adenocarcinomas from
15 patients is shown in FIG. 2. The matrix of reactivity of the
antibodies tested, indicates that individual antibodies reacted to
between 47 and 80% of the tumor specimens tested. No monoclonal
antibodies reacted to all 15 tumors. In tissue sections from
individual patients, the range of reactivity varied from tissues
reactive to all 10 antibodies to tissues reactive to as few as 1 or
2 antibodies. All of the tissue specimens used for determination of
monoclonal antibody reactivity were taken from patients other than
the 10 donors of B-cells for the original fusions.
We compared the pathologic stage of the tumors tested to the
percentage of reactivity with the group of monoclonal antibodies
tested, and found that the tumors with broadest reactivity were
moderately to well differentiated, adenocarcinomas the less common,
poorly differentiated adenocarcinomas were generally nonreactive.
The antibodies typically reacted with metastases.
Monoclonal antibody LiCo 16-88 reacted with an antigen preserved in
paraffin-embedded sections of colorectal carcinoma that was either
absent or greatly reduced in normal colonic mucosa. In addition to
cytoplasmic label, tumor cells exhibited surface-like staining
(FIG. 1D). This binding was specific, as demonstrated by the
absence of staining by normal human immunoglobulin matched in
concentration and isotype to the monoclonal antibody. Also
noteworthy is the observation that this antibody reacted with both
primary tumors and metastases. Antibody LiCo 16-88 reacted with
cryostar sections. As seen in FIG. 1E, intense staining of the
periphery of tumor cells was observed with LiCo 16-88 but not with
normal human immunoglobulin (FIG. 1F ).
The major advantages of a human, compared with a murine, monoclonal
antibody are for in vivo diagnosis (imaging) and therapy. Less than
1% of human monoclonal antibodies isolated from tumor bearing
patients were reported by previous investigators to react with cell
surface antigens (Cote et al., Proc. Nat. Acad. Sci., 80:2026-2030,
1983). These findings suggested that cancer patients may be
tolerant to tumor cell surface antigens. It is significant,
therefore, that one-half of the tissue-positive antibodies isolated
from immunized patients were subsequently found to bind to the
surfaces of tumor cells (Tables 3, 4 and 8). As seen in FIG. 1G,
monoclonal antibody 16-88 reacts with the surface of SW-1463 cells.
The lack of staining of some of the cells may be due to either
clonal or cell cycle variations in the expression of the antigen
(s). Thus, the greatest advantage of this invention, which uses
immunized patients as the source of sensitized B-cells, is the
extremely high frequency of antibodies reactive with cell surface
antigens produced. The antibodies produced according to the
invention have the greatest potential for the diagnosis and
treatment of cancer.
Protein (PBS and 3.0M KCl) and lipid (chloroform-methanol) extracts
were prepared from HT-29 and SW-1463 cells. Thirteen of the
antibodies were found to react with these extracts. The most
striking finding was that all the antibodies react with the protein
extracts, treatment of the extracts with protease significantly
reduced the binding. These results contrast markedly with those
obtained with murine monoclonal antibodies which are often directed
against glycolipid antigens of colon tumors (Morgan et al.,
Hybridoma, 3:3, page 233, 1984), and Lindholm et al., Int. Arch.
Allergy Appl. Immuno., 71:178-181, 1983).
Techniques including the preparation of protein extracts and the
use of immunoadsorbent lectins for the immunization of mice are
required to produce monoclonal antibodies against protein antigens
derived from colon 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 the 28 monoclonal
antibodies examined 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). It is interesting that three of
the human monoclonal antibodies also recognized antigens extracted
by the chloroform-methanol treatment. These antigens may either
represent proteins not denatured by this treatment or alternatively
glycolipids which share a common epitope (i.e., the carbohydrate
moiety) with a glycoprotein.
Reactivity of Human Monoclonal Antibodies to Cell Surface Antigens
of 8 Colon Carcinoma Cell Lines.
Thirty-six human monoclonal antibodies were assessed for reactivity
with tumor cell surface antigens against a panel of 8 human colon
cancer cell lines prepared as air-dried cytocentrifuge specimens.
Thirteen of 36 antibodies recognized antigens expressed on the
surface of at least 2 human colon carcinoma cell lines (FIG. 1H,
Table 3). All 13 surface-reactive antibodies were isotyped as IgM.
These monoclonal antibodies were produced by both heterohybridomas
and diploid B-cell lines.
Experiments using murine antibodies to structural cytoplasmic
antigens, such as actin, confirmed that cytoplasmic structures
could not be detected with properly prepared air-dried cytospin
cell preparations without prior permeabilization of the cell
membrane. The surface localization of the antigens recognized on
the Cytospin-prepared cells for most of the antibodies were
confirmed by indirect immunofluorescence of live cells.
We found no correlation between the reactivity of the monoclonal
antibodies and the immunoglobulin concentration of the
antibody-containing cell supernatant fluids. All cell supernatant
fluids were tested without dilution and without attempt to adjust
them to a constant immunoglobulin concentration. For the most part,
the 13 antibodies reactive with 2 or more cell lines exhibited more
than trace activity: the exceptions were 12-42 and 12-53,
antibodies of the IgG isotype that strongly reacted to only one
cell line. There was some variation in expression of cognate
antigens among the cell lines: LS-174.sup.t bound to 17 monoclonal
antibodies: SW-1463 and HT-29 bound to 12 and 10 antibodies,
respectively: the other cell lines bound to 5 to 9 of the
antibodies: and 7a2 and 16-52 reacted to all 8 cell lines.
Otherwise, the pattern of monoclonal binding indicated a multitude
of recognized specificities.
Reactivity of Human Monoclonal Antibodies to Cell Surface Antigens
of Dissociated Colon Carcinoma Tumor Cells
We confirmed the cell surface reactivity observed with the colon
cell lines in assays on air-dried Cytospin preparations of
enzymatically dissociated colon tumor cells from 9 patients (Table
4). Seventeen of the monclonal antibodies reacted to at least 2 of
the tumor cell preparations. There were some differences between
the cell line data and the tumor cell data: 16-86, which reacted
with 4 out of 8 cell lines, gave positive results with only one
tumor cell preparation, and 16-105 and 12-53, which reacted with 0
out of 8 and 1 out of 8 colon cell lines, respectively, reacted
with 3 or more of the tumor cell preparations. As was seen from the
assays of reactivity with cell lines, the patterns of antibody
binding, which reflect the presence and degree of antigen
expression by the tumor cells, suggest that many different
specificities are recognized by these monoclonal antibodies.
Reactivity of Human Monoclonal Antibodies with Paraffin Sections of
Paired Colon Tumor and Normal Mucosa.
The specificity of 25 of the human monoclonal antibodies reactive
with paraffin sections was tested by indirect immunohistochemistry
against paired sections of colonic tumor and autologous normal
colonic mucosa from 5 patients (Table 5). Eleven of the 25 (44%)
demonstrated no detectable reactivity with normal colonic mucosa in
the 5 patients tested, but all 11 reacted with tumor specimens.
Fourteen of the 25 antibodies, although reactive with the tumor
specimens, also reacted with normal colonic mucosa. Quantitatively,
in these cases reactivity with normal colonic specimens was less
than with tumor specimens. Individual antibodies reacted with 1 to
4 of the normal colonic mucosa specimens tested. Five of 14 of
these cross reactive antibodies only reacted with the normal
colonic mucosa of 1 of the 5 patients. The normal colonic mucosa of
patient 8 reacted with 13 of the 23 antibodies that reacted with
that patient's tumor. Whether the normal colonic mucosa from this
patient was proximal or distal to the tumor is not known. If
patient 8 were eliminated from this analysis only 9 of 24
antibodies tested would have reacted with 1-3 of the normal colonic
mucosa paired samples from 5 patients. Overall, in the total paired
colorectal tumor and normal colonic mucosa specimens tested,
approximately 30% showed cross reactivity with normal colonic
mucosa was seen, although the quantitative reactivity was
significantly less than that observed against the paired tumor
specimen. Moreover, the occurrence of a lower level but detectable
normal cell reactivity may be attributable to the recognition
determinants associated with a deviation from the normal conditions
which does not show as cancerous.
Reactivity of Human Monoclonal Antibodies with Paired Human Colon
Tumor and Mucosa Cell Cytospin Preparations by Direct Binding of
Biotin-Labeled Antibodies
The specificity of antibodies for tumor cells versus normal cells
is difficult to evaluate by indirect staining methods on Cytospin
preparations and cryostat sections. The peroxidase-labeled
antihuman Ig antibodies used to detect the human antibodies also
recognize endogenous human immunoglobulin present on all human
tissues. Normal tissues contain greater amounts of endogenous
immunoglobulin than do corresponding tumor tissues, consequently
the background is higher for normal than for tumor tissue. Direct
labeling of the antibodies overcomes this problem and permits
inclusion of an excess of irrelevant human immunoglobulin with the
monoclonal antibodies to block nonspecific immunoglobulin binding,
another problem associated with indirect techniques.
Five of the surface-reactive human antibodies were purified from
culture medium and labeled with biotin. The 5 were chosen because
they had reacted well in previous assays and produced relatively
high levels of human immunoglobulin. Table 6 shows the results with
the 5 biotin-labeled antibodies in direct assays on air-dried
Cytospin cell preparations of colon tumor and adjacent mucosa cells
obtained from 7 patients. All 5 antibodies reacted with the tumor
cells, confirming the reactivity seen in indirect assays.
Reactivity with normal mucosa cells was weak or non-detectable.
Direct Binding of Biotin-Labeled Monoclonal Antibodies to Frozen
Tissue Sections of Colon Tumor and Normal Colonic Mucosa
Further direct characterization of the 5 biotin-labeled antibodies
with regard to their specificity for tumor versus normal cells was
established with frozen tissue sections of colon tumor and adjacent
normal colonic mucosa (Table 7). Absolute specificity was observed
with 4 of the antibodies as shown by the fact that they strongly
reacted with at least 2 out of 5 colon tumors and did not react
with any of the 4 matched normal colonic mucosa sections. 19b2
reacted strongly with 4 of 5 tumor sections and showed a weak
reaction with 1 of 4 normal colonic mucosa sections. 19b2 also
reacted somewhat with normal colonic mucosa Cytospin cell
preparations (Table 6) and normal colonic mucosa paraffin sections
(Table 5).
Frozen tissue sections of normal breast, stomach, kidney, liver,
muscle and skin (Table 7) showed no staining by biotin-labeled
human antibodies except antibody 1962 which exhibited a low level
of binding to normal stomach tissue. An overall background stain of
connective tissue components was observed. This background staining
was nonspecific and has been-observed by others using
biotin-labeled monoclonal antibodies.
Reactivity of Monoclonal Antibodies with CEA, Erythrocyte and
Leukocyte Antigens.
To further establish the tumor specificity of the monoclonal
antibodies, we tested for reactivity with CEA, human erythrocyte
antigens and human lymphocyte antigens by various techniques. We
found no evidence or reactivity between these antibodies and these
antigens. Anti-CEA activity was assessed by ELISA against two CEA
preparations. The staining patterns of the human monoclonal
antibodies on human colon tumor paraffin sections were different
from those observed with a mouse anti-CEA antibody. None of the 36
human antibodies gave the luminal staining pattern typically seen
with anti-CEA antibodies. Reactivity with human erythrocyte
antigens was measured by indirect immunofluorescence and
hemagglutination against an erythrocyte panel representing all
major and most minor blood group systems. No reactivity was seen.
ELISA, cytotoxicity assays and indirect immunoperoxidase staining
of human lymphocytes showed no evidence of recognition of human
lymphocyte antigens by any of the antibodies.
Functionality of Human Monoclonal Antibodies to Colo-rectal
Cancer
Specificity is a major consideration in the determination of the
usefulness of these tumor-reactive monoclonal antibodies. The lack
of reactivity of some of the monoclonal antibodies with a certain
percentage of the tumor specimens tested is another factor which
must be considered. Thus it is unlikely, based upon these data,
that any single monoclonal antibodies would have all the factors
associated with it that would make it ideal for therapeutic or
diagnostic application. The strategy of using immunized cancer
patients has provided a large number of clones from which certain
selections can be made with regard to range of reactivities, as
well as specificity. By selecting only 2 of the monoclonal
antibodies that we have produced which, based on their
characteristics in a broad in vitro screen, have the greatest
amount of tumor reactivity with the least amount of normal colonic
mucosa reactivity, we can propose and develop cocktails of
antibodies that together promise greater efficacy than any
individual monoclonal antibody. As shown in FIG. 3, 2 monoclonal
antibodies, 6a3-1 and 7a2, paired for their range of reactivity
with both tissue sections and dissociated tumor cells and selected
based on their relative lack of cross reactivity with normal
colonic mucosa, provide an antibody cocktail which will react with
14 of 15 tumor specimens and 9 of 9 dissociated tumor cell
specimens. Other cocktails of this type can be developed: however,
clearly we must have a broad range of monoclonal antibodies to
select from and an extensive in vitro screen for testing a large
number of specimens in a variety of differentiation states in order
to utilize human monoclonal antibodies for therapeutic or
diagnostic purposes.
In addition to providing monoclonal antibodies reactive with tumor
cell surface antigens for the in 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.
Table 3 shows the tissue reactivity of monoclonal antibodies
produced by the monoclonal antibody cell lines prepared according
to these procedures.
The foregoing describes the formation of novel monoclonal
antibodies specific for certain tumors, hybridomas, 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 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 cell lines described in Table 3 were deposited with the
American Type Culture Collection, 12301 Parklawn Drive, Rockville,
Md. 20852, U.S.A., on Jan. 30, 1984. The four individual cell lines
and two mixed cell lines deposited and assigned accession numbers
are identified as follows:
______________________________________ Identification Accession
Number ______________________________________ Human B-Cell Derived
Cell Line, HB 8492 Lico D-23 Human Mouse-Heterohybridomas, HB 8493
LiCo 6a3-1 Human Mouse-Heterohybridomas, HB 8494 LiCo 7a4 Human
B-Cell Derived Cell Line, HB 8495 LiCo 16-88 Human B-Cell Derived
Cell Line, HB 8496 LiCo 16-86 Human Mouse-Heterohybridoma, HB 8497
LiCo H-11 ______________________________________
LiCo D-23, Accession Number HB 8492, consists of a mixture of the
23 cell lines listed in Table 3 as having the clumped cell growth
(diploid) cell type with the exception of 23A4 and 27B1. LiCo H-11,
Accession Number HB 8497, consists of a mixture of the 11 cell
lines listed in Table 3 as having the dispersed cell growth
(hybridoma) cell type with the exception of 28A32.
TABLE 1
Criteria for Successful Vaccines for Active Specific
Immunotherapy
Adjuvant
(a) BCG (Phipps, Tice, Connaught): lyophilized, frozen
(dose-dependence>10.sup.6 (10.sup.7 -10.sup.8)
(b) C. parvum (Wellcome Labs ) (dose-dependence>7 .mu.g (70
.mu.g-700 .mu.g)
Tumor Cells
(a) Enzymatic dissociation
(1) Collagenase type I (1.5-2.0 U/ml HBSS)
(2) DNAase (450 D.U./ml HBSS)
(3) 37.degree. C. with stirring
(b) Cryopreservation
(1) Controlled-rate freezing (-1.degree. C./min ) (7.5% DMSO, 5%
HSA, HBSS)
(2) Viability 80%
(c) X-irradiation
(1) Rendered non-tumorigenic at 12,000-20,000 R.
Components and Administration.sup.a
(a) Ratio of adjuvant to tumor cells -10:1-1:1 (optimum)
(b) 10.sup.7 tumor cells (optimum)
(c) 2-3 i.d. vaccinations at weekly intervals. Third vaccination
contains tumor cells only.
TABLE 2 ______________________________________ DCH Reaction to
Autologous Tumor Cells Pre- Reactivity No. of immunization 6 wk
Stage Patients Reactivity.sup.a and/or 6 mo.
______________________________________ Immunized B2 8 0 4 Patients:
C1, C2 9 2 6 D 7 2 6 Total (%) 24 4 (17%) 16 (67%) Nonimmunized B2
4 1 0 Patients: C1, C2 5 0 1 D 2 0 0 Total (%) 11 1 (9%) 1 (9%)
______________________________________ .sup.a Reactions were
considered positive when the 48hr. induration (the mean of 2
diameters) was more than 5 mm.
TABLE 3
__________________________________________________________________________
Reactivity of Human Monoclonal Antibodies to Cell Surface Antigens
of Eight Colon Carcinoma Cell Lines.sup.a Monoclonal Colon
Carcinoma Cell Lines Antibody Concentration.sup.b Isotype HT-29
SW1463 SW948 SW480 SW403 LS-174.sup.t LoVo WiDr
__________________________________________________________________________
6a3*.sup.c 11 IgM 2+ + - - - 4+ + + 7a2* 23 IgM 4+ 4+ 4+ 4+ 4+ 4+
4+ 4+ 7a4* 18 IgM + 4+ - 2+ - 3+ + - 11A7 3 IgM - - - - - - - -
11B5 7 IgM - - - - - 2+ - - 12-38* 144 IgG - - - - - - - - 12-42*
74 IgG - - - - - - 3+ - 12-47* 25 IgG - - - - - - + - 12-53* 219
IgG - - - - - - - 2+ 15-12 15 IgM - - - - - - - + 15-24 18 IgG - ND
ND - - - - - 15-33 11 IgM - - - - - + - - 15-39 3 IgG - - - - - - -
- 16-4 19 IgM - - - - - + + - 16-50 3 IgM - ND ND - - + ND ND 16-52
4 IgM 2+ 3+ + 3+ 3+ 3+ 4+ 3+ 16-58 14 IgM 3+ 2+ - 4+ 2+ 2+ + -
16-66 7 IgM + 2+ - 4+ 3+ + + - 16-72 5 IgM - - - - - - - - 16-80 8
IgG - - - - - - - - 16-81 6 IgM - - - - - - - - 16-86 9 IgM - 3+ -
- - 4+ 4+ 4+ 16-88 9 IgM 3+ 4+ 3+ - 3+ 4+ 4+ 4+ 16-103 6 IgM - - -
- - + - - 16-105 11 IgM - - - ND - - - - 18-15 16 IgG - - - + - - -
- 18-21* 12 IgM 2+ 2+ - - - 2+ - - 18-22* 7 IgM + + + - + - 2+ +
19b2* 26 IgM 3+ 4+ 2+ - + 4+ 4+ 4+ 20A3 4 IgG - - - - - + - - 20A6
9 IgG - - - - - - - - 20B7 9 IgG - - - - - - - - 21B27* 19 IgM - -
- - - - - - 23A4 8 IgM - - ND ND ND - ND ND 27B1 3 IgM - - ND ND ND
- ND ND 28A32* 3 IgM - 2+ ND ND ND 2+ ND ND
__________________________________________________________________________
.sup.a Intensity of immunoperoxidase staining compared to the
control matched in isotype and concentration to the monoclonal
antibody tested. .sup.b .mu.g/ml. .sup.c "*" Designates hybridomas
culture morphology, other appeared as transformed (diploid) B cell
lines. Cell lines. Human colonic adenocarcinoma cell lines HT29,
SW1463, SW948, SW480, SW403, LoVo, and WiDr were obtained from the
American Type Culture Collection (Rockville, Maryland). The cells
were cultured in the recommended culture medium supplemented with
10% fetal bovine serum. Colo adenocarcinoma cell line LS174.sup.t
obtained from Dr. Jeffrey Schlom, (National Cancer Institute,
Bethesda, Maryland), was cultured in Dulbecco's modified Eagle's
medium. All cell lines were incubated at 37.degree. C. in an
atmosphere of 5% CO.sub.2.
TABLE 4
__________________________________________________________________________
Reactivity of Human Monoclonal Antibodies to Cell Surface Antigens
of Colon Carcinoma Tumor Cells.sup.a Monoclonal Patient Number
Antibodies Concentration Isotype 16 17 18 19 20 21 22 23 24
__________________________________________________________________________
6a3*.sup.c 11 IgM + - - - 2+ 3+ + - - 7a2* 23 IgM 4+ 4+ 4+ 4+ 4+ 4+
- + 4+ 7a4* 18 IgM 2+ - 2+ 2+ - 4+ ND - - 11B5 7 IgM - - - - - + -
- ND 12-38* 144 IgG - - - - - - - - ND 12-42* 74 IgG - - - - - - -
- ND 12-47* 25 IgG - - - - - - ND ND ND 12-53* 219 IgG 2+ - 3+ ND -
- - - 2+ 15-12 15 IgM - - - - + + - - - 15-24 18 IgG - - - - - - ND
- ND 15-33 11 IgM - - - - + 2+ - - - 15-39 3 IgG - - - - - - - - ND
16-4 19 IgM - - - - - 3+ - - ND 16-50 3 IgM - - - - - + ND - ND
16-52 4 IgM 2+ 2+ 2+ 2+ + 2+ + + - 16-58 14 IgM 3+ 3+ 3+ 2+ - + - -
3+ 16-66 7 IgM 4+ 4+ 3+ - + - - - - 16-72 5 IgM - - - - - 2+ - - -
16-80 8 IgG - - - - + - + - - 16-81 6 IgM - + - - + - - - ND 16-86
9 IgM - - - - + - - - - 16-88 4 IgM + + + 3+ 4+ - - + - 16-103 6
IgM - - - - - - ND - ND 16-105 11 IgM - - 2+ + + + ND ND ND 18-15
16 IgG - + - - - + - - ND 18-21* 12 IgM + + + + - + - - - 18-22* 7
IgM 2+ + 2+ 2+ + 2+ - + - 19b2* 26 IgM 2+ 2+ 2+ 2+ 3+ 4+ + 2+ -
20A3 4 IgG - - - - - + - - ND 20A6 9 IgG - - - - - - - - ND 20B7 9
IgG - + - - - - - - ND 21B27* 19 IgM - - - - - - - - ND
__________________________________________________________________________
.sup.a The presence and degree of binding are as explained in the
footnotes to Table 3. .sup.b .mu.g/ml. .sup.c *Designates hybridoma
culture morphology, other grow as transforme (diploid) Bcell
lines.
TABLE 5
__________________________________________________________________________
Reactivity of Human Monoclonal Antibodies on Paraffin Sections of
Colorectal Tumors (T) and Paired Normal Colonic Mucosa (N).sup.a
Patient Number Monoclonal 2 6 7 8 10 Antibodies T N T N T N T N T N
__________________________________________________________________________
6a3 + - - ND 2+ - 2+ - 2+ - 7a2 - ND 3+ - - ND 4+ - - ND 7a4 - ND -
ND - ND 4+ - - ND 11B5 + - 3+ - 3+ - 4+ 2+ 3+ + 12-38 - ND - - 2+ -
2+ - 3+ - 12-42 - ND - ND 2+ - 3+ - 2+ - 12-47 + - - ND + - 2+ - -
ND 12-53 - ND 3+ - - ND + - - ND 15-24-2 - ND 3+ - 4+ 2+ 3+ + - ND
16-4 - ND 2+ - + - 3+ + + - 16-58 - ND 4+ + 4+ - 2+ + - ND 16-66 -
ND 4+ - + + 3+ + + - 16-86 - ND - ND 2+ - + - + - 16-88 + - 2+ - +
- 4+ + + - 18-15 + - 2+ - + - 2+ + + - 18-21 - ND 3+ + 2+ + 3+ + +
+ 18-22 - ND - ND + - - ND + - 19b2 - ND - ND 2+ + 4+ 2+ - ND 20A3
+ - 4+ + + - 2+ - + - 20A6 3+ - 2+ - 2+ - 2+ - 2+ + 20B7 3+ + 2+ -
+ - 3+ + + + 21B27 2+ + 2+ - - ND 3+ + - ND 23A4 - ND 2+ - 2+ - 2+
+ 3+ - 27B1 2+ - 4+ + 3+ - 4+ 2+ 3+ + 28A32 - ND 4+ - + - - ND + -
__________________________________________________________________________
.sup.a Presence and degree of binding are indicated as explained in
the footnotes of Table 3.
TABLE 6 ______________________________________ Reactivity of
Biotin-Labeled Monoclonal Antibodies to Human Colon Tumor (T) and
Normal Mucosa Cell (N) Cytospin Preparations.sup.a Monoclonal
Antibodies Patient 6a3 7a2 7a4 18-22 19b2 Number T N T N T N T N T
N ______________________________________ 18 + - + + + + + + + + 21
3+ - + + 2+ - + + + - 24 2+ - + + 4+ - 2+ - 3+ - 25 + - + - + + 2+
2+ + + 26 - - - - + + - - + - 27 2+ - - - 2+ - 2+ + 2+ 2+ 28 + + +
- + + + - 2+ + ______________________________________ .sup.2 The
presence and degree of binding are indicated as explained in the
footnote to Table 3.
TABLE 7 ______________________________________ Reactivity of
Biotin-Labeled Monoclonal Antibodies with Frozen Section of Colon
Tumors (T) and Normal Tissues (N).sup.a Source Monoclonal
Antibodies of 6a3 7a2 7a4 18-22 19b2 Tissue T N T N T N T N T N
______________________________________ Colon + - - - 2+ - + - 2+ +
Colon 3+ - 2+ - 3+ - + - 2+ - Colon 3+ - + - 3+ - 3+ - 3+ - Colon
2+ - + - - - - - - - Breast - - - - - Breast - - - - - Breast - - -
- - - - - - Stomach - - - - + Kidney - - - - - Liver - - - - -
Muscle - - - - - Skin - - - - - Skin - - - - -
______________________________________ .sup.a The presence and
degree of binding are indicated as explained in the footnotes to
Table 3.
TABLE 8
__________________________________________________________________________
Isolation of Human Monoclonal Antibodies Reactive with Colorectal
Carcinoma No. of Wells No. of Ig.sup.+ No. of Tissue.sup.+ No. of
Cell Surface.sup.+ Isotype.sup.d Culture Pattern.sup.e
Immunization.sup.a Patients Assayed Cell Lines (%).sup.b Cell Lines
Cell Lines (%).sup.c IgG IgM Diploid Hybridoma
__________________________________________________________________________
Pre 2 25 4 0 (0%) 0 (0%) 0 0 1 9 441 65 (15%) 10/65 (15%) 4/10
(40%) 2 8 8 2 2 10 573 154 (27%) 25/154 (9%) 16/25 (64%) 9 16 16 9
3 3 112 11 (10%) 1/11 (10%) 0/1 (0%) 1 1
__________________________________________________________________________
.sup.a PBL were obtained 7 days after each immunization. .sup.b
Production of .gtoreq.1 .mu.g/ml of human immunoglobulin as
measured by ELISA. .sup.c Immunoperoxidase label of unfixed
airdried preparations of SW1463, HT29 or enzymatically dissociated
tumor cells. .sup.d Isotypes were determined by ELISA. .sup. e
Diploid cells, cell growth in clusters. Hybridoma cells, growth a
dispersed cells.
* * * * *