U.S. patent application number 10/197292 was filed with the patent office on 2003-04-17 for immunotherapy of tumor with monoclonal antibody against the 17-1a antigen.
This patent application is currently assigned to Centocor, Inc.. Invention is credited to Carrano, Richard A., Schoemaker, Hubert J.P..
Application Number | 20030072759 10/197292 |
Document ID | / |
Family ID | 25382871 |
Filed Date | 2003-04-17 |
United States Patent
Application |
20030072759 |
Kind Code |
A1 |
Schoemaker, Hubert J.P. ; et
al. |
April 17, 2003 |
Immunotherapy of tumor with monoclonal antibody against the 17-1A
antigen
Abstract
Those skilled in the art will recognize, or be able to ascertain
using no more than routine experimentation, many equivalents to the
specific embodiments of the invention described herein. Such
equivalents are intended to be encompassed by the following
claims.
Inventors: |
Schoemaker, Hubert J.P.;
(Paoli, PA) ; Carrano, Richard A.; (Salisbury,
MD) |
Correspondence
Address: |
HAMILTON, BROOK, SMITH & REYNOLDS, P.C.
530 VIRGINIA ROAD
P.O. BOX 9133
CONCORD
MA
01742-9133
US
|
Assignee: |
Centocor, Inc.
Malvern
PA
|
Family ID: |
25382871 |
Appl. No.: |
10/197292 |
Filed: |
July 16, 2002 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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10197292 |
Jul 16, 2002 |
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08307044 |
Sep 16, 1994 |
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6444207 |
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08307044 |
Sep 16, 1994 |
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07726640 |
Jul 2, 1991 |
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07726640 |
Jul 2, 1991 |
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07384073 |
Jul 20, 1989 |
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07384073 |
Jul 20, 1989 |
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06883572 |
Jul 9, 1986 |
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Current U.S.
Class: |
424/155.1 |
Current CPC
Class: |
A61P 35/00 20180101;
C07K 16/3046 20130101; A61K 38/00 20130101; A61P 37/00 20180101;
C07K 16/303 20130101 |
Class at
Publication: |
424/155.1 |
International
Class: |
A61K 039/395 |
Claims
1. A method of treating gastrointestinal tumor comprising
administering to a patient afflicted with a gastrointestinal tumor,
a murine monoclonal antibody which specifically binds to an epitope
of 17-1A antigen, the antibody being administered parenterally in
multiple doses of 100 milligrams or more per dose for a total dose
of about 0.1-5 grams of antibody.
2. A method of claim 1, wherein the gastrointestinal tumor is
gastrointestinal adenocarcinoma, colorectal or pancreatic
carcinoma.
3. A method of claim 1, wherein a mixture of two or more murine
monoclonal antibodies which specifically bind an epitope of the
17-1A antigen are administered.
4. A method of claim 1, wherein a dose of antibody is infused
intraveneously.
5. A method of claim 1, wherein the total dose is about 1-5 grams.
Description
BACKGROUND
[0001] The tumoricidal activity of the murine monoclonal antibody
17-1A has been characterized in the nude mouse and in humans. See,
e.g., Herlyn, D. and Koprowski, H. (1982) "IgG2a Monoclonal
Antibodies Inhibit Human Tumor Growth Through Interaction with
Effector Cells" Proc. Natl Acad. Sci. USA 79, 4761-4765. Several
cases have been reported where the administration of Mab 17-1A
resulted in a partial or complete regression of metastatic
colorectal or pancreatic carinomas. See Sears, H. F. et al. (1984)
"Effects of monoclonal antibody immunotherapy on patients with
gastrointestinal adonocarcinoma. " J. Biol. Resp. Mod. 3, 138-150;
Sears, H. F., et al., "Phase II Clinical Trial of a Murine
Monoclonal Antibody Cytotixic for Gastrointestinal Adenocarcinoma"
(1985) Cancer Res. 45: 5910-5913. Generally, the antibody has been
administered as single administration of 500 .mu.g or less.
SUMMARY OF THE INVENTION
[0002] This invention pertains to a method of immunotherapy of
gastrointestinal tumors employing multiple, high doses of murine
monoclonal antibody against the gastrointestinal tumor-associated
antigen 17-1A. The method comprises administering to a patient
afflicted with gastrointestinal tumor, murine monoclonal antibody
against the antigen 17-1A in multiple, sequential doses of about
100 mg or more for a total overall dose of from about 0.1 to about
5 grams. Each dose of the murine antibody can be administered at
one to three day intervals up to weekly intervals to achieve and
maintain a "continuous" high level of circulating antibody.
Mixtures ("cocktails") of two or more murine anti-17-1A antibodies
can be given. The multiple, high dose therapy can be performed as
adjuvant therapy to chemotherapy, radiotherapy or surgery.
[0003] The high dose murine antibody therapy is well tolerated in
patients. Further, the anti-murine antibody response which
generally develops in treated humans, surprisingly does not alter
significantly the plasma half life of the murine antibody on repeat
administration. Thus, high blood levels of antibody can be achieved
with sequential injections of high doses to enhance transit of the
antibody from intravascular space into the tumor bed and thus
provide higher concentrations of the therapeutic antibody to the
locus of action.
DETAILED DESCRIPTION OF THE INVENTION
[0004] This invention pertains to therapy of gastrointestinal
tumors with repeated, high dose of murine antibody against the
17-1A antigen associated with most gastrointestinal tumors. This
therapeutic approach is based upon several findings. Murine
anti-17-1A antibody administered in multiple, high dose is
generally well-tolerated by patients. The most common side effect
is mild gastrointestinal symptoms. Allergic responses, however, do
limit repeat therapy in some patients. In addition, although a
human anti-murine antibody response is generally evoked by the
murine antibody, the response does not drastically effect the
pharmacokinetics of the administered murine antibody. This
indicates that sequential, high doses of antibody can be given to
achieve and maintain a continuous high plasma level of antibody.
Maintaining a high circulating level of antibody optimizes transit
of the antibody from intravascular space into the tumor, thereby
enhancing access of the antibody to the tumor for more effective
anti-tumor action. Further, sustained, high blood levels lead to a
prolonged, higher concentration of antibody at the locus of action
for more effective antibody dependent cell mediated cytolysis of
the tumor cells.
[0005] According to the method of this invention, murine antibody
against the 17-1A antigen is administered to patients afflicted
with gastointestinal tumors in multiple doses of about 100 mg or
more, preferably about 400 mg-1 gram, for a total dose of about 0.1
to 5 grams, preferably 1 to 5 grams. The antibody is administered
parenterally preferably by intraveneous infusion. The antibody is
generally administered suspended in a physiologically acceptable
vehicle e.g. normal saline. The antibody doses can be given over
intervals of 1-3 days to intervals of about a week. The dose
regimen for an individual patient will depend, inter alia, on the
patient's clinical status and on his ability to tolerate the dose
without detrimental allergic or anaphylactic reaction. The
objective is to provide antibody at fractional doses which yields a
sustained, high plasma level of antibody over the course of therapy
to provide increased access of the antibody to the tumor site.
[0006] Murine antibodies against 17-1A can be administered
individually or in mixtures (cocktails) of two more murine
anit-17-1A antibodies. Preferably, anti-17-1A antibody having
different epitopic specificity for 17-1A is employed in the
combination in order to increase anti-tumor activity in an additive
or synergistic fashion. Murine antibodies can be selected from the
original 17-1A antibody or other murine antibodies which recognize
similar or different epitopes of the 17-1A antigen, such as the
M72, M74, M77 and M79 antibodies described below.
[0007] Murine antibody against 17-1A antigen can be used in passive
immunotherapy of tumors of the gastrointestinal tract with which
the 17-1A antigen is associated. Examples are gastrointestinal
adenocarcinoma, colorectal carcinoma and pancreatic carcinoma. The
murine antibody treatment can be adjuvant to other forms of
therapy, including chemotherapy, radiotherapy and/or surgery. In
particular, murine antibody therapy can be useful as adjuvant
therapy directed against micro- or mini-metastases which are not
amenable to surgical removal.
[0008] The invention is further illustrated by the following
exemplification.
[0009] Exemplification
[0010] A trial was conducted in 20 patients to examine patient
tolerance to repeated high doses in 17-1A, examine its
pharmacokinetics on repeated administration and to characterize the
human immune response (antibody) to this mouse immunoglobulin.
[0011] Patient Population
[0012] Twenty patients with gastrointestinal malignancy (17 colon;
2 gastric; and 1 pancreatic) were selected on the basis that they
had metastatic disease with small-moderate tumor burden;
performance status greater than 70% (Karnofsky scale) and
objectively measurable disease. Studies were not done to document
17-1A reactivity with individual patient tumor specimens. Seven out
of 20 patients had received prior chemotherapy while 13/20 had no
prior therapy for metastatic disease.
[0013] Treatment Protocol
[0014] The study was conducted the Clinical Research Unit,
Comprehensive Cancer Center, University of Alabama at Burmingham.
All antibody infusions utilized a total dose of 400 mg of 17-1A
diluted in 250 ml of normal saline infused over 30 minutes with
careful monitoring of vital signs. All infusions were preceded by
an intravenous test dose of 0.7 mg followed by 30 minutes of
monitoring prior to administration of the full dose infusion. The
protocol involved the accrual of 4 groups of 5 patients each who
would receive progressively increasing numbers of weekly infusions,
i.e. Group 1-5 patients--single infusion; Group 2-5 patients with 2
infusions--Day 1 and 8; Group 3-5 patients with 3 infusions--Day 1,
8 & 15; Group 4-5 patients with 4 infusions--Day 1, 8, 15 &
22. Because of toxicity noted in Group 3 patients, no patients
received 4 infusions, but these 5 patients were added to Group 2
and had 2 infusions of therapy (Day 1 & 8). All patients were
followed for 6 weeks, following their last infusion with weekly
monitoring of urinalysis, liver and renal function, blood counts
and clinical evaluation.
[0015] Pharmacokinetics
[0016] Pharmacokinetic analysis was done on the first 5 patients at
the time of their single infusion (no prior exposure to 17-1A), the
ten patients who had two infusions were studied at the time of
their second infusion (one prior exposure to 17-1A) and the group 3
patients were studied at the time of their third infusion (two
prior exposures to 17-1A). For pharmacokinetics, blood samples were
drawn prior to infusion, immediately at conclusion of infusion and
at 1/2, 1, 2, 4, 12, 24, 48, 72 hours and 86 hours. Spot samples
were drawn at pre-therapy, 1, 24 & 48 hours post-therapy on
infusions not undergoing a full pharmacokinetic study to confirm
the general pattern of mouse immunoglobulin disappearance. The
plasma level of 17-1A was quantitated using a solid phase
radiometric sandwich assay utilizing latex beads coated with rabbit
anti-mouse gammaglobulin and radiolabeled (.sup.125I) affinity
purified goat antimouse IgG, F(ab').sub.2. The concentration of
17-1A in plasma was quantitated by the amount of latex particle
binding of radiolabeled anti-mouse IgG, F(ab').sub.2 as compared to
a standard curve of known concentrations of 17-1A diluted in normal
plasma. The sensitivity of this assay was 1.0 ng/ml.
[0017] Human Anti-mouse Antibody (HAMA) Response
[0018] Serum samples were drawn on each patient prior to each
infusion and then weekly .times.6. The assay used to determine the
presence of human anti-17-1A was a "double antigen" system
(Addison, G. and Hale, C., Horm. Metab. Res. 3, 59-60 (1971)) using
the concurrent incubation of 17-1A coated latex beads, 100 ul of
test plasma of 1 ug of radiolabeled (.sup.125I) 17-1A (specific
activity of 300-400 cpm/ng). The samples were incubated 90 minutes
at room temperature and the radioactivity associated with the beads
determined by centrifugation of the beads through Percoll as
previously described (Lobuglio, A. et al., New Engl. J. Med. 309,
459-463 (1983)). The cpm of .sup.125I-17-1A bound to the beads by
plasma was converted to ng of 17-1A/ml of plasma by using the known
specific activity of .sup.125I-17-1A. This assay obviously detects
any molecule with more than one binding site for 17-1A (IgG and
IgM). Assay results in normal individuals and cancer patients prior
to 17-1A exposure were 5.+-.4 ng/ml plasma (n=54) with values
ranging from 0 to 16 ng/ml. Values greater than 20 ng/ml were
classified as an antibody response.
[0019] 17-1A Monoclonal Antibody
[0020] The monoclonal antibody was provided by Centocor, Inc. as a
purified suspension of 10 mg/ml in normal saline. It was stored at
4.degree. C. prior to use. The protocol was carried out under
Centocor sponsored IND (#2168).
[0021] Toxicity
[0022] The adverse effects of 17-1A administration are summarized
in Table 1.
1TABLE 1 TOXICITY ASSOCIATED WITH 17-1A INFUSION I. Single dose
(400 mg) - 5 patients 3/5 - none 2/5 - G.I. symptoms II. Two weekly
doses (400 mg) - 10 patients 4/10 - none 5/10 - G.I. symptoms 1/10
- flushing/tachycardia III. Three weekly doses (400 mg) - 5
patients 3/5 - none 2/5 - G.I. symptoms and anaphylaxis (third
dose)
[0023] Ten of 20 patients had no adverse effects including 4
patients who received two infusions and 3 patients who received
three infusions. The most frequently observed side effect was
gastrointestinal (9/20 patients) with nausea and vomiting (4
patients) or diarrhea with or without cramps (7 patients). The
symptoms usually began within an hour of infusion and lasted less
than 24 hours. They were of modest-moderate severity and readily
controlled with anti-emetics of anti-diarrhea medications. The
frequency of gastrointestinal symptoms was not related to the
number of 17-1A infusions. One patient had an episode of flushing
and tachycardia in the midst of her second infusion which
disappeared by simply slowing the infusion rate. This patient had
no other adverse effects with the infusion nor with her prior
infusion.
[0024] Two patients had serious adverse effects. Both patients had
nausea and vomiting associated with their first and second
infusions (Day 1 & 8). They tolerated their test dose of 17-1A
on Day 15 without adverse effects over 30 minutes of observation.
The treatment infusions were then begun and both developed dyspnea,
tachycardia and hypotension judged to be an anaphylactic reaction.
Both infusions were immediately stopped (less than 10% of dose
given) and patients responded well to therapy with corticosteroids,
epinephrine and antihistamines. No patient in the study developed
abnormalities of urinalysis, complete blood count, renal or hepatic
function.
[0025] Pharmacokinetics
[0026] The serial plasma 17-1A levels on each patient were analyzed
and found to fit will with a 1 compartment model of plasma
disappearance. The results for peak plasma concentration, plasma
half-life and area under the curve are summarized in Table 2.
2TABLE 2 PHARMACOKINETICS 17-1A (400 mg) IN MAN Peak Conc.
Half-life AUC Prior Antibody (ug/ml) (hours) (hrs-ug/ml) None (n =
5) 139 .+-. 8 15 .+-. 2 3013 .+-. 175 One (n = 10) 141 .+-. 5 14
.+-. 1 2828 .+-. 93 Two (n = 3) 108 .+-. 2 24 .+-. 2 3771 .+-. 81
Values are expressed as mean .+-. standard error of the mean. AUC =
area under the curve.
[0027] The two patients who had anaphylactic reactions did not
receive their full third dose of 17-1A and therefore had no
pharmacokinetic study. Thus, only 3 patients made up the group with
two prior exposures to 17-1A. The results are similar for all 3
groups of patients. The three patients studied on their third
infusion had a somewhat lower serum peak concentration of 17-1A and
a somewhat longer mean plasma half-life than the groups of patients
with a single or second infusion. Interpretation is limited since
the differences were modest and the group was made up of a small
number of patients.
[0028] Human Anti-mouse Antibody (HAMA) Response
[0029] The patients' serum prior to therapy had little or no
detectable ability to bind .sup.125I-17-1A coated beads. As
summarized in Table 3, almost all patients developed HAMA within 29
days of their first 17-1A exposure (17/20). The majority (11/20)
had HAMA by Day 8 with 8/11 having values of greater than 100 ng/ml
and 2/11 having values of greater than 1000 ng/ml. Peak values were
generally noted on Day 15 or 22 with values falling by day 29 and
beyond. Patients who received one, two or three exposures to 17-1A
had similar degrees by HAMA response as summarized in Table 4.
3TABLE 3 HUMAN ANTI-MOUSE ANTIBODY (HAMA) RESPONSE* Pre-therapy
0/20 had antibody (range - 0-15 ng/ml) Day 8 11/20 had antibody
(range - 36-1106 ng/ml) Day 15 15/20 had antibody (range - 27-5598
ng/ml) Day 22 14/20 had antibody (range - 60-5046 ng/ml) Day 29
15/20 had antibody (range - 23-4900 ng/ml) No antibody 3/20
*Antibody activity expressed as ng of .sup.125I-17-1A bound/ml
Plasma.
[0030]
4TABLE 4 DEGREE OF HUMAN ANTI-MOUSE ANTIBODY (HAMA) RESPONSE*
Exposures Very High (>1000) Moderate (40-999) Poor/none (<40)
Single 3 (60%) 1 (20%) 1 (20%) Double 4 (40%) 4 (40%) 2 (20%)
Triple 2 (40%) 2 (40%) 1 (20%) *Expressed as ng of 17-1A bound/ml
plasma
[0031] The two patients with anaphylactic reactions were
interesting. They has HAMA levels of 1055 and 264 ng/ml on Day 8
and 1716 and 3745 ng/ml on Day 15, respectively. They tolerated
their infusions of antibody on Day 8 without adverse effect except
for nausea and vomiting (similar to what they had on Day 1
infusion) but had anaphylactic reactions on Day 15 at the time of
their third infusion. A total of 11 infusions were administered to
patients when their HAMA levels were greater than 20 ng/ml
(elevated) with five having no side effects, three gastrointestinal
symptoms, one flushing/tachycardia and two anaphylactic reactions.
No patients developed fever, proteinuria or renal impairment.
[0032] It was also interesting that in nine of these 11 infusions,
adequate plasma samples were available to determine peak plasma
concentration and plasma half-life of 17-1A antibody. These values
were not substantially different than infusions in the absence of
detectable HAMA.
[0033] Discussion This phase I/II study of repeated administration
of 400 mg 17-1A monoclonal antibody provides several observations.
In general, the administration of antibody was well tolerated in
patients receiving one or two infusions. The mild gastrointestinal
symptoms were clearly related to antibody infusion and were not a
serious clinical problem. The pathogenesis of these symptoms is not
know but does not seem related to an allergic reaction since they
occurred just as frequently during a patient's first infusion as
compared to third infusion. They may be related to the ability of
this antibody of bind to normal gastrointestinal mucosa (Sears, H.,
et al., Surg. Res. 31, 145-150 (1981)). Two of five patients
receiving three weekly infusions of 17-1A had anaphylactic
reactions. This frequency of a potentially lifethreatening allergic
reaction precluded our testing a four-dose schedule (weekly) and
would deter treatment schedules requiring antibody administration
on Day 15.
[0034] The pharmacokinetic studies indicate that this dose of
antibody can achieve plasma concentrations of 100-200 ug/ml with a
plasma disappearance curve approximating observations with other
mouse monoclonal antibodies (radiolabeled) administered at much
lower doses (Pimm, M., et al., J. Nucl. Med. 26, 1011-1023 (1985)
and Rosenblum, M., et al., Cancer Res. 45, 2382-2386 (1985)). This
plasma half-life results in plasma concentrations of less than 1
ug/ml by day 8. Thus, maintenance of a substantial plasma
concentration of 17-1A would require administration more frequently
than weekly. Prior studies (Pimm, M., et al., J. Nucl. Med. 26,
1011-1023 (1985) and Larson, S., et al., J. Nucl. Med. 24:123-129
(1983)) have suggested that the appearance of HAMA response is
associated with a dramatic alteration in circulating levels of
mouse Ig. Our failure to observe this phenomenon is somewhat
surprising. However, it should be noted that our antibody
measurements are expressed in terms of ng 17-1A bound/ml plasma
with an infusion of 17-1A which readily achieves concentrations of
100-200 ug/ml in the circulation. We are currently modifying our
HAMA assay to allow quantitation of total circulating HAMA. This
may clarify whether the patient's total circulating HAMA is able to
bind only a small fraction of this large circulating dose of
17-1A.
[0035] We did not find evidence of pre-existing human anti-mouse
antibody (17-1A) prior to antibody infusion as reported by others
(Schroff, R., et al., Cancer Res. 45, 879-885 (1985)). We initially
attempted to assay for human anti-mouse antibody using an assay
which detected human immunoglobulin binding to 17-1A coated beads
using radiolabeled monoclonal mouse anti-human Fc antibody. We
found that normal individuals and cancer patients prior to
monoclonal antibody infusion had varying quantities of human
immunoglobulin which bound non-specifically to 17-1A coated beads.
This binding did not have classic competitive inhibition by soluble
antigen (17-1A) and was judged to be a non-specific phenomenon and
not antibody. In contrast, post-immunization plasma immunoglobulin
binding to 17-1A coated beads was readily inhibited by soluble
antigen (17-1A). Thus, we believe that the double antigen assay
system used in this study more clearly reflects immune response to
17-1A. Despite the administration of one or more large doses of
17-1A, human antibody response to this protein was prompt with
antibody frequently detectable by Day 8 and appreciable levels of
antibody achieved by Day 15 & 22. Further studies are underway
to characterize this antibody response in regards to immunoglobulin
subclass and anti-idiotype.
BIOCHEMICAL AND EPITOPE ANALYSIS OF 17-1A ANTIGEN/PRODUCTION OF
MONOCLONAL ANTIBODIES AGAINST 17-1A.
Materials and Methods
[0036] Cells and Tissues
[0037] The human colon carcinoma cell lines DLD-1 and WiDr were
obrtained form the American Type Culture Collection, Rockville, Md.
The colon carcinoma line HT-29 was kindly provided by Dr. J. Fogh,
Sloan Kettering Institute for Cancer Research, NY. Human tissues
were snap-frozen in liquid nitrogen-cooled isopentane immediately
after surgical removal.
[0038] Radiolabelling and Immunoprecipitation
[0039] Cells (5.times.10.sup.7) were surface-labelled by
lactoperoxidase-mediated iodination using .sup.125I as described.
Pink, J. R. L. and Ziegler, A. (1979) in: Research Methods in
Immunology Academic Press, N.Y. pp. 169-180. Immunosorbents
prepared by binding MAbs to protein A-Sepharose (Sigma, St. Louis,
Mo.) were added to labelled cell lysates at 4.degree. C. for 2 hr.
Bound material was eluted by boiling in sample buffer and analyzed
by SDS-polyacrylamide gel electrophoresis (SDS-PAGE) according to
Laemmli. Laemmli, U.K. (1970) Nature, 227: 680-685.
[0040] Selection of MAbs
[0041] Colon carcinoma tissue was obtained from a 51 year old
female patient undergoing lobectomy of the liver because of a
solitary metastasis. The 17-1A positive tumor tissue was carefully
isolated, minced, homogenized and plasma membranes were purified as
described by Touster et al. (1970) J. Cell Biol. 47 604-618. Fusion
of the myeloma P3.times.63Ag8.653 and spleen cells from
(C57BL/6.times.Balb/c)F1 female mice was carried out using standard
procedures (Galfre et al., (1977) Nature 266: 550-552) 3 days after
a single i.p. injection of colon carcinoma plasma membrane
preparations corresponding to 3 mg of protein together with
Bordetella pertussis adjuvant. After fusion the cells were plated
in HAT selective medium (hypoxanthine, aminopterin, thymidine) in
96-well microtiter plates containing mouse peritoneal macrophages.
Supernatants of hybrids were screened by immunoperoxidase staining
of frozen tissue sections derived from the liver metastasis taken
for immunisation. Antibodies reacting with the colon carcinoma
cells of the metastasis and the bile ducts in the adjacent liver,
but not with hepatocytes, were further tested on a panel of
non-malignant epithelial tissues listed in Table 5. Antibodies
showing a 17-1A-like staining pattern in this context were cloned
at least two times by limiting dilution.
5TABLE 5 DISTRIBUTION OF 17-1A ANTIGEN IN NORMAL EPITHELIAL
TISSUES.sup.1 Number tested/ Tissue Number positive Remarks Colon
14/14 strong staining of mucosa Small intestine 5/5 strong staining
of mucosa Stomach 6/9 occasional weak staining of restricted areas
Gall bladder 1/1 strong staining of mucosa Pancreas 1/1 strong
staining of acini, ducts and islets of Langerhans Liver 6/6 strong
staining of bile ducts; hepatocytes negative Kidney 2/2 Loops of
Henle moderately, distal tubules strongly stained; proximal
tubules, glomeruli negative Lung 2/2 strong staining of bronchi;
alveoli moderately stained Thyroid gland 4/4 strong staining of
follicular epithlium Mammary gland 3/3 strong staining of lobules
and ducts Thymus 0/1 Skin 2/2 strong staining of sweat glands;
epidermis negative .sup.1as verified by immunohistochemical
staining with MAbs 17-1A, M72, M74, M77 and M79, which showed a
congruent staining pattern in all of the normal tissues
examined.
[0042] Immunoblotting
[0043] Immunoprecipitates were separated by SDS-PAGE,
electrophoretically transferred to nitrocellulose membranes
(Schleicher & Schull, Dassel, FRG) according to Towbin et al.
(1979) Proc. Natl. Acad. Sci. USA 76: 4350-4354, and transferred
antigen was visualized by an indirect immunoperoxidase technique,
Holzmann, B. et al. (1985) J. Exp. Med. 161: 366-377.
[0044] Flow cytometric analysis
[0045] HT-29 cells were preincubated on ice with MAbs M72, M74, M77
or M79 as unconcentrated, 10.times., or 50.times. concentrated
supernatant, followed by incubation with biotinylated 17-1A
antibody (10 ug/ml) and avidin-phycoerythrin (Becton-Dickinson,
Mountain View, Calif.). The fluorescence profile was analyzed with
an EPICS-V (Coulter Electronics, Hialeah, Fla.).
[0046] Immunohistochemistry
[0047] Frozen tissue sections were prepared and stained by an
indirect immunoperoxidase technique essentially as described
elsewhere. Gottlinger et al., (1985) Int. J. Cancer 35: 199-205.
Briefly, air-dried sections (7 um) were fixed in acetone for 10
min., incubated with MAb (10 ug/ml or undiluted supernatant) for 30
min., washing in PBS and exposed for 30 min. to
peroxidase-conjugated rabbit anti-mouse Ig antiserum (Dianova,
Hamburg, FRG) diluted 1/200 in PBS containing 20% human serum.
After washing in PBS the sections were incubated for 20 min. in
0.004% 3-amino-9-ethylcarbazole in 0.02 M barbital buffer, pH 7.4,
containing 0.001% H.sub.2O.sub.2 and subsequently counterstained
with Mayer's hemalum.
RESULTS
Biochemical Analysis of the 17-1A Ag
[0048] Since the original biochemical analysis of the 17-1A Ag was
only made for one human tumor cell line (HT-29), we investigated
the nature of the 17-1A Ag expressed on two additional human colon
carcimona lines by surface iodination. Precipitation with MAb 17-1A
revealed an identical single protein band in the three cell lines,
DLD-1, WiDr and HT-29, which migrated with an apparent molecular
weight of 37 kD in an SDS-PAGE system. As judged from fluorographic
intensity, the amounts of antigen precipitable from the three cell
lines were quite variable, with the colon carcinoma line DLD-1
providing the highest amount of radiolabelled antigen. Under
reducing conditions, in which 2-mercaptoethanol was added to the
precipitates prior to electrophoresis, a distinct band of 33 kD was
obtained from all three cell lines. In addition, a major component
of about 40 kD component was also found occasionally in HT-29
lysates. This band was apparently absent or could not be
precipitated from WiDr cells.
[0049] Incubation of HT-29 cells with tunicamycin (2 ug/ml) for 24
hr resulted in the appearance of a new band of 30 kD under
non-reducing and of 26 kD under reducing conditions, indicating
that the 17-1A Ag contains 2 N-linked glycosylation sites. The
glycoprotein nature of the 17-1A Ag was further substantiated by
treatment of 17-1A precipitates with neuraminidase, which resulted
in a slight but distinct reduction of the apparent molecular
weight.
Epitope Analysis of the 17-1A Ag by New MAbs
[0050] Four new MAbs directed against the 17-aA Ag (M72, M74, M77,
M79) were obtained by screening supernatants of hybridomas
generated from mice immunized with membrane preparations from colon
carcinoma metastases for a 17-1A-like reactivity on frozen tissue
sections. All four antibodies precipitate proteins of identical
molecular weight as seen with the original 17-1A antibody. In order
to verify the identity of the antigens recognized by the new MAbs
an extensive immunoblotting analysis was performed, whereby 17-1A
immunoprecipitates were transferred to nitrocellulose paper after
separation by SDS-PAGE and tested with the four new reagents. As
shown previously, the four new antibodies bound to a 37 kD protein
was not obtained with isotype-matched control MAbs. To analyze the
epitope specificity of the four new MAbs cross-blocking experiments
were carried out. In a flow cytometry analysis the binding of a
biotinylated 17-1A antibody to HT-29 cells was completely blocked
by preincubation of the tumor cells with MAbs M72 and M74 (Table
6). In contrast, MAbs M77 and M79 showed no significant blocking
activity for the biotinylated 17-1A antibody at all concentrations
tested. We further analyzed whether the epitope specificty could be
related to the idiotypes of the antibodies. All MAbs were analyzed
for reactivity with an anti-idiotypic antiserum generated against
the 17-1A antibody in goats (kindly provided by Dr. Dorothy Herlyn,
The Wistar Institute, Philadelphia). The anti-idotypic antiserum
strongly reacted with the two cross-blocking MAbs (M72 and M74),
but was completely unreactive with the MAbs M77 and M79 (Table 6).
These data suggest that mAbs 17-1A, M72 and M74 recognize the same
or closely related epitopes on the 37 kD glycoprotein, while MAbs
M77 and M79 define additional epitopes on this antigen.
6TABLE 6 IDIOTYPE EXPRESSION AND CROSS-BLOCKING ACTIVITY OF
DIFFERENT MABS DIRECTED AGAINST THE 17-1A ANTIGEN 17-1A M72 M74 M77
M79 Reactivity with +.sup.a ++ + - - goat anti-Id 17-1A
Cross-blocking +.sup.b ++ ++ - - activity .sup.ain an ELISA, where
the MAbs were coated to microtiter plates and subsequent binding of
the goat anti-idiotypic antiserum to the MAbs was revealed by an
indirect immunoperoxidase technique. +: optical density (OD) >
0.8; ++: OD > 1.6; -: OD < 0.05 .sup.bInhibition of binding
of biotinylated 17-1A antibody tested in a flow cytometric analysis
(see Materials and Methods): +: >25%, ++: >80%, -:
<10%
Tissue Distribution of the 17-1A Ag
[0051] Using an indirect immunoperoxidase technique the 17-1A Ag
was identified in various normal human organs and in a variety of
human carcinomas. Concomitantly the four new MAbs were analyzed on
parallel tissue sections. With respect to expression of the 17-1A
Ag on colon tissue, it was found that normal mucosa was stained to
a similar degree as colon carcinoma tissue in the fourteen patients
examined. The 17-1A Ag was clearly detectable on the epithelial
lining of the small intestine, the gall bladder, the bronchi and a
variety of glandular structures, including the tyroid, the mammary
gland, sweat glands and the exocrine as well as endocraine
pancreas. Furthermore, the 17-1A Ag was found to be expressed in
the kidney on distal tubules and the loops of Henle and in the
liver by bile ducts, but not by hepatocytes.
[0052] In the stomach, normal mucosa usually showed a faint
staining restricted to defined areas. However, in five patients
with various degrees of intestinal metaplasia, present in an
islet-like fashion in the gastric mucosa, those lesions were
strongly stained by MAb 17-1A and the four new MAbs. The 17-1A Ag
was also distinctly expressed in 9 of 9 gastric carcinomas
tested.
DISCUSSION
[0053] We have demonstrated that the 17-1A Ag is a glycoprotein
migrating in SDS-PAGE with an apparent molecular weight of 37 kD
under non-reducing conditions. Comparative analysis of three
different colon carcinoma lines did not indicate a hetereogeneity
of the 17-1A Ag when non-reducing conditions were applied. After
reduction of the 17-1A precipitates with 2-mercaptoethanol and
separation by SDS-PAGE a 33 kD band appeared which could be found
with all the cell lines tested. In addition, a 40 kD band could be
seen as a major component in the DLD-1 cell lysate and to a lesser
degree in the lysate of HT-29 cells. This 40 kD band was virtually
absent in lysates from WiDr cells. When precipitates from DLD-1 or
HT-29 cells were processed in single experiments and analyzed in a
parallel fashion under reducing and non-reducing conditions, again
two bands of 33 and 40 kD appeared in the presence, but only a
single band of 37 kD in the absence of 2-mercaptoethanol. Thus,
reduction of the 17-1A Ag may result in the formation of two new
forms of the molecule with different electrophoretic mobilities.
Most probably the presence of intrachain disulfide bonds may
account for this peculiar migration behavior. Alternatively, the
17-1A Ag may actually exist as a dimer of proteins exhibiting the
same migration behavior under non-reducing conditions. The absence
of the 40 kD molecule in WiDr lysates may then be explained by a
differential accessability of this protein for
lactoperoxidase-mediated iodination in different cell lines.
Further analysis using metabolic labelling and cross-linking
chemicals may be required to solve this question. Ross et al. have
recently described a carcinoma-associated surface glycoprotein
defined by their MAb GA733, that consists of 30 kD and 40 kD
subunits. They suggest that the 17-1A antibody recognizes the same
antigen, but binds to a different epitope than MAb GA733. Direct
comparisons with the antibodies described here should reveal the
relationship of these antigens.
[0054] The four new anti-17-1A Ag MAbs which we obtained displayed
a tissue reactivity comparable to the original 17-1A antibody in
normal organs and in tumors. So far no differential epitope
expression was found in a variety of tissues examined. Two of these
MAbs (M72 and M74) recognize a determinant closely related to that
detecterd by MAb 17-1A as judged by cross-blocking experiments,
whereas the MAbs M77 and M79 apparently define at least one
additional epitope since they did not inhibit binding of the
biotinylated 17-1A antibody. Interestingly, the epitope
spcecificity of these MAbs was correlated with their reactivity
with a goat anti-idiotypic antiserum. These MAbs will be of
interest for analyzing their biological activities in conjunction
with the original 17-1A antibody.
[0055] The 17-1A Ag is broadly expressed in non-malignant
epithelial organs and is also present in most carcinomas
originating from 17-1A-positive tissues. Quantitative differences
in expression between normal and malignant tissue were not evident
in a comparative immunohistochemical analysis of colorectal
carcinomas and normal mucosa obtained from the same patietns.
However, a possible structural heterogeneity of this epithelial
antigen on normal and malignant cells is not ruled out by the
present analysis.
* * * * *