U.S. patent number RE39,760 [Application Number 09/633,034] was granted by the patent office on 2007-08-07 for monoclonal antibodies against human colon carcinoma-associated antigens and uses therefor.
This patent grant is currently assigned to International Bio-Immune Systems Inc.. Invention is credited to Myron Arlen, Kwong Y. Tsang.
United States Patent |
RE39,760 |
Tsang , et al. |
August 7, 2007 |
Monoclonal antibodies against human colon carcinoma-associated
antigens and uses therefor
Abstract
Monoclonal antibodies, in particular 33.28 and 31.1, and
chimeric antibodies, in particular mouse/humans chimeric Chi #1
specific for glycoprotein antigens of colon carcinoma-associated
antigens which are immunogenic in humans, are disclosed. Such
antibodies, and fragments and derivatives thereof, are useful in
immunodiagnosis and immunotherapy of human colon, breast, and
ovarian cancer, and for purification of antigens which can serve as
immunotherapeutic agents. Methods of detecting the colon
carcinoma-associated antigen in a sample, and methods for treating
subjects having colon, breast, and ovarian carcinomas are
disclosed.
Inventors: |
Tsang; Kwong Y. (Bethesda,
MD), Arlen; Myron (Great Neck, NY) |
Assignee: |
International Bio-Immune Systems
Inc. (Great Neck, NY)
|
Family
ID: |
38324488 |
Appl.
No.: |
09/633,034 |
Filed: |
August 4, 2000 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
Issue Date |
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08159836 |
Nov 30, 1993 |
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08117430 |
Sep 7, 1993 |
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07670816 |
Mar 18, 1991 |
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07176337 |
Mar 31, 1988 |
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Reissue of: |
08304524 |
Sep 12, 1994 |
05688657 |
Nov 18, 1997 |
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Current U.S.
Class: |
435/7.23;
435/325; 435/328; 435/329; 435/330; 435/332; 435/344; 435/40.51;
435/40.52; 435/7.1; 435/7.2; 530/387.1; 530/387.3; 530/387.5;
530/387.7; 530/388.1; 530/388.8; 530/391.1; 530/391.3;
530/391.7 |
Current CPC
Class: |
C07K
16/3046 (20130101); G01N 33/57419 (20130101); C07K
2317/732 (20130101) |
Current International
Class: |
G01N
33/574 (20060101); C07K 16/18 (20060101); C07K
16/30 (20060101); G01N 33/53 (20060101) |
Field of
Search: |
;530/388.8,387.1,387.3,387.5,387.7,388.1,391.1,391.3,391.7
;435/7.1,7.2,40.51,40.52,325,328,329,330,332,347,7.91,7.92,188 |
References Cited
[Referenced By]
U.S. Patent Documents
Foreign Patent Documents
Other References
Koprowski et al PNAS 74:2985-2988, 1977. cited by examiner .
Paul Fundamental Immunology Raven Press, NY, Chapter 8 p. 242,
1993. cited by examiner .
Harlow et al., Antibodies, A Laboratory Manual, Cold Spring Harbor
Laboratory, pp. 61, 67, 71, 92-93, 1988. cited by examiner .
Hollinshead et al ., Cancer 56:480-89, 1985. cited by examiner
.
Herlyn et al., PNAs 76:1138, 1979. cited by examiner .
Price etal., IRCS Journal of Medical Science 13:366, 1985. cited by
examiner .
Nakao et al., 1997 Am J Gastroenterol. Clinical application of a
new monoclonal antibody (19B7) against PIVKA-II in the diagnosis of
hepatocellular carcinoma and pancreatobiliary malignancies.
92(6):1031-4. cited by other .
Wolf et al., 1993, J. Immunol. 150(8):p142A; Abstract No. 806
Immunohistological and Flowcytometry studies using monoclonal
antibody. cited by other .
Wolf et al., 1993, Immunoreactivity of cell lines and colon
carcinomas with monoclonal antibodies PCA 31.1 and PCA 33.28. Mol.
Biol. Cell 4 (supp): p334A; Abstract 1940. cited by other .
Wolf et al., 1993, Monoclonal antibody 31.1 may differentiate
indolent from aggressive colon cancer. Proc. Am. Assoc. Cancer Res.
34: p452 ; Abstract 2695. cited by other .
Hurwitz et al., 1992, Immunotargeting of a Daunomycin to localized
and metastatic human colon adenocarcinoma in athymic mice. Cancer
Immunol. Immunother. 35:186-192. cited by other .
Kuroki et al., 1992, Reaction profiles of seven enzyme immunoassay
kits for carcinoembryonic antigen (CEA) analysed with purified
preparations of CEA and related normal antigens. Clin. Biochem.
25:29-35. cited by other .
Bartal et al., 1990, Monoclonal antibody defining an antigen
present within a purified tumor membrane vaccine proc. American
Assoc. Cancer Res. 31: p260, Abstract No. 1537. cited by other
.
Tsang, 1987, Monoclonal antibodies to human colon
carcinoma-associated antigens FASEB 46(3):1058, Abstract No. 4322.
cited by other .
Hughes et al., (1986) Antigen expression in normal and neoplastic
colonic mucosa: three tissue-specific antigens using monoclonal
antibodies to isolated colonic glands. Cancer Res. 46:2164-2171
(.sup..sctn.). cited by other .
Proc. Am. Assoc. Cancer Res. 1994, Mar. vol. 35, Abstract 103,
"Comparison of immunostaining of colonic neoplasms with PCA 31.1,
PCA 33.28, B72-3 and anti-CEA monoclonal antibodies". A.H. Bartal,
J. Wolf, H. Lichtig, E. Agranovsky, R. Levy, H. Kerner, V. Bychkov,
O. Saric, M. Wolff, Y. Hirshaut, M. Monizur, M. Arlen and L.
Pertschuk. cited by other .
J. Surg. Oncol. 1993, vol. 54, pp. 103-108, "Monoclonal Antibodies
and Their Role in Modulation of the Immune Response", Myron Arlen,
J.D. and Kwong Y. Tsang, Ph.D. cited by other .
Ann. New York Acad Sci 1993, vol. 690, pp. 374-375; "Identification
and Characterization of a Colon Tumor-Associated Antigen". M.
Arlen, A. C. Hollinshead and K. Y Tsang. cited by other .
Antib. Immunoconjug. Radiopharm 1991, vol. 4, pp. 895-905;
"Monoclonal Antibodies to Immunoreactive Tumor Associated Antigen
(TAA) from Human Colon Carcinoma". M. Arlen, K.Y. Tsang, A. Bartal,
J. Wolf and O. Saric. cited by other .
FASEB J, 1991, vol. J5, No. 4, Abstract 2109; "Construction of a
Mouse/Human Chimeric Light Chain From Murine Monoclonal Antibody
33.28 Against Colorectal Carcinoma-Associated Antigens". D.L. Xu,
S. Wei, K.Y. Tsang. cited by other .
) J. Tumor Marker Oncol. 1990, Vol. 5, No. 4, pp. 313-319; "The
Nature of the Monoclonal Antibodies Derived from Immunogenic
Membrane Antigen of Human Colon Carcinoma Origin". Myron Arlen and
Alfred K. Tsang. cited by other .
FASEB J., 1988, vol. J2, No. 5, Abstract 6815; "Colon Carcinoma
Associated Antigens Characterized by Monoclonal Antibodies". Kwong
Y. Tsang, M.F. LaVia, L. Bishop, D.L. Xu, E. Johnson and M. Arlen.
cited by other .
Bartal, A. H et al, Proc. Am Assoc. Can Res, 31:260 (Ast#1537),
Mar. 1990. cited by other .
Price, M.R et al, IRCS J Med Sci, 13(4):366-367, Apr. 1985. cited
by other .
Herlyn, M et al, PNAS, 76(3):1438-1442, Mar. 1979. cited by other
.
Tsang, K.Y. et al, Fed Proc, 46(3):1058 (Abs#4322), Mar. 1987.
cited by other .
Xu, Danlin, Dissertation (Degree 1990) order #9109383, pp. 1-86,
1990. cited by other .
Kanellos, J et al, JNCI, 75(2):319-332, Aug. 1985. cited by other
.
Shaw, D.R et al, J Immunol, 138:4534-4538, Jun. 15, 1987. cited by
other .
Tsang, K.Y et al, FASEB, J2(5):A6815, 1988. cited by other .
Hollinshead, A., et al., Science. 177: 887-889 (1972). cited by
other .
Von Kleist, S., et al., Proc. Natl. Acad. Sci. USA 69: 2492-2494
(1972). cited by other .
Hollinshead, A.C., J. Nat. Cancer Instit. 52: 327-338 (1974). cited
by other .
Hollinshead, A.C., Cancer, 34: 1235-1243 (1974). cited by other
.
Hollinshead, A.C., Lung Cancer Progress in Therapeutic Research,
pp. 501-520, Muggia, F., Rosencweig, M. (ed.), Raven Press, 1979,
no date. cited by other .
Herlyn, M., et al. Proc. Natl. Acad. Sci. USA 79: 1438-1442 (1979).
cited by other .
Hollinshead, A. et al., Tumor Progression, pp. 289-300, Crispen
(ed) (1980). cited by other .
Herlyn, M., et al. J. Clinical Immunol. 2: 135-140 (1982). cited by
other .
Hollinshead, A.C., et al., Cancer 49: 1387-1404 (1982). cited by
other .
Moldofsky, P.J., et al., Radiology 149: 549-555 (1983). cited by
other .
Buchegger, F., et al., J. Experimental Medicine 158: 413-427
(1983). cited by other .
Takita, J., et al., Cancer Immunol. Immunother. 20:231-235 (1985).
cited by other .
Price, M.R., et al., IRCS Med. Sci. 13: 366-367 (1985). cited by
other .
Kanellos, J., et al., JNCI 75: 319-332 (1985). cited by other .
Hollinshead, A., et al., Cancer 56: 480-489 (1985). cited by other
.
Stewart, T.H.M., et al., Lung Cancer: Current Status and Prospects
for the Future--Ann. Clin. Conf. on Cancer 28:351-374, 1986, no
date. cited by other .
Tsang, K., et al., JNIC 77: 1175-1180 (1986). cited by other .
Douillard, J.Y., et al., Hybridoma 5: 5139-5149 (1986). cited by
other .
Hollinshead, A., Biologic Drugs, Vaccines, Current Status and
Future Directions, pp. 86-103, Springer Verlag (1986). cited by
other .
Tsang, K., et al., Fed Proc. 46:4322 (1987). cited by other .
Shaw, D.R., et al., J. Immunology 138: 4532-4538 (1987). cited by
other .
Tsang, K., et al., Cancer Detection and Prevention 11: 094 (1987).
cited by other .
Greiner, J.W., et al., Science 235: 895-898 (1987). cited by other
.
Tsang, K., et al., Fed. Proc. 6815 (1988). cited by other .
Tsang, K., et al., 7th International Congress of Immunology,
Abstract 125-46 (1989). cited by other .
Bartel, A., et al., Proceedings 31: Abstract 1537 (1990). cited by
other .
Arlen, M. and Tsang, K., J. Tumor Marker Oncology 5: 313-319
(1990). cited by other .
Hirschfield, L.S., et al., Federation of American Societies for
Experimental Biology, Abstract 5708 (1991). cited by other .
Xu, D.L., et al., Fed. Amer. Soc. Exper. Biol., Abstract 2109
(1991). cited by other .
Hollinshead, A.C. and Stewart, T.H.M., Yale Journal of Biology and
Medicine 54: 367-379 (1981). cited by other .
Sears, H.F., et al., J. Clinical Immunol. 2: 141-149 (1982). cited
by other .
Arlen, M., et al., Antibody, Immunoconjugates, and
Radiopharmaceuticals, 4: 895-905 (1991). cited by other .
Arlen, M., et al., Journal of Surgical Oncology 54: 103-108 (1991).
cited by other .
Arlen, M., et al., NY Academy (cancer vaccines) 603-605 (1993).
cited by other .
Fernado, A.D. et al., Miami Symp. Short Reports 3: 88 (1993). cited
by other .
Girardet, C., et al., J. Immunology 136: 1497-1503 (1986). cited by
other .
Chong, G., F. T. Lee, et al. (2005). "Phase I Trial of 131I-huA33
in Patients with Advanced Colorectal Carcinoma." Clin Cancer Res
11(13): 4818-26. cited by other .
Koppe, M. J., R. P. Bleichrodt, et al. (2005). "Radioimmunotherapy
and colorectal cancer." Br J Surg 92(3): 264-276. cited by other
.
Mallegol, J., G. van Niel, et al. (2005). "Phenotypic and
functional characterization of intestinal epithelial exosomes."
Blood Cells Mol Dis 35(1): 11-16. cited by other .
Scott, A. M., F. T. Lee, et al. (2005). "A Phase I Trial of
Humanized Monoclonal Antibody A33 in Patients with Colorectal
Carcinoma: Biodistribution, Pharmacokinetics, and Quantitative
Tumor Uptake." Clin Cancer Res 11(13): 4810-4817. cited by other
.
Deckert, P. M., W. G. Bornmann, et al. (2004). "Specific tumour
localisation of a huA33 antibody--carboxypeptidase A conjugate and
activation of methotrexate-phenylalanine." Int J Oncol 24(5):
1289-95. cited by other .
Joosten, C. E., L. S. Cohen, et al. (2004). "Glycosylation profiles
of the human colorectal cancer A33 antigen naturally expressed in
the human colorectal cancer cell line SW1222 and expressed as
recombinant protein in different insect cell lines." Biotechnol
Prog 20(4): 1273-9. cited by other .
Deckert, P. M., C. Renner, et al. (2003). "A33scFv-cytosine
deaminase: a recombinant protein construct for antibody-directed
enzyme-prodrug therapy." Br J Cancer 88(6): 937-9. cited by other
.
Mao, Z., S. Song, et al. (2003). "Transcriptional regulation of A33
antigen expression by gut-enriched Kruppel like factor." Oncogene
22(28):4434-43. cited by other .
Welt et al., 2003, Phase I Study of Anticolon Cancer Humanized
Antibody A331. Clinical Cancer Res. 9(4):1338:1346. cited by other
.
Welt, S., G. Ritter, et al. (2003). "Preliminary report of a phase
I study of combination chemotherapy and humanized A33 antibody
immunotherapy in patients with advanced colorectal cancer." Clin
Cancer Res 9(4): 1347-53. cited by other .
Johnstone, C. N., S. J. White, et al. (2002). "Analysis of the
regulation of the A33 antigen gene reveals intestine-specific
mechanisms of gene expression." J Biol Chem 277(37): 34531-9. cited
by other .
Orlova, A., J. Hoglund, et al. (2002). "Comparative biodistribution
of the radiohalogenated (Br, I and At) antibody A33. Implications
for in vivo dosimetry." Cancer Biother Radiopharm 17(4): 385-96.
cited by other .
van Niel, G. and M. Heyman (2002). "The epithelial cell
cytoskeleton and intracellular trafficking. II. Intestinal
epithelial cell exosomes: perspectives on their structure and
function." Am J Physiol Gastrointest Liver Physiol 283(2): G251-5.
cited by other .
Barendswaard, E. C., J. L. Humm, et al. (2001). "Relative
therapeutic efficacy of (125)I- and (131)I-labeled monoclonal
antibody A33 in a human colon cancer xenograft." J Nucl Med 42(8):
1251-6. cited by other .
Lee, F. T., A. Rigopoulos, et al. (2001). "Specific localization,
gamma camera imaging, and intracellular trafficking of
radiolabelled chimeric anti-G(D3) and ganglioside monoclonal
antibody KM871 in SK-MEL-28 melanoma xenografts." Cancer Res
61(11): 4474-82. cited by other .
Ritter, G., L. S. Cohen, et al. (2001). "Serological analysis of
human anti-human antibody responses in colon cancer patients
treated with repeated doses of humanized monoclonal antibody A33."
Cancer Res 61(18):6851-9. cited by other .
van Niel, G., G. Raposo, et al. (2001). "Intestinal epithelial
cells secrete exosome-like vesicles." Gastroenterology 121(2):
337-49. cited by other .
Abud, H. E., C. N. Johnstone, et al. (2000). "The murine A33
antigen is expressed at two distinct sites during development, the
ICM of the blastocyst and the intestinal epithelium." Mech Dev
98(1-2): 111-4. cited by other .
Deckert, P. M., A. Jungbluth, et al. (2000). "Pharmacokinetics and
microdistribution of polyethylene glycol-modified humanized A33
antibody targeting colon cancer xenografts." Int J Cancer 87(3):
382-90. cited by other .
Johnstone, C. N., N. C. Tebbutt, et al. (2000). "Characterization
of mouse A33 antigen, a definitive marker for basolateral surfaces
of intestinal epithelial cells." Am J Physiol Gastrointest Liver
Physiol 279(3): G500-10. cited by other .
Rader, C., G. Ritter, et al. (2000). "The rabbit antibody
repertoire as a novel source for the generation of therapeutic
human antibodies." J Biol Chem 275(18): 13668-76. cited by other
.
Ruan, S., J. A. O'Donoghue, et al. (2000). "Optimizing the sequence
of combination therapy with radiolabeled antibodies and
fractionated external beam." J Nucl Med 41(11): 1905-12. cited by
other .
Sakamoto, J., H. Kojima, et al. (2000). "Organ-specific expression
of the intestinal epithelium-related antigen A33, a cell surface
target for antibody-based imaging and treatment in gastrointestinal
cancer." Cancer Chemother Pharmacol 46 Suppl: S27-32. cited by
other .
Barendswaard, E. C., J. A. O'Donoghue, et al. (1999). "131I
radioimmunotherapy and fractionated external beam radiotherapy:
comparative effectiveness in a human tumor xenograft." J Nucl Med
40(10): 1764-8. cited by other .
Miyazono, Y., Y. Kamogawa, et al. (1999). "Effect of
B7.1-transfected human colon cancer cells on the induction of the
autologous tumour-specific cytotoxic T cells." J Gastroenterol
Hepatol 14(10): 997-1003. cited by other .
Barendswaard, E. C., A. M. Scott, et al. (1998). "Rapid and
specific targeting of monoclonal antibody A33 to a colon cancer
xenograft in nude mice." Int J Oncol 12(1): 45-53. cited by other
.
Burgess, A. W. (1998). "Growth control mechanisms in normal and
transformed intestinal cells." Philos Trans R Soc Lond B Biol Sci
353(1370): 903-9. cited by other .
Moritz, R. L., G. Ritter, et al. (1998). "Micro-sequencing
strategies for the human A33 antigen, a novel surface glycoprotein
of human gastrointestinal epithelium." J Chromatogr A 789(1-2):
91-101. cited by other .
Tio, T. L. (1998). "Diagnosis and staging of esophageal carcinoma
by endoscopic ultrasonography." Endoscopy 30 Suppl I: A33-40. cited
by other .
Catimel, B., M. Nerrie, et al. (1997). "Kinetic analysis of the
interaction between the monoclonal antibody A33 and its colonic
epithelial antigen by the use of an optical biosensor. A comparison
of immobilisation strategies." J Chromatogr A 776(1): 15-30. cited
by other .
Heath, J. K., S. J. White, et al. (1997). "The human A33 antigen is
a transmembrane glycoprotein and a novel member of the
immunoglobulin superfamily." Proc Natl Acad Sci USA 94(2): 469-74.
cited by other .
Ji, H., R. L. Moritz, et al. (1997). "Electrophoretic analysis of
the novel antigen for the gastrointestinal-specific monoclonal
antibody, A33." Electrophoresis 18(3-4): 614-21. cited by other
.
Ritter, G., L. S. Cohen, et al. (1997). "Characterization of
posttranslational modifications of human A33 antigen, a novel
palmitoylated surface glycoprotein of human gastrointestinal
epithelium." Biochem Biophys Res Commun 236(3): 682-6. cited by
other .
Tschmelitsch, J., E. Barendswaard, et al. (1997). "Enhanced
antitumor activity of combination radioimmunotherapy (131I-labeled
monoclonal antibody A33) with chemotherapy (fluorouracil)." Cancer
Res 57(11): 2181-6. cited by other .
No Author listed. (1996). "MoAb A33 shows promise in targeting
colon cancer for radioimmunotherapy." Oncology (Huntingt) 10(4):
553. cited by other .
Antoniw, P., A. P. Farnsworth, et al. (1996). "Radioimmunotherapy
of colorectal carcinoma xenografts in nude mice with yttrium-90 A33
IgG and Tri-Fab (TFM)." Br J Cancer 74(4): 513-24. cited by other
.
Daghighian, F., E. Barendswaard, et al. (1996). "Enhancement of
radiation dose to the nucleus by vesicular internalization of
iodine-125-labeled A33 monoclonal antibody." J Nucl Med 37(6):
1052-7. cited by other .
Welt, S., A. M. Scott, et al. (1996). "Phase I/II study of iodine
125-labeled monoclonal antibody A33 in patients with advanced colon
cancer." J Clin Oncol 14(6): 1787-97. cited by other .
King, D. J., P. Antoniw, et al. (1995). "Preparation and
preclinical evaluation of humanised A33 immunoconjugates for
radioimmunotherapy." Br J Cancer 72(6): 1364-72. cited by other
.
Scott, A. M., E. Rosa, et al. (1995). "In vivo imaging and specific
targeting of P-glycoprotein expression in multidrug resistant nude
mice xenografts with [125I]MRK-16 monoclonal antibody." Nucl Med
Biol 22(4): 497-504. cited by other .
Welt et al., 1994, Phase I/II Study of Iodine 131-Labeled
Monoclonal Antibody A33 in Patients With Advanced Colon Cancer. J.
Clin. Oncol. 12:1561-71. cited by other.
|
Primary Examiner: Huff; Sheela J.
Attorney, Agent or Firm: Baker Botts LLP
Parent Case Text
This application is a continuation-in-part of U.S. application Ser.
No. 08/159,836 filed Nov. 30, 1993, which is a continuation-in-part
of U.S. application Ser. No. 08/117,430, filed Sep. 7, 1993, now
abandoned, which is a continuation-in-part of U.S. application Ser.
No. 07/670,816, filed Mar. 18, 1991, now abandoned, which is a
continuation-in-part of U.S. application Ser. No. 07/176,337, filed
Mar. 31, 1988, now abandoned.
Claims
What is claimed is:
1. A monoclonal antibody specific for a purified human colon
carcinoma-associated protein antigen, .[.wherein said antigen has
the following characteristics: (a) said antigen is purified to the
extent that the membrane fractions are free of HL-A antigen and are
substantially free from non-immunogenic glycoprotein fractions; (b)
said antigen is not detectable on normal colon cancer free human
tissues; (c) said antigen is not detectable on human carcinoma
cells other than colon carcinoma cells; (d) said antigen is
specifically immunogenic in humans; and (e) said antigen induces an
immune response in humans having colon carcinoma which is expressed
as cell mediated immunity.]. .Iadd.which is murine monoclonal
antibody 33.28 as produced by hybridoma PCA 33.28, deposited with
the American Type Culture Collection and assigned accession number
PTA-5413..Iaddend.
.[.2. An antibody according to claim 1 which is mouse monoclonal
antibody 33.28 (ATCC HB-12315) or an antibody which binds
specifically to a colon carcinoma-associated epitope that
specifically binds to monoclonal antibody 3328..].
3. An antibody according to claim .[.2.]. .Iadd.1 .Iaddend.wherein
said colon carcinoma-associated antigen is a protein having a
molecular weight of about 61.1 kilodaltons .Iadd.as measured by
gradient polyacrylamide gel electrophoresis..Iaddend.
4. .[.An antibody according claim 1.]. .Iadd.A monoclonal antibody
specific for a purified human colon carcinoma-associated protein
antigen,.Iaddend. which is mouse monoclonal antibody .Iadd.31.1, as
produced by hybridoma PCA .Iaddend.31.1 .[.(ATCC HB-12314).].
.Iadd., deposited with the American Type Culture Collection and
assigned accession number PTA-2497.Iaddend..[.or an antibody which
binds specifically to a colon carcinoma-associated epitope that
specifically binds to monoclonal antibody 31.1.]. .
5. .[.An.]. .Iadd.A monoclonal .Iaddend.antibody .[.according to
claim 4 wherein said colon carcinoma-associated antigen is a
protein having a molecular weight of about 72 kilodaltons.].
.Iadd.which comprises an antigen-binding region obtained from the H
chain of a murine monoclonal antibody 33.28 as produced by
hybridoma PCA 33.28, deposited with the American Type Culture
collection and assigned action number PTA-5413..Iaddend.
6. An antibody according to claim .[.2.]. .Iadd.1 .Iaddend.wherein
said colon carcinoma-associated antigen is a glycoprotien, the
protein component having a molecular weight of 61.1 kilodaltons
.Iadd.as measured by gradient polyacrylamide gel
electrophoresis.Iaddend..
7. An antibody according to claim 1.Iadd., 4 or 5
.Iaddend.immobilized on a solid phase.
8. An antibody according to claim 1.Iadd., 4 or 5 .Iaddend.which is
detectably labeled.
9. An antibody according to claim 8 wherein said detectable label
is a radiolabel.
10. An antibody according to claim 1.Iadd., 4 or 5
.Iaddend.conjugated to a cytotoxic radionuclide.
11. An antibody according to claim 1.Iadd., 4 or 5
.Iaddend.conjugated to a cytotoxic drug.
12. An antibody according to claim 1.Iadd., 4 or 5
.Iaddend.conjugated to a cytotoxic protein.
13. A composition comprising an antibody according to claim 10 in
combination with a pharmaceutically acceptable carrier.
14. A composition comprising an antibody according to claim 11 in
combination with a pharmaceutically acceptable carrier.
15. A composition comprising an antibody according to claim 12 in
combination with a pharmaceutically acceptable carrier.
.[.16. A monoclonal antibody against the monoclonal antibody of
claim 1..].
.[.17. A monoclonal antibody against the monoclonal antibody of
claim 2..].
.[.18. A monoclonal antibody against the monoclonal antibody of
claim 3..].
.[.19. A monoclonal antibody against the monoclonal antibody of
claim 4..].
.[.20. A monoclonal antibody against the monoclonal antibody of
claim 5..].
.[.21. A monoclonal antibody against the monoclonal antibody of
claim 6..].
22. An immunoassay for detecting a colon carcinoma-associated
antigen which binds to mouse monoclonal antibody 33.28 .[.(ATCC
HB-12315).]. .Iadd.as produced by hybridoma PCA 33.28, deposited
with the American Type Culture Collection and assigned accession
number PTA-5413, .Iaddend.in a sample comprising: (a) contacting
said sample with an effective binding amount of the antibody
according to claim 1; and (b) detecting said antigen by detecting
the binding of the antibody to the .[.purified.]. colon
carcinoma.Iadd.-.Iaddend.associated protein antigen.
23. An immunoassay for detecting a colon carcinoma-associated
antigen which binds to mouse monoclonal antibody 31.1 .[.(ATCC
HB-12314).]. .Iadd., as produced by hybridoma PCA 31.1, deposited
with the American Type Culture Collection and assigned accession
number PTA-2497, .Iaddend.in a sample comprising: (a) contacting
said sample with an effective binding amount of the antibody
according to claim .[.1.]. .Iadd.4 or claim 5.Iaddend.; and (b)
detecting said antigen by detecting the binding of the antibody to
the .[.purified.]. colon carcinoma.Iadd.-.Iaddend.associated
protein antigen.
24. A method for diagnosing colon cancer in humans comprising: (a)
removing a histological specimen from a patient suspected of having
a colon cancer; (b) contacting the specimen with monoclonal
antibody 33.28 .[.(ATCC HB-12315).]. .Iadd., as produced by
hybridoma PCA 33.28, deposited with the American Type Culture
Collection and assigned accession number PTA-5413.Iaddend.; (c)
staining the specimen with an immunohistochemical stain; and (d)
detecting the presence of the antigen-antibody complex by the
stain.
25. A method for diagnosing colon cancer in humans comprising: (a)
removing a histological specimen from a patient suspected of having
colon.[.-.]. carcinoma; (b) contacting the specimen with mouse
monoclonal antibody 31.1 .[.(ATCC HB-12314).]. .Iadd., as produced
by hybridoma PCA 31.1, deposited with the American Type Culture
Collection and assigned accession number PTA-2497).Iaddend.; (c)
staining the specimen with an immunohistochemical stain; and (d)
detecting the presence of the antigen-antibody complex.
26. A method according to claim 24 wherein the stain is an
avidin-biotin immunoperoxidase stain.
27. A method according to claim 25 wherein the stain is an
avidin-biotin immunoperoxidase stain.
28. A kit for the immunohistochemical detection of colon carcinoma
comprising: (a) mouse monoclonal antibody 31.1 .[.(ATCC
HB-12314).]. .Iadd., as produced by hybridoma PCA 31.1, deposited
with the American Type Culture Collection and assigned accession
number PTA-2497.Iaddend.; (b) reagents for immunoperoxidase and
secondary antibody; (c) immunoperoxidase; and (d) colorizing
reagents.
29. A kit for the immunohistochemical detection of colon carcinoma
comprising: (a) mouse monoclonal antibody 33.28 .[.(ATCC
HB-12315.]. .Iadd., as produced by hybridoma PCA 33.28, deposited
with the American Type Culture Collection and assigned accession
number PTA-5413.Iaddend.; (b) reagents for immunoperoxidase and
secondary antibody; (c) immunoperoxidase; and (d) colorizing
reagents.
.[.30. A compartmentalized kit for the detection of a human colon
carcinoma-associated antigen, wherein the antigen has the following
characteristics: (a) said antigen is purified to the extent that
the membrane fractions are free of HL-A antigen and are
substantially free from non-immunogenic glycoprotein fractions; (b)
said antigen is not detectable on normal colon cancer free human
tissues; (c) said antigen is not detectable on human carcinoma
cells other than colon carcinoma cells; (d) said antigen is
specifically immunogenic in humans; and (e) said antigen induces an
immune response in humans having colon carcinoma which is expressed
as cell mediated immunity, said kit comprising a first container
adapted to contain an antibody to said antigen or an active
component thereof, and a second container adapted to contain a
second antibody to said antigen or an active component thereof,
said second antibody being labeled with a reporter molecule capable
of giving a detectable signal..].
.[.31. A kit according to claim 30 wherein the reporter molecule is
a radioisotope, an enzyme, a fluorescent molecule, a
chemiluminescent molecule or a bioluminescent molecule..].
.[.32. A kit according to claim 30 wherein the reporter molecule is
an enzyme..].
.[.33. A kit according to claim 30 wherein the kit further
comprises a third container adapted to contain a substrate for the
enzyme..].
.[.34. A compartmentalized kit for the detection of a human colon
carcinoma-associated antigen, wherein the antigen has the following
characteristics: (a) said antigen is purified to the extent that
the membrane fractions are free of HL-A antigen and are
substantially free from non-immunogenic glycoprotein fractions; (b)
said antigen is not detectable on normal colon cancer free human
tissues; (c) said antigen is not detectable on human carcinoma
cells other than colon carcinoma cells; (d) said antigen is
specifically immunogenic in humans; and (e) said antigen induces an
immune response in humans having colon carcinoma which is expressed
as cell mediated immunity, said kit comprising a first container
adapted to contain monoclonal antibody 31.1 (ATCC HB-12314) to said
antigen and a second container adapted to contain a second antibody
to said antigen or an active component thereof, said second
antibody being labeled with a reporter molecule capable of giving a
detectable signal..].
.[.35. A kit according to claim 34 wherein the reporter molecule is
a radioisotope, an enzyme, a fluorescent molecule, a
chemiluminescent molecule or a bioluminescent molecule..].
.[.36. A kit according to claim 32 wherein the reporter molecule is
an enzyme..].
.[.37. A kit according to claim 33 wherein the kit further
comprises a third container adapted to contain a substrate for the
enzyme..].
.[.38. A compartmentalized kit for the detection of a human colon
carcinoma-associated antigen, wherein the antigen has the following
characteristics: (a) said antigen is purified to the extent that
the membrane fractions are free of HL-A antigen and are
substantially free from non-immunogenic glycoprotein fractions; (b)
said antigen is not detectable on normal colon cancer free human
tissues; (c) said antigen is not detectable on human carcinoma
cells other than colon carcinoma cells; (d) said antigen is
specifically immunogenic in humans; and (e) said antigen induces an
immune response in humans having colon carcinoma which is expressed
as cell mediated immunity, said kit comprising a first container
adapted to contain monoclonal antibody 33.28 (ATCC HB-12315) to
said antigen and a second container adapted to contain a second
antibody to said antigen or an active component thereof, said
second antibody being labeled with a reporter molecule capable of
giving a detectable signal..].
.[.39. A kit according to claim 38 wherein the reporter molecule is
a radioisotope, an enzyme, a fluorescent molecule, a
chemiluminescent molecule or a bioluminescent molecule..].
.[.40. A kit according to claim 38 wherein the reporter molecule is
an enzyme..].
.[.41. A kit according to claim 38 wherein the kit further
comprises a third container adapted to contain a substrate for the
enzyme..].
.[.42. The monoclonal antibody of claim 1 which is a chimeric
antibody..].
43. .[.The.]. .Iadd.A .Iaddend.chimeric antibody .[.according to
claim 42.]. which is a chimeric mouse/human antibody Chi #1
.Iadd.as produced by the cell line deposited with the American Type
Culture Collection and assigned accession number
.Iaddend..[.(ATCC.]. CRL-12316.[.).]. .
.[.44. The chimeric antibody according to claim 42 wherein said
colon carcinoma-associated antigen is a protein having a molecular
weight of 72 kilodalton..].
45. A composition comprising the chimeric antibody according to
claim .[.42.]. .Iadd.43 .Iaddend.in combination with a
pharmaceutically acceptable carrier.
.[.46. A monoclonal antibody against the chimeric antibody of claim
42..].
47. An immunoassay for detecting a colon carcinoma-associated
antigen which binds to the mouse/human chimeric antibody Chi #1
.Iadd.as produced by the cell line deposited with the American Type
Culture Collection and assigned accession number
.Iaddend..[.(ATCC.]. CRL-12316.[.) of claim 42.]. in a sample
comprising: (a) contacting said sample with the .Iadd.Chi #1
.Iaddend.antibody .[.according to claim 42.]. ; and (b) detecting
said antigen by detecting the binding of said antibody to the
.[.purified.]. colon carcinoma.Iadd.-.Iaddend.associated protein
antigen.
48. A method for diagnosing colon cancer in humans comprising: (a)
removing a histological specimen from a patient suspected of having
a colon carcinoma; (b) contacting the specimen with a chimeric
antibody .[.which binds to an antigen.]. according to claim .[.1.].
.Iadd.43.Iaddend.; (c) staining the specimen with an
immunohistochemical stain; and (d) detecting the presence of the
antigen-antibody complex by the stain.
49. A method for diagnosing colon cancer in humans comprising: (a)
removing a histological specimen from a patient suspected of having
a colon carcinoma; (b) contacting the specimen with mouse/human
chimeric antibody which binds to an antigen which binds to
mouse/human chimeric antibody Chi #1 .[.(ATCC.]. .Iadd.as produced
by the cell line deposited with the American Type Culture
Collection and assigned accession number .Iaddend.CRL-12316.[.).].
; (c) staining the specimen with an immunohistochemcial stain; and
(d) detecting the presence of the antigen-antibody complex by the
stain.
50. A kit for the immunohistochemical detection of colon carcinoma
comprising: (a) mouse/human chimeric antibody Chi #1 (ATCC
CRL-12316); (b) reagents for immunoperoxidase and secondary
antibody; .Iadd.(c) .Iaddend.immunoperoxidase; and (d) colorizing
reagents.
Description
BACKGROUND OF THE INVENTION
1. Field of the Invention
This invention, in the field of immunology and medicine, relates to
new hybridoma lines and the monoclonal antibodies (mAbs) they
secrete which are specific for clinically defined colon
carcinoma-associated antigens. The mAbs are useful in vivo for
immunodetection and immunotherapy of colon carcinoma as well as for
the detection and purification of colon carcinoma-associated
antigens.
2. Description of the Background Art
During the process of oncogenesis, a number of cell-surface
molecules or markers appear on cells. Such tumor-related markers
include oncofetoproteins, neoglycoproteins, sphignolipids, and
modifications of existing surface proteins. Such new or altered
structures are often shed from the tumor cell surface and appear in
the serum or in other biological fluids. The detection of any of
these substances or "tumor markers" serves as the basis for
diagnosing or monitoring the progress of neoplastic disease.
Early animal studies demonstrated that, among these tumor markers,
a subset of tumor membrane protein or glycoprotein antigens were
immunogenic. Upon appropriate reintroduction into the tumor-bearing
host, typically after surgical removal of the primary tumor, such
antigens could effectively block the establishment of new tumor
growth.
An attempt to use similarly derived tumor-associated antigens in
humans was made by Hollinshead and Stewart using a relatively
purified membrane preparation in patients with lung cancer
(Stewart, T. H. M. et al., Ann. N.Y. Acad. Sci. 277:436 (1976)).
These studies were later expanded to include patients with melanoma
and colon carcinoma, wherein different pooled allogeneic tumor
preparations were administered in combination with complete
Freund's adjuvant (Hollinshead, A. C. et al., Cancer 4:9:1387
(1982); Hollinshead, A. C. et al., Cancer 56:480 (1985)).
The use of Freund's adjuvant was based on observations that normal
tissue antigens with this adjuvant produced severe autoimmune
responses in animal recipients, whereas in the absence of this
adjuvant, no adverse reactions were seen. The adjuvant was thought
to promote antigen processing by host macrophages as well as
prolong the stimulatory action of the antigen at the site of its
deposition (see, for example, Roitt, L. Essential Immunology, 6th
Ed., Blackwell Scientific Publications, Oxford (1988)).
The above observations served as the basis for early clinical
trials using specific human tumor membrane proteins and
glycoproteins as tumor "vaccines." Various of the tumor-associated
antigens which had been isolated and characterized could prolong
survival and, in some cases, produce regression of metastatic
disease.
With the advent of monoclonal antibody (mAb) technology, it has
become possible to obtain pure antibody populations which permit
better purification and characterization of the various tumor
markers and tumor-associated antigens that are useful for
immunodiagnosis or immunotherapy. Many mAbs have been described
that have varying degrees of selectivity for tumor antigens (versus
normal cell surface markers); some of these tumor antigens are
broadly represented across several or many tumor types, whereas
others appear to be truly tumor-specific. A number of these
mAb-tumor antigen systems are described below.
Herlyn et al., Proc. Natl. Acad. Sci. USA 76:1438 (1979), discloses
two mAbs obtained by immunizing mice with human colorectal
carcinoma (CRC) cells. The mAbs have selective reactivity with
human CRC cells. One mAb, 1083-17 (the forerunner of 17-1A), is now
known to react with a 41 kDa glycoprotein (see below).
Herlyn et al., J. Clin. Immunol. 2:135 (1982), described the
detection of a circulating colorectal carcinoma (CRC)-associated
antigen by a mAb developed against a membrane antigen of the SW116
cell line. MAbs 19-9 and 52a, which recognize a
monosialoganglioside antigen (Magnani, J. L. et al., Science 212:55
(1981)), reacted with cells of 8 of 12 CRC lines as well as with
the cells of one gastric carcinoma and one pancreatic carcinoma.
MAb C.sub.414 reacted with four of six CRC cultures and with
gastric tumor cells. The binding of mAbs 19-9 and 52a to tumor
cells were inhibited by a CRC patient's serum. However, CRC sera
inhibited binding less frequently than did sera from patients with
pancreatic or gastric tumors.
Girardet et al., J. Immunol. 136:1497-1503 (1986), disclosed mAbs
against human colon carcinomas. The L-D1 mAb reacted with a 41 kDa
glycoprotein, believed to be the same antigen as that defined by
mAb 1083-17-1A (Herlyn et al., 1979, supra). The L-C5 mAb
precipitated proteins having molecular weights of 43, 45, 47 and 53
kDa from LoVo colon carcinoma cells. L-D1 did react with cervical
carcinoma lines, while L-C5 reacted with breast carcinoma lines.
Their binding to pancreatic carcinomas was not examined.
Greiner et al., Science 235:895-898 (1987), discloses mAb 06.2
which reacts with a 90 kDa glycoprotein allegedly found in 75-80%
of breast carcinomas and more than 90% of colon carcinomas.
Sakamoto et al., U.S. Pat. No. 4,579,827 (Apr. 1, 1986), discloses
a number of mAbs said to be useful for diagnosing or treating human
colon cancer by a number of different approaches. None of these
mAbs is shown to react with a human colon carcinoma-associated
antigen that is a protein of .[.either.]. 61 .[.or 72.]. kDa
molecular weight, distinguishing these antibodies from the
.[.antibodies.]. .Iadd.33.28 antibody .Iaddend.of the present
invention (described below). Furthermore, none of the Sakamoto mAbs
have the degree of colon tumor specificity of the mAbs disclosed in
the present application.
Delaloye et. al., J. Clin. Invest. 77:301 (1986), discloses the use
of a mAb specific for carcinoembryonic antigen (CEA) to detect
colorectal carcinoma in vivo using .sup.123I-labelled fragments and
emission computerized tomography. The mAb described bears no
relation to the mAbs of the present invention.
Douillard et al., Hybridoma 5, Suppl. 1:S139 (1986), describes mAb
17-1A and its cytotoxic properties to gastrointestinal
adenocarcinomas in vitro. 17-1A was used with some degree of
success in immunotherapy trials. This mAb is said to recognize a
38-41 kDa protein and has a broad range of reactivity and lack of
colon tumor specificity, clearly distinguishing it from the
antibodies of the present invention.
Scannon et al., U.S. Pat. No. 4,590,071 (May 20, 1986), discloses
mAbs specific for melanoma antigens conjugated to toxic proteins
such as ricin A chain and the use of these compositions to treat
melanoma. There is no disclosure directed to colon tumor antigens
or antibodies and their uses.
The relatively pure antigen preparation containing the immunogenic
colon carcinoma membrane antigens to which the mAbs of the present
invention are directed was originally described by Hollinshead et
al., Science 177:887-889 (1972).
The clinical evaluation of the above antigen preparation, including
a description of its immunogenicity and potential for enhancing
patient survival through stimulation of specific active immunity,
was described by Hollinshead et al., 1985, supra.
The work of the present inventors leading to the present invention
is briefly described in an abstract by Tsang et al., "Monoclonal
Antibodies to Human Colon Carcinoma Associated Antigens," Intl.
Symp. Biotech. in Clin. Med., Rome, Italy, Apr. 13-15, 1987. This
reference was made available to the public less than one year
before the filing of the ultimate parent application (U.S. Ser. No.
07/176,337) for the present application.
SUMMARY OF THE INVENTION
The present inventors have produced murine mAbs and mouse-human
chimeric antibodies specific for colon carcinoma-associated
antigens which were known to be immunogenic in humans. These
antigens, isolated in the inventors' laboratory, are unique among
the previously described colon cancer antigens in that (1) the
epitopes recognized by the mAbs are of the protein and not the
carbohydrate component of tumor-associated glycoproteins; (2) the
antigens are not expressed in normal tissues; (3) the antigens are
tumor-specific, being present in malignancies of colon, breast, and
ovarian cancer; (4) the antigens are immunogenic in humans, having
the capability of enhancing host anti-tumor immunity by both
cellular as well as humoral responses, thus improving survival in
cancer patients; and (5) the immunogenicity in humans is specific,
in that only colon, breast and ovarian cancer patients, but not
patients with other forms of cancer, show evidence of specific in
vivo or in vitro immunological reactivity to the antigens.
The mAbs and chimeric antibodies of the present invention are
useful for diagnosis or therapy of colon, breast, and ovarian
carcinoma, for example by imaging metastatic tumors, by delivering
cytotoxic agents to the tumors, and by activating host effector
mechanisms such as antibody-dependent cellular cytotoxicity (ADCC)
or complement dependent cytotoxicity (CDC) to directly kill tumor
cells.
The present invention is thus directed to a monoclonal antibody
specific for a human colon carcinoma-associated protein antigen
wherein the antigen is specifically immunogenic in humans, and the
antigen is not detectable on normal human tissues .[.or on human
carcinoma cells other than colon, breast and ovarian carcinoma
cells.]. .
The present invention is also directed to a chimeric antibody
specific for a human colon carcinoma-associated protein antigen
wherein the antigen is not detectable on normal human tissues .[.or
on human carcinoma cells other than colon carcinoma cells.]. .
Mouse hybridoma PCA 31.1 has been deposited at ATCC .Iadd.on Sep.
22, 2000 .Iaddend.and assigned ATCC .[.HB-12314.]. .Iadd.Accession
No. PTA-2497.Iaddend.. Mouse hybridoma PCA 33.28 has been deposited
at ATCC .Iadd.on Aug. 26, 2003 .Iaddend.and assigned ATCC
.[.HB-12315.]. .Iadd.Accession No. PTA-5413.Iaddend.. Cells
transfected with chimeric 31.1 have been deposited at ATCC .Iadd.on
Mar. 13, 1997 .Iaddend.and assigned ATCC .Iadd.Accession No.
.Iaddend.CRL-12316. The above deposits .[.were made.]. .Iadd.are
maintained .Iaddend.at American Type Culture Collection, .[.12301
Parklawn Drive, Rockville, Md. 20862 USA on Mar. 13, 1997.].
.Iadd.10801 University Boulevard, Manassas, Va.
20110-2209.Iaddend..
In one embodiment, the antibody is specific for a CCAA which is a
protein having a molecular weight of about 61 kilodaltons. .[.In
another embodiment, the antibody is specific for a CCAA which is a
protein having a molecular weight of about 72 kilodaltons..]. In a
preferred embodiment, the antibody is the mouse monoclonal antibody
33.28 or 31.1 or an antibody which binds specifically to the same
colon carcinoma-associated epitope as that bound by 33.28 or 31.1.
In another preferred embodiment, the antibody is a mouse/human
chimeric antibody Chi #1 that binds specifically to the same colon
carcinoma-associated epitope as that bound by 31.1.
The present invention is also directed to the above antibody
immobilized on a solid phase.
The present invention includes the above antibody delectably
labelled, for example, with a radiolabel.
In additional embodiments, the above antibody is conjugated to a
cytotoxic radionuclide, a cytotoxic drug, or a cytotoxic
protein.
In yet another embodiment, the present invention is directed to
monoclonal antibodies against the above antibodies, i.e., second
generation monoclonal antibodies.
In a further embodiment, the present invention is directed to third
generation monoclonal antibodies, i.e., monoclonal antibodies
directed against the above second generation monoclonal
antibodies.
The present invention is also directed to the above-discussed colon
carcinoma-associated antigens which are unique in that (1) the
epitopes recognized by the mAbs are of the protein and not the
carbohydrate component of tumor-associated glycoproteins; (2) the
antigens are not expressed in normal tissues; (3) the antigens are
tumor-specific, being present in the malignancies of colon, breast
and ovarian cancer; (4) the antigens are immunogenic in humans,
having the capability of enhancing host anti-tumor immunity, thus
improving survival in cancer patients; and (5) the immunogenicity
in humans is specific, in that only colon, breast and ovarian
cancer patients, but not patients with other forms of cancer, show
evidence of specific in vivo or in vitro immunological reactivity
to the antigens.
To date, all other purified antigens that have been used have
failed to elicit both a cellular and a humoral response.
The present invention also provides a pharmaceutical composition
useful for the immunotherapy of colon, breast and ovarian cancer
comprising an antibody, fragment or derivative, as above,
conjugated to a cytotoxic radionuclide, a cytotoxic drug, or a
cytotoxic protein, in a suitable excipient.
The present invention includes an immunoassay method for detecting
in a sample a colon carcinoma-associated antigen capable of binding
to the 33.28 or 31.1 murine monoclonal antibody or Chi #1,
comprising: (a) contacting the sample with an antibody described
above; and (b) detecting the antigen by detecting the binding of
the antibody.
In another embodiment, the invention provides as imaging method for
detecting a colon carcinoma-associated antigen in a subject,
comprising: (a) contacting the detectably labelled antibody as
described above with the subject; and (b) detecting the
antigen.
The present invention also includes a method of killing cells
carrying a colon carcinoma-associated antigen, comprising: (a)
delivering to the cells an antibody as above, and a cytotoxic
effector agent; and (b) allowing the killing to occur. The effector
agent may be complement, or effector cells active in ADCC.
Alternatively, antibodies labelled conjugated with a cytotoxic
radionuclide, drug or protein may be used directly.
The present invention is further directed to a method of treating a
subject suspected of having a colon, breast and ovarian carcinoma
bearing an antigen which is capable of binding to the 33.28 or 31.1
monoclonal antibody, or Chi #1 chimeric antibody comprising
administering to the subject an effective dose of a pharmaceutical
composition as described above.
Also provided is a method for producing an immunogenic composition
useful for clinical immunotherapy of colon, breast, and ovarian
carcinoma, comprising: (a) preparing a membrane extract of a tumor
or cell line bearing an antigen which is capable of binding to the
33.28 or 31.1 monoclonal antibody or Chi #1 antibody; and (b)
isolating the antigen by affinity purification using an antibody as
described above, thereby producing the immunogenic composition.
In another embodiment, the present invention is directed to the use
of the above antigen to produce a vaccine.
The present invention also includes a method of detecting and
diagnosing colon, breast and ovarian cancer by staining monoclonal
antibody or chimeric antibody bound to the above-described human
colon carcinoma-associated antigen.
In another embodiment, the present invention includes a kit for
selectively characterizing colon, breast, and ovarian
carcinomas.
BRIEF DESCRIPTION OF THE DRAWINGS
FIG. 1 is a tracing showing an HPLC elution profile of the
Hollinshead "vaccine," a partially purified preparation of colon
carcinoma cell membranes.
FIG. 2 is a tracing showing an HPLC elution profile of the colon
carcinoma-associated antigen obtained by affinity purification of
the material contained in peak 4 of FIG. 1.
FIG. 3 is a graph showing the biodistribution of mAb 31.1 in nude
mice bearing a xenografted human tumor, LS-174T.
FIG. 4 is a graph showing the biodistribution of mAb 33.28 in nude
mice bearing a xenografted human tumor, LS-174T.
DESCRIPTION OF THE PREFERRED EMBODIMENTS
The present invention provides antibodies, including monoclonal and
chimeric antibodies, that are specific for, and capable of binding
to, immunogenic human colon carcinoma-associated antigens (CCAA)
which are protein in nature. These antibodies are useful for
diagnostic and therapeutic purposes in subjects having or
developing colon, breast or ovarian carcinoma.
The present invention provides not only mouse mAbs, but also
chimeric antibodies which are constructed from mouse V regions
derived from the mAbs of the present invention. Thus, the chimeric
antibodies maintain the ability to recognize the same CCAA epitopes
as the mAbs.
The term "epitope" refers to that portion of any molecule capable
of being recognized by, and bound by, an antibody. In general,
epitopes consist of chemically active surface groupings of
molecules, for example, amino acids or sugar side chains, and have
specific three-dimensional structural characteristics as well as
specific charge characteristics. The epitopes of interest for the
present invention are epitopes comprising amino acids.
An "antigen" is a molecule or a portion of a molecule capable of
being bound by an antibody which is additionally capable of
inducing an animal to produce an antibody capable of binding to an
epitope of that antigen. An antigen may have one or more than one
epitope. The specific reaction referred to above is meant to
indicate that the antigen will react, in a highly selective manner,
with its corresponding antibody and not with the multitude of other
antibodies which may be evoked by other antigens.
The term "antibody" is meant to include both intact immunoglobulin
molecules as well as fragments and derivatives thereof, such as,
for example, Fab, Fab', F(ab').sub.2 and Fv, which are capable of
binding antigen. These fragments lack the Fc fragment of intact
antibody, clear more rapidly from the circulation, and may have
less non-specific tissue binding than an intact antibody (Wahl et
al., J. Nucl. Med. 24:316-325 (1983)). These fragments are produced
from intact antibodies using methods well known in the art, for
example by proteolytic cleavage with enzymes such as papain (to
produce Fab fragments) or pepsin (to produce F(ab').sub.2
fragments).
A "derivative" of an antibody contains additional chemical moieties
not normally a part of the protein. Covalent modifications of the
protein are included within the scope of this invention. Such
modifications may be introduced into the molecule by reacting
targeted amino acid residues of the antibody with an organic
derivatizing agent that is capable of reacting with selected side
chains or terminal residues. For example, derivatization with
bifunctional agents, well-known in the art, is useful for
cross-linking the antibody or fragment to a water-insoluble support
matrix or to other macromolecular carriers.
By "vaccine" is meant an agent used to stimulate the immune system
of a living organism so that immunological protection against
further harm caused by an infectious agent is provided.
Administration of a vaccine contemplated by the present invention
to the patient may be by any known or standard techniques. These
include oral ingestion, intestinal intubation, or broncho-nasal
spraying. Other methods of administration, such as intravenous
injection, that allow the carrier microbe to reach the human or
animal's bloodstream may be acceptable when the carrier microbe is
unable to reproduce.
The antibodies of the present invention are novel in that they are
the first known mAbs and chimeric antibody specific for CCAA
wherein the tumor antigens are known to be immunogenic in humans.
That is, the antigens recognized by the antibodies of the present
invention induce an immune response in patients with colon, breast,
and ovarian cancer, but not in other individuals, such as patients
with other types of cancer. The immunogenicity of these antigens is
expressed chiefly as cell-mediated immunity, measurable either by
assay of delayed cutaneous hypersensitivity in vivo ("skin tests"),
or by various in vitro assays of specific lymphocyte reactivity,
such as lymphocyte proliferation or lymphocyte migration inhibition
assays. For general principles of immunogenicity and description of
various assays of specific immunological reactivity, see: Roitt,
I., Essential Immunology, 6th Ed., Blackwell Scientific
Publications, Oxford (1988); Roitt, I. et al., Immunology, C. V.
Mosby Co., St. Louis, Mo. (1985); Klein, J., Immunology, Blackwell
Scientific Publications, Inc., Cambridge, Mass. (1990); Klein, J.,
Immunology: The Science of Self-Nonself Discrimination, John Wiley
& Sons, New York, N.Y. (1982); Paterson, P. Y., Textbook of
Immunopathology, Grune and Stratton, New York, (1986), which are
hereby incorporated by reference.
In a preferred embodiment, the antibody of the present invention is
a murine mAb designated 33.28. In another preferred embodiment, the
antibody is a murine mAb designated 31.1. In yet another embodiment
the antibody is a chimeric antibody which recognizes an epitope
recognized by 33.28. In another embedment, the antibody is a
chimeric antibody which recognizes an epitope recognized by
31.1.
The chimeric antibodies of the invention comprise individual
chimeric heavy (H) and light (L) immunoglobulin chains. The
chimeric H chain comprises an antigen-binding region derived from
the H chain of a non-human antibody specific for the epitope
recognized by 33.28 or 31.1, which is linked to at least a portion
of a human H chain C region (C.sub.H).
A preferred chimeric L chain comprises an antigen-binding region
derived from the L chain of either the 33.28 or 31.1 mAb, linked to
at least a portion of a human L chain C region (C.sub.L).
Alternatively, a preferred chimeric H chain comprises an
antigen-binding region derived from the L chain of either the 33.28
or 31.1 mAb, linked to at least a portion of a human L chain C
region (C.sub.H).
As used herein, the term "antigen-binding region" refers to that
portion of an antibody molecule which contains the amino acid
residues that interact with an antigen and confer on the antibody
its specificity and affinity for the antigen. The antibody region
includes the "framework" amino acid residues necessary to maintain
the proper conformation of the antigen-binding residues.
As used herein, the term "chimeric antibody" includes monovalent,
divalent or polyvalent immunoglobulins. A monovalent chimeric
antibody is a dimer (HL) formed by a chimeric H chain associated
through disulfide bridges with a chimeric L chain. A divalent
chimeric antibody is tetramer (H.sub.2L.sub.2) formed by two HL
dimers associated through at least one disulfide bridge. A
polyvalent chimeric antibody can also be produced, for example, by
employing a C.sub.H region that aggregates (e.g., from an IgM H
chain, or .mu. chain).
The invention also provides for "derivatives" of the monoclonal or
chimeric antibodies, which term includes those proteins encoded by
truncated or modified genes to yield molecular species functionally
resembling the immunoglobulin fragments. The modifications include,
but are not limited to, addition of genetic sequences coding of
cytotoxic proteins such as plant and bacterial toxins. The
fragments and derivatives can be produced from prokaryotic or
eukaryotic hosts, as described herein by recombinant means.
Alternatively, the fragments and derivatives may be produced by
chemical means, such as proteolytic cleavage of intact
immunoglobulin molecules, or other chemical modifications or
derivatizations known in the art. Such derivatized moieties may
improve the solubility, absorption, biological half-life, and the
like. The moieties may alternatively eliminate or attenuate any
undesirable side effect of the antibody protein. Moieties capable
of mediating such effects are disclosed, for example, in
Remington's Pharmaceutical Sciences, 16th ed., Hack Publishing Co.,
Easton, Pa. (1980).
Antibodies, fragments or derivatives having chimeric H chains and L
chains of the same or different V region binding specificity can be
prepared by appropriate association of the individual polypeptide
chains, as taught, for example by Sears et al., Proc. Natl. Acad.
Sci. USA 72:353.varies.357 (1975). With this approach, hosts
expressing chimeric H chains (or their derivatives) are separately
cultured from hosts expressing chimeric L chains (or their
derivatives) and the immunoglobulin chains are separately recovered
and then associated. Alternatively, the hosts can be co-cultured
and the chains allowed to associate spontaneously in the culture
medium, followed by recovery of the assembled immunoglobulin,
fragment or derivative.
Murine hybridomas which produce mAb specific for CCAA, such as the
33.28 and 31.1 mAbs of the present invention, are formed by the
fusion of a mouse fusion partner cell, such as SP2/0, and spleen
cells from mice immunized against the CCAA.
Mice may be immunized with crude or semi-purified preparations
containing the antigens of interest, such as, for example, the
Hollinshead "vaccine," which is a partially purified membrane
preparation of colon-carcinoma cells (Hollinshead et al., supra).
To immunize the mice, a variety of different conventional protocols
may be followed. For example, mice may receive primary and boosting
immunizations of antigenic preparations.
The cell fusions are accomplished by standard procedures well known
to those skilled in the field of immunology (Kohler and Milstein,
Nature 256:495-497 (1975) and U.S. Pat. No. 4,376,110; Hartlow, E.
et al., supra; Campbell, A., "Monoclonal Antibody Technology," In:
Laboratory Techniques in Biochemistry and Molecular Biology, Volume
13 (Burdon, R., et al., eds. ), Elsevier, Amsterdam (1984); Kennett
et al., Monoclonal Antibodies (Kennett et al., eds. pp. 365-367,
Plenum Press, N.Y., 1980); de St. Groth, S. F., et al., J. Immunol.
Meth. 35:1-21 (1980); Galfre, G. et al., Methods Enzymol. 73:3-46
(1981); Goding, J. W. 1987, Monoclonal Antibodies: Principles and
Practice, 2nd ed. Academic Press, London, 1987).
Fusion partner cell lines and methods for fusing and selecting
hybridomas and screening for mAbs are well known in the art
(Hartlow, E. et al., supra; Kawamoto, T. et al., Meth. Enzymol.
121:266-277 (1986); Kearney, J. F. et al., J. Immunol.
123:1548-1550 (1979); Kilmartin, J. V. et al., J. Cell Biol.
93:576-582 (1982); Kohler, G. et al., Eur. J. Immunol. 6:292-295
(1976); Lane, D. P. et al., J. Immunol. Meth. 47:303-307 (1981);
Mueller, U. W. et al., J. Immunol. Meth. 87:193-196 (1986);
Pontecorvo, G., Somatic Cell Gener. 1:397-400 (1975); Sharo, J., et
al., Proc. Natl. Acad. Sci. USA 7:6:1420-1424 (1979); Shulman, M.
et al., Nature 276:269-270 (1978); Springer, T. A. (ed), Hybridoma
Technology in the Biosciences and Medicine, Plenum Press, New York,
1985; and Taggart, R. T. et al., Science 219:1228-1230 (1982)).
The mAbs of the present invention may be produced in large
quantities by injecting hybridoma cells secreting the antibody into
the peritoneal cavity of mice and, after appropriate time,
harvesting the ascites fluid which contains a high titer of the
mAb, and isolating the mAb therefrom. Alternatively, the mAbs may
be produced by culturing hybridoma cells in vitro and isolating the
secreted mAb from the cell culture medium.
Human genes which encode the C regions of the chimeric antibodies
of the present invention are derived from cells which express, and
preferably, produce, human immunoglobulins. The human C.sub.H
region can be derived from any of the known classes or isotypes of
human H chains, including gamma, .mu., .alpha., .delta. or
.epsilon.. Since the H chain isotype is responsible for the various
effector functions of an antibody, the choice of C.sub.H region
will be guided by the desired effector functions, such as
complement fixation, or activity in antibody-dependent cellular
cytotoxicity (ADCC). Preferably, the C.sub.H region is derived from
gamma 1 (IgG1), gamma 3 (IgG3), gamma 4 (IgG4), and .mu. (IgM).
The human C.sub.L region can be derived from either human L chain
isotype, kappa or lambda.
Genes encoding human immunoglobulin C regions are obtained from
human cells by standard cloning techniques (Sambrook, J. et al.,
Molecular Cloning: A Laboratory Manual, 2nd Edition, Cold Spring
Harbor Press, Cold Spring Harbor, N.Y. (1989)). Human C region
genes are readily available from known clones containing genes
representing the two classes of L chains and the five classes of H
chains. Chimeric antibody fragments, such as F(ab').sub.2 and Fab,
can be prepared by designing a chimeric H chain gene which is
appropriately truncated. For example, a chimeric gene encoding the
H chain portion of an F(ab').sub.2 fragment would include DNA
sequences encoding the CH.sub.1 domain and hinge region of the H
chain, followed by a translational stop codon to yield the
truncated molecule.
Generally, the chimeric antibodies of the present invention are
produced by cloning DNA segments encoding the H and L chain
antigen-binding regions of a CCAA-specific antibody, preferably
non-human, most preferably 33.28 or 31.1, and joining these DNA
segments to DNA segments encoding human C.sub.H and C.sub.Lregions
to produce chimeric immunoglobulin-encoding genes.
Thus, in a preferred embodiment, a fused gene is created which
comprises a first DNA segment that encodes at least the
antigen-binding region of non-human origin, such as a functionally
rearranged V region with joining (J) segment, linked to a second
DNA segment encoding at least a part of a human C region. This
fusion can be accomplished by the polymerase chain reaction, as
reported by Fernando et al., Miami Symp. Short Reports 3: 88,
1993.
The DNA encoding the antibody-binding region may be genomic DNA or
cDNA. A convenient alternative to the use of chromosomal gene
fragments as the source of DNA encoding the murine V region
antigen-binding segment is the use of cDNA for the construction of
chimeric immunoglobulin genes, as reported by Liu et al., Proc.
Natl. Acad. Sci., USA 84:3439 (1987) and J. Immunology 139:3521
(1987), which references are hereby incorporated by reference. The
use of cDNA requires that gene expression elements appropriate for
the host cell be combined with the gene in order to achieve
synthesis of the desired protein. The use of cDNA sequences is
advantageous over genomic sequences (which contain introns), in
that cDNA sequences can be expressed in bacteria or other hosts
which lack appropriate RNA-splicing systems.
Therefore, in an embodiment utilizing cDNA encoding the antibody V
region, the method of producing the chimeric antibody involves
several steps, outlined below: 1. Isolation of messenger RNA (mRNA)
from the cell line producing the monoclonal antibody, cloning and
cDNA production therefrom; 2. Preparation of a full length cDNA
library from purified mRNA from which the appropriate V region gene
segments of the L and H chain genes can be: (i) identified with
appropriate probes, (ii) sequenced, and (iii) made compatible with
a C gene segment; 3. Preparation of C region gene segments by cDNA
preparation and cloning; 4. Construction of complete H or L chain
coding sequences by linkage of the cloned specific V region gene
segments to cloned human C region gene, as described above; 5.
Expression and production of chimeric L and H chains in selected
hosts, including prokaryotic and eukaryotic cells.
One common feature of all immunoglobulin H and L chain genes and
their encoded mRNAs is the J region. H and L chain J regions have
different sequences, but a high degree of sequence homology exists
(greater than 80%) among each group, especially near the C region.
This homology is exploited in this method and consensus sequences
of H and L chain J regions may be used to design oligonucleotides
for use as primers for introducing useful restriction sites into
the J region for subsequent linkage of V region segments to human C
region segments.
C region cDNA vectors prepared from human cells can be modified by
site-directed mutagenesis to place a restriction site at the
analogous position in the human sequence. For example, one can
clone the complete human kappa chain C (C.sub.k) region and the
complete human gamma-1 C region (C.sub.gamma-1). In this case, the
alternative method based upon genomic C region clones as the source
for C region vectors would not allow these genes to be expressed in
bacterial systems where enzymes needed to remove intervening
sequences are absent. Cloned V region segments are excised and
ligated to L or H chain C region vectors. Alternatively, the human
C.sub.gamma-1 region can be modified by introducing a termination
codon thereby generating a gene sequence which encodes the H chain
portion of an Fab molecule. The coding sequences with linked V and
C regions are then transferred into appropriate expression vehicles
for expression in appropriate hosts, prokaryotic or eukaryotic.
Two coding DNA sequences are said to be "operably linked" if the
linkage results in a continuously translatable sequence without
alteration or interruption of the triplet reading frame. A DNA
coding sequence is operably linked to a gene expression element if
the linkage results in the proper function of that gene expression
element to result in expression of the coding sequence.
Expression vehicles include plasmids or other vectors. Preferred
among these are vehicles carrying a functionally complete human
C.sub.H or C.sub.L chain sequence having appropriate restriction
sites engineered so that any V.sub.H or V.sub.L chain sequence with
appropriate cohesive ends can be easily inserted therein. Human
C.sub.H or C.sub.L chain sequence-containing vehicles thus serve as
intermediates for the expression of any desired complete H or L
chain in any appropriate host.
A chimeric mouse-human antibody will typically be synthesized from
genes driven by the chromosomal gene promoters native to the mouse
H and L chain V regions used in the constructs; splicing usually
occurs between the splice donor site in the mouse J region and the
splice acceptor site preceding the human C region and also at the
splice regions that occur within the human C.sub.H region;
polyadenylation and transcription termination occur at native
chromosomal sites downstream of the human coding regions.
Gene expression elements useful for the expression of cDNA genes
include: (a) viral transcription promoters and their enhancer
elements, such as the SV40 early promoter (Okayama, H. et al., Mol.
Cell. Biol. 3:280 (1983)), Rous sarcoma virus LTR (Gorman, C. et
al., Proc. Natl. Acad. Sci., USA 79:6777 (1982)), and Moloney
murine leukemia virus LTR (Grosschedl, R. et al., Cell 41:885
(1985)); (b) splice regions and polyadenylation sites such as those
derived from the SV40 late region (Okayama et al., supra); and (c)
polyadenylation sites such as in SV40 (Okayama et al., supra).
Immunoglobulin cDNA genes may be expressed as described by Liu et
al., Supra, and Weidle et al., Gene 51:21 (1987), using as
expression elements the SV40 early promoter and its enhancer, the
mouse immunoglobulin H chain promoter enhancers, SV40 late region
mRNA splicing, rabbit .beta.-globin intervening sequence,
immunoglobulin and rabbit .beta.-globin polyadenylation sites, and
SV40 polyadenylation elements. For immunoglobulin genes comprised
of part cDNA, part genomic DNA (Whittle et al., Protein Engineering
1:499 (1987)), the transcriptional promoter may be human
cytomegalovirus (CMV), the promoter enchancers derived from CMV and
mouse/human immunoglobulin, and mRNA splicing and polyadenylation
regions derived from the native chromosomal immunoglobulin
sequences.
In one embodiment, for expression of cDNA genes in rodent cells,
the transcriptional promoter is a viral LTR sequence, the
transcriptional promoter enchancers are either or both the mouse
immunoglobulin heavy chain enhancer and the viral LTR enhancer, the
splice region contains an intron of greater than 31 bp, and the
polyadenylation and transcription termination regions are derived
from the native chromosomal sequence corresponding to the
immunoglobulin chain being synthesized. In other embodiments, cDNA
sequences encoding other proteins are combined with the
above-recited expression elements to achieve expression of the
proteins in mammalian cells.
Each fused gene is assembled in, or inserted into, an expression
vector. Recipient cells capable of expressing the chimeric
immunoglobulin chain gene product are then transfected singly with
a chimeric H or chimeric L chain-encoding gene, or are
co-transfected with a chimeric H and a chimeric L chain gene. The
transfected recipient cells are cultured under conditions that
permit expression of the incorporated genes and the expressed
immunoglobulin chains or intact antibodies or fragments are
recovered from the culture. In one embodiment, the fused genes
encoding the chimeric H and L chains, or portions thereof, are
assembled in separate expression vectors that are then used to
co-transfect a recipient cell.
Each vector may contain two selectable genes, a first selectable
gene designed for selection in a bacterial system and a second
selectable gene designed for selection in a eukaryotic system,
wherein each vector has a different pair of genes. This strategy
results in vectors which first direct the production, and permit
amplification, of the fused genes in a bacterial system.
Subsequently, the genes so produced and amplified in a bacterial
host are subsequently used to co-transfect a eukaryotic cell, and
allow selection of a co-transfected cell carrying the desired
transfected genes.
Examples of selectable genes of use in a bacterial system are the
gene that confers resistance to ampicillin and the gene that
confers resistance to chloramphenicol. Preferred selectable genes
for use in eukaryotic transfectants include the xanthine guanine
phosphoribosyl transferase gene (designated gpt) and the
phosphotransferase gene from Tn5 (designated neo). Selection of
cells expressing gpt is based on the fact that the enzyme encoded
by this gene utilizes xanthine as a substrate for purine,
nucleotide synthesis, whereas the analogous endogenous enzyme
cannot. In a medium containing (1) mycophenolic acid, which blocks
the conversion of inosine monophosphate to xanthine monophosphate,
and (2) xanthine, only cells expressing the E gene can survive. The
product of the neo blocks the inhibition of protein synthesis by
the antibiotic G418 and other antibiotics of the neomycin
class.
The two selection procedures can be used simultaneously or
sequentially to select for the expression of immunoglobulin chain
genes introduced on two different DNA vectors into a eukaryotic
cell. It is not necessary to include different selectable markers
for eukaryotic cells; an H and an L chain vector, each containing
the same selectable marker can be co-transfected. After selection
of the appropriately resistant cells, the majority of the clones
will contain integrated copies of both H and L chain vectors.
Alternatively, the fused genes encoding the chimeric H and L chains
can be assembled on the same expression vector.
For transfection of the expression vectors and production of the
chimeric antibody, the preferred recipient cell line is a myeloma
cell. Myeloma cells can synthesize, assemble and secrete
immunoglobulins encoded by transfected immunoglobulin genes and
possess the mechanism or glycosylation of the immunoglobulin. A
particularly preferred recipient cell is the Ig-non-producing
myeloma cell SP2/0 (ATCC #CRL 8287). SP2/0 cells produce only
immunoglobulin encoded by the transfected genes. Myeloma cells can
be grown in culture or in the peritoneal cavity of a mouse, where
secreted immunoglobulin can be obtained from ascites fluid. Other
suitable recipient cells include lymphoid cells such as B
lymphocytes of human or non-human origin, hybridoma cells of human
or non-human origin, or interspecies heterohybridoma cells.
The expression vector carrying a chimeric antibody construct of the
present invention may be introduced into an appropriate host cell
by any of a variety of suitable means, including such biochemical
means as transformation, transfection, conjugation, protoplast
fusion, calcium phosphate-precipitation, and application with
polycations such as diethylaminoethyl (DEAE) dextran, and such
mechanical means as electroporation, direct microinjection, and
microprojectile bombardment (Johnston et al., Science 240:1538
(1988)). A preferred way of introducing DNA into lymphoid cells is
by electroporation (Potter et al., Proc. Natl. Acad. Sci. USA
81:7161 (1984); Yoshikawa, K. et al., Jpn. J. Cancer Res.
77:1122-1133). In this procedure, recipient cells are subjected to
an electric pulse in the presence of the DNA to be incorporated.
Typically, after transfection, cells are allowed to recover in
complete medium for about 24 hours, and are then seeded in 96-well
culture plates in the presence of the selective medium. G418
selection is performed using about 0.4 to 0.8 mg/ml G418.
Mycophenolic acid selection utilizes about 6 .mu.g/ml plus about
0.25 mg/ml xanthine. The electroporation technique is expected to
yield transfection frequencies of about 10.sup.-5 to about
10.sup.-4 for Sp2/0 cells. In the protoplast fusion method,
lysozyme is used to strip cell walls from catarrhal harboring the
recombinant plasmid containing the chimeric antibody gene. The
resulting spheroplasts are fused with myeloma cells with
polyethylene glycol.
The chimeric immunoglobulin genes of the present invention can also
be expressed in nonlymphoid mammalian cells or in other eukaryotic
cells, such as yeast, or in prokaryotic cells, in particular
bacteria.
Yeast provides substantial advantages for the production of
immunoglobulin H and L chains. Yeasts carry out post-translational
peptide modifications including glycosylation. A number of
recombinant DNA strategies now exist which utilize strong promoter
sequences and high copy number plasmids which can be used for
production of the desired proteins in yeast. Yeast recognizes
leader sequences of cloned mammalian gene products and secretes
peptides bearing leader sequences (i.e., pre-peptides) (Hitzman, et
al., 11th International Conference on Yeast, Genetics and Molecular
Biology, Montpelier, France, Sep. 13-17, 1982).
Yeast gene expression systems can be routinely evaluated for the
levels of production, secretion and the stability of chimeric H and
L chain proteins and assembled chimeric antibodies. Any of a series
of yeast gene expression systems incorporating promoter and
termination elements from the actively expressed genes coding for
glycolytic enzymes produced in large quantities when yeasts are
grown in media rich in glucose can be utilized. Known glycolytic
genes can also provide very efficient transcription control
signals. For example, the promotor and terminator signals of the
phosphoglycerate kinase (PGK) gene can be utilized. A number of
approaches may be taken for evaluating optimal expression plasmids
for the expression of cloned immunoglobulin cDNAs in yeast (see
Glover, D. M., ed., DNA Cloning, Vol. II, pp. 45-66, IRL Press,
1985).
Bacterial strains may also be utilized as hosts for the production
of antibody molecules or antibody fragments described by this
invention. E. coli K12 strains such as E. coli W3110 (ATCC 27325),
and other enterobacteria such as Salmonella typhimurium or Serratia
marcescens, and various Pseudomonas species may be used.
Plasmid vectors containing replicon and control sequences which are
derived from species compatible with a host cell are used in
connection with these bacterial hosts. The vector carries a
replication site, as well as specific genes which are capable of
providing phenotypic selection in transformed cells. A number of
approaches may be taken for evaluating the expression plasmids for
the production of chimeric antibodies or antibody chains encoded by
the cloned immunoglobulin cDNAs in bacteria (see Glover, D. M.,
ed., DNA Cloning, Vol. I, IRL Press, 1985).
Other preferred hosts are mammalian cells, grown in vitro or in
vivo. Mammalian cells provide post-translational modifications to
immunoglobulin protein molecules including leader peptide removal,
folding and assembly of H and L chains, glycosylation of the
antibody molecules, and secretion of functional antibody
protein.
Mammalian cells which may be useful as hosts for the production of
antibody proteins, in addition to the cells of lymphoid origin
described above, include cells of fibroblast origin, such as Vero
(ATCC CRL 81) or CHO-K1 (ATCC CRL 61).
Many vector systems are available for the expression of cloned H
and L chain genes in mammalian cells (see Glover, D. M., ed., DNA
Cloning, Vol. II, pp. 143-238, IRL Press, 1985). Different
approaches can be followed to obtain complete
H.sub.2L.sub.2antibodies. As discussed above, it is possible to
co-express H and L chains in the same cells to achieve
intracellular association and linkage of H and L chains into
complete tetrameric H.sub.2L.sub.2 antibodies. The co-expression
can occur by using either the same or different plasmids in the
same host. Genes for both H and L chains can be placed into the
same plasmid, which is then transfected into cells, thereby
selecting directly for cells that express both chains.
Alternatively, cells may be transfected first with a plasmid
encoding one chain, for example the L chain, followed by
transfection of the resulting cell line with an H chain plasmid
containing a second selectable marker. Cell lines producing
H.sub.2L.sub.2 molecules via either route could be transfected with
plasmids encoding additional copies of H, L, or H plus L chains in
conjunction with additional selectable markers to generate cell
lines with enhanced properties, such as higher production of
assembled H.sub.2L.sub.2 antibody molecules or enchanced stability
of the transfected cell lines.
In addition to mAbs or chimeric antibodies, the present invention
is also directed to an anti-idiotypic (anti-Id) antibody specific
for V region epitopes of the mAb antibody or chimeric antibody of
the invention. An anti-Id antibody is an antibody which recognizes
unique determinants generally associated with the antigen-binding
region of another antibody. The antibody specific for CCAA, such as
33.28, is termed the idiotypic or Id antibody. The anti-Id can be
prepared by immunizing an animal of the same species and genetic
type mouse strain) as the source of the Id antibody with the Id
antibody or the antigen-binding region thereof. The immunized
animal will recognize and respond to the idiotypic determinants of
the immunizing antibody and produce an anti-Id antibody. The
anti-Id antibody may also be used as an "immunogen" to induce an
immune response in yet another animal, producing a so-called
anti-anti-Id antibody. The anti-anti-Id may be epitopically
Identical to the original antibody which induced the anti-Id. Thus,
by using antibodies to the idiotypic determinants of a mAb, it is
possible to identify other clones expressing antibodies of
identical specificity.
Accordingly, the mAbs or chimeric antibodies of the present
invention may be used to induce anti-Id antibodies in suitable
animals, such as BALB/c mice. Spleen cells from such immunized mice
can be used to produce anti-Id hybridomas secreting anti-Id mAbs.
Further, the anti-Id mAbs can be coupled to a carrier such as
keyhole limpet hemocyanin (KLH) and used to immunize additional
BALB/c mice. Sera from these mice will contain anti-anti-Id
antibodies that have the binding properties of the original mAb
specific for a CCAA epitope.
The antibodies of the present invention, including their
antigen-binding fragments and derivatives, have a multitude of uses
relating to the diagnosis, monitoring and therapy of colon, breast,
and ovarian cancer. Such uses are summarized in Schlom, J., Canc.
Res., 46:3225-3238 (1986), which is hereby incorporated by
reference.
In diagnosis, the antibodies may be used in immunoassays (described
below) to screen body fluids, such as serum, sputum, effusions,
urine, cerebrospinal fluid, and the like, for the presence of CCAA.
The antibodies may be used for scanning or radioimaging, when
labelled with an appropriate radiolabel, to detect primary or
metastatic foci of tumor cells. Furthermore, the antibodies are
useful in lymphoscintigraphy to detect lymph node involvement in
the disease.
The antibodies of the present invention are also useful for
immunopathological analysis, such as the differential diagnosis of
tumor type, the subclassification of the tumor based on its
expression of CCAA. Such determinations would be important in
assessment of metastatic potential, predicted responses to therapy
and prognosis.
In particular, because of the specificity of the mAbs and chimeric
antibodies of the present invention, they may permit the definition
of defining subpopulations of tumor cells among the heterogeneous
cells present in a growing tumor. These antibodies could therefore
be used in the typing and cross-matching of the tumor cell "lines"
comprising the tumor by means of flow cytometry, both at the time
of surgery and prior to therapy. An analysis of the tumor cell
subpopulations with the antibodies of this invention, and a battery
of additional mAbs, is used to define (a) which antigen preparation
would be the most appropriate for specific active immunotherapy,
(b) which mAb or chimeric antibody would be efficacious for ADCC,
and (c) which antibody or combination of mAbs should be used for
imaging the patient at a later date in search for recurrent or
metastatic tumors.
In addition to their diagnostic utility, the antibodies of the
present invention are useful for monitoring the progression of
disease by screening body fluids for CCAA, radioimaging of tumor,
or the detection of occult metastasis through aspiration cytology,
lymph node or bone marrow biopsy, or cytology of body fluids.
A summary of the ways in which the antibodies of the present
invention may be used therapeutically includes direct cytotoxicity
by the antibody, either mediated by complement (CDC) or by effector
cells (ADCC), conjugated to anti-tumor drugs, toxins,
radionuclides. The antibodies can be used for ex vivo removal of
tumor cells from the circulation or from bone marrow.
Some of these approaches are described in more detail below. Armed
with the teachings provided herein, one of ordinary skill in the
art will know how to use the antibodies of the present invention
for diagnostic, monitoring or therapeutic purposes without undue
experimentation.
The preferred animal subject of the present invention is a mammal.
By the term "mammal" is meant an individual belonging to the class
Mammalia. The invention is particularly useful in the treatment of
human subjects.
By the term "treating" is intended the administering to subjects of
the antibodies of the present invention or a fragment or derivative
thereof for purposes which may include prevention, amelioration, or
cure of colon, breast, and ovarian cancer.
The pharmaceutical compositions of the present invention may be
administered by any means that achieve their intended purpose.
Amounts and regimens for the administration of antibodies, their
fragments or derivatives can be determined readily by those with
ordinary skill in the clinical art of treating colon, breast, and
ovarian cancer and related disease.
For example, administration may be by parenteral, subcutaneous,
intravenous, intramuscular, intraperitoneal, transdermal, or buccal
routes. Alternatively, or concurrently, administration may be by
the oral route. The dosage administered will be dependent upon the
age, health, and weight of the recipient, kind of concurrent
treatment, if any, frequency of treatment, and the nature of the
effect desired.
Compositions within the scope of this invention include all
compositions wherein the antibody, fragment or derivative is
contained in an amount effective to achieve its intended purpose.
While individual needs vary, determination of optimal ranges of
effective amounts of each component is within the skill of the art.
The effective dose is a function of the individual chimeric or
monoclonal antibody, the presence and nature of a conjugated
therapeutic agent (see below), the patient and his clinical status,
and can vary from about 10 ng/kg body weight to about 100 10 mg/kg
body weight. The preferred dosages comprise 0.1 to 10 mg/kg body
weight.
In addition to the pharmacologically active compounds, the new
pharmaceutical compositions may contain suitable pharmaceutically
acceptable carriers comprising excipients and auxiliaries which
facilitate processing of the active compounds into preparations
which can be used pharmaceutically. Preferably, the preparations,
contain from about 0.01 to 99 percent, preferably from about 20 to
75 percent of active compound(s), together with the excipient.
Preparations of the antibody, fragment or derivative of the present
invention for parenteral administration, such as in detectably
labelled form for imaging or in a free or conjugated form for
therapy, include sterile aqueous or non-aqueous solutions,
suspensions, and emulsions. Examples of non-aqueous solvents are
propyleneglycol, polyethyleneglycol, vegetable oil such as olive
oil, and injectable organic esters such as ethyloleate. Aqueous
carriers include water, alcoholic/aqueous solutions, emulsions or
suspensions, including saline and buffered media, parenteral
vehicles including sodium chloride solution, Ringer's dextrose,
dextrose and sodium chloride, lactated Ringer's, or fixed oils.
Intravenous vehicles include fluid and nutrient replenishers, such
as those based on Ringer's dextrose, and the like. Preservatives
and other additives may also be present, such as, for example,
antimicrobials, antioxidants, chelating agents, and inert gases and
the like. See, generally, Remington's Pharmaceutical Science, 16th
ed., Mack Publishing Co., Easton, Pa., 1980.
In particular, the antibodies, fragments and derivatives of the
present invention are useful for treating a subject having or
developing colon, breast, and ovarian adenocarcinoma. Such
treatment comprises parenterally administering a single or multiple
doses of the antibody, fragment or derivative, or a conjugate
thereof.
The antibodies of this invention can be adapted for therapeutic
efficacy by virtue of their ability to mediate ADCC and/or CDC
against cells having CCAA associated with their surface. For these
activities, either an endogenous source or an exogenous source of
effector cells (for ADCC) or complement components (for CDC) can be
utilized.
The antibodies of this invention, their fragments, and derivatives
can be used therapeutically as immunoconjugates (see for review:
Dillman, R. O., Ann. Int. Med. 111:592-603 (1989)). They can be
coupled to cytotoxic proteins, including, but not limited to,
Ricin-A, Pseudomonas toxin. Diphtheria toxin, and tumor necrosis
factor. Toxins conjugated to antibodies or other ligands are known
in the art (see, for example, Olsnes, S. et al., Immunol. Today
10:291-295 (1989)). Plant and bacterial toxins typically kill cells
by disrupting the protein synthetic machinery.
The antibodies of this invention can be conjugated to additional
types of therapeutic moieties including, but not limited to,
diagnostic radionuclides and cytotoxic agents such as cytotoxic
radionuclides, drug and proteins. Examples of radionuclides which
can be coupled to antibodies and delivered in vivo to sites of
antigen include 212.sub.Bi, 131.sub.I, 186.sub.Re, and 90.sub.Y,
which list is not intended to be exhaustive. The radionuclides
exert their cytotoxic effect by locally irradiating the cells,
leading to various intracellular lesions, as is known in the art of
radiotherapy.
Cytotoxic drugs which can be conjugated to antibodies and
subsequently used for in vivo therapy include, but are not limited
to, daunorubicin, doxorubicin, methotrexate, and Mitomycin C.
Cytotoxic drugs interface with critical cellular processes
including DNA, RNA, and protein synthesis. For a fuller exposition
of these classes of drugs which are known in the art, and their
mechanisms of action, see Goodman, A. G., et al., Goodman and
Gilman's THE PHARMACOLOGICAL BASIS OF THERAPEUTICS, 7th Ed.,
Macmillan Publishing Co., 1985.
The antibodies of this invention may be advantageously utilized in
coordination with other monoclonal or chimeric antibodies, or with
lymphokines or hemopoietic growth factors, etc., which serve to
increase tee number or activity of effector cells which interact
with the antibodies.
The antibodies, fragments, or derivatives of this invention,
attached to a solid support, can be used to remove soluble colon
carcinoma-associated antigens from fluids or tissue or cell
extracts. In a preferred embodiment, they are used to remove
soluble tumor antigens from blood or blood plasma products. In
another preferred embodiment, the antibodies are advantageously
used in extracorporeal immunoadsorbent devices, which are known in
the art (see, for example, Seminars in Hematology, Vol. 26 (2
Suppl. 1) (1989)). Patient blood or other body fluid is exposed to
the attached antibody, resulting in partial or complete removal of
circulating CCAA (free or in immune complexes), of CCAA-bearing
cells, following which the fluid is returned to the body. This
immunoadsorption can be implemented in a continuous flow
arrangement, with or without interposing a cell centrifugation
step. See, for example, Terman, D. S. et al., J. Immunol.
117:1971-1975 (1976).
The present invention also provides the above antibodies, fragments
and derivatives, detectably labelled, as described below.
The antibodies of the present invention are useful for immunoassays
which detect or quantitate CCAA or cells bearing CCAA in a sample.
Such an immunoassay typically comprises incubating a biological
sample in the presence of a detectably labelled antibody of the
present invention capable of identifying the tumor antigen, and
detecting the labelled antibody which is bound in a sample.
Thus, in this aspect of the invention, a biological sample may be
treated with nitrocellulose, or other solid support or carrier
which is capable of immobilizing cells, cell particles or soluble
proteins or glycoproteins. The support may then be washed with
suitable buffers followed by treatment with the detectably labelled
antibody of the present invention. The solid phase support may then
be washed with the buffer a second time to remove unbound antibody.
The amount of bound label on said solid support may then be
detected by conventional means.
By "solid phase support" or "carrier" is intended any support
capable of binding antigen or antibodies. Well-known supports or
carriers include glass, polystyrene, polypropylene, polyethylene,
dextran, nylon, amylases, natural and modified celluloses,
polyacrylamides, agaroses, and magnetic. The nature of the carrier
can be either soluble to some extent or insoluble for the purposes
of the present invention. The support material may have virtually
any possible structural configuration so long as the coupled
molecule is capable of binding to CCAA or the antibody specific for
CCAA. Thus, the support configuration may be spherical, as in a
bead, or cylindrical, as in the inside surface of a test tube, or
the external surface of a rod. Alternatively, the surface may be
flat such as a sheet, test strip, etc. Preferred supports include
polystyrene beads. Those skilled in the art will know many other
suitable carriers for binding antibody or antigen, or will be able
to ascertain the same by use of routine experimentation.
The binding activity of a given lot of antibody may be determined
according to well-known methods. Those skilled in the art will be
able to determine operative and optimal assay conditions for each
determination by employing routine experimentation.
One of the ways in which the antibody of the present invention can
be detectably labelled is by linking the same to an enzyme and use
in an enzyme immunoassay (EIA) or enzyme-linked immunosorbent assay
(ELISA). This enzyme, when subsequently exposed to its substrate,
will react with the substrate generating a chemical moiety which
can be detected, for example, by spectrophotometric, fluorometric
or by visual means. In an alternate embodiment, the enzyme is used
to label a binding partner for the antibody of the invention. Such
a binding partner may be an antibody against the constant or
variable region of the antibody of the invention, such as a
heterologous anti-mouse immunoglobulin antibody. Alternatively, the
binding partner may be a non-antibody protein capable of binding to
the antibody of the present invention, such as staphylococcal
protein A, or streptococcal protein G.
Enzymes which can be used to detectably label the CCAA-specific
antibodies of the present invention, or the binding partners for
these antibodies, include, but are not limited to, malate
dehydrogenase, staphylococcal nuclease, delta-5-steroid isomerase,
yeast alcohol dehydrogenase, alpha-glycerophosphate dehydrogenase,
triose phosphate isomerase, horseradish peroxidase, alkaline
phosphatase, asparaginase, glucose oxidase, beta-galactosidase,
ribonuclease, urease, catalase, glucose-6-phosphate dehydrogenase,
glycoamylase and acetylcholinesterase.
By radioactively labelling the antibody of the present invention or
the binding partner, it is possible to detect CCAA through the use
of a radioimmunoassay (RIA) (see, for example, Work, T. S. et al.,
Laboratory Techniques and Biochemistry in Molecular Biolog. North
Holland Publishing Company, N.Y. (1978)). The radioactive isotope
can be detected by such means as the use of a gamma counter or a
scintillation counter or by autoradiography. Isotopes which are
particularly useful for the purpose of the present invention are
well known in the art.
It is also possible to label the antibodies or binding partners
with a fluorescent compound. When the fluorescently labelled
antibody is exposed to light of the proper wave length, its
presence can then be detected due to fluorescence. Among the most
commonly used fluorescent labelling compounds are fluorescein
isothiocyanate, rhodamine, phycoerythrin, phycocyanin,
allophycocyanin, o-phthaldehyde and flourescamine.
The antibodies can also be detectably labelled using
fluorescence-emitting metals such as .sup.152Eu, or others of the
lanthanide series. These metals can be attached to the antibody
using such metal chelating groups as diethylenetriaminepentaacetic
acid (DTPA) or ethylenediaminetetraacetic acid (EDTA).
The antibodies of the present invention also can be detectably
labelled by coupling to a chemiluminescent compound. The presence
of the chemiluminescently labelled antibody is then determined by
detecting the presence of luminescence that arises during the
course of a chemical reaction. Examples of particularly useful
chemiluminescent labelling compounds are luminol, isoluminol,
imidazole, acridinium salt and oxalate ester.
Likewise, a bioluminescent compound may be used to label the
antibody, fragment or derivative of the present invention.
Bioluminescence is a type of chemiluminescence found in biological
systems, in which a catalytic protein increases the efficiency of
the chemiluminescent reaction. The presence of a bioluminescent
protein is determined by detecting the presence of luminescence.
Important bioluminescent compounds for purposes of labelling are
luciferin, luciferase and sequorin.
Detection of the antibody, fragment or derivative may be
accomplished by a scintillation counter, for example, if the
detectable label is a radioactive gamma emitter, or by a
fluorometer, for example, if the label is a fluorescent material.
In the case of an enzyme label, the detection can be accomplished
by colorimetric methods which employ a substrate for the enzyme.
Detection may also be accomplished by visual comparison of the
extent of enzymatic reaction of a substrate in comparison with
similarly prepared standards.
In situ detection may be accomplished by removing a histological
specimen from a patient, and providing the labelled antibody, or
the unlabelled antibody plus a labelled binding partner to such a
specimen. Through the use of such a procedure, it is possible to
determine not only the presence of the antigen but also its
distribution in the examined tissue. Using the present invention,
those of ordinary skill will readily perceive that any of a wide
variety of histological methods (such as staining procedures) can
be modified in order to achieve such in situ detection. Such
methods include, for example, immunohistochemical staining
procedures. In a preferred embodiment, an avidin-biotin
immunoperoxidase staining system can be used, and a kit utilizing
this system is also contemplated.
The kit employing mAbs or chimeric antibodies of the present
invention can be used for immunohistochemical evaluation of colon,
breast, and ovarian carcinoma. Indications for tissue study are to
evaluate subpopulations of tumor cells that express the antigens
defined by mAbs 31.1 and 33.28.
The colon kit is comprised of the reagents necessary for
immunohistochemical analysis as follows: a) mAbs 31.1, 33.28 or
mouse/human chimeric antibody Chi #1, and the mAb for
carcinoembryonic antigen (CEA), the latter representing the
standard monoclonal used for colon tissue immunohistochemistry; b)
reagents for immunoperoxidase (blocking reagent) in the form of,
for example, goat serum; and secondary antibody, such as, for
example, goat anti-mouse antibody; c) immunoperoxidase; and d)
reagents to produce the brown coloration.
Similar kits can be employed for the immunohistochemical analysis
of breast and ovarian carcinoma.
The immunoperoxidase technique to be employed is that of
Sternberger. The primary antibody (mAb or chimeric antibody) serves
as an antigen which can bind more than one secondary antibody. The
secondary antibodies form a "bridge" between the primary antibody
and the horseradish peroxidase-antiperoxidase complexes.
The kit contemplated herein can be used to study fully developed
colon, carcinoma, polyps in transformation to define the extent of
malignant transformation, benign polyps to see if a site of
transformation has been missed and inflammatory bowel disease to
evaluate any sites of undetected transformation. Similar kits can
be employed to study breast and ovarian carcinomas.
Another kit similar to the above kit is also contemplated which
uses all five mAbs to colon carcinoma in order to evaluate all
subpopulations of tumors and as such has the capability to type and
cross-match the lesions.
The antibody, fragment or derivative of the present invention may
be adapted for utilization in an immunometric assay, also known as
a "two-site" or "sandwich" assay. In a typical immunometric assay,
a quantity of unlabelled antibody (or fragment of antibody), is
bound to a solid support that is insoluble in the fluid being
tested and a quantity of detectably labelled soluble antibody is
added to permit detection and/or quantitation of the ternary
complex formed between solid-phase antibody, antigen, and labelled
antibody.
Typical, and preferred, immunometric assays include "forward"
assays in which the antibody bound to the solid phase is first
contacted with the sample being tested to extract the tumor antigen
from the sample by formation of a binary solid phase antibody-CCAA
complex. After a suitable incubation period, the solid support is
washed to remove the residue of the fluid sample, including
unreacted tumor antigen, if any, and then contacted with the
solution containing an unknown quantity of labelled antibody (which
functions as a "reporter molecule"). After a second incubation
period to permit the labelled antibody to complex with the CCAA
bound to the solid support through the unlabelled antibody, the
solid support is washed a second time to remove the unreacted
labelled antibody. This type of forward sandwich assay may be a
simple "yes/no" assay to determine whether CCAA is present or may
be made quantitative by comparing the measure of labelled antibody
with that obtained for a standard sample containing known
quantities of the antigen. Such "two-site" or "sandwich" assays are
described by Wide (Radioimmune Assay Method, Kirkham, ed., E. &
S. Livingstone, Edinburgh, 1970, pp. 199-206).
Other type of "sandwich" assays, which may also be useful with
CCAA, are the so-called "simultaneous" and "reverse" assays. A
simultaneous assay involves a single incubation step wherein the
antibody bound to the solid support and labelled antibody are both
added to the sample being tested at the same time. After the
incubation is completed, the solid support is washed to remove the
residue of fluid sample and uncomplexed labelled antibody. The
presence of labelled antibody associated with the solid support is
then determined as it would be in a conventional "forward" sandwich
assay.
In the "reverse" assay, stepwise addition first of a solution of
labelled antibody to the fluid sample, followed by the addition of
unlabelled antibody bound to a solid support after a suitable
incubation period, is utilized. After a second incubation, the
solid phase is washed in conventional fashion to free it of the
residue of the sample being tested and the solution of unreacted
labelled antibody. The determination of labelled antibody
associated with a solid support is then determined as in the
"simultaneous" and "forward" assays. In one embodiment, a
combination of antibodies of the present invention specific for
separate epitopes may be used to construct a sensitive three-site
immunoradiometric assay.
For purposes of in vivo imaging of colon, breast, and ovarian
cancer using the antibodies of the present invention, there are
many different labels and methods of labelling known to those of
ordinary skill in the art. Examples of the types of labels which
can be used in the present invention include radioactive isotopes,
paramagnetic isotopes, and compounds which can be imaged by
positron emission tomography (PET). Those of ordinary skill in the
art will know of other suitable labels for binding to the
antibodies used in the invention, or will be able to ascertain
such, using routine experiments. Furthermore, the binding of these
labels to the antibody can be done using standard techniques common
to those of ordinary skill in the art.
For diagnostic in vivo imaging, the type of detection instrument
available is a major factor in selecting a given radionuclide. The
radionuclide chosen must have a type of decay which is detectable
by a given type of instrument. In general, any conventional method
for visualizing diagnostic imaging can be utilized in accordance
with this invention.
Another important factor in selecting a diagnostic radionuclide for
in vivo imaging is that the half-life of a radionuclide be long
enough so that it is still detectable at the time of maximum uptake
by the target issue, but short enough so that deleterious radiation
of the host is minimized. In one preferred embodiment, a
radionuclide used for in vivo imaging does not emit particles, but
produces a large number of photons in a 140-200 keV range, which
may be readily detected by conventional gamma cameras.
For in vivo diagnosis, radionuclides may be bound to the antibody
either directly or indirectly by using an intermediary functional
group. Intermediary functional groups which are often used to bind
radioisotopes which exist as metallic ions to the antibodies are
the chelating agents, diethylene triamine pentaacetic acid (DTFA)
and ethylene diamine tetraacetic acid (EDTA). Examples of metallic
ions which can be bound to the antibodies of the present invention
are .sup.99mTc, .sup.123I, .sup.111In, .sup.131I, .sup.97Ru,
.sup.67Cu, .sup.67Ga, .sup.125I, .sup.68Ga, .sup.72As, .sup.89Zr,
and .sup.201TI.
The specifically exemplified mAbs 33.28 and 31.1, and the chimeric
antibody Chi #1 may be used to facilitate the production of
additional mAbs which bind the same or immunologically
cross-reactive colon carcinoma-associated antigens. First, these
antibodies may be conjugated to a chromatographic support, and used
to immunopurify colon carcinoma-associated antigens. These purified
antigens, in turn, may be used to stimulate an immune response in
suitable animals. Secondly, spleen cells from the responsive
animals may be fused to immortalizing cells, and the resulting
hybridomas screened for secretion of antibodies which bind to the
purified antigen and/or whose binding to colon carcinoma-associated
antigen is competitively inhibited by antibody 33.28 or 31.1, or
chimeric antibody Chi #1.
Having now generally described the invention, the same will be
further understood by reference to certain specific examples which
are included herein for purposes of illustration only and are not
intended to be limiting unless otherwise specified.
EXAMPLE I
Preparation and Characterization of the Colon Carcinoma-Associated
Antigen (CCAA)
The antigenic preparation was obtained from pooled colon carcinoma
membranes according to the method described by Hollinshead et al.,
Cancer 56:480 (1985). This antigenic material was purified to the
extend that the membrane fractions were free of HL-A antigens and
were separated from much of the non-immunogenic glycoprotein
fractions. In its final form the antigenic preparation was shown to
be immunogenic in a specific manner in humans as evidenced by its
capability of eliciting a delayed hypersensitivity reaction only in
patients with active colon, breast, and ovary carcinoma.
Tumor cell suspensions in saline were prepared from fresh operating
room specimens. Single cell suspensions, obtained by conventional
means, were centrifuged for 10 minutes at about 400.times.g and the
supernatant was retained. The cell pellet was resuspended and
recentrifuged. The membrane material was examined by electron
microscopy to assure that only membrane material (and no intact
cells) was present, and the protein content was measured by the
Lowry method.
The membrane material was next subjected to sequential low
frequency sonication and resuspended as a soluble pool of membrane
proteins. The soluble sonicates were separated by gel filtration on
Sephadex-6200. Fractions of 2 ml were collected and the absorbance
profile at 220 and 280 .mu.m was recorded. Fractions comprising
individual protein peaks were pooled, and the pools were
concentrated by Diaflo ultrafiltration. Sephadex-G200 fractions IB
and IIA, as defined by Hollinshead et al., (supra), were further
purified by gradient polyacrylamide gel electrophoresis (PAGE). The
fractions were tested for their ability to elicit positive delayed
cutaneous hypersensitivity reactions in patients with colon
carcinoma. Those fractions with immunogenic activity were said to
contain colon carcinoma-associated antigens (CCAA) and were
employed as immunogens and screening agents in the preparation of
the mAbs.
By gradient PAGE, a double-banded antigen distinct from that of
carcinoembryonic antigen (Gold, P. et al., J. Exp. Med. 122:467-481
(1965); Hollinshead, A. et al., Cancer 56:480 (1985)) was
identified and isolated. The bands comprising this antigen migrated
6.3 and 6.6 cm. distant from tracking dye. Biochemical analysis of
the antigen proved that it was glycoprotein. The molecular weight
of the antigen was estimated based on the electrophoretic mobility
of transferrin (6.4-6.5 cm) which has a molecular weight of 76.5
kDa.
EXAMPLE II
Preparation and Screening of Monoclonal Antibodies
Monoclonal antibodies (mAbs) against human colon
carcinoma-associated antigens (CCAA) were obtained by the
production and cloning of hybrids resulting from the fusion of
mouse myeloma cells Sp2/0-Ag14 with spleen cells from BALB/c mice
which had been immunized with the CCAA described above.
Five hybrid clones were established, as described below, and
designated as 31.2, 31.1, 77, 33.23 and 33.28. All five mAbs
reacted strongly with the CCAA and with two colon carcinoma cell
lines (SW480 and SW620) when assayed by ELISA. Two of the mAbs,
31.1. and 33.28, were studied in greatest detail.
A. Immunization and Cell Fusion
BALB/c mice were immunized by intrapertioneal injection of 50 .mu.g
of the CCAA described above emulsified in complete Freud's
adjuvant, as described by Hollinshead in clinical trials
(Hollinshead et al., supra). Ten days later the mice received an
intravenous booster injection of the same amount of CCAA in saline.
Mice were sacrificed three days later and their spleen cells
obtained. Cell fusion was performed by incubation 5.times.10.sup.7
mouse spleen cells with 10.sup.7 sP2/0-Ag14 myeloma cells in 40%
polyethylene glycol (MW=1500)
B. Screening of Hybrid Clones
An enzyme-linked immunosorbent assay (ELISA), described by Tsang et
al., JNCI77:1175 (1986), was used for the detection of hybridoma
clones producing antibodies specific for the CCAA. CCAA (100
n/well) was immobilized on polystyrene microplates. Antibodies
present in the test supernatants were allowed to bind to the
immobilized antigen. The presence of the bound murine mAbs was
detected with peroxidase-conjugated second antibodies, specific for
mouse immunoglobulins, followed by the chromogenic substrate for
peroxidase, O-phenyldiamine. Wells showing color reactions yielding
Absorbances .gtoreq.0.500 units were scored as positive. Negative
controls gave values of 0.01 to 0.09 units.
Hybridoma wells scoring as positive by ELISA were further screened
by indirect immunofluorescene, using various tumor cells and normal
cells as identified in Table 1, below. All of the tumor cell lines
were obtained from the ATCC. Cells were incubated with hybridoma
culture supernatants at an appropriate dilution (1:2) in phosphate
buffered saline (PBS) for 1 hour at 4.degree. C. The cells were
washed and incubated with a fluorescein-labelled goat anti-mouse
immunoglobulin antibody. The cells were then washed three times
with PBS and examined by fluorescence microscopy. The results
appear in Table 1.
TABLE-US-00001 TABLE 1 INDIRECT IMMUNOFLUORESCENCE REACTIVITY OF
ANTI-COLON CARCINOMA-ASSOCIATED ANTIGEN(S) (COAA) MoAbS WITH HUMAN
CULTURE CELLS.sup.A REACTIVITY OF MoAbS.sup.B CELLS 31.2 77 31.1
33.28 33.23 TUMOR LINES SW948 (COL) - + - - - HCT116 (COL) - - - +
- WIDR (COL) + + + + + COLO320 (COL) + + + - - HS619 (COL) - - - -
- HS853 (COL) - - - - - CACO-2 (COL) + + - - - SK-CO-1 (COL) + + -
+ + HT-29 (COL) + + - + + SW1116 (COL) + - - + + SW480 (COL) + + +
+ + SW620 (COL) + + + + + 231 (BE) - - - - - CAMA-1 (BE) - - - - -
PAN-1 (PAN) - - + - - MIA (PAN) - - + - - HS766T (PAN) - - - - -
M-14 (MEL) - - - - - HT1080 (FIB) - - - - - LM (OS) - - - - - TE-85
(OS) - - - - - NORMAL SKIN - - - - - FIBROBLAST BONE MARROW - - - -
- CELL NORMAL HUMAN - - - - - PBMC .sup.ACULTURE SUPERNATANT WAS
DILUTED 1:2 WITH PBS. .sup.BPOSITIVE (+) AND NEGATIVE (-)
REACTIVITIES WERE DEFINED BY INTENSITY OF THE MEMBRANE FLUORESCENCE
AS COMPARED TO THE BACKGROUND. C. COL: COLON CARCINOMA; BR: BREAST
CARCINOMA; PAN: PANCREATIC CARCINOMA; FIB: FIBROSOMCOMA.
EXAMPLE III
Analysis of Monoclonal Antibodies and their Reactivity
The anti-CCAA mAbs produced and detected as above were also tested
for reactivity with fresh human tissue. Cryostat sections of the
tissue types listed in Table 2, below, were fixed with 3.5%
formaldehyde in PBS and then washed three times with PBS. For
indirect immunofluorescence studies, the sections were incubated
with the mAbs and then stained with a fluorescein-labelled second
antibody as above.
As is shown in Table 2, mAbs 31.1 and 33.28 were highly specific
for colon carcinoma cells. This indicates that an antigen (CCAA)
which was highly specific for colon carcinoma and, furthermore, was
immunogenic in colon carcinoma patients (positive DH reactions),
and served as a successful immunogen in mice for the devnd
phycoerythrin excitation was used. Trigger regions were established
by examining cells by forward versus 90.degree. light scatter. As
shown in Table 4, both 31.1 and 33.28 bound to colon carcinoma
cells; neither mAb bound significantly to PBMC.
TABLE-US-00002 TABLE 2 Indirect Immunofluorescence of Anti-CCAA
Mabs with Flesh Human Tissues.sup.a Reactivity of Mabs Tissues
33.28 31.1 Tumor Colon Carcinoma 3/3 3/3 Pancreatic Carcinoma 0/2
0/2 Melanoma 0/2 0/1 Breast Carcinoma 0/2 0/1 Normal Placenta 0/1
0/1 Liver 0/1 0/1 Colon 0/3 0/3 Spleen 0/1 0/1 Thymus 0/1 0/1
Muscle 0/1 0/1 .sup.aAscitic fluid was diluted 1:50 with PBS.
Cryostat sections (4-6 .mu.M thick) were fixed with 3.5%
formaldehyde in PBS for 10 minutes and then washed three times in
PBS. Sections were stored at -70.degree. C. unless used
immediately. Results are expressed as number of positive/negative
of tissue tested.
Table 3 shows the results of an immunoabsorption analysis of the
Mabs. Three colon carcinoma cell lines (HT-29, WIDR and SW 620) and
an osteosarcoma cell line (LM) were used to absorb fluorescein
isothiocyanate (FITC)-conjugated Mabs 31.1 or 3.28. Ascites fluid
from mice in which the hybridomas were growing, diluted 1:50, was
added to the absorbing cells. The mixtures were incubated for 1 hr
at 4.degree. C. Either 2.times.10.sup.7 cells (Table 3, Part A) or
10.sup.4 cells (Table 3, Part 5) were used to absorb the antibodies
(Table 3) The osteosarcoma cell line did not absorb out 31.1 or
33.28 activity, while the colon carcinoma cell lines did.
TABLE-US-00003 TABLE 3 Immunoabsorption Analysis of mAbs Absorbing
Cells HT-29 WIDR SW620 LM mAb A B A B A B A B 33.28 - + - + - + + +
31.1 - + - + - + + +
Cytofluorometric analysis was used to measure the binding of 31.1.
33.28 and a control mAb to HT29, WIDR and SW480 tumor cells and to
peripheral blood mononuclear cells (PBMCs) An Ortho Spectrum III
Cytofluorograph, equipped with an argon laser capable of
fluorescein and phycoerythrin excitation was used. Trigger regions
were established by examining cells by forward versus 90.degree.
light scatter. As shown in Table 4, both 31.1 and 33.28 bound to
colon carcinoma cells; neither mAb bound significantly to PBMC.
TABLE-US-00004 TABLE 4 SUMMARY OF CYTOFLUOROMETRIC ANALYSIS % of
cells stained with mAb; Cells 33.28 31.1 Control HT29 51.0 53.9 9.2
WiDr 21.0 ND 8.1 SW480 37.0 32.0 3.8 PBMC 2.1 2.1 ND
The heavy and light chain isotypes of the mAbs were determined by
immunodiffusion. The 31.1 mAb was found to be an IgG1 with a kappa
light chain. The 33.28 mAb was found to be an IgG2a with a kappa
light chain (Table 5). This is in strong contrast to the prior art
mAb 19.9 (Herlyn, M. et al., J. Biol. Chem. 257:14365-14369 (1982))
which is of the IgG1 class (Herlyn, D. et al., Proc. Natl. Acad.
Sci. USA 79:4761-4765 (1982)). Importantly, antibodies of the IgG2a
class are expected to be more useful for immunotherapeutic purposes
(Colcher, D. et al., Proc. Natl. Acad. Sci. USA 78:3199-3203
(1981)). Although the 19.9 mAb has reactivity to colon tumors, it
was derived by immunization with pancreatic carcinoma antigens, and
is therefore cross-reactive with colon. This is analogous to the
situation with the B72.3 mAb ([citation]), which is a
colon-reactive antibody obtained by immunization with breast cancer
tissue.
TABLE-US-00005 TABLE 5 Isotyping of Monoclonal Antibodies Culture
Light Chains Supernatant IgG1 IgG2a IgG2b IgM Kappa Lambda 31.2 - +
- - + - 31.1 + - - - + - 77 - - + - + - 33.23 - + - - + - 33.28 - +
- - + -
EXAMPLE IV
Characterization of the Colon Carcinoma-Associated Antigen
The molecular mass of the antigens to which the above mAbs bound
was determined by Western blot analysis using soluble protein
extracted from colon carcinoma cell lines SW480 and SW620. The
33.28 .[.and 31.1 mAbs.]. .Iadd.antibody .Iaddend.reacted with
molecules having .Iadd.an .Iaddend.apparent molecular .[.weights.].
.Iadd.weight .Iaddend.of 61.1 kDa.[.and 72 kDa, respectively.]. ,
from both of these cells lines. .[.These mAbs.]. .Iadd.The 33.28
and 31.1 mAbs .Iaddend.did not react with material from human PBMCs
or from human tumor cell lines of other histologic types in Western
blot analysis.
In order to define better the specificity of the mAbs of the
present invention for the immunizing CCAA and to establish whether
the mAbs reacted with an immunogenic component of the cell membrane
preparation which has been used in clinical immunotherapy trials
(Hollinshead et al., supra), the original immunogenic preparation
described above was performed by high performance liquid
chromatography (HPLC).
The analysis revealed 4 distinct peaks, each of which was tested
for immune reactivity (elicitation of DH) in patients with colon
carcinoma by skin test (FIG. 1). Among the 10 patients with colon
carcinoma tested only the material in peak #4 induced a cutaneous
DH reaction. The peak #4 antigen was found to react with mAb 33.28,
while mAb 31.1 reacted with peak #3, the next most prominent
peak.
The references cited above are all incorporated by reference
herein.
EXAMPLE V
Affinity Purification of Colon Carcinoma-Associated Antigen
The mAb 33.28 was used in affinity chromatography to isolate
antigen extracted from cells of the HT-29 line. Five mg of purified
33.28 IgG was coupled to CNBr-activated sepharose 4B. The column
was pre-eluted with 0.05M diethylamine, pH 11.5, and then
equilibrated with 0.14M NaCl/0.01M Tris (pH 8.0). CCAA preparation
was applied to the column, and the column was eluted with 0.05M
diethylamine, pH 11.5. The eluted fractions were neutralized by the
addition of IM Tris-HCl, pH 8.0.
The material bound and eluted from the 33.28 affinity matrix was
then subjected to HPLC. The eluted CCAA preparation was adjusted in
starting buffer (0.01M sodium phosphate buffer, pH 7.0), applied to
a Synchropak Wax weak anion exchange HPLC column (250.times.4.6 mm)
and eluted with a gradient of 0 to 1M NaCl in 0.01M sodium
phosphate buffer, pH 6.0, at a flow rate of 1 ml/min. Anion
exchange chromatography was performed using a Hewlett-Packard HPLC
(HP 1090, Hewlett-Packard, Arondale, Pa.).
Results appear in FIG. 2. The antigenic material derived from HT-29
cells isolated by mAb 33.28 gave a peak that matched peak #4
described above and had similar immunogenic activity in humans,
indicating the utility of mAb 33.28 for isolation of a colon cancer
preparation which is immunogenic for humans.
EXAMPLE VI
ADCC Activity of mAbs 33.28 and 31.1
In order to be therapeutically useful, a mAb specific for an
immunogenic tumor antigen should have the following properties: (a)
high tumor tissue specificity, (b) absence of cross-reactivity to
normal human tissue, and (c) a biological activity associated with
destruction of tumors, such as antibody-dependent cellular
cytotoxicity (ADCC).
The ADCC activity of mAbs 33.28 and 31.1 was tested on the colon
carcinoma line WiDR as target cell. The melanoma cell line, M-14,
served as a specificity control. ADCC was assayed using a
conventional 4 hr. .sup.51Cr release assay using normal human PBMC
as effector cells, and the results are shown as percent isotope
release (% lysis) (Table 6). The background lysis was 8.3%. At an
effector:target ratio of 100:1, mAb 33.28 caused 40.3% lysis of
tumor cells, and 31.1 induced 51.8% lysis.
TABLE-US-00006 TABLE 6 ADCC Activity of mAbs 33.28 and 31.1 % Lysis
of Target Cells at E:T Ratios: Antibody WiDR M-14 or Control 25 50
100 25 50 100 33.28 23.1 40.3 45.3 6.9 8.4 9.0 31.1 14.3 26.7 51.8
7.5 6.4 8.7 OSA1 10.0 9.2 12.2 11.4 14.8 10.9 NMS 12.2 11.7 13.1
14.2 15.0 11.1 PBS 8.2 5.1 7.6 11.0 14.2 10.5 ADCC was assayed by a
4 hour .sup.51Cr release assay. Background .sup.51Cr release was
8.3%. E:T Ratio indicates effector cell-to-target ratios. The mAb
or serum was tested at a 1:100 dilution; OSA1 - mAb to-osteosarcoma
associated antigens; NMS - normal mouse serum; WiDr - colon
carcinoma cell line; M14 - melanoma cell line.
EXAMPLE VII
Detection of Circulating CCAA with mAbs 33.28 and 31.1
The mAbs of the present invention were tested for their ability to
detect circulating CCAA in 79 unknown serum samples (Table 7). The
assay was based on the ability of the serum samples to inhibit
binding of the mAb to the CCAA in ELISA. None of the 50 normal
serum samples gave false positive results. Nine of the ten serum
samples from patients with active colon carcinoma were positive.
None of the sera from disease-free colon cancer patients one year
post-resection were positive.
TABLE-US-00007 TABLE 7 Detection of Circulating Colon
Carcinoma-Associated Antigen No. of sera inhibiting binding of
mAbs; 33.28 31.1 DONOR No. of <15% >15% <15% >15%
CONDITION Samples (Neg) (Pos) (Neg) (Pos) Colon 10 3 7 2 8
Carcinoma Colon 4 4 0 4 0 Carcinoma (Resected) Breast 9 9 0 9 0
Carcinoma Melanoma 5 5 0 5 0 Prostate 1 1 0 1 0 Cancer Normal Serum
50 50 0 50 0 Colon carcinoma-associated antigens was detected by
ELISA. 100 .mu.l of serum were used in each assay.
EXAMPLE VIII
Comparison of Specificity with Other mAbs Reactive with Colon
Tumors
Further studies of tumor specificity were conducted using ELISA
(Table 8). The mAb 31.1 was compared with CC49, a colorectal
carcinoma-specific mAb purified from B-72.3, and a control mouse
myeloma protein. 31.1 was shown to react with a narrower range of
colorectal carcinomas than did CC49. However, it had a higher
degree of specificity, having lower or no cross-reactivity with
stomach tumors or normal colon tissue.
TABLE-US-00008 TABLE 8 ELISA ON NORMAL AND TUMOR TISSUES USING MAbs
31.1 CC49 AND MOPC-21 Tissues 31.1 CC49 MOPC-21 Colorectal
Carcinomas 1. COCA2A - +++ - 2. COCA2 - +++ - 3. COCA3 + ++ - 4.
COCA4 +++ +++ - 5. G820 .+-. ++ - 6. G853 +++ ++ - 7. G817 +++ +++
- 8. G781 - - - Other Carcinomas 1. Breast CA1 - - - 2. Breast CA2
- - - 3. Lung CA1 - - - 4. Lung CA2 - - - 5. Ovarian CAD106 - - -
6. Ov CA5 - - - 7. Ov CAV5 - - - 8. Ov CAV45 - - - 9. Ov CAV43 - -
- 10. Stomach CA14A ++ +++ 11. Stomach CA12A - +++ - 12. Stomach
CA15A - - - Other Normal Tissues 1. Endometrium E21 - - - 2.
Endometrium EC19 - - - 3. Endometrium EC17 - + - 4. Endometrium
EC18 - - - (RBC) 5. Red blood cells 1 - - - 6. RBC 2 - - - 7. RBC 3
- - - 8. RBC 4 - - - 9. RBC 5 - - - 10. RBC 6 - - - 11. RBC 7 - - -
12. RBC 8 - - - 13. RBC 9 - - - 14. RBC 10 - - - 15. RBC 11 - - -
16. Granulocytes - - - 17. 385 - - - 18. 386 - - - 19. Normal
spleen 3 - - - 20. 392 (N. Spleen) - - - 21. 395 (N. Liver) - - -
22. 387 (N. Kidney) - - - 23. 398 (N. Spleen) - - - 24. 390 (N.
Liver) - - - 25. N. Spleen #1 - - - 26. N. Spleen #2 - - - 27. 800
(N. Colon) - ++ - 28. N. Colon (GW) - ++ - 29. N. Colon (Meloy) - -
- 30. N. Colon - - - 31. G1155B (N. Colon) - - - 32. G1164B (N.
Colon) - - - 33. N. Colon .+-. - - 34. Normal Stomach A - - - 35.
N. Stomach B - - - 36. N. Stomach C - - - 37. Normal Lung - - - 38.
Normal Liver - - - CC49 - NCI monoclonal antibody to colorectal
carcinomas MOPC-21 - negative control myeloma protein All
monoclonal antibodies were used at 40 ng/well, POGAM at 1:3000
dilution
EXAMPLE IX
In Vivo Localization of mAbs 33 28 and 31.1 to Tumors
The in vivo behavior of the mAbs of the present invention was
examined by pharmacokinetic studies using .sup.125I-labelled mAb
and athymic nude mice bearing LS-174T colon tumor xenografts. Mice
implanted with the A375 melanoma were employed as controls. The
relative concentration of mAb in tumor as compared to adjacent
normal tissue such as liver and spleen is represented by the
radiolocalization index (concentration of radiolabelled material in
tumor/concentration in surrounding tissue). The biodistribution of
.sup.125I-labelled 31.1 and 33.28 mAbs is shown in Table 9. Both
mAbs were able to significantly concentrate within the tumor,
compared to localization in normal tissues (spleen and liver). This
selective accumulation was six-fold at 96 hours and about 12-fold
at 168 hrs. The radiolocalization indices for both mAbs to tumor as
compared to blood, liver and spleen are shown in FIG. 3 and FIG.
4.
TABLE-US-00009 TABLE 9 Biodistribution of .sup.125I-mAbs in
Tumor-Bearing Athymic Nude Mice 96 hours 168 hours Tissue LS174T
A375 LS174T A375 A. mAb 31.1 Blood 7.30 NA 4.67 4.42 Tumor 21.92 NA
25.43 2.91 Liver 3.74 NA 2.16 1.24 Spleen 3.68 NA 2.41 1.32 B. mAb
33.28 Blood 7.81 NA 5.58 3.95 Tumor 13.12 NA 15.50 2.24 Liver 2.55
NA 1.74 1.68 Spleen 2.31 NA 1.70 1.92 Results are expressed in %
injected dose/gram of tissue. LS174T = colon carcinoma; A375 =
melanoma.
EXAMPLE X
Immunohistochemical Studies
The ability to detect tumor markers in the serum, image the related
neoplastic process and define the cell population of that neoplasm
by immunohistochemistry depends on the ability of specific mAbs to
selectively characterize a tumor population.
The mAbs 31.1 and 33.28 were tested in more than 50 colon
carcinomas by means of immunoperoxidase staining. They have been
found to be highly reactive with the colon neoplasm and did not
interact with the adjacent normal tissues. When polyps were
evaluated, the wholly benign lesions such as villo-tubular adenomas
showed no reactivity. Villous adenomas undergoing transformation
reacted only at the site of malignancy. When similar tissues were
evaluated using the more common carbohydrate-antigen derived mAbs,
normal adjacent colon tissue reacted equally with the neoplastic
portion of the specimen. With the 31.1 and 33.28 mAbs, each
appeared to stain different cell populations within the tumor. This
suggests that surface antigens representing different oncogene
products were being defined.
EXAMPLE XI
Selective Binding to Subpopulations of Epithelial Cells in
Paraffin-Embedded Benign and Malignant Mammary Lesions
Forty-one formalin-fixed, paraffin-embedded benign and malignant
breast specimens were studied with mAbs 31.1 and 33.28, using the
avidin-biotin staining method. Without enzymatic pretreatment,
positive staining of epithelium was observed on the cell surface
and in the cytoplasm with both antibodies. 7/21 (33%) duct
carcinogens were positive with mAb 31.1 as were 5/20 (25%) samples
of benign breast disease. 10/21 (48%) duct carcinomas were positive
with mAb 33.28 together with 7/20 (35%) specimens of benign mammary
disease. 10 to 75% of the cell population was positively stained.
These results indicate that the antigens defined by mAbs 31.1 and
33.28 are expressed in a select group of women with breast disease
and would be useful for diagnosis of said disease.
EXAMPLE XII
Selective Binding to Subpopulations of Epithelial Cells in Fresh
Frozen Benign and Malignant Ovarian Tumors
Fresh frozen tissue biopsies obtained from twenty-one ovarian
tumors subjected to immunocytochemical analysis were studied with
mAbs 31.1 and 33.28 using the avidin biotin indirect
immunoperoxidase assay. Focal positive staining was observed in 4/7
papillary mucous, 1/1 mucinous and 1/2 endometroid adenocarcinoma.
None of the nonepithelial ovarian tumors stained positive using
these monoclonal antibodies. These results indicate that the
antigens as defined by mAbs 31.1 and 33.28 are expressed in a
select group of woman with ovarian cancer and would be useful for
diagnosis of said disease.
EXAMPLE XIII
Immunoreactivity of Human Carcinoma Tissues and Cell Lines with
mAbs 31.1 and 33.28
The mAbs 31.1 and 33.28 were used to screen a panel of cell lines
including colon adenocarcinoma, lymphoma, leukemia and
neuroblastoma lines.
Using the avidin-biotin immunoperoxidase staining system, the mAbs
31.1 and 33.28 were shown to strongly bind to colon adenocarcinoma
cell lines WIDR and HT-29. Immunoreactions were not observed with
KG1-a, HL-60, Molt-3 and JUKRAT cell lines. Both antibodies reacted
weakly with one lymphoma line (JY). The mAb 33.28 reacted weakly
with one leukemic line (K562) and a neuroblastoma line (U87. MG).
These results confirm and extend previous flow cytometry and
immunofluorescent results in which it has been reported that strong
binding reactions were observed with these mAbs with colon
adenocarcinoma cell lines and reactions were not observed with
other tumor cell lines. Using flow cytometry, mAb 31.1 reacted with
85% of HT-29 and WIDR colon carcinoma cells but not with SKBR-3
breast cancer cells.
Both antibodies were extensively shown to bind distinctively to
colon carcinoma tissues (mAb 33.28-84%, mAb 31.1-64%), and not to
normal tissues or malignant tissues including neuroblastoma tissues
( 0/3), lymphomas ( 0/3) and leukemic infiltrates ( 0/3) tested.
These results suggest that these antibodies can serve as a useful
research tool in evaluating tumor markers in cancer and cell
biology research.
EXAMPLE XIV
Expression and Characterization of Chimeric Antibodies Against
Human Colorectal Carcinoma-Associated Antigen
A chimeric mouse/human heavy chain gene was constructed by splicing
the exon of the 31.1 antibody heavy chain variable region gene to
the exon of the human gamma1 chain contstant region gene using the
polymerase chain reaction. Subsequently, the 31.1 chimeric gene was
cloned into a retroviral expression vector pLgptCXII and
transfected into the packaging cell line PA317. The transfected
cells (PA317H) were cultivated with another packaging cell line
PA317L, which contained an irrelevant mouse/human chimeric light
chain gene in retroviral expression vector pLneoCXII, and
SP2/0-Ag14 cells. The transduced SP2/0-Ag14 cells yielded a
complete chimeric antibody. Chi #1 which reacted with horseradish
peroxidase-conjugated igG of goat anti-human IgG Fc in ELISA
analyses, which indicated that the constant region of Chi #1 was
human. Cytofluorometry analysis indicated that Chi #1 stained human
colorectal carcinoma cell lines HT-29 and LS174T but not a human
lung carcinoma cell line A-427. Antibody-dependent cell-mediated
cytoxicity (ADCC) assay indicated that Chi #1 lysed Ls174T cells.
These results shows that Chi #1 retained the antigen-binding
specificity of the parental 31.1 mouse monoclonal antibody,
suggesting the useful of this chimeric antibody in ascertaining
prognosis of colon carcinoma.
Having now fully described this invention, it will be appreciated
by those skilled in the art that the same can be performed within a
wide range of equivalent parameters, concentrations, and conditions
without departing from the spirit and scope of the invention and
without undue experimentation.
While this invention has been described in connection with specific
embodiments thereof, it will be understood that it is capable of
further modifications. This application is intended to cover any
variations, uses, or adaptations of the inventions following, in
general, the principles of the invention and including such
departures from the present disclosure as come within known or
customary practice within the art to which the invention pertains
and as may be applied to the essential features hereinbefore set
forth as follows in the scope of the appended claims.
* * * * *