Skip to main content
PMC home page
The Journal of Experimental Medicine logoLink to The Journal of Experimental Medicine
. 1992 Jan 1;175(1):217–225. doi: 10.1084/jem.175.1.217

Development of humanized bispecific antibodies reactive with cytotoxic lymphocytes and tumor cells overexpressing the HER2 protooncogene

PMCID: PMC2119072  PMID: 1346155

Abstract

The HER2 protooncogene encodes a 185-kD transmembrane phosphoglycoproteins, human epidermal growth factor receptor 2 (p185HER2), whose amplified expression on the cell surface can lead to malignant transformation. Overexpression of HER2/p185HER2 is strongly correlated with progression of human ovarian and breast carcinomas. Recent studies have shown that human T cells can be targeted with bispecific antibody to react against human tumor cells in vitro. We have developed a bispecific F(ab')2 antibody molecule consisting of a humanized arm with a specificity to p185HER2 linked to another arm derived from a murine anti-CD3 monoclonal antibody that we have cloned from UCHT1 hybridoma. The antigen-binding loops for the anti-CD3 were installed in the context of human variable region framework residues, thus forming a fully humanized BsF(ab')2 fragment. Additional variants were produced by replacement of amino acid residues located in light chain complementarity determining region 2 and heavy chain framework region 3 of the humanized anti-CD3 arm. Flow cytometry analysis showed that the bispecific F(ab')2 molecules can bind specifically to cells overexpressing p185HER2 and to normal human peripheral blood mononuclear cells bearing the CD3 surface marker. In additional experiments, the presence of bispecific F(ab')2 caused up to fourfold enhancement in the cytotoxic activities of human T cells against tumor cells overexpressing p185HER2 as determined by a 51Cr release assay. These bispecific molecules have a potential use as therapeutic agents for the treatment of cancer.

Full Text

The Full Text of this article is available as a PDF (1.0 MB).

Selected References

These references are in PubMed. This may not be the complete list of references from this article.

  1. Beverley P. C., Callard R. E. Distinctive functional characteristics of human "T" lymphocytes defined by E rosetting or a monoclonal anti-T cell antibody. Eur J Immunol. 1981 Apr;11(4):329–334. doi: 10.1002/eji.1830110412. [DOI] [PubMed] [Google Scholar]
  2. Brüggemann M., Winter G., Waldmann H., Neuberger M. S. The immunogenicity of chimeric antibodies. J Exp Med. 1989 Dec 1;170(6):2153–2157. doi: 10.1084/jem.170.6.2153. [DOI] [PMC free article] [PubMed] [Google Scholar]
  3. Chang C. N., Kuang W. J., Chen E. Y. Nucleotide sequence of the alkaline phosphatase gene of Escherichia coli. Gene. 1986;44(1):121–125. doi: 10.1016/0378-1119(86)90050-8. [DOI] [PubMed] [Google Scholar]
  4. Chothia C., Lesk A. M. Canonical structures for the hypervariable regions of immunoglobulins. J Mol Biol. 1987 Aug 20;196(4):901–917. doi: 10.1016/0022-2836(87)90412-8. [DOI] [PubMed] [Google Scholar]
  5. ELLMAN G. L. Tissue sulfhydryl groups. Arch Biochem Biophys. 1959 May;82(1):70–77. doi: 10.1016/0003-9861(59)90090-6. [DOI] [PubMed] [Google Scholar]
  6. Ellison J. W., Berson B. J., Hood L. E. The nucleotide sequence of a human immunoglobulin C gamma1 gene. Nucleic Acids Res. 1982 Jul 10;10(13):4071–4079. doi: 10.1093/nar/10.13.4071. [DOI] [PMC free article] [PubMed] [Google Scholar]
  7. Fanger M. W., Segal D. M., Romet-Lemonne J. L. Bispecific antibodies and targeted cellular cytotoxicity. Immunol Today. 1991 Feb;12(2):51–54. doi: 10.1016/0167-5699(91)90156-N. [DOI] [PubMed] [Google Scholar]
  8. Garrido M. A., Valdayo M. J., Winkler D. F., Titus J. A., Hecht T. T., Perez P., Segal D. M., Wunderlich J. R. Targeting human T-lymphocytes with bispecific antibodies to react against human ovarian carcinoma cells growing in nu/nu mice. Cancer Res. 1990 Jul 15;50(14):4227–4232. [PubMed] [Google Scholar]
  9. Hale G., Dyer M. J., Clark M. R., Phillips J. M., Marcus R., Riechmann L., Winter G., Waldmann H. Remission induction in non-Hodgkin lymphoma with reshaped human monoclonal antibody CAMPATH-1H. Lancet. 1988 Dec 17;2(8625):1394–1399. doi: 10.1016/s0140-6736(88)90588-0. [DOI] [PubMed] [Google Scholar]
  10. Hudziak R. M., Lewis G. D., Shalaby M. R., Eessalu T. E., Aggarwal B. B., Ullrich A., Shepard H. M. Amplified expression of the HER2/ERBB2 oncogene induces resistance to tumor necrosis factor alpha in NIH 3T3 cells. Proc Natl Acad Sci U S A. 1988 Jul;85(14):5102–5106. doi: 10.1073/pnas.85.14.5102. [DOI] [PMC free article] [PubMed] [Google Scholar]
  11. Hudziak R. M., Schlessinger J., Ullrich A. Increased expression of the putative growth factor receptor p185HER2 causes transformation and tumorigenesis of NIH 3T3 cells. Proc Natl Acad Sci U S A. 1987 Oct;84(20):7159–7163. doi: 10.1073/pnas.84.20.7159. [DOI] [PMC free article] [PubMed] [Google Scholar]
  12. Lanzavecchia A., Scheidegger D. The use of hybrid hybridomas to target human cytotoxic T lymphocytes. Eur J Immunol. 1987 Jan;17(1):105–111. doi: 10.1002/eji.1830170118. [DOI] [PubMed] [Google Scholar]
  13. Liu M. A., Kranz D. M., Kurnick J. T., Boyle L. A., Levy R., Eisen H. N. Heteroantibody duplexes target cells for lysis by cytotoxic T lymphocytes. Proc Natl Acad Sci U S A. 1985 Dec;82(24):8648–8652. doi: 10.1073/pnas.82.24.8648. [DOI] [PMC free article] [PubMed] [Google Scholar]
  14. Maurer R., Meyer B., Ptashne M. Gene regulation at the right operator (OR) bacteriophage lambda. I. OR3 and autogenous negative control by repressor. J Mol Biol. 1980 May 15;139(2):147–161. doi: 10.1016/0022-2836(80)90302-2. [DOI] [PubMed] [Google Scholar]
  15. Nitta T., Sato K., Yagita H., Okumura K., Ishii S. Preliminary trial of specific targeting therapy against malignant glioma. Lancet. 1990 Feb 17;335(8686):368–371. doi: 10.1016/0140-6736(90)90205-j. [DOI] [PubMed] [Google Scholar]
  16. Nolan O., O'Kennedy R. Bifunctional antibodies: concept, production and applications. Biochim Biophys Acta. 1990 Aug 1;1040(1):1–11. doi: 10.1016/0167-4838(90)90139-7. [DOI] [PubMed] [Google Scholar]
  17. Orlandi R., Güssow D. H., Jones P. T., Winter G. Cloning immunoglobulin variable domains for expression by the polymerase chain reaction. Proc Natl Acad Sci U S A. 1989 May;86(10):3833–3837. doi: 10.1073/pnas.86.10.3833. [DOI] [PMC free article] [PubMed] [Google Scholar]
  18. Palm W., Hilschmann N. Die Primärstruktur einer kristallinen monoklonalen Immunglobulin-L-Kette vom kappa-Typ, Subgruppe I(Bence-Jones Protein Rei.), Isoleirung und Charakteristierung der tryptischen Peptide; die vollständige Aminosäuresequenz des Proteins. Ein Beitrag zue Aufklärung der räumlichen Struktur der Antikörper, insbesondere der Haftstelle. Hoppe Seylers Z Physiol Chem. 1975 Feb;356(2):167–191. [PubMed] [Google Scholar]
  19. Perez P., Hoffman R. W., Shaw S., Bluestone J. A., Segal D. M. Specific targeting of cytotoxic T cells by anti-T3 linked to anti-target cell antibody. Nature. 1985 Jul 25;316(6026):354–356. doi: 10.1038/316354a0. [DOI] [PubMed] [Google Scholar]
  20. Picken R. N., Mazaitis A. J., Maas W. K., Rey M., Heyneker H. Nucleotide sequence of the gene for heat-stable enterotoxin II of Escherichia coli. Infect Immun. 1983 Oct;42(1):269–275. doi: 10.1128/iai.42.1.269-275.1983. [DOI] [PMC free article] [PubMed] [Google Scholar]
  21. Riechmann L., Clark M., Waldmann H., Winter G. Reshaping human antibodies for therapy. Nature. 1988 Mar 24;332(6162):323–327. doi: 10.1038/332323a0. [DOI] [PubMed] [Google Scholar]
  22. Sanger F., Nicklen S., Coulson A. R. DNA sequencing with chain-terminating inhibitors. Proc Natl Acad Sci U S A. 1977 Dec;74(12):5463–5467. doi: 10.1073/pnas.74.12.5463. [DOI] [PMC free article] [PubMed] [Google Scholar]
  23. Scholtissek S., Grosse F. A cloning cartridge of lambda t(o) terminator. Nucleic Acids Res. 1987 Apr 10;15(7):3185–3185. doi: 10.1093/nar/15.7.3185. [DOI] [PMC free article] [PubMed] [Google Scholar]
  24. Shalaby M. R., Ammann A. J. Suppression of immune cell function in vitro by recombinant human transforming growth factor-beta. Cell Immunol. 1988 Apr 1;112(2):343–350. doi: 10.1016/0008-8749(88)90303-6. [DOI] [PubMed] [Google Scholar]
  25. Shepard H. M., Lewis G. D., Sarup J. C., Fendly B. M., Maneval D., Mordenti J., Figari I., Kotts C. E., Palladino M. A., Jr, Ullrich A. Monoclonal antibody therapy of human cancer: taking the HER2 protooncogene to the clinic. J Clin Immunol. 1991 May;11(3):117–127. doi: 10.1007/BF00918679. [DOI] [PubMed] [Google Scholar]
  26. Slamon D. J., Clark G. M., Wong S. G., Levin W. J., Ullrich A., McGuire W. L. Human breast cancer: correlation of relapse and survival with amplification of the HER-2/neu oncogene. Science. 1987 Jan 9;235(4785):177–182. doi: 10.1126/science.3798106. [DOI] [PubMed] [Google Scholar]
  27. Slamon D. J., Godolphin W., Jones L. A., Holt J. A., Wong S. G., Keith D. E., Levin W. J., Stuart S. G., Udove J., Ullrich A. Studies of the HER-2/neu proto-oncogene in human breast and ovarian cancer. Science. 1989 May 12;244(4905):707–712. doi: 10.1126/science.2470152. [DOI] [PubMed] [Google Scholar]
  28. Titus J. A., Garrido M. A., Hecht T. T., Winkler D. F., Wunderlich J. R., Segal D. M. Human T cells targeted with anti-T3 cross-linked to antitumor antibody prevent tumor growth in nude mice. J Immunol. 1987 Jun 1;138(11):4018–4022. [PubMed] [Google Scholar]
  29. Verhoeyen M., Milstein C., Winter G. Reshaping human antibodies: grafting an antilysozyme activity. Science. 1988 Mar 25;239(4847):1534–1536. doi: 10.1126/science.2451287. [DOI] [PubMed] [Google Scholar]

Articles from The Journal of Experimental Medicine are provided here courtesy of The Rockefeller University Press

RESOURCES