Novel antibodies reactive with human carcinomas

Abstract

The present invention relates to novel antibodies, antibody fragments and antibody conjugates and single-chain immunotoxins reactive with human carcinoma cells. More particularly, the antibodies, conjugates and single-chain immunotoxins of the invention include: a murine monoclonal antibody, BR96; a human / murine chimeric antibody, ChiBR96; a F(ab')2 fragment of BR96; ChiBR96-PE, ChiBR96-LysPE40, ChiBR96 F(ab')2-LysPE40 and ChiBR96 Fab'-LysPE40 conjugates and recombinant BR96 sFv-PE40 immunotoxin. These molecules are reactive with a cell membrane antigen on the surface of human carcinomas. The BR96 antibody and its functional equivalents, displays a high degree of selectivity for carcinoma cells and possess the ability to mediate antibody-dependent cellular cytotoxicity and complement-dependent cytotoxicity activity. In addition, the antibodies of the invention internalize within the carcinoma cells to which they bind and are therefore particularly useful for therapeutic applications, for example, as the antibody component of antibody-drug or antibody-toxin conjugates. The antibodies also have a unique feature in that they are cytotoxic when used in the unmodified form, at specified concentrations.

Description

[0001] This application is a continuation-in-part of U.S. Ser. No. 08 / 057,444, filed May 5, 1993, which is a file wrapper continuation application of U.S. Ser. No: 07 / 544,246 filed Jun. 26, 1990, which was a continuation-in-part of U.S. Ser. No: 07 / 374,947, filed Jun. 30, 1989, now abandoned, the entire disclosure of these applications being incorporated by reference herein.[0002] The present invention relates to novel antibodies reactive with carcinoma cells. More particularly, the invention relates to a murine monoclonal antibody and a chimeric monoclonal antibody, including immunoconjugates and recombinant immunotoxins made therefrom, that react with cell membrane antigens associated with a large variety of carcinomas including carcinomas of the colon, breast, ovary and lung. The murine monoclonal antibody is highly specific for carcinomas, showing no to very low reactivity with normal animal tissues or other types of tumors such as lymphomas or sarcomas.[0003] 1. Monoclonal Anti...

AI Technical Summary

Benefits of technology

: Initial studies with various previously known immunoconjugates have been disappointing particularly with solid tumors. In our effort to improve antibody based therapy of carcinomas, we have developed and examined novel immunoconjugates and the anti-cancer drug doxorubicin (DOX).[0383] BR96 is important for several reasons. It can trigger irreversible changes in membrane structure which leads to tumor cell death, most likely through the loss of osmotic control (J. Garrigues, U. Garrigues, I. Hellstrom, K. E. Hellstrom, Am. J Pathol. 142, 607 (1993)). Further, it is an internalizing MAb that cycles in a nondegraded form between the intracellular compartment and the medium for extended periods of time. The latter characteristic makes BR96 an attractive candidate for targeting to tumors various agents for selective concentration in antigen positive cells.[0384] The antigen for BR96 is abundantly expressed (>200,000 molecules / cell) on human carcinoma lines. BR96 binds, according to immunohistology, the majority of human carcinomas of the breast, lung and colon. Although BR96, like essentially all MAbs to human tumors, is not truly tumor-specific, it offers advantages over most other antibodies which recognize the Le.sup.y determinant (K. Lloyd, G. Larson, N. Stromberg, J. Thurin, K. A. Karlsson, Immunogenetics 17, 537 (1983); P. M. Pour, V. E. Tempero, C. Cordon-Cardo, P. Avner, Cancer Res. 48, 5422 (1988); J. Sakamoto et al., ibid. 49, 745 (1989); T. F. Orntoft, H. Wolf, H. Clausen, E. Dabelsteen, S. I. Hakomori, Int. J. Cancer 43, 774 (1989)).[0385] BR96 is more tumor selective and the normal tissues to which it binds primarily comprise differentiated cells of the esophagus, stomach, and intestine as well as acinar cells of the pancreas (I. Hellstrom, H. J. Garrigues, U. Garrigues, K. E. Hellstrom, Cancer Res. 50, 2183 (1990)).[0386] BR96 is rapidly internalized into lysosomes and endosomes after binding to cells expressing the antigen (J. Garrigues et al. 1993).[0387] The antibodies mediate antibody-dependent cellular cytotoxicity "antibody-dependent cellular cytotoxicity," "complement-mediated cytotoxicity," and "complement-dependent cytotoxicity."[0388] The antibodies can kill antigen-positive tumor cells in the unconjugated form if present at a sufficient concentration. The antibody conjugates and recombinant immunotoxins are useful as reagents for killing tumor cells. The antibodies are also useful in diagnostic methods, such as the detection of carcinomas by in vitro or in vivo technology.

Problems solved by technology

Antibodies to tumor-associated antigens which are not able to internalize within the tumor cells to which they bind are generally not useful to prepare conjugates with antitumor drugs or toxins, since these would not be able to reach their site of action within the cell.

However, because the transferrin-receptor is also expressed on many normal tissues, and often at high levels, the use of an anti-transferrin-recepto-r antibody in an antibody-drug or antibody-toxin conjugate may have significant toxic effects on normal cells.

The utility of this antibody for selective killing or inhibition of tumor cells is therefore questionable.

However, murine monoclonal antibodies may be recognized as foreign substances by the human immune system and neutralized such that their potential in human therapy is not realized.

Antibodies lacking antibody-dependent cellular cytotoxicity and complement-dependent cytotoxicity activity in vitro, on the other hand, are commonly ineffective in vivo unless used as carriers of antitumor agents.

However, with chemotherapy only modest progress has been made for treating the majority of carcinomas, including carcinomas of breast, lung, and colon.

However, activity of these MAbs was usually assessed against newly implanted rather than established tumors and was typically superior to matching, but not optimal, doses of the unconjugated drug.

Such antibodies are of particular interest since they can interfere with some key event in the survival of neoplastic cells.

Tumors that are detected early on such as acute lymphocytic leukemia and lymphomas are highly susceptible to drugs.

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