Novel antibody conjugates reactive with human carcinomas

a technology of human carcinoma and antibody conjugates, which is applied in the field of murine monoclonal antibodies and chimeric monoclonal antibodies, can solve the problems of inconvenient preparation of conjugates with antitumor drugs or toxins, anti-transferrin-receptor antibodies in antibody-drug or antibody-toxin conjugates that may have significant toxic effects on normal cells, and the utility of this antibody for selective killing or inhibition of tumor cells is questionabl

Inactive Publication Date: 2006-01-26
BRISTOL MYERS SQUIBB CO
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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-receptor 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.

Method used

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  • Novel antibody conjugates reactive with human carcinomas
  • Novel antibody conjugates reactive with human carcinomas
  • Novel antibody conjugates reactive with human carcinomas

Examples

Experimental program
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Effect test

preparation 1

2,5-Dihydro-2,5-Dioxo-1H-Pyrrolo-1-Hexanoic Acid Hydrazide and its Trifluoroacetic Acid Salt (“Maleimidocaproyl Hydrazide”)

[0283] Maleimidocaproic acid (2.11 g, 10 mmol) [see, e.g., D. Rich et al., J. Med. Chem., 18, 1004 (1975) and, O. Keller, et al., Helv. Chim. Acta, 58 531 (1975)] was dissolved in dry tetrahydrofuran (200 mL) The solution was stirred under nitrogen, cooled to 4° C. and treated with N-methylmorpholine (1.01 g, 10 mmol) followed by dropwise addition of a solution of isobutyl chloroformate (1.36 g, 10 mmol) in THF (10 mL). After 5 min a solution of t-butyl carbazate (1.32 g, 10 mmol) in THF (10 mL) was added dropwise. The reaction mixture was kept at 4° C. for a half hour and at room temperature for 1 hour. The solvent was evaporated and the residue partitioned between ethyl acetate and water. The organic layer was washed with dilute HCl solution, water and dilute bicarbonate solution, dried over anhydrous sodium, sulfate and the solvent evaporated. The material w...

preparation 2

Maleimidocaproylhydrazone of Adriamycin

[0286] A mixture of adriamycin hydrochloride (44 mg, 0.075 mmol), maleimidocaproyl hydrazide (23 mg, 0.102 mmol), prepared according to the procedure outlined in Preparation 1, and 2-3 drops of trifluoroacetic acid in absolute methanol (25 mL) was stirred or 15 hours under nitrogen and protected from light. At the end of this period no free adriamycin was detected by HPLC (mobile phase 0.01 molar ammonium acetate:acetonitrile, (70:30)). The solution was concentrated at room temperature under vacuum to 10 mL and diluted with acetonitrile. The clear solution was concentrated to a small volume, the solid was collected by centrifugation, and the product was dried under high vacuum to yield the title compound. The NMR was consistent with structure. High Resolution MS, calc'd. for C33H42N4O13: 751.2827; Found 757 2804.

[0287] The hydrazone also was formed by using adriamycin and the trifluoroacetic acid salt of the hydrazide. Thus, the salt (40 mg, ...

example 1

Preparation of the BR96 Monoclonal Antibody

[0345] The BR96 monoclonal antibody of the invention was produced using hybridoma fusion techniques as described previously by M. Yeh et al., Proc. Natl. Acad. Sci. USA. (1979), supra and Yeh et al., Int. J. Cancer (1982), supra. Briefly, a three month-old BALB / c mouse was immunized using as the immunogen explanted cultured cells from a human breast adenocarcinoma, designated 3396 or H3396 (from adenocarcinoma of the breast from a patient which had been established in culture at Bristol-Myers Squibb Co., Seattle, Wash.). Methods for establishing and maintaining cell lines from carcinomas isolated from patients are fully described in Yeh et al., Proc. Natl. Acad. Sci. USA 76:2927-2931 (1979) The mouse received injections on five occasions: on the first four occasions, the mouse received one intraperitoneal injection and 1 subcutaneous injection split between 4 sites on the mouse. On the fifth occasion, the mouse was given only one intraper...

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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

CROSS-REFERENCE TO RELATED APPLICATIONS [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.TECHNICAL FIELD OF THE INVENTION [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 othe...

Claims

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Application Information

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Patent Type & Authority Applications(United States)
IPC IPC(8): A61K39/395A61K39/21A61K38/00A61K47/48A61K49/00A61K51/10C07K14/415C07K16/30C12N15/10G01N33/574
CPCA61K38/00G01N33/57484A61K47/48415A61K47/48484A61K47/48569A61K49/0058A61K51/1045A61K51/1087A61K2123/00C07K14/415C07K16/30C07K2317/24C07K2317/54C07K2317/55C07K2317/56C07K2317/622C07K2317/732C07K2317/734C07K2317/77C07K2319/00C12N15/10A61K47/48407A61K47/6809A61K47/6811A61K47/6829A61K47/6851
Inventor HELLSTROM, INGEGERGHELLSTROM, KARLBRUCE, KIMSCHREIBER, GEORGE
Owner BRISTOL MYERS SQUIBB CO
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