Novel antibodies reactive with human carcinomas

Inactive Publication Date: 2004-03-04
BRISTOL MYERS SQUIBB CO
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Benefits of technology

0262] One skilled in the art will recognize that a bifunctional-chimeric antibody can be prepared which would have the benefits of lower immunogenicity of the chimeric or humanized antibody, as well as the flexibility, especially for therapeutic treatment, of the bifunctional antibodies described above. Such bifunctional-chimeric antibodies can be synthesized, for in

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

Method used

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  • Novel antibodies reactive with human carcinomas
  • Novel antibodies reactive with human carcinomas
  • Novel antibodies reactive with human carcinomas

Examples

Experimental program
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Effect test

example 2

[0396] Characterization of the BR96 Monoclonal Antibody

[0397] Isotype Determination

[0398] To determine the class of immunoglobulin produced by the BR96 hybridoma, the following techniques were utilized:

[0399] (a) Ouchterlony Immunodiffusion

[0400] An aliquot of supernatant of the hybridoma cells was placed into the center well of a 25% agar plate. Monospecific rabbit anti-mouse Ig isotype antibodies (Southern Biotechnology, Birmingham, Ala.) were placed in the outer wells and the plate was incubated for 24-28 h at room temperature. Precipitation lines were then read.

[0401] (b) ELISA Isotyping

[0402] Dynatech Immulon 96-well plates were coated with goat anti-mouse Ig antibodies at 1 .mu.g / ml concentration, 50 .mu.l / well in PBS and left covered overnight at 4.degree. C. The plates were washed with PBS / Tween 20, 0.05% and blocked with medium at 100 .mu.l / well for 1 h at room temperature. After washing the plates, supernatants from the BR96 hybridoma were added and incubated at room tempe...

example 3

[0416] Internalization of the BR96 Monoclonal Antibody within Carcinoma Cells

[0417] Studies were conducted to measure internalization of the BR96 monoclonal antibody within antigen-positive carcinoma cells. According to one procedure, BR96 was conjugated to the ricin A chain toxin to form an immunotoxin, BR96-RA, whose internalization by carcinoma cells was then determined. Uptake of the conjugate by the carcinoma cells was assessed by determining to what extent the tumor cells were killed by ricin A chain.

[0418] Conjugation of the antibody to the toxin was carried out as follows: Deglycosylated ricin-A chain (Inland Labs, Austin, Tex.) (see, also, Blakey et al., Cancer Res., 47:947-952 (1987)) was treated with dithiothreitol (5 mM) prior to gel filtration on G-25 Sephadex using PBS, pH 7.2 as eluant. This was added in a 2:1 molar ratio to the antibody in PBS, the antibody having been previously modified with N-succinimidyl-3-(2-pyridyldithio) propionate (SPDP) (Pierce, Rockford, Il...

example 4

[0424] Cytotoxicity of Unmodified BR96 Monoclonal Antibody

[0425] Three types of experiments were performed to follow up on the unexpected observation that monoclonal antibody BR96 appeared to be cytotoxic by itself (i.e., in unmodified state) when tested in a FACS assay. So as to avoid an effect of complement in serum, all sera used were heat inactivated (56.degree. C. for 30 min); in addition, some of the experiments with FACS analysis (as described below) were performed on cells which were grown in serum-free medium and tested in the absence of serum.

[0426] First, living suspended cells from a variety of antigen positive carcinoma lines (3396, 2987, 3619) were treated with monoclonal antibody BR96. Cells (5.times.10.sup.5) were incubated on ice for 30 min with 100 .mu.l of BR96 or control monoclonal antibody at a concentration of 60, 30, 15, 7.5 and 3.8 .mu.g / ml in culture medium (IMDM, 15% FBS). After washing the cells twice with culture medium, the cells were suspended in 500 .m...

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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

[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...

Claims

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Application Information

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IPC IPC(8): A61K38/00A61K47/48A61K49/00A61K51/10C07K14/415C07K16/30C12N15/10G01N33/574
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, KARL ERIKBRUCE, KIM FOLGERSCHREIBER, GEORGE J.
Owner BRISTOL MYERS SQUIBB CO
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