Methods of inhibiting the growth of cells bearing LewisY...

Details Methods of inhibiting the growth of cells bearing LewisY... Methods of inhibiting the growth of cells bearing LewisY...

1999-01-08

2001-09-11

Huff, Sheela (Department: 1642)

Drug, bio-affecting and body treating compositions

Conjugate or complex of monoclonal or polyclonal antibody,...

Conjugated to proteinaceous toxin or fragment thereof

C424S178100, C424S181100, C424S156100, C530S387300, C530S388800, C530S388850, C530S391100, C530S391700

Reexamination Certificate

active

06287562

ABSTRACT:

BACKGROUND OF THE INVENTION
The subject invention relates to tumor-specific recombinant antibody fragments, to molecules incorporating such fragments such as immunotoxins and to uses thereof. Exemplary embodiments of the invention include immunotoxins comprising Pseudomonas exotoxins fused to the Fv regions of monoclonal antibodies B1, B3, and B5 which have tumor specificity and which may be used in the treatment of mammalian cancer.
Monoclonal antibodies B1, B3, and B5 are recently isolated murine antibodies directed against a carbohydrate antigen in the Lewis
Y
(Le
Y
) family (Pastan et al.
Cancer Res.,
51: 3781-3787 (1991)). The Le
y
antigens are found on the surface of many mucinous carcinomas of the colon, stomach, ovaries, breast, lung as well as some epidermal carcinomas. Because they react with only a limited number of normal tissues, these antibodies are ideal candidates for use in the treatment and diagnosis of cancer.
In order to create a cytotoxic agent that specifically attacks cancer cells, an antibody or its fragments may be used as the targeting moiety of an immunotoxin. In such immunotoxins, the targeting moiety typically replaces the cell binding domain of a cytotoxin molecule (e.g. domain I of Pseudomonas exotoxin (PE) or the B chain of Diphtheria toxin) and acts to specifically direct the cytotoxin to its target cell (as determined by the specificity of the targeting moiety). As a result, only cells which are recognized by the targeting moiety are efficiently killed and cells which are not recognized are spared (for a review see Brinkmann and Pastan,
Biochem. Biophys. Acta.,
1198: 27-45 (1994)).
Immunotoxins were first made by chemically coupling antibodies to cytotoxic molecules. Thus, for example, monoclonal antibody B3 has been chemically coupled to at least two different forms of Pseudomonas exotoxin (PE) (U.S. Pat. No. 4,545,985). One of these is the full length toxin (PE) and the other a truncated derivative (PE40) (Kondo et al.,
J. Biol. Chem.,
263: 9470-75 (1988) and Pai et al., supra). Both of these immunotoxins have been shown to be selectively cytotoxic to tumor cells that contain the B3 antigen on their surface, and these immunotoxins have been shown to cause complete tumor regression in mice bearing human tumor xenografts (Pai et al.,
Proc. Natl. Acad. Sci. USA,
88: 3358-62 (1991)).
Although chemically coupled immunotoxins are useful they have several undesirable properties. For example, the chemical modifications can change the antibody and affect its binding to the antigen. Furthermore, the purified immunotoxins are a heterogeneous mixture of antibody-toxin molecules connected to each other via different positions on the antibody and the toxin. Thus, Pseudomonas exotoxin, for example, can be coupled either to the light- or heavy-chain of the antibody and to different positions on each of these chains.
To overcome the limitations of chemically conjugated immunotoxins, chimeric immunotoxins have been made as recombinant, single chain, antibody-toxin fusion proteins. It has been shown that certain single chain antigen binding proteins made from the Fv portions of the heavy and light chain of antibodies held together by a polypeptide linker can have the same binding properties as their full length two chain counterparts (Bird et al.,
Science,
242: 423-26 (1988) and Huston et al.,
Proc. Natl. Acad. Sci. USA,
85: 5879-83 (1988)). It has also been shown that, in some cases, fusion proteins composed of single chain antibodies linked to toxins may retain the binding capacity of the single chain antibody as well as the activity of the toxin (Chaudhary et al.,
Nature,
339: 394-97 (1989); Batra et al.,
J. Biol. Chem.,
265: 15198-15202 (1990); Batra et al.,
Proc. Natl. Acad. Sci. USA
86: 8545-8549 (1989); Chaudhary et al.,
Proc. Natl. Acad. Sci. USA
87: 1066 1070 (1990)).
Receptor proteins have often been used as immunotoxin targets because they are cell surface proteins which are often overexpressed in various cancers (Brinkmann and Pastan,
Biochem. Biophys. Acta.,
1198: 27-45 (1994)) and thus provide cancer-specific targets. For example, single chain immunotoxins have been made consisting of the Fv domain of an antibody directed at the interleukin 2 receptor (Chaudhary et al.,
Nature,
339: 394-97 (1989) and Batra et al.,
J. Biol. Chem.
265: 15198-15202 (1990)) or at the transferrin receptor (Batra et al.,
Proc. Natl. Acad. Sci. USA
86: 8545-49 (1989)) fused to truncated forms of PE or diphtheria toxin (Chaudhary et al.,
Proc. Natl. Acad. Sci. USA
87:9491-94 (1990)). Although receptor proteins are overexpressed on many cancers, they may still be present on healthy cells and therefore often do not provide the defined cancer specificity desired for an immunotoxin.
Since the number of antibodies that react preferentially with carcinomas is limited, the identification and characterization of additional “cancer specifics” antibodies that would react with all or most of the cells in a tumor and with relatively few normal cells and tissues is desirable. In addition, recombinant immunotoxins are known to degrade over time both in vitro and in vivo. It would be desirable to obtain immunotoxins that show a reduced rate of degradation and therefore require less frequent administration. Finally, with repeated use, murine antibodies and fusion proteins containing murine antibodies, like any other foreign protein, may ultimately prove immunogenic and invoke an immune response in the treated organism. It would be desirable to produce targeting moieties and immunotoxins having reduced antigenic potential. As will be explained herein, these advantages and others are provided by the present invention.
SUMMARY OF THE INVENTION
The present invention provides for recombinant single chain antibodies and fusion proteins, such as immunotoxins, employing these antibodies. In particular, this invention provides for recombinantly produced humanized single-chain antibodies comprising humanized variable light and heavy (Fv) regions of antibodies that have the binding specificity of monoclonal antibody B1, B3 or B5. These antibodies provide carcinoma-specific targeting moieties suitable for use in cytotoxic fusion proteins. Particularly preferred are humanized single-chain Fv regions of B1, B3 or B5.
In one embodiment, the single-chain antibody is a humanized B3(Fv). Particularly preferred is an antibody comprising a humanized variable heavy chain, more specifically a humanized variable heavy chain having the amino acid sequence designated HumB3V
H
in FIG.
11
. Another preferred variant is an antibody comprising a humanized variable light chain, more specifically a humanized variable light chain having the amino acid sequence designated HumB3V
L
in FIG.
11
. Yet another preferred humanized antibody is one comprising both a humanized variable light chain and a humanized variable heavy chain. Particularly preferred is an antibody comprising a humanized variable heavy chain having the amino acid sequence designated HumB3V
H
in
FIG. 11 and a
humanized variable light chain having the amino acid sequence designated HumB3V
L
in FIG.
11
. Still yet another preferred humanized antibody is one comprising a humanized variable heavy chain having the amino acid sequence designated HumB3V
L
in
FIG. 11 and a
humanized variable light chain having the amino acid sequence designated HumB3V
L
in
FIG. 11
in which the serine at the position designated as 82b in
FIG. 11
, is replaced with arginine.
In any of the single chain antibodies described above the variable heavy region and the variable light region may be joined by a linker. One particularly preferred linker is (Gly
4
Ser)
3
.
This invention also provides for single-chain fusion proteins incorporating any of the above described single-chain antibodies. The fusion proteins comprise the single chain antibodies recombinantly fused to an effector molecule. The effector molecule may be a cytotoxin such as Pseudomonas exotoxin and more preferably is either PE38, PE40, PE38KDEL, or PE38REDL. Thus preferred

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