Toxin conjugates

Details Toxin conjugates Toxin conjugates

2000-02-08

2003-10-28

Ponnaluri, Padmashri (Department: 1627)

Drug, bio-affecting and body treating compositions

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

Conjugated to proteinaceous toxin or fragment thereof

C424S178100, C424S193100, C424S236100, C530S391700

Reexamination Certificate

active

06638509

ABSTRACT:

TECHNICAL FIELD
The present invention relates to toxin conjugates in which a toxin is bound through a spacer to a residue derived from a compound which has an affinity for a target cell, for example, a residue derived from an antibody or antibody fragment which is specific to a cancer. The toxin conjugate obtained by the present invention inhibits the growth of a target cell selectively and efficiently, and is useful as an active ingredient of an antitumor agent.
BACKGROUND ART
Anthracycline anticancer compounds so far known include daunomycin (U.S. Pat. No. 3,590,028) and adriamycin (U.S. Pat. No. 3,590,028), which are in wide clinical use as anticancer agents. However, side effects of these compounds have been reported; for example, adriamycin is known to have side effects such as cardial toxicity and marrow depression [Cancer Chemotherapy and Pharmacology, 4, 5-10 (1980)]. Alleviation of such side effects is a big problem to be solved, and comprehensive research has so far been made to this end. Specifically, in recent years, research on drug delivery systems has been pursued aiming at alleviation of toxicity, maintenance of concentration in blood and improvement of affinity for a cancer cell. For example, the modification with a copolymer of divinyl ether-maleic anhydride (Japanese Published Unexamined Patent Application No. 67490/85), and the modification with dextran [Cancer Treatment Reports, 66, 107 (1982)] have been reported.
Further, antibody conjugates (toxin conjugates) having a specificity to a cancer cell have been studied. Some examples of such conjugates are shown below [Bioconjugate Chem., 1, 13 (1990)].
stracture
toxin
vinblastine

risin A diphtheria toxin A abrin A

vinblastine hydrazide methotrexate hydrazide

anthracycline

chelates of indium and yttrium

metal chelates

anthracycline
There are some other reports relating to antibody conjugates [Japanese Published Unexamined Patent Application No. 67433/85; Japanese Published Unexamined Patent Application No. 35575/88; Japanese Published Unexamined Patent Application No. 150282/88; Japanese Published Unexamined Patent Application No. 246336/88; Biochem. J., 173, 723 (1978); Cancer Res., 50, 6600 (1990); Science, 261, 212 (1993); Bioconjugate Chem., 4, 275 (1993); Bioconjugate Chem., 4, 251 (1993); Bioconjugate Chem., 5, 88 (1994); Bioconjugate Chem., 5, 31 (1994); and Bioconjugate Chem., 5, 246 (1994)].
There are also known examples in which low molecular weight polyethylene glycol is used as a spacer [Proc. Natl. Acad. Sci. USA, 88, 9287 (1991); PCT National Publication No. 508856/93; and Bioconjugate Chem., 4, 455 (1993)], and examples of the modification of an antibody with polyethylene glycol (WO 93/08838 and WO 86/04145). Further, the use of a spacer containing a peptide has been reported [U.S. Pat. No. 4,671,958; PCT National Publication No. 502886/93; and Bioconjugate Chem., 4, 10 (1993)].
DISCLOSURE OF THE INVENTION
The present inventors made intensive studies in search of an excellent toxin conjugate which kills tumor cells selectively. As a result, the inventors have found that a conjugate having a spacer which is specifically cleaved when introduced into a specific cell can be obtained by chemically binding a toxin to a compound which has a specific affinity for a cancer cell through a novel spacer comprising polyethylene glycol and dipeptide. Thus the present invention has been completed.
The present invention relates to a toxin conjugate in which a residue derived from a compound having an affinity for a target cell is bound to a toxin through a spacer comprising polyalkylene glycol and dipeptide.
Typical examples of the conjugates of the present invention are toxin conjugates represented by general formula (A):
Z&Parenopenst;X
1
—CH
2
(OCH
2
CH
2
)
n
OCH
2
CO—R
1
—R
2
—W—Y
1
)
m
  (A)
wherein Z represents a residue derived from a compound having an affinity for a target cell; Y
1
represents a toxin; R
1
and R
2
, which may be the same or different, each represents an amino acid residue; Alk represents alkylene; n represents an integer of 1-1000; and m represents an integer of 1-100. Although X
0
, W
0
and W
1
are not specifically defined, examples of their representations are as follows: X
0
represents —COAlk
1
-, —SAlk
1
-, —COOAlk
1
-, —CONHAlk
1
-, —COAlk
1
CO—,
W
0
represents CO, -Alk
1
CO—, or -Alk
1
S—; and W
1
represents a single bond, S, —OAlk
1
CO—, —NHAlk
1
CO—, —NHAlk
1
NH—,
In the above formulae, Alk
1
and Alk
2
, which may be the same or different, each represents a straight-chain or branched alkylene having 1-8 carbon atoms, such as methylene, ethylene, propylene, isopropylene, butylene, isobutylene, pentylene, hexylene, heptylene, and octylene.
Particularly, preferred toxin conjugates are compounds represented by general formula (I):
Z&Parenopenst;X
1
—CH
2
(OCH
2
CH
2
)
n
OCH
2
CO—R
1
—R
2
—W—Y
1
)
m
  (I)
wherein X
1
represents CO, S or
W represents a single bond or
and Z, Y
1
, R
1
, R
2
, n and m have the same meanings as defined above. The compounds represented by general formula (I) are hereinafter referred to as Compounds (I), and the same applies to the compounds of other formula numbers.
In the definitions of the above-described groups, the alkylene moiety of the alkylene and the polyalkylene glycol means a straight-chain or branched alkylene having 1-8 carbon atoms such as methylene, ethylene, propylene, isopropylene, butylene, isobutylene, pentylene, hexylene, heptylene, and octylene. Examples of the compounds which have an affinity for a target cell are compounds having a structure capable of binding to X
1
such as COOH, NH, SH, and OH, e.g., receptor ligands such as epidermal growth factors (EGF) and transferrin having an affinity for a target cell, adhesion molecules represented by the arginine-glycine-aspartic acid sequence, and proteins and peptides such as antibodies and antibody fragments. Preferred examples are antibodies and antibody fragments. The antibodies include polyclonal antibodies and monoclonal antibodies produced according to known methods which belong to immunoglobulin (Ig) classes such as IgG, IgA, IgM, and IgE, and immunoglobulin subclasses, for example, IgG
1
, IgG
2
, IgG
3
, and IgG
4
in the case of IgG. Preferred examples are KM-641 antibody which is an antibody against ganglioside GD
3
which is highly expressed in a cancer cell (Japanese Published Unexamined Patent Application No. 176791/93), KM-231 (AMC-462) antibody which is an antibody against sialyl Lewis a (Japanese Published Unexamined Patent Application No. 021562/88), and NL-1 antibody which is an antibody against common human acute lymphatic leukemia cell antigen (CALLA) [Proc. Natl. Acad. Sci. USA 79, 4386-4390 (1982)]. Examples of the antibody fragments are F(ab′)
2
obtained by treating the above-mentioned antibodies with a proteolytic enzyme such as pepsin, Fab′ obtained by reducing F(ab′)
2
with mercaptan, and Fab obtained by degrading the antibodies with a proteolytic enzyme such as papain, trypsin, chymotrypsin, and plasmin. F(ab′)
2
, Fab′, and Fab are known as well as methods for producing them [Immunochemistry, Yuichi Yamamura et al., p. 461, Asakura Shoten (1973)]. Examples of the toxins are toxins having a structure capable of condensing with a carboxyl group of the terminal amino acid R
2
or capable of attaching to a double bond of maleinimide, such as NH, SH and OH, e.g., anthracycline compounds such as adriamycin (U.S. Pat. No. 3,590,028) and daunorubicin (U.S. Pat. No. 3,616,242), duocarmycin derivatives such as DC-88A derivatives (Japanese Published Unexamined Patent Application No. 288879/90) and the compounds described in Reference Examples, mitomycin A, mitomycin C, and protein toxins such as ricin A, diphtheria toxin, and Pseudomonas exotoxin. Examples of the amino acid residues are an alanine residue, a leucine residue, a glycine residue, a proline residue and a valine residue.
The abbreviations used herein

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