Chemistry: natural resins or derivatives; peptides or proteins; – Peptides of 3 to 100 amino acid residues – 4 to 5 amino acid residues in defined sequence
Reexamination Certificate
1997-10-31
2004-07-06
Low, Christopher S. F. (Department: 1653)
Chemistry: natural resins or derivatives; peptides or proteins;
Peptides of 3 to 100 amino acid residues
4 to 5 amino acid residues in defined sequence
C514S002600, C514S018700, C514S019300, C530S331000, C530S399000, C530S345000, C424S178100, C424S179100, C424S181100
Reexamination Certificate
active
06759509
ABSTRACT:
BACKGROUND OF THE INVENTION
Bifunctional compounds which link cytotoxic reagents to antibodies (i.e., “linkers”) are known in the art. These compounds have been particularly useful in the formation of immunoconjugates directed against tumor associated antigens. Such immunoconjugates allow the selective delivery of toxic drugs to tumor cells. (See e.g., Hermentin and Seiler, “Investigations With Monoclonal Antibody Drug Conjugates,” Behring Insti. Mitl. 82:197-215 (1988); Gallego et al., “Preparation of Four Daunomycin-Monoclonal Antibody 791T/36 Conjugates With Anti-Tumor Activity”. Int. J. Cancer 33:737-44 (1984); Arnon et al., “In Vitro and In Vivo Efficacy of Conjugates of Daunomycin With Anti-Tumor Antibodies,” Immunological Rev. 62:5-27 (1982).
Greenfield et al. have described the formation of acid-sensitive immunoconjugates containing the acylhydrazide conjugated via an acylhydrazone bond to the 13-keto position of an anthracycline molecule, and conjugation of this anthracycline derivative to an antibody molecule (Greenfield et al., European Patent Publication EP 0 328 147, published Aug. 16, 1989, which corresponds to pending U.S. Ser. No. 07/270,509, filed Nov. 16, 1988 and U.S. Ser. No. 07/155,181, filed Feb. 11, 1988, now abandoned). This latter reference also discloses specific thioether-containing linkers and conjugates, including hydrazone thioether containing immunoconjugates.
Kaneko et al. (U.S. Pat. No. 5,137,877 which is equivalent to European Patent Publication, EP A 0 457 250, published Nov. 21, 1991) have also described the formation of conjugates containing anthracycline antibiotics attached to a bifunctional linker by an acylhydrazone bond at the C-13 position of an anthracycline molecule. In their invention the linkers contain a reactive pyridinyidithio- or an ortho-nitrophenyldithio- group, by which the linker reacts with a suitable group attached to a cell reactive ligand, to form the completed conjugate.
Conjugates which rely on simple acid hydrolysis may release the drug prematurely. Accordingly, it would be desirable to have conjugates that release active drug in a more site-specific fashion. European Patent Publication 94107501.2 discloses lysosomal enzymes-cleavable antitumor drug conjugates which are selectively activatible at the site of the tumor. However, one of the problems with prior art immunoconjugates is the relatively low ratio of drug to targeting ligand (e.g., immunoglobulin) achievable. It would be highly desirable to have immunoconjugates, activatible at the tumor site, which provide a higher ratio of drug to targeting ligand.
SUMMARY OF THE INVENTION
The present invention provides novel branched peptide linkers. The novel linkers are used to prepare novel drug/linker molecules and biologically active conjugates composed of a targeting ligand, a therapeutically active drug, and a branched peptide linker. The novel conjugates are selectively activatible at the site of of a selected target cell population recognized by the targeting ligand.
As used herein the term “drug/linker” or “linker/drug” refers to the branched peptide linker molecule coupled to two or more therapeutically active drug molecules, and the term “conjugate” refers to the drug/linker molecule coupled to the targeting ligand.
The branched peptide linker contains a protein peptide spacer and may also contain a self-immolating spacer which spaces the protein peptide sequence and the drug. The linkers of the invention are branched so that more than one drug molecule per linker are coupled to the targeting ligand. The number of drugs attached to each linker varies by a factor of 2 for each generation of branching. Thus, the number of drug molecules per molecule of linker can be 2, 4, 8, 16, 32, 64, etc. The factor of branching can be expressed mathematically as 2
n
, wherein n is a positive integer. Thus, a singly branched linker will have a first generation of branching or 2
1
, i.e., contains a potential of two drug molecules per linker. A doubly branched linker will have a second generation of branching or 2
2
, i.e., contains a potential of four drug molecules per linker.
As n rises from n=1, there is a tendency for the solubility of the immunoconjugate to diminish. Solubility can be enhanced by using more water-soluble peptides, or addition of a water-solubilizing moiety such as polyethylene glycol or charged species, e.g., &bgr;-alanine, to the drug in such a way that it is released from the drug by either the low pH or the enzymes of the liposomal milieu.
The present invention is directed to a branched peptide linker for linking a thiol group derived from a targeting ligand to two or more drug moieties which comprises a compound having a terminus containing a thiol acceptor for binding to a thiol group (also called a sulfhydryl group) derived from a targeting ligand, at least one point of branching which is a polyvalent atom, such as a carbon atom or a nitrogen atom, allowing for a level of branching of 2
n
, wherein n is a positive integer, at least two amino acid moieties per branch providing at least one enzymatic site per branch, and at least two other termini containing groups capable of forming covalent bonds with chemically reactive functional groups derived from a drug moiety. It is preferred that n is 1, 2, 3, or 4; more preferably 1, 2, or 3; and most preferably 1 or 2. It is also preferred that the targeting ligand is an antibody or fragment thereof.
As used in the preceeding paragraph, the phrase “thiol group derived from the targeting ligand” means that the thiol group is already present on the targeting ligand or that the targeting ligand is chemically modified to contain a thiol group, which modification optionally includes a thiol spacer group between the targeting ligand and the thiol group. Likewise, the phrase “chemically reactive functional group derived from a drug moiety” means that the chemically reactive functional group is already present on the drug or the drug is chemically modified to contain such chemically reactive functional group. Such chemically reactive functional groups are groups that are capable of forming covalent bonds with a linker terminus. Examples of such chemically reactive functional groups include primary or secondary amino, hydroxyl, sulfhydroxyl, carboxyli, aldehyde, ketone, and the like.
Also provided by the invention are intermediates for preparing the linkers, drug/linkers, and/or conjugates; and a method for treating or preventing a selected disease state which comprises administering to a patient a conjugate of the invention.
The selected target cell population recognized by the targeting ligand is preferably a tumor.
An aspect of the invention provides tumor-specific conjugates which are highly selective substrates for drug-activating enzymatic cleavage by one or more tumor-associated enzymes.
A further aspect of the invention provides tumor-specific drug conjugates wherein the activating enzyme is one which is present in the tumor in sufficient amounts to generate cytotoxic levels of free drug in the vicinity of the tumor.
Another aspect of the invention provides tumor-specific drug conjugates which are stable to adventitious proteases in blood.
A still further aspect of the present invention provides a tumor-specific conjugate in accordance with the preceding aspects, which is considerably less toxic than the activated drug.
In another aspect the present invention provides methods for delivering the conjugates to target cells in which a modification in biological process is desired, such as in the treatment of diseases such as cancer.
The present invention also provides a method for delivering to the site of tumor cells in a warm-blooded animal an active antitumor drug by administering to said warm-blooded animal the conjugate according to this invention.
The above and other aspects of the present invention are achieved by derivatizing a drug (e.g., an an antitumor agent) linked to a ligand through a peptide linker, made up of a protein peptide sequence and a self-immolating spacer, at a reactive site a
Dubowchik Gene M.
Firestone Raymond A.
King Dalton
Bristol--Myers Squibb Company
Lange Keith R.
Low Christopher S. F.
Lukton David
Sher Audrey F.
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