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WO1998019705A1 - Sequences de liaison peptidiques ramifiees - Google Patents

Sequences de liaison peptidiques ramifiees Download PDF

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Publication number
WO1998019705A1
WO1998019705A1 PCT/US1997/019851 US9719851W WO9819705A1 WO 1998019705 A1 WO1998019705 A1 WO 1998019705A1 US 9719851 W US9719851 W US 9719851W WO 9819705 A1 WO9819705 A1 WO 9819705A1
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WIPO (PCT)
Prior art keywords
compound
formula
integer
lys
group
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Application number
PCT/US1997/019851
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English (en)
Inventor
Dalton King
Raymond A. Firestone
Gene M. Dubowchik
Original Assignee
Bristol-Myers Squibb Company
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Publication date
Application filed by Bristol-Myers Squibb Company filed Critical Bristol-Myers Squibb Company
Priority to EP97946428A priority Critical patent/EP0941120A4/fr
Priority to JP52160698A priority patent/JP2001505194A/ja
Priority to AU51597/98A priority patent/AU5159798A/en
Priority to CA002264610A priority patent/CA2264610A1/fr
Publication of WO1998019705A1 publication Critical patent/WO1998019705A1/fr

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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K5/00—Peptides containing up to four amino acids in a fully defined sequence; Derivatives thereof
    • C07K5/04—Peptides containing up to four amino acids in a fully defined sequence; Derivatives thereof containing only normal peptide links
    • C07K5/06—Dipeptides
    • C07K5/06086—Dipeptides with the first amino acid being basic
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
    • A61K47/51—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent
    • A61K47/62—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being a protein, peptide or polyamino acid
    • A61K47/64—Drug-peptide, drug-protein or drug-polyamino acid conjugates, i.e. the modifying agent being a peptide, protein or polyamino acid which is covalently bonded or complexed to a therapeutically active agent
    • A61K47/641—Branched, dendritic or hypercomb peptides
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
    • A61K47/51—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent
    • A61K47/62—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being a protein, peptide or polyamino acid
    • A61K47/65—Peptidic linkers, binders or spacers, e.g. peptidic enzyme-labile linkers
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P43/00—Drugs for specific purposes, not provided for in groups A61P1/00-A61P41/00
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K5/00—Peptides containing up to four amino acids in a fully defined sequence; Derivatives thereof
    • C07K5/02—Peptides containing up to four amino acids in a fully defined sequence; Derivatives thereof containing at least one abnormal peptide link
    • C07K5/0202—Peptides containing up to four amino acids in a fully defined sequence; Derivatives thereof containing at least one abnormal peptide link containing the structure -NH-X-X-C(=0)-, X being an optionally substituted carbon atom or a heteroatom, e.g. beta-amino acids
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K5/00—Peptides containing up to four amino acids in a fully defined sequence; Derivatives thereof
    • C07K5/04—Peptides containing up to four amino acids in a fully defined sequence; Derivatives thereof containing only normal peptide links
    • C07K5/06—Dipeptides
    • C07K5/06008—Dipeptides with the first amino acid being neutral
    • C07K5/06017—Dipeptides with the first amino acid being neutral and aliphatic
    • C07K5/06026—Dipeptides with the first amino acid being neutral and aliphatic the side chain containing 0 or 1 carbon atom, i.e. Gly or Ala
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K5/00—Peptides containing up to four amino acids in a fully defined sequence; Derivatives thereof
    • C07K5/04—Peptides containing up to four amino acids in a fully defined sequence; Derivatives thereof containing only normal peptide links
    • C07K5/06—Dipeptides
    • C07K5/06008—Dipeptides with the first amino acid being neutral
    • C07K5/06017—Dipeptides with the first amino acid being neutral and aliphatic
    • C07K5/06034—Dipeptides with the first amino acid being neutral and aliphatic the side chain containing 2 to 4 carbon atoms
    • C07K5/06052—Val-amino acid
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K5/00—Peptides containing up to four amino acids in a fully defined sequence; Derivatives thereof
    • C07K5/04—Peptides containing up to four amino acids in a fully defined sequence; Derivatives thereof containing only normal peptide links
    • C07K5/06—Dipeptides
    • C07K5/06008—Dipeptides with the first amino acid being neutral
    • C07K5/06078—Dipeptides with the first amino acid being neutral and aromatic or cycloaliphatic
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides

Definitions

  • bifunctional compounds which link cytotoxic reagents to antibodies 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.
  • linkers i.e., "linkers”
  • Such immunoconjugates allow the selective delivery of toxic drugs to tumor cells.
  • the linkers contain a reactive pyridinyldithio- 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.
  • 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.
  • the present invention provides novel branched peptide linkers.
  • 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.
  • novel conjugates are selectively activatible at the site of of a selected target cell population recognized by the targeting ligand.
  • drug/linker or “linker/drug” refers to the branched peptide linker molecule coupled to two or more therapeutically active drug molecules
  • 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.
  • 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.
  • 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.
  • 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., ⁇ -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.
  • 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.
  • 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, carboxyl, 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.
  • 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.
  • a drug e.g., an an antitumor agent
  • a ligand 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 appropriate for inhibiting the pharmacological activity of the antitumor agent to thereby convert the antitumor agent into a pharmacologically inactive peptidyl derivative conjugate.
  • 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 appropriate for inhibiting the pharmacological activity of the antitumor agent to thereby convert the antitumor agent into a pharmacologically inactive peptidyl derivative conjugate.
  • the peptide linker has at least two amino acid residue sequences specifically tailored so as to render the peptidyl derivatives _ a selective substrates for drug-activating enzymatic cleavage by one or more lysosomal proteases, such as cathepsin B, C, D, or L.
  • the enzymatic cleavage reaction will remove the peptide linker moiety from the drug conjugate and effect release of the antitumor agents in pharmacologically active form selectively at the tumor site.
  • the conjugates of the present invention show significantly less systemic toxicity than biparte conjugates and free drug.
  • the conjugates of the invention retain both specificity and therapeutic drug activity for the treatment of a selected target cell population. They may be used in a pharmaceutical composition, such as one comprising a pharmaceutically effective amount of a compound of Formula (III) below, associated with a pharmaceutically acceptable carrier, diluent or excipient.
  • the present invention is directed to a linker molecule of the formula
  • A is a thiol acceptor
  • W is a bridging moiety; c is an integer of 0 to 1 ; a is an integer of 2 to 12;
  • Q is O, NH, or N-lower alkyl; p is an integer of 0 or 1 ; d is an integer of 0 or 1 ;
  • E is a polyvalent atom; each b is an integer of 1 to 10; each X is of the formula -CO-Y-Z m -Gn wherein
  • Y is two amino acid residues in the L form
  • Z is one or two amino acid residues; m is an integer of 0 or 1 ;
  • G is a self-immolative spacer; and n is a integer of 0 or 1 ; provided that when G is 0 then -Y-Z m is ala-leu-ala-leu or gly-phe-leu-gly; or each X is of the formula
  • each X 2 is of the formula -CO-Y-Zm-Gn; and wherein Y, Z, G, Q, E, m, d, p, a, b and n are as defined above; or each X 2 is of the formula
  • each ⁇ 3 is of the formula -CO-Y-Z m -Gn; and wherein Y, Z, G, Q, E, m, d, p, a, b and n are as de Lned ⁇ _ above; or each ⁇ 3 is of the formula
  • the present invention is also directed to a drug/linker molecule of the formula
  • A is a thiol acceptor
  • W is a bridging moiety; c is an integer of 0 to 1 ; a is an integer of 2 to 12;
  • Q is O, NH, or N-lower alkyl; p is an integer of 0 or 1 ; d is an integer of 0 or 1 ; E is a polyvalent atom; each b is an integer of 1 to 10; each X is of the formula
  • Z is one or two amino acid residues; m is an integer of 0 or 1 ;
  • G is a self-immolative spacer
  • n is a integer of 0 or 1 ; provided that when G is 0 then crizo-Z m - _ a is ala-leu-ala-leu or gly-phe-leu-gly; and D is a Drug moiety having a backbone and at least one chemically reactive functional group pendant thereto chemically reacted to the self-immolative spacer or terminal amino acid residue to form a covalent bond, said functional group selected from the group consisting of a primary or secondary amine, hydroxyl, sulfhydryl, carboxyl, aldehyde or ketone; or each X is of the formula jj* (CH 2 ) b -X 1
  • each X 1 is of the formula -CO-Y-Zm-Gn-D; wherein Y, Z, G, D, Q, E, m, d, p and n are as defined above; or each X " ! is of the formula
  • each X 2 is of the formula -CO-Y-Z m -Gn-D; and wherein Y, Z, G, D, Q, E, m, d, p, a, b and n are as defined above; or each X 2 is of the formula
  • each ⁇ 3 is of the formula -CO-Y-Z -Gn-D; and wherein Y, Z, G, D, Q, E, m, d, p, a, b and n are as .defined above; or each X 3 is of the formula O
  • each X 4 is of the formula -CO-Y-Z m -Gn-D; and wherein Y, Z, G, D, Q, E, m, d, p, a, b and n are as defined above. Furthermore, the present invention is directed to conjugate of the formula
  • A is a thiol acceptor
  • W is a bridging moiety; c is an integer from 0 to 1 ; a is an integer of 2 to 12;
  • Q is O, NH, or N-lower alkyl; p is an integer of 0 or 1 ; d is an integer of 1 or 2;
  • E is a polyvalent atom; each b is an integer of 1 to 10; each X is of the formula
  • Y is two amino acid residues in the L form
  • Z is one or two amino acid residues; m is an integer of 0 or 1 ;
  • G is a self-immolative spacer; and n is a integer of 0 or 1 ; provided that when G is 0 then -Y-Z m - is ala-leu-ala-leu or gly-phe-leu-gly; D is a drug moiety having a backbone and at least one chemically reactive functional group pendant thereto reacted to the self-immolative spacer to form a covalent bond, said funtional group selected from the group consisting of a primary or secondary amine, hydroxyl, carboxyl, sulfhydryl, aldehyde, ketone; or X is of the formula jj> (CH 2 ) b -X 1
  • each X 1 is of the formula -CO-Y-Z m -GrrD; and wherein Y, Z, G, D, Q, E, m, d, p, a, b and n are as defined above; or each ⁇ 1 is of the formula
  • each X 2 is of the formula -CO-Y-Z m -Gn-D; and wherein Y, Z, G, D, Q, E, m, d, p, a, b and n are as defined above; or each X 2 is of the formula
  • each X 3 is of the formula -CO-Y-Z m -Gn-D; wherein Y, Z, G, D, Q, E, m, d. p, a, b and n are as defined above; or each X 3 is of the formula
  • end X 4 is of the formula -CO-Ym-Zm-Gn-D; wherein Y, Z, G, D, Q, E, m, d, p, a, b and n are as defined above.
  • lower alkyl is an alkyl group having 1 to 3 carbon atoms. It is preferred that the polyvalent atom is carbon or nitrogen.
  • the drug moiety is an anthracycline antibiotic and the targeting ligand is an antibody.
  • anthracycline is bound to the linker at an amino sugar group of the anthriacycline. It is preferred that the sugar moiety is daunosamine.
  • the antibody then is bound, through the linker, to the anthracycline compound. In an especially preferred embodiment, this linkage occurs through a reduced disulfide group (LJ a free sulfhydryl group (-SH)) on an antibody).
  • LJ reduced disulfide group
  • -SH free sulfhydryl group
  • the anthracycline drug moiety is doxorubicin; the thiol acceptor is a Michael Addition acceptor (from which a Michael Addition Adduct is derived), more preferably is a maleimido- group; and the antibody moiety is a chimeric or humanized antibody, and the point of attachment of the linker to the drug is at the amino group of the sugar moiety of the drug.
  • the conjugates of the invention retain both specificity and therapeutic drug activity for the treatment of a selected target cell population. They may be used in a pharmaceutical composition, such as one comprising a pharmaceutically effective amount of a compound of Formula (III) associated with a pharmaceutically acceptable carrier, diluent or excipient.
  • a pharmaceutical composition such as one comprising a pharmaceutically effective amount of a compound of Formula (III) associated with a pharmaceutically acceptable carrier, diluent or excipient.
  • the present invention provides novel drug-linker-ligand conjugates composed of a ligand capable of targeting a selected cell population, and a drug connected to the ligand by a branched peptide linker.
  • the linker contains a thiol acceptor such as a Michael Addition accceptor, a bridging moiety, a point of branching, and a peptide sequence per each branch containing at least two amino acid moieties which provides an enzymatic cleavage site for an enzyme such as cathepsin B, C, D, or L.
  • the branched peptide linker may also contain a self-immolating spacer, which spaces the drug and the protein peptide sequence.
  • the targeting ligand molecule can be an immunoreactive protein such as an antibody, or fragment thereof, a non-immunoreactive protein, or peptide ligand such as bombesin or, a binding ligand recognizing a cell associated receptor such as a lectin, or any protein or peptide that possesses a reactive sulfhydryl group (-SH) or can be modified to contain such a sulfhydryl group.
  • the thiol acceptor carboxylic is linked to the ligand via a thioether bond, and the drug is linked to the linker via a functional group selected from primary or secondary amine, hydroxyl, sulfhydryl, carboxyl, aldehyde or ketone.
  • ⁇ DBU ⁇ diazobicycloundecene
  • DCC dicyclohexylcarbodiimide
  • DCI direct chemical ionization
  • DCU dicyclohexylurea
  • DIEA diisopropylethylamine
  • DMAP 4-dimethylaminopyridine
  • DME 1 ,2-dimethoxyethane
  • DOX doxorubicin
  • DTT dithiothreitol
  • EEDQ N-ethoxycarbonyl-2-ethoxy-1 ,2- dihydroquinoline
  • EtOAc ethyl acetate
  • FAB fast atom bombardment
  • Fmoc fluorenylmethoxycarbonyl
  • GABA ⁇ -aminobutyric acid
  • HOBt N- hydroxybenzotriazole
  • HRMS high resolution mass spectroscopy
  • LDL low density lipoprotein
  • MC 6-maleimidocaproyl
  • MP 3-maleimid
  • the peptide linker of the present invention contains two, three or four amino acid residues per branch, together with the self-immolative spacer if present, that provides one or more enzyme cleavage sites.
  • the amino acid moieties collectively form a peptide sequence.
  • the amino acid residues making up the peptide residues are each selected, independently, from the group of amino acids, preferably naturally occurring amino acids.
  • the naturally occurring amino acids are alanine (Ala), arginine (Arg), asparagine (Asn), aspartic acid (Asp), cysteine (Cys), glutamic acid (Glu), glutamine (Gin), glycine (Gly), histidine (His), isoleucine (He), leucine (Leu), lysine (Lys) methionine (Met), ornithine (Orn), phenylalanine (Phe), proline (Pro), serine (Ser), threonine (Thr), tryptophan (Trp), tyrosine (Tyr), and valine (Val).
  • Preferred naturally occuring amino acids are Ala, Val. Leu, Lys, lie, Met, Phe, Trp, and Pro. Certain non-naturally occuring amino acids also can be part of the peptide residue. Such non-naturally occuring amino acids include citrulline (Cit) and protected amino acids such as naturally occuring amino acids protected with groups such as acetyl, formyl, tosyl, nitro and the like. When specific amino acids are indicated herein as part of a peptide sequence, they are in the L form _ unless specified otherwise. The amino acid residues making up the "Y" moiety must be in the L form. The amino acid residue(s) making up the "Z” moiety can be either in the L or D form.
  • More preferred amino acids include Lys, Lys protected with acetyl or formyl, Arg, Arg arginine protected with tosyl or nitro groups, His, Orn, Orn protected with acetyl or formyl, Phe, Val, Ala, and Cit. Most preferred are Lys and Cit..
  • amino acid residue sequence is specifically tailored so that it will be selectively enzymatically cleaved from the resulting peptidyl derivative drug-conjugate by one or more of the tumor-associated proteases.
  • the amino acid residue chain length of each branch of the peptide linker preferably ranges from that of a dipeptide to that of a tetrapeptide.
  • the first amino acid residue i.e., the first amino acid making up "Y", read left to right
  • the second amino acid residue i.e., the second amino acid making up "Y", read left to right
  • has a hydropholic side chain e.g., Phe, Val, Ala, Leu or lie.
  • Phe-Lys Val-Lys, Phe-Phe-Lys, Lys-Phe-Lys, Gly-Phe-Lys, Ala-Lys, Val-Cit, Phe-Cit, Leu-Cit, lle-Cit, Trp-Cit, Phe-Ala, Gly-Phe-Leu-Gly, Ala-Leu-Ala-Leu, Phe-Ng-tosyl-Arg, Phe-Ng-Nitro-Arg, Lys-Lys, Lys-Cit, and Cit-Cit.
  • peptide sequences include Phe-Lys, Val-Lys, Val-Cit, and D-Phe-Phe-Lys.
  • peptide linker molecules suitable for use in the present invention can be designed and optimized in their selectivity for enzymatic cleavage by a particular tumor-associated protease.
  • the preferred peptide linkers for use in the present invention are those which are optimized toward the protease, cathepsin B, C, D, and L.
  • the molecules in accordance with the present invention may employ an intermediate self-immolative spacer moiety which spaces and covalently links together the drug moiety and the protein peptide moiety.
  • a self-immolative spacer may be defined as a bifunctional chemical moiety which is capable of covalently linking together two spaced chemical moieties into a normally stable tripartate molecule, releasing one of said spaced chemical moieties from the tripartate molecule by means of enzymatic cleavage; and following said enzymatic cleavage, spontaneously cleaving from the remainder of the molecule to release the other of said spaced chemical moieties.
  • the self- immolative spacer is covalently linked at one of its ends to the protein peptide moiety and covalently linked at its other end to the chemical reactive site of the drug moiety whose derivatization inhibits pharmacological activity, so as to space and covalently link together the protein peptide moiety and the drug moiety into a tripartate molecule which is stable and pharmacologically inactive in the absence of the target enzyme, but which is enzymaticaliy cleavable by such target enzyme at the bond covalently linking the spacer moiety and the protein peptide moiety to thereby effect release of the protein peptide moiety from the tripartate molecule.
  • G is a self-immolative spacer moiety which spaces and covalently links together the drug moiety and the amino acid, in which the spacer is linked to the drug moiety via the T moiety (as used in the following formulas "T” represents a nucleophilic atom which is already contained in the Drug), and which may be represented by the structures of Formulae, (IV), (V), (VI), (Vll).or (VIII):
  • T is O, N or S, and R 1 is C1 -C5 alkyl
  • R 2 is H or Ci -C ⁇ alkyl
  • C1 -C5 alkyl is meant to include a branched or unbranched hydrocarbon chain having, unless otherwise noted, one to five carbon atoms, including but not limited to methyl, ethyl, isopropyl, ⁇ -propyl, sec-butyl, isobutyl, n-butyl and the like.
  • a preferred G self-immolative spacer moiety suitable for use in the present invention is PABC represented by the Formula (IVa):
  • GABA Another preferred G self-immolative spacer moiety suitable for use in the present invention is GABA represented by the Formula (Va):
  • Yet another preferred G self-immolative spacer moiety suitable for use in the present invention is ⁇ , ⁇ -dimethyl GABA represented by the Formula (Vb):
  • Formula (Vb) Another preferred G self-immolative spacer moiety suitable for use in the present invention is ⁇ , ⁇ -dimethyl GABA represented by the Formula (Vc):
  • the thiol acceptor "A" is linked to the ligand via a sulfur atom derived from the ligand.
  • the thiol acceptor becomes a thiol adduct after bonding to the ligand through a thiol group via a thioester bond.
  • the thiol acceptor can be , for example, an alpha- , ⁇ substitited acetyl group. Such a group has the formula
  • leaving groups include Cl, Br, I, mesylate, tosylate, and the like. If the thiol acceptor is an alpha- substituted acetyl group, the thiol adduct after linkage to the ligand forms the bond -S-CH2-
  • the thiol acceptor is a Michael Addition acceptor.
  • a representative Michael Addition acceptor of this invention has the formula
  • Michael Addition acceptor After linkage the thiol group of the ligand, the Michael Addition acceptor becomes a Michael Addition adduct, such as of the formula A
  • the bridging group ia a bifunctional chemical moiety which is capable of covalently linking together two spaced chemical moieties into a stable tripartate molecule.
  • Examples of bridging groups are described in S.S. Wong, Chemistry of Protein Conjugation and Crosslinking, CRC Press, Florida, (1991 ); and G.E. Means and R.E. Feeney, Bioconjugate Chemistry, vol. 1 , pp.2-12, (1990), the disclosures of which are incorporated herein by reference.
  • the bridging group "W” covalently links the thiol acceptor to a keto moiety.
  • An example of a bridging group has the formula — (CH 2 ) f ⁇ (Z) g — (CH 2 ) h wherein f is an integer of 0 to 10, h is an integer of 0 to 10, g is an integer of 0 or 1 , provided that when g is 0, then f + h is 1 to 10, Z is S, O, NH, SO2, phenyl, naphthyl, a polyethylene glycol, a cycloaliphatic hydrocarbon ring containing 3 to 10 carbon atoms, or a heteroaromatic hydrocarbon ring containing 3 to 6 carbon atoms and 1 or 2 heteroatoms selected from O, N. or S.
  • Preferred cycloaliphatic moieties include cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, and the like.
  • Preferred heteroaromatic moieties include pyridyi, polyethlene glycol (1 -20 repeating units), furanyl, pyranyl, pyrimidinyl, pyrazinyl, pyridazinyl, oxazinyl, pyrrolyl, thiazolyl, morpholinyl, and the like.
  • f + h is an integer of 2 to 6 preferably 2 to 4 and more preferably 2.
  • f is 0, 1 or 2
  • h is 0, 1 or 2.
  • Preferred bridging groups coupled to thiol acceptors are shown in the Pierce Catalog, pp. E-12, E-13, E-14.E-15, E-16, andE-17 (1992).
  • the drug conjugates of the present invention are effective for the usual purposes for which the corresponding drugs are effective, and have superior efficacy because of the ability, inherent in the ligand, to transport the drug to the desired cell where it is of particular benefit.
  • the drug moiety is not to be construed as limited to classical chemical therapeutic agents.
  • the drug moiety may be a protein or polypeptide possessing a desired biological activity.
  • proteins may include, for example, a protein such as tumor necrosis factor.
  • the preferred drugs for use in the present invention are cyt ⁇ toxic drugs, particularly those which are used for cancer therapy. Such drugs include, in general, DNA damaging agents, anti-metabolites, natural products and their analogs.
  • cytotoxic agents include, for example, the enzyme inhibitors such as dihydrofolate reductase inhibitors, and thymidylate synthase inhibitors, DNA intercalators, DNA cleavers, topoisomerase inhibitors, the anthracycline family of drugs, the vinca drugs, the mitomycins, the bleomycins, the cytotoxic nucleosides, the pteridine family of drugs, diynenes, the podophyllotoxins, differentiation inducers, and taxanes.
  • the enzyme inhibitors such as dihydrofolate reductase inhibitors, and thymidylate synthase inhibitors
  • DNA intercalators DNA cleavers, topoisomerase inhibitors, the anthracycline family of drugs, the vinca drugs, the mitomycins, the bleomycins, the cytotoxic nucleosides, the pteridine family of drugs, diynenes, the podophyllotoxins, differentiation inducers,
  • Particularly useful members of those classes include, for example, methotrexate, methopterin, dichloromethotrexate, 5-fluorouracil, 6- mercaptopurine, cytosine arabinoside, melphalan, leurosine, leurosideine, actinomycin, daunorubicin, doxorubicin, mitomycin C, mitomycin A, carminomycin, aminopterin, tallysomycin, podophyllotoxin and podophyllotoxin derivatives such as etoposide or etoposide phosphate, vinblastine, vincristine, vindesine, taxol, taxotere, retinoic acid, butyric acid,
  • N 3 -acetyl spermidine, camptothecin, and their analogues N 3 -acetyl spermidine, camptothecin, and their analogues.
  • D is a drug moiety having pendant to the backbone thereof a chemically reactive functional group my means of which the drug backbone is bonded to the protein peptide linker, said functional group selected from the group consisting of a primary or secondary amine, hydroxyl, sulfhydryl, carboxyl, aldehyde or a ketone.
  • amino containing drugs are mitomycin-C, mitomycin-A, daunorubicin, doxorubicin, aminopterin, actinomycin, bleomycin, 9-amino camptothecin, N 8 -acetyl spermidine, 1 -(2- chloroethyl)-1 ,2-dimethanesulfonyl hydraxide, tallysomycin, cytarabine and derivatives thereof.
  • alcohol group containing drugs are , ⁇ etoposide, camptothecin, taxol, esperamicin, 1 ,8-dihydroxy- bicyclo[7.3.1 ]trideca-4-9-diene-2,6-diyne-13-one, (U.S. Patent 5,198,560), podophyllotoxin, anguidine, vincristine, vinblastine, morpholine-doxorubicin, n-(5,5-diacetoxy-pentyl)doxorubicin, and derivatives thereof.
  • sulfhydryl containing drugs are esperamicin and 6-mercaptopuhne, and derivatives thereof.
  • carboxyl containing drugs are methotrexate, camptothecin (ring-opened form of the lactone), butyric acid, retinoic acid, and derivatives thereof.
  • aldehyde and ketone containing drugs are anguidine and anthracyclines such as doxorubicin, and derivatives thereof.
  • R1 is hydrogen or methyl
  • R1 is hydroxy, amino, C1 -C3 alkylamino. di(C ⁇ -C3 alkyl)amino, C4-C6 polymethylene amino,
  • R1 s amino or hydroxy
  • R 2 hydrogen or methyl
  • R 3 hydrogen, fluoro, chloro, bromo or iodo
  • R 4 s hydroxy or a moiety which complete a salt of the carboxylic acid.
  • R 2 is hydrogen
  • R1 is hydrogen or wherein
  • R 3 is NH2, OH, OCH3, NH(C ⁇ -C3 akyl) or N(C ⁇ -C3 alkyl)2
  • R 4 is OH, or NH2
  • R5 is methyl or thienyl
  • R6 is hydrogen or methyl, or a phosphate salt thereof.
  • (C1 -C3 akyl) means a straight or branched carbon chain having from one to three carbon atoms; examples include methyl, ethyl, n-propyl and isopropyl.
  • R 1 is H, CH3 or CHO; when R 2 and R 3 are taken singly, R 3 is H, and one of R 4 and $2 is ethyl and the other is H or OH: when R 2 and R 3 are taken together with the carbons to which they ⁇ are attached, they form an oxirane ring in which case R 4 is ethyl;
  • R 5 is hydrogen, (C1 -C3 alkyl)-CO, or chlorosubstituted (C1 -C2 alkyl)-CO.
  • C1 -C3 alkyl means a straight or branched carbon chain having from one to three carbon atoms; examples include methyl, ethyl, n-propyl and isopropyl.
  • R 2 is hydrogen, methyl, bromo, fluoro, chloro, or iodo
  • R 3 is -OH or -NH2
  • R 4 is hydrogen, bromo, chloro, or iodo.
  • R 2 is hydrogen or hydroxy
  • R 2 ' is hydrogen, hydroxy, or acetoxy
  • R 7 is hydrogen or hydroxy
  • R 3 is hydrogen, hydroxy, or acetoxy
  • R 4 is aryl, substituted aryl, C1 -6 alkyl, C2-6 alkenyl, C2-6 alkynyl or t-butoxy;
  • R 5 is C1 -6 alkyl, C2-6 alkenyl, C2-6 alkynyl, or -Z-R 6 ;
  • Z is a direct bond, C-
  • R 6 is aryl, substituted aryl, C3-6 cycloalkyl, thienyl or furyl.
  • alkyl means a straight or branched saturated carbon chain having from one to six carbon atoms; examples include methyl, ethyl, n-propyl, isopropyl, n-butyl, sec-butyl, isobutyl, t-butyl, n-pentyl, sec- butyl, isopentyl, and n-hexyl.
  • Alkenyl means a straight or branched carbon chain having at least one carbon-carbon double bond, and having from two to six carbon atoms; examples include ethenyl, propenyl, isopropenyl, butenyl, isobutenyl, pentenyl, and hexenyl.
  • Alkynyl means a straight or branched carbon chain having at least one carbon-carbon triple bond, and from two to six carbon atoms; examples include ethynyl, propynyl, butynyl, and hexynyl.
  • Aryl means aromatic hydrocarbon having from six to ten carbon atoms; examples include phenyl and naphthyl.
  • Substituted aryl means aryl substituted with at least one group selected from C1 -6 alkanoyloxy, hydroxy, halogen, C1-6 alkyl, trifluoromethyl, C1-6 alkpxy, aryl, C2-6 alkenyl, C1-6 alkanoyl, nitro, amino, and amido.
  • R1 is OH or O; R isHorO;
  • Anguidine can be targeted at the C-3, C-4, C-8 or C-15 positions, as an ester or hydrazone
  • PHENYLENE DIAMINE MUSTARD (12): H2N-C6H4-N(CH2CH2CI)2
  • R1 is -CH3, -CH2OH, -CH2 ⁇ CO(CH2)3CH3 or -
  • R 2 is -OCH3, -OH or -H
  • R 3 is -NH2, -NHCOCF3, 4-morpholinyl, 3-cyano-4-morpholinyl, 1 - piperidinyl, 4-methoxy-1 -piperidinyl, benzylamine, dibenzylamine, cyanomethylamine, 1 -cyano-2-methoxyethyl amine, or NH-(CH2)4-
  • R 4 is -OH, -OTHP, or -H; and R5 is -OH or -H provided that R5 is not -OH when R4 is -OH or -
  • structural Formula (13) includes compounds which are drugs, or are derivatives of drugs, which have acquired in the art different generic or trivial names.
  • Table I which follows, represents a number of anthracycline drugs and their generic or trivial names and which are especially preferred for use in the present invention.
  • Doxorubicin (also referred to herein as "DOX") is that anthracyclin shown in the formula of Table I in which Ri is -CH2OH, R 2 is - OCH3, R 3 is -IMH2, R 4 is -OH, and R 5 is -H.
  • n Daunomycin is an alternate nane for datmorubicin
  • the most highly preferred drugs are taxol, mitomycin C, and the __ anthracycline antibiotic agents of Formula (13), described previously.
  • the Ligand includes within its scope any molecule that specifically binds or reactively associates or complexes with a receptor or other receptive moiety associated with a given target cell population.
  • This cell reactive molecule to which the drug reagent is linked via the linker in the conjugate, can be any molecule that binds to, complexes with or reacts with the cell population sought to be therapeutically or otherwise biologically modified and, which possesses a free reactive sulfhydryl (-SH) group or can be modified to contain such a sulfhydryl group.
  • the cell reactive molecule acts to deliver the therapeutically active drug moiety to the particular target cell population with which the ligand reacts.
  • Such molecules include, but are not limited to, large molecular weight proteins such as, for example, antibodies, smaller molecular weight proteins, polypeptides or peptide ligands, and non-peptidyl ligands.
  • the non-immunoreactive protein, polypeptide, or peptide ligands which can be used to form the conjugates of this invention may include, but are not limited to, transferrin, epidermal growth factors ("EGF"), bombesin, gastrin, gastrin-releasing peptide, platelet-derived growth factor, IL-2, IL-6, tumor growth factors ("TGF”), such as TGF- ⁇ and TGF- ⁇ , vaccinia growth factor (“VGF”), insulin and insulin-like growth factors I and II.
  • Non-peptidyl ligands may include, for example, carbohydrates, lectins, and apoprotein from low density lipoprotein.
  • the immunoreactive ligands comprise in antigen-recognizing immunoglobulin (also referred to as "antibody"), or an antigen-recognizing fragment thereof.
  • immunoglobulins are those immunoglobulins which can recognize a tumor-associated antigen.
  • immunoglobulin may refer to any recognized class or subclass of immunoglobulins such as IgG, IgA, IgM, IgD, or IgE. Preferred are those immunoglobulins which fall within the IgG class of immunoglobulins.
  • the immunoglobuin can be derived from any species. Preferably, however, the immunoglobulin is of human, murine, or rabbit origin. Furthermore ; the immunoglobulin may be polyclonal or monoclonal, preferably monoclonal.
  • immunoglobulin fragments may include, for example, the Fab', F(ab')2, F v or Fab fragments, or other antigen recognizing immunoglobulin fragments.
  • immunoglobulin fragments can be prepared, for example, by proteolytic enzyme digestion, for example, by pepsin or papain digestion, reductive alkylation, or recombinant techniques. The materials and methods for preparing such immunoglobulin fragments are well-known to those skilled in the art. See generally, Parham, J. Immunology, 131 , 2895 (1983); Lamoyi et al., J.
  • the immunoglobulin can be a "chimeric antibody” as that term is recognized in the art.
  • the immunoglobulin may be a "bifunctional” or “hybrid” antibody, that is, an antibody which may have one arm having a specificity for one antigenic site, such as a tumor associated antigen while the other arm recognizes a different target, for example, a hapten which is, or to which is bound, an agent lethal to the antigen-bearing tumor cell.
  • the bifunctional antibody may be one in which each arm has specificity for a different epitope of a tumor associated antigen of the cell to be therapeutically or biologically modified.
  • the hybrid antibodies have a dual specificity, preferably with one or more binding sites specific for the hapten of choice or more or more binding sites specific for a target antigen, for example, an antigen associated with a tumor, an infectious organism, or other disease state.
  • Biological bifunctional antibodies are described, for example, in European Patent Publication, EPA 0 105 360, to which those skilled in the art are referred.
  • Such hybrid or bifunctional antibodies may be derived, as noted, either biologically, by cell fusion techniques, or chemically, especially with cross-linking agents or disulfide bridge-forming reagents, and may be comprised of whole antibodies and/or fragments thereof.
  • Methods for obtaining such hybrid antibodies are disclosed, for example, in PCT Application WO83/03679, published October 27, 1983, and published _ European Application EPA 0 217 577, published April 8, 1987, both of which are incorporated herein by reference.
  • bifunctional antibodies are those biologically prepared from a "poiydoma” or “quadroma” or which are synthetically prepared with cross-linking agents such as bis-(maleimido)-methyl ether (“BMME”), or with other cross-linking agents familiar to those skilled in the art.
  • cross-linking agents such as bis-(maleimido)-methyl ether (“BMME”), or with other cross-linking agents familiar to those skilled in the art.
  • the immunoglobulin may be a single chain antibody ("SCA”). These may consist of single chain Fv fragments (“scFv”) in which the variable light (“VL”) and variable heavy (“VH”) domains are linked by a peptide bridge or by disulfide bonds. Also, the immunoglobulin may consist of single VH domains (dAbs) which possess antigen-binding activity. See, e.g.. G. Winter and C. Milstein, Nature. 349, 295 (1991 ); R. Glockshuber et al., Biochemistry 29, 1362 (1990); and E. S. Ward et al.. Nature 341 , 544 (1989).
  • SCA single chain antibody
  • chimeric monoclonal antibodies preferably those chimeric antibodies having specificity toward a tumor associated antigen.
  • the term "chimeric antibody” refers to a monoclonal antibody comprising a variable region, e , binding region, from one source or species and at least a portion of a constant region derived from a difference source of species, usually prepared by recombinant DNA techniques.
  • Chimeric antibodies comprising a murine variable region and a human constant region are especially preferred in certain applications of the invention, particularly human therapy, because such antibodies are readily prepared and may be less immunogenic than purely murine monoclonal antibodies.
  • Such murine/human chimeric antibodies are the product of expressed immunoglobulin genes comprising DNA segments encoding murine immungobulin constant regions.
  • Other forms of chimeric antibodies encompassed by the invention are those in which the class or subclass has been modified or changed from that of the original antibody.
  • Such "chimeric" antibodies are also referred to as "class-switched antibodies”.
  • Methods for producing chimeric antibodies involve conventional recombinant DNA and gene transfection techniques now well known in the art. See, e.g., , orrison, ⁇ S. L. et al., Proc. Nat'l Acad. Sci, 81 6851 (1984).
  • chimeric antibody is the concept of "humanized antibody”, that is those antibodies in which the framework or “complementarity determining regions (“CDR") have been modified to comprise the CDR of an immunoglobulin of different specificitry as compared to that of the parent immunoglobulin.
  • CDR complementarity determining regions
  • a murine CDR is grafted into the framework region of a human antibody to prepare the "humanized antibody”. See, e.g., L. Riechmann et al., Nature 332, 323 (1988): M. S. Neuberger et al., Nature 314, 268 (1985).
  • Particularly preferred CDR's correspond to those representing sequences recognizing the antigens noted above for the chimeric and bifunctional antibodies.
  • the reader is referred to the teaching of EPA 0 239 400 (published September 30, 1987), incorporated herein by reference, for its teaching of CDR modified antibodies.
  • 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 instance, by chemical synthesis using cross-linking agents and/or recombinant methods of the type described above.
  • the present invention should not be construed as limited in scope by any particular method of production of an antibody whether bifunctional, chimeric, bifunctional-chimeric, humanized, or an antigen-recognizing fragment or derivative thereof.
  • the invention encompasses within its scope immunoglobulins (as defined above) or immunoglobulin fragments to which are fused active proteins, for example, an enzyme of the type disclosed in Neuberger, et al.. PCT application, WO86/01533, published March 13, 1986. The disclosure of such products is incorporated herein by reference.
  • bifunctional, “fused”, “chimeric” (including humanized), and “bifunctional-chimeric” (including humanized) antibody constructions also include, within their individual contexts constructions comprising antigen recognizing fragments. As one skilled in the art will recognize, such_ fragments could be prepared by traditional enzymatic cleavage of intact bifunctional, chimeric, humanized, or chimeric-bifunctional antibodies.
  • the noted constructions can be prepared with immunoglobulin fragments used as the starting materials; or, if recombinant techniques are used, the DNA sequences, themselves, can be tailored to encode the desired "fragment” which, when expressed, can be combined in vivo or in vitro, by chemical or biological means, to prepare the final desired intact immunoglobulin "fragment". It is in this context, therefore, that the term "fragment" is used.
  • the immunoglobulin (antibody), or fragment thereof, used in the present invention may be polyclonal or monoclonal in nature.
  • Monoclonal antibodies are the preferred immunoglobulins, however.
  • the preparation of such polyclonal or monoclonal antibodies now is well known to those skilled in the art who, of course, are fully capable of producing useful immunoglobulins which can be used in the invention. See, e.g., G. Kohler and C. Milstein, Nature 256, 495 (1975).
  • hybridomas and/or monoclonal antibodies which are produced by such hybridomas and which are useful in the practice of the present invention are publicly available from sources such as the American Type Culture Collection ("ATCC") 12301 Parklawn Drive, Rockville, Maryland 20852 or, commercially, for example, from Boehringer-Mannheim Biochemicals, P.O. Box 50816, Indianapolis, Indiana 46250.
  • ATCC American Type Culture Collection
  • Particularly preferred monoclonal antibodies for use in the present invention are those which recognize tumor associated antigens.
  • Such monoclonal antibodies are not to be so limited, however, and may include, for example, the following: Antigen Site Monoclonal Recognized Antibodies Reference
  • the ligand containing conjugate is derived from chimeric antibody BR96, "ChiBR96", disclosed in U.S. Serial No. 07/544,246, filed June 26, 1990, and which is equivalent to PCT Published Application, WO 91/00295, published January 10, 1991.
  • ChiBR96 is an internalizing murine/human chimeric antibody and is reactive, ad noted, with the fucosylated Lewis Y antigen expressed by human carcinoma cells such as those derived from breast, lung, colon, and ovarian carcinomas.
  • the hybridoma expressing chimeric BR96 and identified as ChiBR96 was deposited on May 23, 1990, under the terms of the Budapest Treaty, with the American Type Culture Collection ("ATCC"), 12301 Parklawn Drive, Rockville, Maryland 20852. Samples of this hybridoma are available under the accession number ATCC 10460. ChiBR96 is derived, in part, from, its source parent, BR96. The hybridoma expressing BR96 was deposited, on February 21 , 1989, at the ATCC, under the terms of the Budapest Treaty and is available under the accession number HB 10036. The desired hybridoma is cultured and the resulting antibodies are isolated from the cell culture supernatant using standard techniques now well known in the art. See, e.g.. "Monoclonal Hybridoma Antibodies: Techniques and Applications", Hurell (ed.) (CRC Press, 1982).
  • immunoglobulin or “antibody” encompasses within its meaning all of the immunoglobulin/antibody forms or constructions noted above.
  • linkers, drug/linkers, and conjugates of the invention can be made techniques taught herein, known in the art, or can be made via routine experimentation using as guidance the techniques taught herein and/or known in the art.
  • the attachment of the drug to the linker is accomplished by reacting a nucleophilic atom of the drug (O, N or S) to an electrophilic atom (C, S, P) on either the self-immolating spacer or the carboxy terminus of the peptide. This is illustrated as follows:
  • Phe-Lys(Mtr)-PABOH 10a DCC, NHS, DIEA, DME
  • Example 7b Fmoc-Val-OSu 7b This was prepared from Fmoc-Val (7.02 g, 20.7 mmol) as described above for 7a.
  • Example 8a Fmoc-Phe-Lys(Mtr) A suspension of Lys(Mtr) 2 (4.686 g, 1 1 .20 mmol) and NaHC ⁇ 3 (941 .0 mg, 1 equiv) in water (100 mL) and DME (50 mL) was treated with a solution of Fmoc-Phe-OSu 7a (1 1 .20 mmol) in DME (50 mL). THF (25 mL) was then added to aid solubility. The mixture was stirred at rt for 2 days and then as much DME as possible was removed on the rotovap (bath at 30°C).
  • Example 8b Fmoc-Val-Lys(Mtr) This was prepared from Fmoc-Val-OSu 7b (20.7 mmol) and Lys(Mtr) 2 (9.09 g, 1 .05 equiv) as described above for 8a (15.28 g, 100%).
  • H-NMR CDCI3/CD3OD
  • ⁇ 0.85 m, 6H
  • 1 .20 m, 2H
  • 1 .59 (m, 4H) 1 .99 (m, 1 H)
  • 2.41 m, 2H), 3.71 (s, 3H), 4.21 (m, 5H), 6.78 (d, 2H), 7.29 (m, 16H), 7.51 (bit, 2H), 7.71 (d, 2H).
  • HRMS Calcd: 740.3700. Found: 740.3712.
  • Example 8c , . ⁇ _-
  • Example 10a Phe-Lys(Mtr)-PAB-OH Fmoc-Phe-Lys(Mtr)-PAB-OH 9a (4.2857 g, 4.80 mmol) in methylene chloride (35 mL) at rt was treated with diethylamine (50 mL). The mixture was sonicated briefly and stirred at rt for 4 h. after which time no starting material was observed by TLC.
  • Example 10c Ala-Lys(Mtr)-PABOH
  • a solution of Fmoc-Ala-Lys(Mtr)-PABOH 9c (1 1 .0073 g, 13.47 mmol) in DMF (50 mL) at rt was treated with diethylamine (25 mL). After 2.5 h the mixture was concentrated under high vacuum and the residue was flushed with methylene chloride (2x100 mL) and then toluene (150 mL).
  • N-carbobenzyloxy-N 1 , N'-bis-t-butoxypropionylethylenediamine 13 (18.3640 g, 40.76 mmol) was passed through a bed of Raney nickel in a fritted glass funnel.
  • the filtrate was degassed with nitrogen and 10% Pd-C (1 g) and 12M HCl (6.8 mL, 2 equiv) were added.
  • the mixture was hydrogenated on a Parr shaker at 50 psi for 16 h and then degassed once again with nitrogen.
  • the catalyst was filtered off on a bed of celite.
  • N-Maleoyl-N', N'-bis-(carboxyethyl)ethylenediamine p-toluenesulfonic acid N-Maleoyl-N', N'-bis-t-butoxypropionylethylenediamine 15 (3.9934 g, 10.07 mmol) in methylene chloride (100 mL) at rt was treated with p- toluenesuifonic acid monohydrate (5.75 g, 3 equiv). The reaction was stirred at rt overnight. The solvent was evaporated at rt and the gummy residue dried in vacuo for 3 h. NMR showed complete loss of t-butyl ester signal. The crude product was carried on without further purification. 1 H-NMR
  • MEt-IDP-(Lys(Mtr)-Lys(Mtr)-PABOH)2 18a (2.3623 g, 1 .127 mmol) and bis- p-nitrophenylcarbonate (1 .5424 g, 4.5 equiv) in dry methylene chloride (60 mL) were treated with freshly activated 4A powdered sieves (8.5 g) and DIEA
  • Example 20a MEt-I DP-(Lys(Mtr)-Lys(Mtr)-PABC-DOX)2
  • Example 23 N, N-Bis-(t-butoxycarbonylmethyl)ethylenediamine dihydrochloride A solution of N-carbobenzyloxy-N', N'-bis-(t-butoxyacetyl)ethylenediamine 22 (15.02 g, 35.54 mmol) in ethanol (75 mL) was filtered through a bed a Raney nickel. Acetic acid (6.1 mL, 3 equiv) was added and the mixture was hydrogenated as described above for 4. the crude product was carried on without further purification.
  • Example 24 gave ⁇ _
  • N, N-Bis-(t-butoxyacetyl)ethylenediamine dihydrochloride 23 (ca. 35.54 mmol) in CHCI3 (150 mL) at rt was treated with maleic anhydride (3.83 g, 1 .1 equiv). The mixture was stirred for 14 h and then the solvent was evaporated. The residue was flushed with CHCI3 (200 mL) and then dissolved in acetonitrile (200 mL) under argon. The stirred mixture was cooled to 0°C and treated with trimethylsiiyl chloride (18 mL, 4 equiv) and triethylamine (20 mL, 4 equiv).
  • Example 28a M Et-I DA-(Phe-Lys(Mtr)-PABC-DOX)2
  • a solution of MEt-IDA-(Phe-Lys(Mtr)-PABC-PNP)2 27a (410.3 mg, 0.217 mmol) and doxorubicin hydrochloride (283.0 mg, 2.25 equiv) in NMP (10 mL) at rt was treated with DIEA (0.094 mL, 2.5 equiv). After 48 h the mixture was poured into ethyl acetate. The solution was washed with water (3x) and evaporated in vacuo. In each case some orange solid, that was shown by TLC to contain product, was formed.
  • Example 28b M Et-IDA-(Val-Lys(Mtr)-PABC-DOX)2 This was prepared from MEt-IDA-(Val-Lys(Mtr)-PABC-PNP)2 27b (6.351 g,
  • Example 30 MEt-IDA-(Phe-Lys(Mtr)-PABC-M MC)2
  • Example 32 2'-Methoxytrityl-Paclitaxel
  • paclitaxel (0.51 g, 0.597 mmol) and p-methoxytrityl chloride (4.63 g, 25 equiv) in methylene chloride (14 mL) under nitrogen at rt
  • pyridine (1 .23 mL, 25 equiv)
  • the solvent was evaporated and the residue dissolved in ethyl acetate.
  • the solution was washed with cold pH 5 buffer (2x100mL), water and brine, dried and evaporated.
  • the residue was chromatographed on silica, eluting with 3% methanol/methylene chloride, to give the product as a white solid (482 mg,
  • paclitaxel-2'-Mtr 32 (789.3 mg, 0.701 mmol) in methylene chloride (5 mL) at 0°C under argon was treated with DIEA (0.122 mL, 1 equiv), pyridine (0.057 mL, 1 equiv) and diphosgene (0.043 mL, 0.5 equiv).
  • Example 37 N-Maleoyl-N', N'-bis-(t-butoxycarbonylmethyl)- propylenediamine This was prepared from N, N-bis-(t-butoxycarbonyl- methyl)propylenediamine diacetic acid 36 (12.14 mmol) as described above for 24. The crude product was chromatographed on silica, eluting with 3:1 hexane/ethyl acetate, to give the product as a pale-yellow oil (2.0653 g, 44% (3 steps)).
  • Example 47 M P-IDA-(Phe-Lys(Mtr)-PABC-PNP)2 This was prepared from MP-IDA-(Phe-Lys(Mtr)-PABOH)2 46 (225.4 mg,
  • Example 51 Z- ⁇ Ala-OSu A mixture of Z- ⁇ Ala 50 (37.42 g, 0.168 mol) and N-hydroxysuccinimide
  • Example 52 Z- ⁇ Ala-IDA
  • iminodiacetic acid (16.84 g, 84.32 mmol) and lithium hydroxide monohydrate (10.62 g, 2 equiv) in water (240 mL) at 0°C
  • a solution of Z- ⁇ Ala-OSu 51 27.01 g, 0.667 equiv
  • DME DME
  • Example 55 ⁇ Ala-IDA-( ⁇ Ala-O-t-Bu)2 Z- ⁇ Ala-IDA-(0-t-Bu)2 54 (16.93 g, 28.56 mmol) in absolute ethanol (100 mL) was degassed by bubbling nitrogen through it for 30 min. Palladized charcoal (10%, 1 g) was carefully added and the mixture was hydrogenated on a Parr apparatus at 50 psi for 16 h. The catalyst was then filtered off on _ , bed of celite and the filtrate concentrated in vacuo. The residue was flushed with methylene chloride, giving a colorless foam (13.11 g, 100%).
  • the aqueous phase was extracted with more ethyl acetate.
  • the combined organic phases were dried over sodium sulfate and evaporated.
  • the residue was chromatographed on silica, eluting with 1 ) 5%, 2) 7%, and 3) 10% methanol/methylene chloride, to give the product as a pale-yellow gum (4.83 g, 66%).
  • the crude reaction mixture containing MP-IDA-( ⁇ Ala)2 58 (ca. 0.319 mmol) was diluted with DMF (5 mL) and then treated with Phe-Lys(Mtr)-PABOH 10a (429.1 mg, 2.0 equiv). After stirring at rt for 5 h the solvents were removed under high vacuum and the residue was triturated with ether. The resulting solid was collected by filtration and then chromatographed on silica, eluting with 5% methanol/ethyl acetate to remove impurities, and 10% methanol/methylene chloride to elute the product (220.1 mg, 40%).
  • MTEG- ⁇ -Ala Hydrazide (254 mg, 866 umole) was dissolved in 10 ml methanol along with 50 ul TFA. This solution was added to a suspension of M-Et-IDA-(Phe-Lys-PABC-DOX)2 (276 mg, 108 umole) in 40 ml anhydrous methanol. The reaction was stirred for 3.5 hr. at room temperature. The reaction mixture was rotary evaporated to the point of precipitation, then 1 ml CH2CI2 was added to redissolve. This was added dropwise to 250-ml ether,, precipitating a red solid.
  • the protected amino alcohol 64 (6.601 g, 37.67 mmol) in methylene chloride (45 mL) at 0°C under argon was treated with methanesulfonyl chloride (2.92 mL, 1.0 equiv) and triethylamine (5.25 mL, 1.0 equiv), dropwise over 30 min. The mixture was stirred for 16 h and then the solvent was evaporated. The residue was partitioned between ether (200 mL) and water (250 mL) and the organic phase was washed with 10% citric acid, water and brine, dried over sodium sulfate and evaporated.
  • the diester 66 (4.957 g, 15.62 mmol) in ether (100 mL) under argon at 0°C was treated with lithium borohydride (2M in THF, 24 mL, 3 equiv) and then with methanol (1 .9 mL, 3 equiv). The mixture was heated at reflux for 2.5 h and then carefully quenched with methanol (25 mL) followed by acetic acid (2.7 mL, 3 equiv) upon cooling to rt. The mixture was evaporated and flushed with methylene chloride (2x). The residue was dissolved in methanol (100 mL) and the mixture heated at reflux overnight and then evaporated.
  • the aminodiol 68 in dry acetonitrile under argon at 0°C is treated with maleic anhydride (1 equiv) and DIEA (1 equiv).
  • the mixture is allowed to stir at rt for 4 h.
  • the reaction is re-cooled to 0°C and treated with trimethylsilyl chloride (3 equiv) and DIEA (3 equiv).
  • the mixture is heated at reflux for 4 h and then, after cooling to rt, is evaporated.
  • the residue is partitioned between ethyl acetate and water.
  • the organic phase is washed with J 0% _ citric acid, water and brine, dried over sodium sulfate and evaporated.
  • the residue is chromatographed on silica gel to give the product.
  • a solution of the diol 69 in dry methylene chloride is added dropwise to a vigorously stirred solution of 1 M phosgene in toluene (20 equiv) at 0°C. After addition is complete the mixture is stirred at rt for 16 h and then the solvents are removed on the rotary evaporator. The residue is flushed with dry methylene chloride and dried in vacuo. The crude bis-chloroformate is used without further purification.
  • Adpoc substrate 77 is dissolved in 5% trifluoroacetic acid/methylene chloride containing anisole (100 equiv). After 30 min the solvents are removed under high vacuum at rt and the residue dried in vacuo for several hours. The crude product is carried on immediately without further purification.
  • the di-t-butyl ester 79 is dissolved in 20% trifluoroacetic acid and the mixture is stirred at rt for 5 h. The solvents are evaporated and the residue flushed with methylene chloride several times. The residue is triturated with ether and the resulting solid product is collected by filtration, washed with ether, and dried in vacuo.
  • a stirred solution of the diacid 80 in dimethoxyethane at 0°C is treated with n-hydroxysuccinimide (2.2 equiv) and 0.5 M DCC in methylene chloride (2.2 equiv). The mixture is allowed to warm to rt. After 4 h a solution of Phe- Lys(MMT)-PABOH 10a (2 equiv) in dimethoxyethane is added and the mixture is stirred at rt for 16 h. The mixture is then filtered and the filtrate evaporated. The residue is partitioned between ethyl acetate and water. The organic phase is washed with water and brine, dried over sodium sulfate and evaporated. The product is purified by chromatography on silica gel.
  • Example 84 MPr-Mal-(Phe-Lys(MMT)-PABC-PNP) 2
  • a stirred mixture of 81 , bis-p-nitrophenylcarbonate (6 equiv) and freshly activated 4A powdered sieves in dry methylene chloride under argon at rt is treated with DIEA (6 equiv).
  • DIEA 6 equiv
  • the mixture is filtered and the filtrate evaporated.
  • the residue is dried in vacuo for several hours and then dissolved in a minimum volume of methylene chloride. To this is added two-times the volume of ether.
  • the resulting solid is collected by filtration, washed with 2:1 ether/methylene chloride, and dried in vacuo.
  • the di-t-butyl ester 22 is dissolved in 20% trifluoroacetic acid and the mixture is stirred at rt for 5 h. The solvents are evaporated and the residue flushed with methylene chloride several times. The residue is triturated with ether and the resulting solid product is collected by filtration, washed with ether, and dried in vacuo.
  • Example 88 N-Carbobenzyloxy-N', N'-bis-(2-hydroxyethyl)- ethylenediamine 86
  • a solution of the bis-carboxylic acid 85 in diglyme at 0°C is treated with N- hydroxysuccinimide (2.2 equiv) and DCC (2.2 equiv). The mixture is allowed to warm to rt and then stirred for 16 h. The resulting solid is removed by filtration. The filtrate is cooled to 0°C under an argon atmosphere and then treated with 0.5 M sodium borohydride in diglyme. The mixture is allowed to warm to rt for 16 h and is then poured into ice water. The resulting mixture is extracted with ethyl acetate. The organic phase is washed with water and brine, dried over sodium sulfate and evaporated to give the product which is carried on without further purification.
  • Example 89 Bis-(2-hydroxyethyl)ethylenediamine hydrochloride
  • the aminodiol 87 in dry acetonitrile under argon at 0°C is treated with maleic anhydride (1 equiv) and DIEA (1 equiv).
  • the mixture is allowed to stir at rt for 4 h.
  • the mixture is re-cooled to 0°C and treated with trimethylsilyl chloride (3 equiv) and DIEA (3 equiv).
  • the reaction is heated at reflux for 4 h and then, after cooling to rt, is evaporated.
  • the residue is partitioned between ethyl acetate and water.
  • the organic phase is washed with 10% citric acid, water and brine, dried over sodium sulfate and evaporated.
  • the residue is chromatographed on silica gel to give the product.
  • Example 91 Bis-(2-chlorocarbonyloxyethyl)-2'-maleimidoethylamine
  • a solution of the diol 88 in dry methylene chloride is added dropwise to a vigorously stirred solution of 1 M phosgene in toluene (20 equiv) at 0°C. After addition is complete the mixture is stirred at rt for 16 h and then the solvents are removed on the rotary evaporator. The residue is flushed with dry methylene chloride and dried in vacuo. The crude bis-chloroformate is used without further purification.
  • Example 92 , ⁇ __
  • Example 94 MEt-IBHE-(Phe-Lys(MMT)-PABC-DOX) 2
  • DIEA 1.1 equiv
  • the solution is washed with water (4x) and brine, dried over sodium sulfate and evaporated. The residue is chromatographed on silica gel to give the product.
  • Example 96 1 14 (257 mg, 0.25 mmole) and Doxorubicin HCl (377 mg, 0.65 mmole) are stirred in 25 ml dry methanol for 24 hour. The reaction is concentrated by rotary evaporation to 4 ml, then purified in two equal portions on Sephadex LH-20 (1 " x 18") with methanol. Fractions containing pure product are pooled, rotary evaporated, and dried under high vacuum to yield 1 15 (222 mg, 50%).
  • BR96 and IgG were thiolated (reduced) by a variation of previously reported method .
  • 1.27 g BR96 (75 ml at 105.7 uM, 7.93 umole) was de-oxygenated by several cycles of alternating vacuum and Ar atmosphere. This was then treated with 7.4 mM DTT (8.6 ml, 63.6 umole in Ar-bubbled PBS, pH 7.0) and stirred at 37°C under Ar for 3 hr. Removal of low molecular weight compounds was accomplished by ultrafiltration against PBS, pH 6.0 in an Amicon stirred cell at 4°C.
  • a 400 ml Amicon cell was fitted with an Amicon YM30 filter (molecular weight cut-off 30,000), and charged to 40 psi with Ar. Cell eluant was monitored for thiol content with Ellman's reagent 2 until a baseline reading at 412 nm was obtained. Concentration of protein and thiol groups were determined according to the previously reported method 1 . In this example, 1.10 g reduced BR96 (85 ml at 80.8 uM MAb, 619.1 uM thiol) was obtained, for a yield of 87% and a thiol titer of 7.7 mole thiol groups/mole BR96.
  • Conjugate was purified at 4°C by percolation (approximately 2 ml/min.) through a 1 " x 24" Bio-Beads column (initially prepared by swelling and packing in methanol, then equilibrated in H2O, and finally PBS, pH 7.4).
  • Conjugate was frozen in liquid N2 and stored at -80°C.

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Abstract

La présente invention concerne des conjugués contenant un ligand ciblé tel qu'un anticorps, un médicament thérapeutiquement actif, et une séquence de liaison peptidique ramifiée. Cette séquence de liaison peptidique ramifiée contient au moins deux fractions acide aminé qui constituent un site de clivage d'enzyme. Le nombre de médicaments qu'il est possible de lier aux séquences de liaison ramifiées est multiplié par 2 à chaque génération de ramification.
PCT/US1997/019851 1996-11-05 1997-10-31 Sequences de liaison peptidiques ramifiees WO1998019705A1 (fr)

Priority Applications (4)

Application Number Priority Date Filing Date Title
EP97946428A EP0941120A4 (fr) 1996-11-05 1997-10-31 Sequences de liaison peptidiques ramifiees
JP52160698A JP2001505194A (ja) 1996-11-05 1997-10-31 分枝ペプチド・リンカー
AU51597/98A AU5159798A (en) 1996-11-05 1997-10-31 Branched peptide linkers
CA002264610A CA2264610A1 (fr) 1996-11-05 1997-10-31 Sequences de liaison peptidiques ramifiees

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US3036796P 1996-11-05 1996-11-05
US60/030,367 1996-11-05

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WO2000064486A2 (fr) * 1999-04-28 2000-11-02 Vectramed, Inc. Conjugues de medicaments polymeres actives par voie enzymatique
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US6261537B1 (en) 1996-10-28 2001-07-17 Nycomed Imaging As Diagnostic/therapeutic agents having microbubbles coupled to one or more vectors
US6264917B1 (en) 1996-10-28 2001-07-24 Nycomed Imaging As Targeted ultrasound contrast agents
US6331289B1 (en) 1996-10-28 2001-12-18 Nycomed Imaging As Targeted diagnostic/therapeutic agents having more than one different vectors
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WO2003051393A2 (fr) * 2001-12-17 2003-06-26 University Of Wales College Of Medicine Reactifs clivables de liberation specifique a des sites pathologiques
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EP0961619A4 (fr) * 1996-09-27 2001-09-26 Bristol Myers Squibb Co Promedicaments hydrolysables pour la liberation de medicaments anticancereux dans des cellules metastatiques
US6264917B1 (en) 1996-10-28 2001-07-24 Nycomed Imaging As Targeted ultrasound contrast agents
US6680047B2 (en) 1996-10-28 2004-01-20 Amersham Health As Diagnostic/therapeutic agents
US6331289B1 (en) 1996-10-28 2001-12-18 Nycomed Imaging As Targeted diagnostic/therapeutic agents having more than one different vectors
US6261537B1 (en) 1996-10-28 2001-07-17 Nycomed Imaging As Diagnostic/therapeutic agents having microbubbles coupled to one or more vectors
WO1999025729A1 (fr) * 1997-11-18 1999-05-27 Chugai Seiyaku Kabushiki Kaisha Composes possedant une activite antitumorale
US6835807B1 (en) * 1998-05-22 2004-12-28 Daiichi Pharmaceuticals Co., Ltd. Drug complex and drug delivery system
US7041818B2 (en) 1998-10-30 2006-05-09 Daiichi Pharmaceutical Co., Ltd. DDS compound and method for measurement thereof
US6811996B1 (en) * 1998-10-30 2004-11-02 Daiichi Pharmaceutical Co., Ltd. DDS compounds and method for assaying the same
JP4808846B2 (ja) * 1999-02-24 2011-11-02 ザ ユーエイビー リサーチ ファンデイション 癌の標的付けられた治療のためのタキサン誘導体
JP2002537347A (ja) * 1999-02-24 2002-11-05 ザ ユーエイビー リサーチ ファンデイション 癌の標的付けられた治療のためのテキサン誘導体
WO2000064486A3 (fr) * 1999-04-28 2001-04-26 Veritas Medical Technologies I Conjugues de medicaments polymeres actives par voie enzymatique
WO2000064486A2 (fr) * 1999-04-28 2000-11-02 Vectramed, Inc. Conjugues de medicaments polymeres actives par voie enzymatique
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WO2001038318A1 (fr) * 1999-11-24 2001-05-31 Immunogen, Inc. Agents cytotoxiques comprenant des taxanes et leur utilisation therapeutique
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AU5159798A (en) 1998-05-29

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