EP0540612A1 - Reactifs heterobifonctionnels et conjugues avec unites d'oxa-alkylene pour des structures de pontage amphiphiles - Google Patents

Reactifs heterobifonctionnels et conjugues avec unites d'oxa-alkylene pour des structures de pontage amphiphiles

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Publication number
EP0540612A1
EP0540612A1 EP91913626A EP91913626A EP0540612A1 EP 0540612 A1 EP0540612 A1 EP 0540612A1 EP 91913626 A EP91913626 A EP 91913626A EP 91913626 A EP91913626 A EP 91913626A EP 0540612 A1 EP0540612 A1 EP 0540612A1
Authority
EP
European Patent Office
Prior art keywords
group
sea
conjugate
mab
groups
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Ceased
Application number
EP91913626A
Other languages
German (de)
English (en)
Inventor
Hubert Agback
Leif Ahrgren
Martin Haraldsson
Eva Kerblom
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
Pfizer Health AB
Original Assignee
Kabi Pharmacia AB
Pharmacia AB
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Priority claimed from SE9002490A external-priority patent/SE9002490D0/xx
Priority claimed from SE9002484A external-priority patent/SE9002484L/xx
Application filed by Kabi Pharmacia AB, Pharmacia AB filed Critical Kabi Pharmacia AB
Publication of EP0540612A1 publication Critical patent/EP0540612A1/fr
Ceased legal-status Critical Current

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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07D—HETEROCYCLIC COMPOUNDS
    • C07D209/00—Heterocyclic compounds containing five-membered rings, condensed with other rings, with one nitrogen atom as the only ring hetero atom
    • C07D209/02—Heterocyclic compounds containing five-membered rings, condensed with other rings, with one nitrogen atom as the only ring hetero atom condensed with one carbocyclic ring
    • C07D209/44—Iso-indoles; Hydrogenated iso-indoles
    • C07D209/48—Iso-indoles; Hydrogenated iso-indoles with oxygen atoms in positions 1 and 3, e.g. phthalimide
    • 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
    • A61K47/56—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 an organic macromolecular compound, e.g. an oligomeric, polymeric or dendrimeric molecule
    • A61K47/59—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 an organic macromolecular compound, e.g. an oligomeric, polymeric or dendrimeric molecule obtained otherwise than by reactions only involving carbon-to-carbon unsaturated bonds, e.g. polyureas or polyurethanes
    • A61K47/60—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 an organic macromolecular compound, e.g. an oligomeric, polymeric or dendrimeric molecule obtained otherwise than by reactions only involving carbon-to-carbon unsaturated bonds, e.g. polyureas or polyurethanes the organic macromolecular compound being a polyoxyalkylene oligomer, polymer or dendrimer, e.g. PEG, PPG, PEO or polyglycerol
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
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    • 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
    • A61K47/56—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 an organic macromolecular compound, e.g. an oligomeric, polymeric or dendrimeric molecule
    • A61K47/61—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 an organic macromolecular compound, e.g. an oligomeric, polymeric or dendrimeric molecule the organic macromolecular compound being a polysaccharide or a derivative thereof
    • 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
    • A61K47/68—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 an antibody, an immunoglobulin or a fragment thereof, e.g. an Fc-fragment
    • A61K47/6801—Drug-antibody or immunoglobulin conjugates defined by the pharmacologically or therapeutically active agent
    • A61K47/6803—Drugs conjugated to an antibody or immunoglobulin, e.g. cisplatin-antibody conjugates
    • A61K47/6811—Drugs conjugated to an antibody or immunoglobulin, e.g. cisplatin-antibody conjugates the drug being a protein or peptide, e.g. transferrin or bleomycin
    • A61K47/6817—Toxins
    • A61K47/6829—Bacterial toxins, e.g. diphteria toxins or Pseudomonas exotoxin A
    • 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
    • A61K47/68—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 an antibody, an immunoglobulin or a fragment thereof, e.g. an Fc-fragment
    • A61K47/6889—Conjugates wherein the antibody being the modifying agent and wherein the linker, binder or spacer confers particular properties to the conjugates, e.g. peptidic enzyme-labile linkers or acid-labile linkers, providing for an acid-labile immuno conjugate wherein the drug may be released from its antibody conjugated part in an acidic, e.g. tumoural or environment
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07C—ACYCLIC OR CARBOCYCLIC COMPOUNDS
    • C07C217/00—Compounds containing amino and etherified hydroxy groups bound to the same carbon skeleton
    • C07C217/02—Compounds containing amino and etherified hydroxy groups bound to the same carbon skeleton having etherified hydroxy groups and amino groups bound to acyclic carbon atoms of the same carbon skeleton
    • C07C217/04—Compounds containing amino and etherified hydroxy groups bound to the same carbon skeleton having etherified hydroxy groups and amino groups bound to acyclic carbon atoms of the same carbon skeleton the carbon skeleton being acyclic and saturated
    • C07C217/06—Compounds containing amino and etherified hydroxy groups bound to the same carbon skeleton having etherified hydroxy groups and amino groups bound to acyclic carbon atoms of the same carbon skeleton the carbon skeleton being acyclic and saturated having only one etherified hydroxy group and one amino group bound to the carbon skeleton, which is not further substituted
    • C07C217/08—Compounds containing amino and etherified hydroxy groups bound to the same carbon skeleton having etherified hydroxy groups and amino groups bound to acyclic carbon atoms of the same carbon skeleton the carbon skeleton being acyclic and saturated having only one etherified hydroxy group and one amino group bound to the carbon skeleton, which is not further substituted the oxygen atom of the etherified hydroxy group being further bound to an acyclic carbon atom
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07C—ACYCLIC OR CARBOCYCLIC COMPOUNDS
    • C07C233/00—Carboxylic acid amides
    • C07C233/01—Carboxylic acid amides having carbon atoms of carboxamide groups bound to hydrogen atoms or to acyclic carbon atoms
    • C07C233/16—Carboxylic acid amides having carbon atoms of carboxamide groups bound to hydrogen atoms or to acyclic carbon atoms having the nitrogen atom of at least one of the carboxamide groups bound to a carbon atom of a hydrocarbon radical substituted by singly-bound oxygen atoms
    • C07C233/17—Carboxylic acid amides having carbon atoms of carboxamide groups bound to hydrogen atoms or to acyclic carbon atoms having the nitrogen atom of at least one of the carboxamide groups bound to a carbon atom of a hydrocarbon radical substituted by singly-bound oxygen atoms with the substituted hydrocarbon radical bound to the nitrogen atom of the carboxamide group by an acyclic carbon atom
    • C07C233/18—Carboxylic acid amides having carbon atoms of carboxamide groups bound to hydrogen atoms or to acyclic carbon atoms having the nitrogen atom of at least one of the carboxamide groups bound to a carbon atom of a hydrocarbon radical substituted by singly-bound oxygen atoms with the substituted hydrocarbon radical bound to the nitrogen atom of the carboxamide group by an acyclic carbon atom having the carbon atom of the carboxamide group bound to a hydrogen atom or to a carbon atom of an acyclic saturated carbon skeleton
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07C—ACYCLIC OR CARBOCYCLIC COMPOUNDS
    • C07C271/00—Derivatives of carbamic acids, i.e. compounds containing any of the groups, the nitrogen atom not being part of nitro or nitroso groups
    • C07C271/06—Esters of carbamic acids
    • C07C271/08—Esters of carbamic acids having oxygen atoms of carbamate groups bound to acyclic carbon atoms
    • C07C271/10—Esters of carbamic acids having oxygen atoms of carbamate groups bound to acyclic carbon atoms with the nitrogen atoms of the carbamate groups bound to hydrogen atoms or to acyclic carbon atoms
    • C07C271/16—Esters of carbamic acids having oxygen atoms of carbamate groups bound to acyclic carbon atoms with the nitrogen atoms of the carbamate groups bound to hydrogen atoms or to acyclic carbon atoms to carbon atoms of hydrocarbon radicals substituted by singly-bound oxygen atoms
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07C—ACYCLIC OR CARBOCYCLIC COMPOUNDS
    • C07C43/00—Ethers; Compounds having groups, groups or groups
    • C07C43/02—Ethers
    • C07C43/03—Ethers having all ether-oxygen atoms bound to acyclic carbon atoms
    • C07C43/14—Unsaturated ethers
    • C07C43/178—Unsaturated ethers containing hydroxy or O-metal groups
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07C—ACYCLIC OR CARBOCYCLIC COMPOUNDS
    • C07C43/00—Ethers; Compounds having groups, groups or groups
    • C07C43/02—Ethers
    • C07C43/03—Ethers having all ether-oxygen atoms bound to acyclic carbon atoms
    • C07C43/14—Unsaturated ethers
    • C07C43/178—Unsaturated ethers containing hydroxy or O-metal groups
    • C07C43/1785—Unsaturated ethers containing hydroxy or O-metal groups having more than one ether bound
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07C—ACYCLIC OR CARBOCYCLIC COMPOUNDS
    • C07C43/00—Ethers; Compounds having groups, groups or groups
    • C07C43/02—Ethers
    • C07C43/03—Ethers having all ether-oxygen atoms bound to acyclic carbon atoms
    • C07C43/14—Unsaturated ethers
    • C07C43/178—Unsaturated ethers containing hydroxy or O-metal groups
    • C07C43/1787—Unsaturated ethers containing hydroxy or O-metal groups containing six-membered aromatic rings and having unsaturation outside the aromatic rings
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07C—ACYCLIC OR CARBOCYCLIC COMPOUNDS
    • C07C69/00—Esters of carboxylic acids; Esters of carbonic or haloformic acids
    • C07C69/66—Esters of carboxylic acids having esterified carboxylic groups bound to acyclic carbon atoms and having any of the groups OH, O—metal, —CHO, keto, ether, acyloxy, groups, groups, or in the acid moiety
    • C07C69/67—Esters of carboxylic acids having esterified carboxylic groups bound to acyclic carbon atoms and having any of the groups OH, O—metal, —CHO, keto, ether, acyloxy, groups, groups, or in the acid moiety of saturated acids
    • C07C69/708—Ethers
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07D—HETEROCYCLIC COMPOUNDS
    • C07D207/00—Heterocyclic compounds containing five-membered rings not condensed with other rings, with one nitrogen atom as the only ring hetero atom
    • C07D207/46—Heterocyclic compounds containing five-membered rings not condensed with other rings, with one nitrogen atom as the only ring hetero atom with hetero atoms directly attached to the ring nitrogen atom
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
    • C07K16/28—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/44—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material not provided for elsewhere, e.g. haptens, metals, DNA, RNA, amino acids
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K2039/505—Medicinal preparations containing antigens or antibodies comprising antibodies
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K2039/60—Medicinal preparations containing antigens or antibodies characteristics by the carrier linked to the antigen
    • A61K2039/6031—Proteins
    • A61K2039/6037—Bacterial toxins, e.g. diphteria toxoid [DT], tetanus toxoid [TT]
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K2039/60—Medicinal preparations containing antigens or antibodies characteristics by the carrier linked to the antigen
    • A61K2039/6031—Proteins
    • A61K2039/6068—Other bacterial proteins, e.g. OMP
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K2039/62—Medicinal preparations containing antigens or antibodies characterised by the link between antigen and carrier
    • A61K2039/627—Medicinal preparations containing antigens or antibodies characterised by the link between antigen and carrier characterised by the linker

Definitions

  • conjugate is a common designation for
  • the conjugate of the invention complies with the general formula A-B-A' wherein A and A' comprise residues from organic compounds F and F', respectively. At least one of the compounds is a polymer, the polymeric structure of which being present also in the residue. B represents an organic bridge linking A and A' together and comprising the structure -(OCH 2 CK2) n -. A and A' may comprise several identical residues originating from the compounds to be conjugated (F and F').
  • hetero- or homobifunctional reagents of the type Z-B'-Z' where Z and Z' are reactive functional groups that are inert with respect to reaction with each other (in pairs they are nucleophilic or electrophilic) and B' is an inert bridge.
  • inert is contemplated that the bridge is stabile and has no groups that are able to neutralize the reactivity of Z and/or Z' .
  • PEG polyethylene glycol
  • the structure -(OCH 2 CH 2 ) n - is also present in certain known amino-PEG-carboxylic acids. For instance
  • Slama and Rando did not manifold the -OCH 2 CH 2 - structure by increasing the integer n. For unknown reason they instead duplicated the complete structure -NHCH 2 CH 2 (OCH 2 CH 2 ) n OCH 2 CO- by making n' equal 2.
  • the present invention provides conjugate substances in which the spectrum of individual molecules have an improved uniformity and a good bioavailability, particularly in hydrophilic aqueous media. This goal is achieved by
  • the free valencies in formula (I) link to A and A', respectively. This takes place either directly or through further divalent inert structures that are comprised within the bridge -B-.
  • the length of -B- is usually shorter than 180 atoms, such as ⁇ 100 atoms, but longer than 13, preferably longer than 16 atoms.
  • n is an integer > 0, e.g. 1-20, and such that n is uniform for bridges linking identical positions together in individual molecules of the conjugate (conjugate
  • n is 1 or 2. From the synthetic point of view n is preferably 10 or ⁇ 10. In order for the conjugate to express the unique amphiphilic properties of PEG, the integer n should be higher than 2, preferably higher than 3 or higher than 4. Thus based on different combinations of criteria the intervals for the integer n may be 1-20, 1-10, 1-9, 2-20, 2-10, 2-9, 3-20, 3-10, 3-9, 4-20, 4-10, 4-9, 520, 5-10, and 5-9.
  • disulfide group or a thioether group. If S r is binding directly to A, one of the sulphur atoms may originate from F.
  • R is preferably alkylene (having 1-4 carbon atoms, often 1 or 2 carbon atoms), that possibly is substituted with one or more (1-3, in the preferred case ⁇ 2) hydroxy (OH) groups. At most one oxygen atom is bound to one and the same carbon atom in R. With respect to availability in hydrophilic media R may in many cases be equivalently substituted for a higher alkylene selected from the group comprising straight, branched and cyclic alkylene, with the provision that the higher alkylene shall exhibit
  • hydrophilic substituents compensating for an enhanced hydrophobicity
  • the bridge -B- must not contain any aromatic ring.
  • the polymer may be a synthetic one or a biopolymer.
  • the term polymer stands for a compound in which three or more, preferably more than 10, repeating monomeric units bind sequentially to each other.
  • the term polymer also
  • inorganic polymers such as glass and other polymeric silicates.
  • Examples of synthetic polymers are poly(hydroxyalkyl (meth) acrylate), poly((meth)acrylamide), poly(vinyl alcohol), etc and derivatized forms of these polymers.
  • biopolymer means a polymer in which the basic skeleton is of biological origin.
  • Biopolymers that have been derivatized. Biopolymers exhibit as a rule nucleic acid or
  • Proteins including polypeptides (e.g. albumin and immunoglobulins) and polysaccharides, e.g. soluble and insoluble ones
  • Polymers of immediate interest e.g. those of
  • polypeptide and/or polysaccharide structure usually have functional groups such as hydroxy (-OH), carboxy (-COOH), amino (primary or secondary) and/or mercapto (-SH).
  • functional groups such as hydroxy (-OH), carboxy (-COOH), amino (primary or secondary) and/or mercapto (-SH).
  • chemic 1 modification can as a rule be carried out to the effect that the group will be inserted.
  • the compounds F and F' are selected according to the properties that they shall confer to the conjugate.
  • the compounds may thus be: carrier compounds, bioaffinity compounds, analytically detectable compounds, compounds that are insoluble or soluble in aqueous media,
  • toxins therapeutically active compounds (drugs), enzymes, immune stimulators, toxins etc.
  • Particularly important toxins are the peptide cytotoxins that exert their effect
  • peptide toxins are those that exert their effect through immune stimulation (e.g. by activating cytotoxic T-cells via simultaneous binding to T-cells and cells carrying Class II MHC antigens).
  • immune stimulation e.g. by activating cytotoxic T-cells via simultaneous binding to T-cells and cells carrying Class II MHC antigens.
  • Bioaffinity compounds i.e. compounds that exert
  • biospecific affinity are particularly interesting, because they as a part of a conjugate may be utilized as a targeter for their bioaffinity counterparts.
  • Examples are antibodies and their antibody active fragments and corresponding antigens/haptens, Fc-fragments/Fc-parts of immunoglobulins (Ig) and corresponding receptors (for instance IgG binds to Protein A and G ), avidin/strepavidin and biotin, lectins and corresponding carbohydrate structures, enzymes and respective substrate, coenzyme, cofactor, and cosubstrate, etc.
  • Antibodies of various classes in particular IgG
  • subclasses and various specificities may be one part of the conjugate according to the invention.
  • the specificities of the antibodies (and fragment thereof) may e.g. be for tumour cells and/or tumour antigens/haptens, hormones, hormone receptors etc.
  • Particularly interesting insoluble polymers are those ones that are used as adsorbents in connection with
  • conjugates between an analytically detectable compound and a compound (targeter) showing biospecific affinity are of great importance for detecting and localizing the
  • the analytically detectable compounds may be radioactive, enzymatically active
  • particulate e.g. latex
  • bioaffinity compounds may be conjugated to drugs and other substances that exert a therapeutic effect.
  • Z 1 ' (II) m and n have the same meaning as above for formula (I).
  • m and n are uniform, i.e. the reagent substance is not a mixture of compounds having different m and different n.
  • R is an alkylene group of the same meaning as above.
  • Z 1 - is an HS-reactive electrophili ⁇ group, thiol (-SH) or protected thiol (e.g. Acs-), with the provision that a thiol group and a hydroxy group must not be bound to one and the same carbon atom in R.
  • HS-reactive electrophilic groups are:
  • halogen that is bound to a saturated carbon atom, preferably in the form of an alfa-halo-alkylcarbonyl (e ⁇ g- Z 1 CH 2 CO-, where Z 1 preferably is bromo or iodo);
  • activated thiol preferably a so called reactive disulfide (-SSR 1 ) that is bound to a saturated carbon atom;
  • Reactive disulfides are well known in the context of synthetic chemistry (See EP-A-063,109, 064,040, and
  • R 1 is defined by the chemical reaction between -S-S-R 1 and HS- releasing R 1 -SH that is thermodynamically stabilized to be withdrawn from further thiol-disulfide exchange reactions.
  • Many thiol compounds (R 1 'SH) comply with this criterion by spontaneously tautomerizing to a thione form in aqueous solutions, i.e. the thione form is more stabile than the corresponding thiol form.
  • One prerequisite for this type of tautomerization may be that the sulfur atom of the thiol group is bound to a carbon atom that constitutes a part of an aromatic ring that (a) is heterocyclic having the thiol sulfur atom located at a distance of an odd number of atoms from a heteroatom in the ring, or (b) is non-heterocyclic and substituted with electron-withdrawing groups.
  • R 1 are 5-nitro-2-pyridyl, 5-carboxy-2-pyridyl, 2-pyridyl, 4-pyridyl, 2-benzthiazolyl, 4-nitro-3-carboxyphenyl and the N-oxides of the pyridyl groups just mentioned.
  • Z 1 ' is activated carboxy, i.e. an electrophilic group.
  • Examples are carboxylic acid halides (-COCl, -COBr, and -COI), mixed carboxylic acid anhydrides (-COOOCR 1 ), reactive esters, such as N-succinimidyloxycarbonyl,
  • R 1 and R 2 may be lower alkyl (C 1 -C 6 ) and R 2 also benzyl or C 2 -C 3 alkylene with one of its valencies substituting H in NH (giving cyclic structures such as in oxazolin-2-yl that possibly may be substituted with lower alkyl (C 1 -C 6 ) or benzyl in its 3- and/or 4-position).
  • the Z 1 ' terminal may be reacted selectively with a compound F' showing a nucleophilic group selected among:
  • R 1 is selected from hydrogen and lower alkyl (C 1 -C 6 )) and in hydrazine/hydrazide, i.e. NH 2 NH 2 and compounds in which NH 2 NH- and NH 2 NH-CO- are bound to an
  • Z 1 may then be transformed (reduced) to a thiol group in a thiol-disulfide exchange reaction. In the latter case F' will become thiolated.
  • the Z 1 terminal may selectively be reacted with a compound F having a thiol group (SH) or a HS-reactive electrophilic group. If F has a thiol group reaction can take place directly.
  • compound F will be bound to R in formula (II) via a thioether (-S-) or disulfide (-S-S-) group.
  • the use of the reagent (II) is carried out in a manner known per se.
  • the reaction medium is selected so that side reactions of Z 1 and Z 1 ' are avoided.
  • F and/or F' are biopolymers it is preferred to run the reaction in aqueous media, and in order to achieve selectivity pH shall be 89.5 for reaction at Z 1 ' and ⁇ 8 for reaction at Z 1 .
  • Aprotic media are often inert against Z 1 ' which means that they generally speaking are the preferred ones.
  • the result will be a conjugate in which F and F' are linked together through a bridge complying with formula I.
  • Extended bridges can be introduced by reacting the reagent of formula (II) at either its Z 1 or Z 1 ' terminal by suitable bifunctional compounds. For instance, if Z 1 ' is reacted with an alkylene diamine, alkylene dihydrazine, alkylene dihydrazide etc and only one of their H 2 N- groups is consumed, the remaining free NH 2 -group can be used for reaction with other compounds, e.g. F or F' (exhibiting karboxy, optionally after activation).
  • Elongation of the bridge may also be accomplished by reacting the compounds F and/or F' with appropriate
  • bifunctional reagents of the type A-B'-Z' (see page 1) prior to linking them together by the use of the reagent of the invention.
  • B' may be selected from the same group as R above. If each of the compounds F and F' are initially reacted with the same reagent Z-B'-Z' at the Z ' group and then linked together through the so introduced Z group the group B' will appear twice in the conjugate (head to head linking).
  • Known techniques encompass a large number of
  • bifunctional reagents that are useful for chain elongation, either starting from a reagent of formula (II) or from one of the compounds F and F'.
  • Specific examples are N-succinimidyl 4-(N-malein-imidyl)-butyrate, N-succinimidyl iodoodacetate, N-succinimidyl S-acetyl-2-mercaptoacetate, and N-succinimidyl 3-(pyridyl-2-dithio)propionate, alkylene diamine, alkylene diacylhydrazide etc. Alkylene has the same meaning as previously.
  • conjugates a vicinal diol, e.g in a carbohydrate
  • carbohydrate structures are the glycoproteins.
  • the group -CONHNH 2 may be present in a bifunctional reagent Z-B'-Z', optionally after reaction with a polymer.
  • Chain elongation by starting from a reagent of the present invention may result in conjugates in which -B- is: (1) -COR'-S r -RCONHCH 2 CH 2 (OCH 2 CH 2 ) n O(CH 2 ) m COY-;
  • N is usually bound to a sp 2 -hybridized
  • This structure may have been formed by reaction between an aldehyde group and a NH 2 -NHCO- group,
  • N is bound as above in (2 ) ,
  • R and R' are alkylene selected in the same way as R in formula (I).
  • the reagent (Formula II) can be prepared starting from compounds complying with formula III:
  • n an integer 1-20, such as 2-20.
  • n is an integer 2-20, preferably 3-20.
  • hexanoylamido including acylamido groups that have electron-withdrawing substituents on the alpha carbon atom of the acyl moiety and then particularly CF 3 CONH-, CH 3 COCH 2 CONH- etc; phtalimidyl which possibly is ring substituted; carbamato (particularly R 1 'OCONH- and (R 1 'OCO) (R 2 'OCO)N-, such as N-(t-butyloxycarbonyl) amino (Boc), N-(benzyloxycarbonyl) amino and di(N- (benzyloxycarbonyl))amino (Z and diZ, respectively) which possibly are ring substituted; alkyl amino in which the carbon atom binding to the nitrogen atom is alpha to an aromatic system, such as N-monobenzylamino and
  • Si silicon atom
  • R 1 ' and R 2 ' stand for lower alkyl, particularly secondary and tertiary alkyl groups, and a methyl group that is substituted with 1-3 phenyl groups that possibly are ring substituted.
  • Lower alkyl and lower acyl groups have 1-6 carbon atoms.
  • Y is carboxy (-COOH including -COO-) or a group that is transformable to carboxy, preferably by hydrolysis or oxidation.
  • the most important groups are the ester groups in which the carbonyl carbon atom or the corresponding atom in orto esters binds to the methylene group in the right terminal of formula (I).
  • R 3 ' has the meaning as previously given for R 1.
  • Y are -CHO, -CN, -COKR 1 'R 2 ' , where R 1 ' and R 2 ' have the same meaning as previously, and -CONH 2 .
  • the compound of the invention may be synthesized from known starting materials by combining methods that are known per se. Appropriate synthetic routes are:
  • Convenient starting materials that have the repeating unit -OCH 2 CH 2 - are commercially available. Examples are oligoethylene glycols having 2 to 6 repeating units. Other suitable compounds with identical terminal groups are corresponding dicarboxylic acids and diamines.
  • Convenient starting materials that have different terminal groups are omega-hydroxy monocarboxylic acids in which the terminal groups are spaced apart by a pure polyethyleneoxide bridge. Such compounds having up to 5 repeating units have been described in the prior art
  • Williamson's ether synthesis can be applied to the
  • the method means alkylation of an alcohol (QZ" + HOQ' —> Q-O-Q' or the other way round QOH + Z"Q' —> Q-O-Q').
  • Williamson's ether synthesis may also be applied to the synthesis of the type of bisymmetric chains that is
  • Y' is not Y
  • Y' one select Y' preferably among groups that are stabile against strong bases.
  • R 5 ' and R 6 ' are hydrogen, lower alkyl (C 1 C 6 ) or phenyl that possibly is substituted.
  • Z" is a leaving group of moderate to high reactivity and may be halo, alkanesulfonate, arenesulfonate, preferably toluenesulfonate (tosylate), and perfluoroalkanesulfonate, preferably trifluoromethanesulfonate etc.
  • Williamson's ether synthesis may be carried out in inert solvents and normally in the presence of a base - often strong bases, such as sodium hydride etc. By combining components of appropriate lengths one can in principle develop any chain -(OCH 2 CH 2 -) n by a sequence of elongation steps.
  • the chain can also be elongated with one OCH 2 CH 2 unit through Michael addition of HOQ or HOQ' to a compound
  • X"-C(X'") CH 2 , where Q and Q', respectively', have the same meaning as previously.
  • X'" may be hydrogen and X" may be a group that is easily transformable to amino or substituted amino, e.g. nitro.
  • X" and X'", respectively, may be groups that after the transformation enable the group
  • X"-C(X'")CH 2 - to be converted to the group HOOCCH 2 - or a derivative thereof.
  • X" is methylsulfinyl and X'" methylthio, and this requires hydrolysis of the product obtained in the addition step, said hydrolysis giving an aldehyde that subsequently may be oxidized to the corresponding carboxylic acid.
  • the conditions for Michael addition are similar to those ones for Williamson's ether synthesis.
  • a third alternative for chain elongation is reduction of thione ester by the use of Raney Nickel or corresponding reagents having a high affinity for sulfur.
  • Suitable thiono esters comply with the formula:
  • Thione esters can be synthesized through rearrangement of the corresponding thiol esters by the action of an alkylating agent, e.g. methyl iodide, dimethyl sulfate or diazo methane.
  • Thiol esters may be synthesized by reaction of a carboxylic acid halide with a thiol compound.
  • Another alternative is reaction of a carboxylic acid ester with a sulfur transferring reagent that is selective for double bonded oxygen when present in a carboxylic acid ester.
  • reagents examples include phosphorous pentasulfide or preferably Lawesson's reagent (2,4-bis(4-methoxyphenyl)-1,2,3,4-dithiaphosphetane-2,4-bis-sulfide).
  • Lawesson's reagent 2,4-bis(4-methoxyphenyl)-1,2,3,4-dithiaphosphetane-2,4-bis-sulfide.
  • X'- is XCH 2 - or a group that in one or more steps is transformable to XCH 2 -
  • -Y' is Y or a group that, in one or more steps is transformable to Y
  • reaction conditions that give the desired transformation, and end product or where appropriate give an intermediary product that can be used in a coupling step.
  • the transformation can take place, e.g. by reaction with a sulfonyl halogenide in the presence of a base and followed by reaction with ammonia and, if
  • R 4 ' contains at least one alpha positioned phenyl
  • the removal can be performed by catalytic hydrogenolysis.
  • the exposed hydroxymethyl group may then be transformed according to what has been said above.
  • the oxidations may be run in the presence of an appropriate tertiary alcohol, for instance t-butanol, in such a way that the corresponding ester will be formed directly.
  • Symmetrical ethers can be synthesized from shorter polyethylenoxide ethers by chain elongation, for instance by the use of Williamson's ether synthesis. Transformation of one of the two identical groups X 1 , where X 1 is X' or Y' as given previously, to another group can be performed by partial reaction of the desired kind and subsequent
  • the separation is carried out after the reduction step by a sequence of ion exchange separation steps.
  • the monoacylated product is then separated from the starting material and the diacylated product, whereafter its aminomethyl group is oxidized, for instance through the corresponding aldehyde, to a carboxy group, and its acyl group removed by hydrolysis.
  • X 1 is a group of moderate to high lipophilicity, for instance trityloxymethyl or allyloxymethyl, partial removal or transformation of one of the groups will facilitate separation by liquid partition due to a significant
  • Analogous critera are valid if the appropriately lipophilic group has been introduced onto to a symmetric polyether having hydrophilic groups X 1 , for instance oligoethylene glycol that is monosubstituted with trityl.
  • Examples are diethylene glycol or triethylene glycol.
  • D Splitting of a bisymmetric chain into two identical fragments.
  • the splitting is carried out by oxidation of the double bond; in case A directly to two identical carboxy groups and in case B to two identical aldehyde groups that in turn is transformed to aminomethyl groups by reductive amination.
  • A is a possibly substituted lower alkylidene
  • the group A is removed by acid hydrolysis, whereafter the intermediary 1,2-diol formed is oxidized, for instance by periodate and subsequent transformation of the aldehyde group formed to an aminomethyl group or to a carboxy group in a manner known per se.
  • Part II illustrates the synthesis of heterobifunctional reagents complying with formula II and of conjugates having a bridge structure according to formula I, and
  • Part III sums up the results obtained for conjugates between the T-cell immune stimulator (superantigen)
  • SEA staphylococcal enterotoxin A
  • Examples 5 and 6 (Part 2) illustrate the synthesis of a conjugate between an immunoglobulin and a peptide.
  • the mixture was filtered and the filtrate evaporated nearly to dryness, whereupon it was dissolved in water (200 ml).
  • the water phase was extracted with 1,1,1-trichloro-ethane (3x50ml).
  • the pooled organic phases were washed with water (20 ml).
  • the product was extracted from the pooled water phases with dichloromethane (50 ml) that after evaporation gave an oil (55 g).
  • the mixture was filtered and the filtrate evaporated nearly to dryness, whereupon it was dissolved in water (200 ml).
  • the water phase was extracted with 1,1,1-trichloro-ethane (3x50ml).
  • the pooled organic phases were washed with water (20 ml).
  • the product was extracted from the pooled water phases with dichloromethane (50 ml) that after evaporation gave an oil.
  • Triphenylmethyl chloride 28 g, 0.1 mole was added to a solution of pyridine (8 ml, 0.1 mole) in diethylene glycol (100 ml, 0.94 mole) and the mixture was stirred at 50°C for 20 hours. The crystals formed were filtrated off and washed with water. Yield 31 g. M.p. 103-105°C.
  • Triphenylmethyl chloride (187 g, 0.67 mole) was added to a solution of pyridine (54 ml, 0.67 mole) in triethylene glycol (1,000 ml, 7.3 mole) and the mixture was stirred at 60°C for 16 hours.
  • Triphenylmethyl chloride (129 g, 0.46 mole) was added to a solution of pyridine (38 ml, 0.48 mole) in tetraethylene glycol (800 ml, 4.2 mole) and the mixture was stirred at 80°C for 20 hours. Water (800 ml) was added and the mixture was extracted with dichloromethane (3x200 ml). The pooled organic phases were washed with water (150 ml), whereafter the product was obtained as a syrup upon evaporation. Small amounts of the disubstituted and the unreacted materials were present in the product. Yield 200 g.
  • p-Toluenesulfonyl chloride (5 g, 26 mmole) was added to a solution of 7,7,7-triphenyl-3,6-dioxa-heptanol (7) (3.5, 10 mmole) in pyridine (10 ml) . The mixture was stirred at room temperature for 30 minutes, whereupon water (1 ml) was added and the stirring continued for 10 minutes more.
  • p-Toluenesulfonyl chloride (5 g, 26 mmole) was added to a solution of 10,10,10-triphenyl-3,6,9-trioxa-decanol (8) (4.8 g, 12 mmole) in pyridine (10 ml). The mixture was stirred at room temperature for 1 hour, whereupon water (1 ml) was added and the stirring continued for 10 minutes more. The mixture was diluted with dichloromethane (50 ml) and washed with 1 M hydrochloric acid (2x100 ml), water (50 ml) and NaHC0 3 (saturated, 50 ml). The organic phase was evaporated and the product was purified on silica gel
  • Isopropyl 17-amino-3,6,9,12,15-pentaoxaheptadecanoate (see part I of the experimental part) (1.1 g, 3.2 mmole) was dissolved in 3 ml of 1 M sodium hydroxide solution and left at room temperature for 30 min. 1.5 ml of 6 M hydrochloric acid was added and the mixture was evaporated to dryness. The residue was taken up in dichloromethane and filtered to give 545 mg of 17-amino-3,6,9,12,15-pentaoxaheptadecanoic acid after evaporation of the solvent. 460 mg (1,39 mmoles) of this compound were dissolved in 10 ml of borate buffer pH 8.4.
  • the solution was deaerated with nitrogen gas.
  • a solution of 432 mg (1.52 mmoles) of N-succinimidyl 2-iodoacetate in 5 ml of dioxane was added dropwise during 1 min pH was kept at 8.4 by addition of 5 M NaOH.
  • the reaction solution was stirred for 15 min during inlet of nitrogen gas. According to thin layer chromatography (eluent: CH 2 Cl 2 -MeOH 60:35) the reaction was completed in some few minutes. After 15 min the pH of the reaction solution was adjusted to 3 and the solution was frozen and lyophilized.
  • reaction mixture was fractionated on a reversed phase column PEP-RPC HR 30/26 (Pharmacia Biosystems AB) using a gradient of 0-13 % acetonitrile with 0.1 % trifluoroacetic acid followed by isocratic separation at 13 % acetonitrile, 0.1 % TFA.
  • Fractions from the desired peak were pooled and lyophilized giving 351 mg of 17-iodoacetylamino-3,6,9,12,15-pentaoxa-heptadecanoic acid (A). Yield: 76 %.
  • the structure of the product was established by the aid of its NMR spectrum. 1 H NMR spectrum (D 2 O) expressed as ⁇ -values:
  • 146 ⁇ l of a dioxane solution containing 3.6 mg (6.4 ⁇ mole) of N- hydroxysuccinimide ester of 17-iodoacetylamino- 3,6,9,12,15-pentaoxaheptadecanoic acid (B) was injected into the buffer solution and the reaction was completed during stirring for 25 min. at room temperature.
  • the reaction vial was covered with folie to exclude light.
  • a monoclonal antibody (Mab C242) of the immunoglobulin class IgG 1 was reacted with 15, 20 and 22 times molar excess of N-hydroxysuccinimide ester of 17- iodoacetyl-amino-3,6,9,12,15-pentaoxaheptadecanoic acid (B) respectively according to the procedure described in example 2.A giving nona, dodeca and tetradeca(17-iodoacetylamino)-3,6,9,12,15- pentaoxaheptadecanoylamino)-Mab C242. (C) C. Monoclonal antibody Mab C
  • a monoclonal antibody (Mab C) of the immunoglobulin class IgG 2a was reacted with 14 and 18 times molar excess of N-hydroxysuccinimide ester of 17- iodoacetylamino-3,6,9,12,15-pentaoxaheptadecanoic acid (B) respectively according to the procedure described in example 2A giving tetra and hepta(17-iodoacetylamino-3,6,9,12,15-pentaoxa-heptadecanoylamino)- Mab C. (C)
  • SEA freezed dried product from Toxin Technology Inc. (2 mg, 72 nmole) was dissolved in 722 ⁇ l milli-Q water and added to a 15 ml polypropylene tube. 100 ⁇ l of 0.1 M borate buffer pH 8.6 was added and then 2160 nmoles of Eu 3+ -chelate reagents
  • Native SEA freeze dried product from Toxin Technology Inc
  • rSEA recombinant prepared SEA
  • the degree of substitution was determined with UV- analysis according to Carlsson et al (Biochem. J. 173(1978)723-737) to be 1.9 mercaptopropionyl group per SEA.
  • the conjugate obtained was composed of 6% Mab C215 with three SEA, 15% with two SEA, 28% with one SEA and 51% unsubstituted Mab C215.
  • the same reaction was run but the reaction product was treated 4 h with 0.2 M hydroxylamine before column fractionation to remove unstable bonds between Mab C242 and the spacer and between SEA and the mercaptopropionyl group.
  • the conjugate formed had the composition 1% Mab C242 with four SEA, 12% with three SEA, 27% with two SEA, 36% with one SEA and 24% of unsubstituted Mab.
  • the same reaction was run with 0.2 M hydroxylamine present to remove unstable bonds between Mab C and spacer and between SEA and the mercaptopropionyl group.
  • the conjugate formed had the following
  • composition 11% Mab C with three SEA, 24% with two SEA, 30% with one SEA, 18% of unsubstituted Mab C and 17% in a dimeric form.
  • Example 5 Coupling of the peptide sequence 145-165 derived from the human alloantigen HLA-A2.1 to the monoclonal antibody Mab C215 (H)
  • Tridecane (17-iodoacetylamino-3,6,9,12,15-pentaoxaheptadecanoylamino)-immunoglobulin G2a (4 mg, 25.6 nmole) dissolved in 1.6 ml 0.1 M phosphate buffer pH 7.5 containing 0.9 % sodium chloride was added to a 5 ml Reacti vial.
  • This compound was prepared similar to compound C, Example 2A,using less excess of the reagent B). The solution was deaerated by nitrogen gas.
  • the HLA-A2.1 peptide MetTyrGlyCysAspValGlySerAspTrpArgPheLeuArgGlyTyr (4.7 mg, 2.1 ⁇ mole) was suspended in 0.2 ml of
  • reaction solution was adjusted to 4.5 with 5 M hydrochloric acid and the solution was frozen and lyophilized.
  • the reaction mixture was fractionated on a reversed phase column PEP RPC HR 16/10 (Pharmacia Biosystems AB) using a gradient of 0.17 % acetonitrile with 0.1 % TFA followed by isocratic separation at 17 % acetonitrile with 0.1 % TFA. Fractions with the desired compound J were pooled and lyophilized. The fractionation was repeated 13 times. Yield: 39 mg.
  • reaction solution was fractionated on a Superdex 200 HR 16/65 column.
  • the product was eluated with 2 mM phosphate buffer pH 7.5 with 0.9 % NaCl. Fractions with the desired product Q were pooled and analysed as described in example 4B.
  • the conjugate was composed of 9% Mab C242 with two SEA, 25% with one SEA and 66% of unsubstituted Mab C242.
  • the bacterial toxin used in the following experiments was Staphylococcus enterotoxin A (SEA) obtained from Toxin Technologies (WI; USA) or produced as a recombinant protein from E. Coli.
  • SEA Staphylococcus enterotoxin A
  • C215 is an lgG2a mAb raised against human colon carcinoma cell line and reacts with a 37kD protein antigen on several human colon cell lines. References to these mAbs have been given above.
  • the conjugates were prepared as described in the preceding part.
  • SEA-C215 mAb conjugate and unconjugated SEA and C215 mAb against colon carcinoma cells lacking MHC Class II or expressing low but undetectable amounts of MHC Class II we employed various human SEA expanded T cell lines as effector cells and a panel of colon carcinoma cells and MHC Class II + Raji cells as target cells.
  • HLA-DR HLA-DR
  • HLA-DP HLA-DP
  • HLA-DQ FACS analysis.
  • the SEA expanded T cell lines were established from peripheral blood by weekly restimulations with mitomycin C treated MHC
  • T cell lines were strongly cytotoxic towards Raji or BSM cells coated with SEA but not to uncoated cells or cells coated with staphylococcal enterotoxin B (SEB).
  • SEB staphylococcal enterotoxin B
  • HLA-DR transfected L-cells (Dohlsten et al., Immunology 71 (1990) 96-100. These T cell lines could be activated to kill C215 + MHC Class II- colon carcinoma cells by the C215-SEA conjugate. In contrast unconjugated SEA and C215 mAb were unable to induce more than marginal T cell killing against the C215 MHC Class II- colon carcinoma cells.
  • the staphylococcal enterotoxin antibody conjugate dependent cell-mediated cytotoxicity was dependent on binding of the SEA-C215 mAb conjugate to the C215 + tumor cells.
  • FCS fetal calf serum
  • SEA-C242, SEA-C215 and SEA-anti-Thy-1.2 mAb conjugates bind to cells expressing the relevant epitopes of the mAbs, respectively, and to MHC Class II cells.
  • Unconjugated SEA on the other hand only binds to MHC Class II cells.
  • Unconjugated C215, C242 and Thy-1.2 mAbs bind to the relevant cells but not to Raji cells.
  • Colo205 and WiDr cells in the presence of SEA-C215 mAb conjugate but not in the presence of unconjugated SEA and C215 mAb (Fig. 1).
  • the lysis of colon carcinoma cells was seen at 10-100 ng/ml of SEA-C215 mAb conjugate.
  • High levels of lysis at various effector to target ratios were seen with SEA-215 mAb conjugate against SW620 (Fig. 1).
  • unconjugated SEA or C215 mAb mediated no cytotoxicity against SW620 cells at all tested effector to target ratios.
  • SEA-C215 mAb conjugate to induce T cell dependent lysis of MHC Class II SW620 colon carcinoma cells was seen in both CD4 and CD8 + T cell populations (Table 2).
  • SEA did not activate any of these T cell subsets to mediate killing of SW620 cells but induced lysis of MHC Class II + Raji cells (Table 2).
  • the SEA-C215 mAb conjugate induced lysis of SW620 and Raji cells by a SEA expanded T cell line, but not by a SEB expanded T cell line (Fig. 3).
  • the specificity of the SEA and SEB lines is indicated by their selective response to SEA and SEB, respectively, when exposed to Raji cells
  • Fig. 1 The SEA-C215 mAb conjugate directs CTLs against MHC class II- colon carcinoma cells.
  • Upper left panel demonstrates the effect of SEA responsive CTLs against SW620 cells at various effector to target ratios in the absence (-) or presence of SEA-C215 mAb conjugate, SEA, C215 and a mixture of C215 and SEA (C215+SEA) at a concentration of 1 ⁇ g/ml of each additive.
  • the other panels demonstrates the capacity of SEA-C215 mAb conjugate, and SEA to target SEA responsive CTLs against the C215+MHC class II- colon carcinoma cell lines SW620, Colo205 and WiDr, MHC class II + C215 + interferon treated Colo205 cells and C215- MHC class II + Raji cells. Effector to target ratio was 30:1. Addition of unconjugated C215 mAb, at several concentrations, did not induce any CTL targeting against these cell lines.
  • SEA-C215 mAb conjugate and SEA-C242 mAb conjugate induced CTL targeting against colon carcinoma cells depends on the antigen selectivity of the mAb. Lysis of Colo205 cells by a SEA responsive CTL line in the presence of SEA-C215 mAb and SEA-C242 mAb conjugate (3 ⁇ g/ml) is blocked by addition of unconjugated C215 and C242 mAbs
  • SEA-C215 mAb conjugate coated colon carcinoma cells is mediated by SEA but not SEB responding CTLs.
  • Autologous SEA and SEB selective T cell lines were used at an effector to target ratio of 10:1 against SW620 and Raji target cells in the absence (control) or presence of SEA-C215 mAb conjugate, a mixture of unconjugated C215 mAb and SEA (C215+SEA) and unconjugated C215 mAb and SEB (C215+SEB) at a concentration of 1 ⁇ g/ml of each additive.
  • Fig. 4 Cytotoxicity induced by the SEA-C242 mAb conjugate and SEA-Anti-Thy-1.2 mAb conjugate against their target cells (Colo205 tumour cells and EL-4 tumour cells, respectively). Table 1
  • SEA-C215 mAb conjugate bind to C215 colon carcinoma cells and MHC Class II + Raji cells
  • CD4 and CD8 CTLs lyse colon carcinoma cells presenting the C215-SEA conjugate.
  • SEA-3 The CTLs (SEA-3) were used at effector to target ratios of 30:1 in the absence (control) or presence of SEA and C215-SEA at 1 ⁇ g/ml.

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Abstract

Substance de conjugué (A-B-A'), dans laquelle A et A' sont des restes de composés organiques F et F'; l'un d'eux au moins est un polymère (porteur) et lesdits composés ont des propriétés qui sont conservées dans le conjugué, et -B- est un pont qui lie par covalence A à A'. Le pont -B- comprend la structure -SrRCONHCH2CH2(OCH2CH2)nO(CH2)mCOY- (I); (i) n est souvent un nombre entier compris entre 1 et 20 qui est uniforme pour des ponts effectuant la liaison de position située de manière identique dans des molécules individuelles de la substance; (ii) m est égal à 1 ou 2; (iii) R est un groupe alkylène (1 à 4 atomes de carbone) qui peut être substitué par 1 ou plusieurs (1-3) groupes hydroxy (OH); (iv) = soufre sous la forme d'un thio-éther (r=1) ou bisulfure (r=2), et Sr se liant à des atomes de carbone saturés dans les deux directions; (v) Y représente -NH-, -NHNH- ou -NHN=CH- qui, dans leurs extrémités de gauche se lient à CO et dans leurs extrémités de droite se lient à un atome de carbone saturé ou à un groupe carbonyle (uniquement -NHNH-). Un réactif de couplage bifonctionnel ayant la formule Z1RCONHCH2CH2(OCH2CH2)nO(CH2)mZ'1 (III); (i) n est un nombre entier souvent compris entre 1 et 20; (ii) m est un nombre entier égal à 1 ou 2, de préférence 1; (iii) Z1 est un thiol ou électrophile réagissant à SH (SH-) ou un thiol protégé; (iv) R est un groupe alkylène (1 à 4 atomes de carbone) qui est éventuellement substitué par un ou plusieurs (1-3) groupes hydroxy (OH); et (v) Z'1 est un carboxy octivé. Un polyéther ayant la formule XCH2CH2(OCH2CH2-)nOCH2Y (IV) dans laquelle n est un nombre entier compris entre 2 et 20, de préférence entre 3 et 10. X représente H2N- ou H2N- substitué qui est transformable en H2N-, de préférence par hydrolyse ou réduction. Y représente un carboxy ou un groupe qui est transformable en carboxy.
EP91913626A 1990-07-20 1991-07-16 Reactifs heterobifonctionnels et conjugues avec unites d'oxa-alkylene pour des structures de pontage amphiphiles Ceased EP0540612A1 (fr)

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CA2087163A1 (fr) 1992-01-21
IE912556A1 (en) 1992-01-29
JPH06500077A (ja) 1994-01-06
AU657483B2 (en) 1995-03-16
AU8239491A (en) 1992-02-18
PT98395A (pt) 1992-06-30
WO1992001474A1 (fr) 1992-02-06

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