EP0537222A1 - Compose immunoreactif - Google Patents

Compose immunoreactif

Info

Publication number
EP0537222A1
EP0537222A1 EP91911923A EP91911923A EP0537222A1 EP 0537222 A1 EP0537222 A1 EP 0537222A1 EP 91911923 A EP91911923 A EP 91911923A EP 91911923 A EP91911923 A EP 91911923A EP 0537222 A1 EP0537222 A1 EP 0537222A1
Authority
EP
European Patent Office
Prior art keywords
igm
iga
compound according
immunoreactive compound
fragment
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.)
Withdrawn
Application number
EP91911923A
Other languages
German (de)
English (en)
Inventor
Ebo Sybren Bos
Petrus Johannes Boon
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.)
Akzo NV
Original Assignee
Akzo NV
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
Application filed by Akzo NV filed Critical Akzo NV
Publication of EP0537222A1 publication Critical patent/EP0537222A1/fr
Withdrawn legal-status Critical Current

Links

Classifications

    • 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
    • A61K47/30—Macromolecular organic or inorganic compounds, e.g. inorganic polyphosphates
    • A61K47/42—Proteins; Polypeptides; Degradation products thereof; Derivatives thereof, e.g. albumin, gelatin or zein
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/574—Immunoassay; Biospecific binding assay; Materials therefor for cancer
    • G01N33/57484—Immunoassay; Biospecific binding assay; Materials therefor for cancer involving compounds serving as markers for tumor, cancer, neoplasia, e.g. cellular determinants, receptors, heat shock/stress proteins, A-protein, oligosaccharides, metabolites
    • 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
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/06—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies from serum
    • C07K16/065—Purification, fragmentation
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/531—Production of immunochemical test materials
    • G01N33/532—Production of labelled immunochemicals

Definitions

  • the present invention relates to an immunoreactive compound and a pharmaceutical preparation comprising the same.
  • Such an immunoreactive compound can particularly be used in immunotherapy and in diagnosis.
  • Immunotherapy is one of the promising possibilities to fight a number of diseases.
  • the principle of immunotherapy itself is old. It comprises a targeting moiety which delivers an active substance to the immediate vicinity of the target.
  • Ligand- receptor interactions or antibody-antigen interactions are suitable couples of targeting moiety and target, but others can of course be envisioned by the person skilled in the art.
  • pretargeting strategies include, but are not limited to, prodrug activation, whereby an enzyme is coupled to a targeting moiety, which is administered before or together with a prodrug which is less toxic than its parent drug and which enzyme converts the prodrug into the parent drug at the target site.
  • antibodies coupled to a label For anti-tumour therapy and tumour localization, and for cancer diagnosis generally use is made of antibodies coupled to a label.
  • a label can be e.g. a toxic compound such as adriamycin, verrucarin, calicheamycin, mitomycin, ricin a, or any other suitable toxic compound, or an isotope or, as described above an enzyme.
  • the antibodies generally are targeted against a particular antigen of the tumour.
  • the antibodies used for this purpose in most cases are monoclonal antibodies of murine origin. Murine monoclonal antibodies are easy to obtain according to well established methods, and against virtually any antigen.
  • tumour material is administered to mice these develop antibodies against almost any antigen of the tumour material, including the normal antigens of these cells. In this way it is difficult to obtain antibodies specific for tumour cells only.
  • murine antibodies may be directed to epitopes that are not tumour-specific according to the human immune repertoire.
  • a solution has been sought in the use of antibody fragments mainly containing the antigen binding domain of the murine antibody, which may overcome the second, but certainly not the first problem.
  • Human anti-tumour antibodies can suitably be obtained according to the method described in EP 0151030.
  • a problem is, however, that this method mainly yields immunoglobulins of the IgM and/or IgA type. These are in fact pentameric or dimeric, i.e. they are composed of five or two monomers interconnected via S-S bridges, whereas each of these IgM/IgA monomers is composed of two heavy and two light chains and contains two antigen binding sites. Each of these monomers roughly equals an IgG molecule in size. Hence a complete IgM molecule is about five times the size of an intact IgG molecule.
  • IgM's are characterized by antigen-affinities which are at the low end of the IgG affinity range.
  • the present invention is concerned with the restoration of the antigen affinity of fragments of IgM.
  • the affinity of antigen binding IgM or IgA fragments can be restored by coupling them to at least one polypeptide.
  • Such a polypeptide can advantageously be a human protein such as human serum albumin, or a (human) enzyme, or it can be a synthetic polypeptide with a low immunogenicity such a poly-L-glutamic acid or poly-L-lysine.
  • the IgM or IgA fragment either can be an IgM or an IgA monomer, which can be obtained by reducing the S-S bonds between the monomers, or can be an antigen binding fragment obtained after enzymatic cleavage of the IgM or the IgA, e.g. by use of pepsin or papain.
  • F(ab')2 Digestion with pepsin delivers an antibody fragment generally indicated as F(ab')2 which in turn is composed of two antigen bindings parts interconnected by S-S bonds. Reduction of these bonds yields two F(ab') fragments.
  • Both IgM or IgA monomers and F(ab ! ) fragments ideally can be bound to the polypeptide(s) via their sulphur atom.
  • binding of the IgM fragment to the polypeptide can be by any other suitable bond, as long as the antigen binding characteristics are not hampered. In this respect it is also convenient to establish a binding via glycosyl groups if present at the constant region of the IgM or IgA fragment.
  • the bond between the IgM or IgA fragment and the polypeptide can either be a direct link or an indirect link via a linking group and/or a spacer.
  • the bond between the IgM or IgA fragment and the polypeptide can be established by making available on both components a group suitable for linking, optionally reacting either or both linking groups with a linker and/or spacer, and thereafter reacting the components to form the desired immunoreactive compound.
  • the polypeptide can be labelled with one or more therapeutically or diagnostically useful groups prior to or after the coupling to the IgM or IgA fragment.
  • Suitable therapeutically useful groups are e.g. cytotoxic drugs, (optionally chelated) radioactive atoms, or enzymes for the conversion of prodrugs into active drugs.
  • the polypeptide is an enzyme itself, which is able to convert a prodrug into a drug at the target site, there is an additional advantage, because the size of the immunoreactive compound plays an important role in its applicability.
  • Suitable diagnostically useful groups are e.g. (optionally chelated) radioactive atoms.
  • the IgM or IgA fragment advantageously is obtained from human IgM or IgA. This IgM or IgA is directed against an antigen specific for, or derived from the tumour, which may be found either in or on or outside the tumour cells.
  • IgM (3-5 mg/ml) was incubated in 10 mmol/1 cysteine in PBS (6.7 mmol/1 K/Na phosphate buffer PH 6.5; 0.13 mol/1 NaCl) for 3 h at 37 °C. Buffers were saturated with nitrogen and the reaction vessel was closed gas-tight. After incubation, the reaction mixture was chromatographed on Sephadex G25, equilibrated with 1 mmol/1 cysteine in PBS. Monomers were precipitated with (NH 4 ) 2 S ⁇ 4 at 50% saturation and dissolved in a minimal volume of 1 mmol/1 cysteine in PBS PH 7.5.
  • the monomer solution was applied on a Fractogel TSK HW 55 (S) column equilibrated in 1 mmol/1 cysteine in PBS PH 7.5.
  • the bed volume of the column was 45 times that of the volume applied and the elution rate was 0.06 bed volume/h.
  • Solid DTNB dithionitrobenzoic acid, Ellman's reagent
  • HSA(-DTPA) was dissolved to a concentration of 5- 10 mg/ml in 0.1 mol/1 sodium phosphate; 0.1 mol/1 NaCl; 5 mmol/1 EDTA PH 7.5.
  • DTT dithio-threitol
  • Solution B Solution B
  • HSA(-DTPA) was removed by (NH ) 2 S0 4 precipitation at 50% saturation. Conjugate and non- conjugated monomers were precipitated, whereas HSA(- DTPA) remains in solution.
  • the precipitate was washed several times with 50 % saturated (NH ) 2 S0 4 and was dissolved in a minimal volume of 0.1 mol/1 sodium phosphate, 5 mmol/1 EDTA PH 7.5. The solution was chromatographed on Sephadex G25 equilibrated in the EDTA phosphate buffer (buffer A) (devoid of NaCl 1 ! ) .
  • the protein-containing fraction was then applied on a Q-Sepharose (Fast Flow) column equilibrated in buffer A in order to separate non-conjugated monomers from the immunoconjugate by anion-exchange chromatography.
  • the Q-Sepharose column was washed with buffer A until the A 8u nad returned to baseline level. Proteins retained by the column were eluted by stepwise increasing the NaCl concentration from 0 - 0.6 mol/1 NaCl.
  • IgM was digested with pepsin according to the method of Putnam. Briefly, whole IgM at a concentration of 2-5 mg/ml in 0.1 mol/1 sodium acetate buffer PH 4.0 was incubated with pepsin (0.08-0.2 mg/ml) for 8 h at 4 °C. The reaction mixture was chromatographed on Fractogel H 55S equilibrated in buffer A in order to purify the F(ab') 2 fragments formed from whole, undigested IgM and low molecular weight fragments. In this way, chromatographically pure F(ab') 2 was isolated at a 40-60% yield.
  • F(ab') 2 was reduced with DTT and coupled to HSA(- DTPA) in the way described for the monomers.
  • Purification of F(ab ! )-HSA(-DTPA) was achieved by anion exchange chromatography on Q-sepharose and gel filtration on Fractogel as described above. The final prep was sterilised by filtration and stored at 4 °C.
  • E ampl 3
  • IgM Ffab o fragments IgM monomer-HSA(-DTPA) and IgM Fab'-HSAf- DTPA)-immunoconiugates.
  • Immunoreactivity was determined by either an antigen binding assay or competitive EIA.
  • dilution series to be tested are incubated with a given amount of peroxidase-labelled whole IgM for 3 h at ambient temperature in an microtitre plate coated with 0.1 ⁇ g/ l antigen solution. After incubation, the contents of the wells is discarded and the plates are washed three times with PBS-Tween buffer. Enzyme activity is detected with a substrate solution containing tetra ethylbenzidine as hydrogen donor. The enzyme reaction was stopped with 2 mol/1 H S0 and the absorbance was read at 450 nm.
  • purified HSA(-DTPA) conjugates of monomers exhibit an immunoreactivity identical to that of whole IgM and F(ab')-HSA(-DTPA) conjugates show some immunoreactivity comparable to that of purified monomers.
  • covalent attachment of a (carrier) protein e.g. through disulphide or thioether bridges, is able to restore the immunoreactivity of IgM monomers or fragments considerably, even to full extent in case of monomers.
  • the reaction mixture was incubated for 16 hours at room temperature in the dark.
  • the conjugate was recovered by addition of an equal volume of 100 % satured ammonium sulph.
  • the precipitate was washed three times with 50 % saturated ammonium sulph, before being dissolved in an appropiate buffer.
  • the enzyme was activated by SPDP us described in method A.
  • the activated enzyme was chromati chargedd on a Sephadex G-25 (M) column equilibrated in 0,1 M NaAc pH 4,5; 0,1 M NaCl.
  • To the enzyme containing fractions 1/20 volume of 1M DTT was added and incubation was performed for at least 30 minutes at room temperature in the dark.
  • the monomerie IgM was chromatographed over Sephadex G-25 (M) into : 50 mM Tris-HCL pH 8 140 mM NaCl
  • the immuno reactivity of the enzyme conjugates was determined by incubation of the conjugate with a icrotiter plate coated with crude antigen mixture of a tumour cell line expressing antigens that are recognized by Monoclonal antibodies 16.88 and 81AV78 (shown in fig. 1).
  • Mab 16.88 recognices specifically a tumour-associated epitope on cytokeratins, whereas Mab 81MV78 reacts with a tumour-associated antigen at the surface of the cells.
  • Myeloma is an antibody (IgM) recognizing an antigen not present in the mixture of crude antigens.

Abstract

On décrit des nouveaux composés immunoréactifs comprenant un ou plusieurs fragments de fixation de l'antigène de l'IgM ou l'IgA couplés à une molécule vectrice, telle que la sérumalbumine humaine (SAH), une enzyme ou un polypeptide synthétique de faible immunogénicité. Le couplage de ces fragments au vecteur permet de ramener leur relativement faible affinité aux antigènes à un niveau comparable au niveau d'affinité de l'IgM ou de l'IgA natif.
EP91911923A 1990-07-03 1991-06-28 Compose immunoreactif Withdrawn EP0537222A1 (fr)

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
EP90201781 1990-07-03
EP90201781 1990-07-03

Publications (1)

Publication Number Publication Date
EP0537222A1 true EP0537222A1 (fr) 1993-04-21

Family

ID=8205056

Family Applications (1)

Application Number Title Priority Date Filing Date
EP91911923A Withdrawn EP0537222A1 (fr) 1990-07-03 1991-06-28 Compose immunoreactif

Country Status (7)

Country Link
EP (1) EP0537222A1 (fr)
JP (1) JPH05507924A (fr)
KR (1) KR930701201A (fr)
CA (1) CA2086531A1 (fr)
FI (1) FI925804A0 (fr)
HU (1) HUT63343A (fr)
WO (1) WO1992000763A1 (fr)

Families Citing this family (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
GB9824632D0 (en) * 1998-11-10 1999-01-06 Celltech Therapeutics Ltd Biological compounds

Family Cites Families (11)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
AU556548B2 (en) * 1980-03-03 1986-11-06 Milton David Goldenberg Tumor localization and therapy with labeled antibodies and antibody fragments specific to tumor-associated markers
CA1168150A (fr) * 1981-12-18 1984-05-29 The Governors Of The University Of Alberta Conjugats d'albumine et d'agents therapeutiques
NZ210867A (en) * 1984-01-31 1989-01-06 Litton Bionetics Inc Tumour-specific monoclonal antibodies, production thereof and use
EP0293524A1 (fr) * 1987-06-02 1988-12-07 Vasocor Immunoessai de plaque athérosclérotique
EP0295719A3 (fr) * 1987-06-19 1989-12-20 The Agouron Institute Antigène SM-D, le clonant de l'antigène SM-D et la détection de lupus érythémateux systémique en utilisant cet antigène SM-D
EP0303088B1 (fr) * 1987-08-10 1992-11-11 Miles Inc. IgM purifiée
US4937183A (en) * 1988-02-03 1990-06-26 Cytogen Corporation Method for the preparation of antibody-fragment conjugates
EP0398872A1 (fr) * 1988-02-12 1990-11-28 HighTech Receptor AB Substance de liaison d'immunoglobuline, sous-fragments, leur procede de preparation, kit de reactif et organismes de liaison d'immunoglobuline
US4983529A (en) * 1988-06-10 1991-01-08 Abbott Laboratories Immunoassay for HIV-I antigens using F(AB')2 fragments as probe
KR900005995A (ko) * 1988-10-31 1990-05-07 우메모또 요시마사 변형 인터류킨-2 및 그의 제조방법
EP0465513A1 (fr) * 1989-03-27 1992-01-15 Centocor, Inc. COMPOSITIONS SERVANT A STABILISER DES ANTICORPS D'IMMUNOGLOBULINE IgM

Non-Patent Citations (1)

* Cited by examiner, † Cited by third party
Title
See references of WO9200763A1 *

Also Published As

Publication number Publication date
HU9204170D0 (en) 1993-04-28
FI925804A (fi) 1992-12-21
WO1992000763A1 (fr) 1992-01-23
FI925804A0 (fi) 1992-12-21
CA2086531A1 (fr) 1992-01-04
HUT63343A (en) 1993-08-30
KR930701201A (ko) 1993-06-11
JPH05507924A (ja) 1993-11-11

Similar Documents

Publication Publication Date Title
AU622415B2 (en) Immunoconjugates joined by thioether bonds having reduced toxicity and improved selectivity
AU636872B2 (en) Cross-linked antibodies and processes for their preparation
JP2942356B2 (ja) 毒素および薬剤を含む治療用複合体
US4350626A (en) Antitumor hybrid and process for the preparation thereof
AU659427B2 (en) Site-specific conjugation of immunoglobulins and detectable labels
EP0055575B1 (fr) Hybride protéique cytotoxique et procédé pour sa préparation
EP0055115B1 (fr) Hybride protéique cytotoxique et son procédé de préparation
JPS6221000B2 (fr)
JPS58208238A (ja) 共有結合によつて酵素と抗体を結合させる新しい共役物とこの共役物を用いた医薬会合
US5354554A (en) Crosslinked antibodies and processes for their preparation
US5134071A (en) Polymerization and copolymerization of proteins
IE67320B1 (en) Cross-linked antibodies and processes for their preparation
Esswein et al. Construction and chemotherapeutic potential of carboxypeptidase-A/monoclonal antibody conjugate
US5714149A (en) Crosslinked antibodies and processes for their preparation
EP0537222A1 (fr) Compose immunoreactif
AU8063591A (en) Immunoreactive compound
JPH04266829A (ja) 免疫結合体を形成するための抗体の化学修飾
JPS62175500A (ja) 修飾糖タンパク質、当該修飾糖タンパク質の製造方法、当該修飾糖タンパク質を含む免疫毒素および当該免疫毒素を有効成分として含む抗ガン剤組成物
JPH0428719B2 (fr)
EP0650735A2 (fr) Kit et méthode de préciblage
Wines et al. The Fab/c fragment of IgG produced by cleavage at cyanocysteine residues
WO1996000087A2 (fr) Materiel de preciblage et nouveaux conjugues de preciblage
AU678474B2 (en) Kit and method for pretargeting
JPH0428720B2 (fr)
AU4605401A (en) Multimeric erythropoietin with increased biological activity

Legal Events

Date Code Title Description
PUAI Public reference made under article 153(3) epc to a published international application that has entered the european phase

Free format text: ORIGINAL CODE: 0009012

17P Request for examination filed

Effective date: 19921203

AK Designated contracting states

Kind code of ref document: A1

Designated state(s): AT BE CH DE DK ES FR GB GR IT LI LU NL SE

STAA Information on the status of an ep patent application or granted ep patent

Free format text: STATUS: THE APPLICATION HAS BEEN WITHDRAWN

18W Application withdrawn

Withdrawal date: 19940204