WO2001080904A2 - Moyen permettant de diagnostiquer et de traiter des carcinomes - Google Patents

Moyen permettant de diagnostiquer et de traiter des carcinomes Download PDF

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Publication number
WO2001080904A2
WO2001080904A2 PCT/DE2001/001530 DE0101530W WO0180904A2 WO 2001080904 A2 WO2001080904 A2 WO 2001080904A2 DE 0101530 W DE0101530 W DE 0101530W WO 0180904 A2 WO0180904 A2 WO 0180904A2
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WO
WIPO (PCT)
Prior art keywords
gamma
binding
composition according
glutamyl transferase
fischer
Prior art date
Application number
PCT/DE2001/001530
Other languages
German (de)
English (en)
Other versions
WO2001080904A3 (fr
Inventor
Stefan Dübel
Andreas Schmiedl
Original Assignee
Fischer, Peter
SCHERBERICH, Jürgen, E.
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 Fischer, Peter, SCHERBERICH, Jürgen, E. filed Critical Fischer, Peter
Priority to AU2001265749A priority Critical patent/AU2001265749A1/en
Publication of WO2001080904A2 publication Critical patent/WO2001080904A2/fr
Publication of WO2001080904A3 publication Critical patent/WO2001080904A3/fr

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Classifications

    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/40—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against enzymes
    • 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
    • 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
    • G01N33/57407—Specifically defined cancers
    • G01N33/57438—Specifically defined cancers of liver, pancreas or kidney
    • 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
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/60—Immunoglobulins specific features characterized by non-natural combinations of immunoglobulin fragments
    • C07K2317/62—Immunoglobulins specific features characterized by non-natural combinations of immunoglobulin fragments comprising only variable region components
    • C07K2317/622—Single chain antibody (scFv)
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2333/00—Assays involving biological materials from specific organisms or of a specific nature
    • G01N2333/90—Enzymes; Proenzymes
    • G01N2333/91—Transferases (2.)
    • G01N2333/91045—Acyltransferases (2.3)
    • G01N2333/91074—Aminoacyltransferases (general) (2.3.2)
    • G01N2333/9108—Aminoacyltransferases (general) (2.3.2) with definite EC number (2.3.2.-)

Definitions

  • gamma-glutamyltransferase gamma-glutamyltranspeptidase, GGT, EC 2.3.2.2.
  • the monoclonal antibody (138H11) against human, renal gamma-glutamyl transferase (GGT, EC 2.3.2.2)) was produced by Fischer & Scherberich [3,4].
  • the mAb 138H11 reacts specifically with GGT human tissue on a large number of frozen sections.
  • the mAb 138H11 thus enabled a differential diagnostic classification (according to Störkel and Thoenes) of epithelial kidney tumors in two groups: one over 98% positive (clear cell and chromophilic kidney Ca) and one 100% negative (oncocytomas, chromophobic and duct Bellini Ca) [2,3,5,6].
  • the mAb 138H11 also reacted with proximal tubular cells of the kidney.
  • the polarization of the GGT distribution was striking: almost exclusively the luminal (apical) part of the cells (brush border) contained immunoreactive GGT.
  • An extremely weak but specific marking in the basal cell pole was only visible in individual kidney sections.
  • the tumor cells of the clear cell renal carcinomas no longer showed polarization; the entire cell membrane and z. T. also the cytoplasm reacted with mAb 138H11 [5].
  • mAb 138H11 only reacted with the GGT in the biliary tubules. A positive reaction was also found in gliomas [7], hepatocellular carcinomas and in gastric carcinomas as well as in RCC liver metastases [2,3].
  • the monoclonal antibody 138H11 therefore recognizes the target antigen not because of increased antigen expression in the tumor, but because of the changed target antigen localization on tumor cells [8] (FIG. 1) [2,10].
  • Calicheamicin- ⁇ is a biological highly active and selective Enediyne molecule, which has been designed in such a way that, in contrast to natural calicheamicin, it is biochemically stable and is therefore very suitable for coupling reactions with mAbs.
  • Mouse IgGl antibodies such as 138H11 are particularly problematic because they do not indicate complement activation, an important component of tumor cell destruction.
  • the therapeutic efficiency of mouse antibodies can be significantly increased by chemically coupling toxins, cytostatics or radionuclides as in the known chemoimmunoconjugate with calicheamicin theta, the disadvantages described above remain. The stability and quality control of such chemical couplings is also very problematic.
  • An essential part of the invention is the use of a recombinantly produced ligand, in particular a recombinant antibody or antibody fragment.
  • a particularly preferred recombinant antibody is shown in FIG. 2.
  • the recombinant therapeutic agent against gamma-glutamyltransferase produced according to the invention therefore enables in particular:
  • the invention enables the construction of the constructs exemplified in the examples, such as chimeric antibodies, fusion proteins, bispecific antibodies, immunocytokines etc., from the DNA of the antigen binding site of the mAb 138H11.
  • Whose DNA or amino acid sequence also enables direct humanization of the GGT binding partner z. B. by CDR crafting or the like.
  • Recombinant fragments can also bring significant advantages such as faster blood clearing and better tumor penetration for radioimmuno diagnostics and therapy, since their size can be varied depending on the application, e.g. for rapid excretion by the kidney, which is not possible with complete IgG molecules.
  • the agent can be used to treat tumors (especially renal cell carcinoma) by using the recombinant antibody fragment as a fusion protein, e.g. B. with Interleukin 2 (IL2) is produced (Fig. 3).
  • IL2 Interleukin 2
  • Systemic administration of IL2 is one of the few treatments for renal cell carcinoma that have been shown to have an impact on the tumor.
  • the use of the fusion protein instead of native IL2 increases the local concentration of IL2 on the tumor and therefore leads locally to an increase in the immunostimulating effect compared to the systemic administration of IL2. This can significantly reduce systemic toxicity.
  • the two gene fragments for the variable regions (Vh and VI) of the antibody against GGT are obtained via a polymerase chain reaction with oligonucleotide primers which bind specifically to the antigen-binding (V-) regions of antibodies.
  • the amplified genes of the two V regions are provided with the interfaces for further cloning by this PCR and then joined together in the form of an scFv antibody fragment, ie their open reading frames are linked without a stop codon by a DNA fragment which is a peptide encoded by 15-18 amino acid residues.
  • the corresponding scFv gene fragment is then also linked to the gene for IL2 in the same reading frame.
  • a stop codon is encoded, followed directly by the recognition sequence for Xbal.
  • This gene cassette is cloned into the bacterial expression vector pOPElOl via the restriction sites Ncol and Xbal. After transfection of the construct produced in this way in E. coli, the promoter of the pOPElOl vector is switched on by administration of IPTG for three hours, during which the cells are to be shaken at 28 ° C.
  • the scFv-IL2 fusion protein accumulates in the periplasm of the E. co / z ' cells during this time.
  • the cells are harvested by centrifugation at 5000 xg for 10 minutes and transferred to a buffer which enables the outer membrane of the E. coli to dissolve (50 mM Tris (hydroxymethyl) aminomethane / ⁇ Cl pH 8.0 with 20% w / v) sucrose and 1 mM EDTA), and incubated for 20 min at 0 ° C with shaking.
  • the supernatant obtained after centrifugation at 30,000 xg for 30 min contains approx. 50-1000 ⁇ g / ml of the scFv-IL2 fusion proteins, which are further purified, characterized and prepared for therapeutic use via a sequence of affinity chromatography and ion exchange chromatography according to the prior art can.
  • Example 2 Example 2:
  • the agent can be used for the therapy of tumors (in particular kidney cell carcinomas) by coupling the recombinant antibody fragment with an enzyme which converts a systemically applied, less toxic precursor substance into a tumor-damaging substance ("ADEPT").
  • ADPT an enzyme which converts a systemically applied, less toxic precursor substance into a tumor-damaging substance
  • CPG2 gamma-glutamyl transferase and carboxypeptidase G2
  • CPG2 carboxypeptidase G2
  • the patient is given time to excrete excess molecular complexes before the precursor substance (the "prodrug", in the case of CPG2 these are benzoic acid mustard-derived prodrugs) is given.
  • the precursor substance the "prodrug", in the case of CPG2 these are benzoic acid mustard-derived prodrugs
  • the agent can be used to treat tumors (especially renal cell carcinomas) by presenting the recombinant antibody fragment on the surface of T lymphocytes. This can be done by genetic fusion to the zeta chain of the T cell receptor of the T lymphocyte (FIG. 5), or by loading modified T lymphocytes with recombinant antibody fragments via specially introduced surface molecules of the T lymphocytes.
  • the latter surface molecules can be anchor molecules expressed by the T-lymphocytes, in particular anti-peptide "tag” or anti-hapten antibodies, the recombinant antibody fragment against gamma-glutamyl transferase with the corresponding peptide "tag” or the corresponding one Hapten was modified (Fig. 6).
  • T lymphocytes by treatment with agents which anchor the recombinant antibody fragment against gamma-glutamyl transferase in the surface of the T lymphocytes.
  • agents which anchor the recombinant antibody fragment against gamma-glutamyl transferase in the surface of the T lymphocytes This is possible by producing fusion proteins from the recombinant antibody fragment against gamma-glutamyl transferase with a surface protein of the Newcastle Disease Virus (in particular hemagglutinin). These viruses fuse with the membrane of the target cell and integrate their hemagluttinin into the target cell membrane, and thus also the recombinant antibody fragment against gamma-glutamyl transferase.
  • This procedure has the advantage that no transformation of the T lymphocytes is necessary.
  • bispecific antibodies Another possibility of anchoring the recombinant antibody fragments against gamma-glutamyl transferase in the surface of T-lymphocytes is the production of bispecific antibodies (FIG. 7). With one binding arm they bind gamma glutamyl transferase, with the other an epitope on the T lymphocytes. The latter binding to the T lymphocytes can be mediated either by an antibody fragment or by a natural ligand of the epitope. This epitope in turn on the T- Lymphocytes can either be contained in a natural surface molecule of the T lymphocytes (in particular CD4, CD5, TCR, CD28), or it can have got onto the surface of the T lymphocytes by fusion of virus particles, in particular NDV viruses.
  • Several bispecific constructs can be used together to generate the primary and secondary activation signal for the T lymphocyte. The use of bispecific antibodies also has the advantage that no transformation of the T lymphocytes is necessary.
  • the recombinant antibody is integrated into the liposomes in a known, chemical way or incorporated into the genome of the viruses (e.g. retroviruses, adenoviruses) from a molecular biological point of view.
  • the coding sequences of the antibody are integrated into a suitable expression plasmid.
  • Acidification with anti-idiotypic antibodies or their antigenic components also as DNA or RNA (see Example 4), which are obtained against GGT binding partners in vitro, in vivo, from antibody libraries, transgenic animals etc. or when applied to humans. This leads to an activation of the tumor patient's immune system, which is directed against the tumor and can thereby control or destroy it.
  • GGT binding partners for therapy (e.g. purging for bone marrow or stem cell transplantation, for in vttro gene transfer) or diagnostics (detection of "minimal residual disease” (MRD), relapse etc.)
  • the binding partner of the GGT can be coupled, for example, to magnetic beads, with which a tumor can be used to enrich individual tumor cells from a large excess of irrelevant cells.

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  • Health & Medical Sciences (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Immunology (AREA)
  • Chemical & Material Sciences (AREA)
  • Engineering & Computer Science (AREA)
  • Molecular Biology (AREA)
  • Urology & Nephrology (AREA)
  • Biomedical Technology (AREA)
  • Hematology (AREA)
  • General Health & Medical Sciences (AREA)
  • Biochemistry (AREA)
  • Medicinal Chemistry (AREA)
  • Physics & Mathematics (AREA)
  • General Physics & Mathematics (AREA)
  • Organic Chemistry (AREA)
  • Food Science & Technology (AREA)
  • Cell Biology (AREA)
  • Oncology (AREA)
  • Analytical Chemistry (AREA)
  • Biotechnology (AREA)
  • Hospice & Palliative Care (AREA)
  • Microbiology (AREA)
  • Pathology (AREA)
  • Biophysics (AREA)
  • Genetics & Genomics (AREA)
  • Proteomics, Peptides & Aminoacids (AREA)
  • Gastroenterology & Hepatology (AREA)
  • Peptides Or Proteins (AREA)
  • Medicines Containing Antibodies Or Antigens For Use As Internal Diagnostic Agents (AREA)
  • Preparation Of Compounds By Using Micro-Organisms (AREA)
  • Medicines That Contain Protein Lipid Enzymes And Other Medicines (AREA)

Abstract

L'invention concerne un moyen permettant de diagnostiquer et de traiter des carcinomes, notamment des carcinomes de cellules rénales, sur la base de substances recombinantes qui sont des partenaires de liaison de la gamma-Glutamyltransférase (=Gamma-Glutamyltranspeptidase, GGT, EC 2.3.2.2.). Ce moyen a, comme domaines d'application, la médecine et l'industrie pharmaceutique. L'invention vise à développer des procédés fiables de diagnostic et de traitement de carcinomes de cellules rénales à partir de procédés actuels et à l'aide d'anticorps natifs comme mAK 138H11. L'invention concerne principalement l'utilisation d'un ligand, obtenu par recombinaison, contre la gamma-glutamyltransférase, notamment d'un anticorps ou fragment d'anticorps recombinant. La figure 2 présent un anticorps recombinant particulièrement préféré.
PCT/DE2001/001530 2000-04-22 2001-04-23 Moyen permettant de diagnostiquer et de traiter des carcinomes WO2001080904A2 (fr)

Priority Applications (1)

Application Number Priority Date Filing Date Title
AU2001265749A AU2001265749A1 (en) 2000-04-22 2001-04-23 Means for diagnosing and treating carcinomas

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
DE10020034.6 2000-04-22
DE2000120034 DE10020034A1 (de) 2000-04-22 2000-04-22 Mittel zur Diagnose und Therapie von Karzinomen

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WO2001080904A2 true WO2001080904A2 (fr) 2001-11-01
WO2001080904A3 WO2001080904A3 (fr) 2002-04-04

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AU (1) AU2001265749A1 (fr)
DE (1) DE10020034A1 (fr)
WO (1) WO2001080904A2 (fr)

Citations (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US4511651A (en) * 1982-07-30 1985-04-16 American Monitor Corporation Reagent composition and assay for the determination of γ-glutamyltransferase activity
EP0207406A2 (fr) * 1985-06-20 1987-01-07 Fuji Photo Film Co., Ltd. Feuille de réactifs et élément analytique intégrant à plusieurs couches pour la mesure de l'activité de la gamma-glutamyle transferase
WO1998017804A2 (fr) * 1996-10-17 1998-04-30 Institut Pasteur ANTIGENE D'HELICOBACTER (η-GLUTAMYLTRANSFERASE) ET SEQUENCES CODANT CE DERNIER
US5854006A (en) * 1997-03-28 1998-12-29 The University Of Virginia Gamma-glutamyl transpeptidase-specific antibody, prodrugs for the treatment of gamma-glutamyl transpeptidase-expressing tumors, and methods of administration thereof

Family Cites Families (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
DE19720152A1 (de) * 1997-05-02 1998-11-05 Max Delbrueck Centrum Retrovirales Vektorsystem zur Optimierung des Gentransfers und der Genexpression in primären humanen T-Lymphozyten

Patent Citations (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US4511651A (en) * 1982-07-30 1985-04-16 American Monitor Corporation Reagent composition and assay for the determination of γ-glutamyltransferase activity
EP0207406A2 (fr) * 1985-06-20 1987-01-07 Fuji Photo Film Co., Ltd. Feuille de réactifs et élément analytique intégrant à plusieurs couches pour la mesure de l'activité de la gamma-glutamyle transferase
WO1998017804A2 (fr) * 1996-10-17 1998-04-30 Institut Pasteur ANTIGENE D'HELICOBACTER (η-GLUTAMYLTRANSFERASE) ET SEQUENCES CODANT CE DERNIER
US5854006A (en) * 1997-03-28 1998-12-29 The University Of Virginia Gamma-glutamyl transpeptidase-specific antibody, prodrugs for the treatment of gamma-glutamyl transpeptidase-expressing tumors, and methods of administration thereof

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WO2001080904A3 (fr) 2002-04-04
DE10020034A1 (de) 2001-10-31
AU2001265749A1 (en) 2001-11-07

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