EP1218414A2 - Polypeptides pour la detection et l'elimination de cellules reagissant de maniere positive a l'antigene ca19-9 - Google Patents

Polypeptides pour la detection et l'elimination de cellules reagissant de maniere positive a l'antigene ca19-9

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Publication number
EP1218414A2
EP1218414A2 EP00979484A EP00979484A EP1218414A2 EP 1218414 A2 EP1218414 A2 EP 1218414A2 EP 00979484 A EP00979484 A EP 00979484A EP 00979484 A EP00979484 A EP 00979484A EP 1218414 A2 EP1218414 A2 EP 1218414A2
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European Patent Office
Prior art keywords
antigen
polypeptide
seq
polypeptide according
antibody
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EP00979484A
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German (de)
English (en)
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Hinrich Abken
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Individual
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Individual
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Priority to EP00979484A priority Critical patent/EP1218414A2/fr
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    • 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
    • C07K16/30—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants from tumour cells
    • C07K16/303—Liver or Pancreas
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • 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
    • C07K16/30—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants from tumour cells
    • C07K16/3046—Stomach, Intestines
    • 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

Definitions

  • the invention relates to polypeptides which have binding specificity for the CA19-9 antigen, their production and use for the detection and elimination of CA19-9 antigen positive cells, i. H. for tumor diagnosis and therapy.
  • the invention further relates to radiolabeled derivatives of these polypeptides, their preparation and their use in radioimmunoassays, radio scintigraphy and radioimmunotherapy, and coupling products of these polypeptides with indicator substances or pharmacologically active substances.
  • Efforts to improve this situation are hampered by the lack of a reagent that can be used in vivo imaging techniques to detect and localize primary tumors and metastases.
  • a therapeutic approach is further complicated by the fact that a reagent for the specific see in vivo drug targeting of pancreatic and gastric carcinoma is not available.
  • the CA19-9 antigen is a monosialoganglioside which is detected in immunohistochemical analysis with the help of the antibody 1116-NS-19-9 (mouse IgG1) in numerous gastric and pancreatic carcinomas (Koprowski et al., Somatic Cell Genet. 5 , 957-971, 1979; Koprowski et al., U.S. Pat. 4,471,057; hybridoma clone ATCC HB-8059).
  • the multiple application of the anti-CA19-9 antibody for in vivo purposes is limited. Since the antibody 1116-NS-19-9 (ATCC HB-8059) is a mouse antibody, "HAMA” immune reactions ("human anti-mouse anti-bodies”) can be expected in repeated applications. Further attempts to make the antibody usable by modifications for diagnostic or therapeutic in vivo use have so far been unsuccessful.
  • the object of the invention was to provide reagents for the detection (in vivo targeting) of gastric and pancreatic carcinoma.
  • VH variable region of the heavy chain
  • (B) consisting of the variable region of the light chain (VL) of an anti-CA19-9 antibody or a fragment thereof with binding specificity for the CA19-9 antigen, wherein the oligopeptides (A) and (B) directly or through a bridge peptide (C) are linked together;
  • a radiolabeled polypeptide the polypeptide being as defined in (1)
  • a method for producing the radiolabeled polypeptide defined in (7) comprising reacting the polypeptide defined in (1) with the radioisotope;
  • FIG. 1 shows the DNA and the amino acid sequence (frame: one-letter code) of the CA19-9-binding polypeptide.
  • the VH and VL regions and the bridge peptide are labeled.
  • the integrated peptide (DNA bp 341-433) is a preferred bridge peptide and can be replaced by other bridge peptides.
  • a microtiter plate was coated with the monoclonal antibody NS19-9 (2 ⁇ g / ml) and then with lysates of the CA19-9 positive tumor cell line SW948 ( ⁇ ) or the CA19-9 negative tumor cell line H716 ( D) incubated. Dilutions of the SEQ ID NO: 2-scFv-M13 phage antibody were transferred to the antigen-coated plate and bound phage antibodies were measured photometrically at O.D. using a peroxidase-coupled anti-M13 antibody and indicator color reaction. 405 nm detected.
  • a microtiter plate was coated with anti-CA19-9 antibody NS19-9 (2 ⁇ g / ml NS19-9 mAb) and then incubated with 100 ⁇ l of supernatant from the CA19-9 secreting tumor cells H498.
  • the SEQ ID NO: 2-scFv-M13 phage antibody was incubated in the presence of various dilutions of the monoclonal anti-CA19-9 antibody NS19-9 ( ⁇ ) or an antibody of the same isotype (IgG) with irrelevant specificity (D) (maximum concentration) 10 ⁇ g / ml).
  • the photometric detection (OD 405 nm) of the bound phage antibody SEQ ID NO: 2-scFv-M13 was carried out using a peroxidase-coupled anti-M13 antibody and a color indicator reaction.
  • a microtiter plate was coated with the monoclonal antibody NS19-9 (2 ⁇ g / ml) and then with culture medium containing CA19-9 antigen from the CA19-9 positive tumor cell line H498 ( ⁇ ) or with the culture supernatant of CA19-9 negative tumor line HL60 (D) incubated.
  • the fusion protein SEQ ID NO: 2-scFv-CH2CH3 was added in dilutions to the antigen-coated plate, and the bound fusion protein was subsequently used with the aid of a biotin-coupled anti-human IgG (Fc) antibody which is directed against the CH2CH3 domain of the fusion protein , and a streptavidin-coupled peroxidase and color indicator reaction were detected photometrically (OD 405 nm).
  • Fc biotin-coupled anti-human IgG
  • OD 405 nm streptavidin-coupled peroxidase and color indicator reaction
  • the photometric detection (O.D. 405 nm) of the bound fusion protein SEQ ID NO: 2-scFv-CH2CH3 was carried out using a biotin-coupled anti-human IgG (Fc) antibody and a streptavidin-coupled peroxidase.
  • the data are mean values from two independent test approaches.
  • the fusion protein SEQ ID NO: 2-scFv-CH2CH3 was found on the indirectly immobilized CA19-9 antigen in the presence of dilutions of the culture medium of the CA19-9 positive tumor cell line H498 (•), which secreted large amounts of CA19-9 antigen, or the culture supernatant of the Incubated CA19-9 negative tumor line HL60 (o).
  • the photometric detection (OD 405 nm) of the bound fusion protein SEQ ID NO: 2-scFv-CH2CH3 was carried out using a biotin-coupled anti-human IgG (Fc) antibody and a streptavidin-coupled peroxidase. The results are averages from two independent experimental approaches. 5: FACS analysis of the binding of the fusion protein SEQ ID NO: 2-CH2CH3 on CA19-9 positive tumor cells
  • a microtiter plate was coated with the monoclonal antibody NS19-9 (2 ⁇ g / ml) and then with CA19-9 antigen-containing culture supernatant of the CA19-9 positive tumor cell line H498 ( «, s) or with the culture supernatant the CA19-9 negative tumor line H716 (D, ⁇ ).
  • the fusion protein SEQ ID NO: 2-CH2CH3-IL2 was incubated in the indicated dilutions on immobilized CA19-9 antigen.
  • Bound fusion protein was (a) photometrically detected using a biotin-coupled anti-human IgG (Fc) antibody that binds the CH2CH3 domain of the fusion protein, or (b) using a biotin-linked anti-human IL2 antibody and subsequent color indicator reaction ( OD 405 nm).
  • Fc biotin-coupled anti-human IgG
  • the two oligopeptides preferably originate from the same antibodies.
  • one or both of the oligopeptides be derived from the antibody 1116-NS-19-9.
  • the hybridoma cells that produce these antibodies can be obtained from the American Type Culture Collection, Rockville; MD, available under reference number ATCC HB-8059.
  • both the oligopeptide (A) and the oligopeptide (B) can be the N-terminal component of the polypeptide according to the invention.
  • the oligopeptides (A) and (B) are linked to one another via a bridge peptide (C) (hereinafter also referred to as "linker” or linker peptide ").
  • bridge peptide (C) All peptides which allow a folding and arrangement of the oligopeptides (A) and (B) with respect to one another can be used as the bridge peptide (C), so that efficient antigen binding is ensured.
  • Preferred bridge peptides consist of a short, flexible oligopeptide with 5 to 50, in particular with 20 to 50, amino acid residues.
  • Ser and Gly (SG) -rich bridge peptides which, for. B. store up to 10 amino acid residues.
  • These SG-rich bridge peptides are preferably modular and include SG-rich elements, e.g. T. separated by pro. The SG-rich elements are between 2 and 20 amino acids long. One SG element is sufficient to hold the above-mentioned. To perform functions.
  • a proline residue in the middle of the bridge peptide is used to create an angle in the polypeptide chain.
  • Other amino acids such as e.g. B. Gly, Ser, Asp and Asn can
  • bridge peptides preferably have the following structure
  • A, B, D, E are independently a bond, an amino acid residue or a polypeptide with up to 5 amino acid residues,
  • C is selected from Pro, Gly, Ser, Asp and Asn and in particular Pro is, n, m, o and p are independently integers from 0 to 10 and x and y are independently integers from 1 to 5.
  • a particularly preferred bridge peptide (C) is shown in SEQ ID NO: 3.
  • the polypeptide (1) according to the invention has the amino sequence of SEQ ID NO: 2, which consists of VH and VL of the 1116-NS-19-9 antibody in the arrangement VH linker peptide-VL.
  • scFv polypeptide chain in the arrangement VH-Iinker-VL or VL-Iinker-VH.
  • VH immunoglobulin heavy chain
  • VL cDNA for the of the light chain
  • This DNA can then be expressed in a suitable host organism / cell system.
  • Suitable systems in the sense of the present invention are bacterial expression systems, e.g. B. coli HB2151 using the vector pCANBAB 5E or eurkaryotic expression systems, e.g. B. 293T cells using the vector pRSV (with Ig kappa leader peptide).
  • polypeptide according to the invention shown here by way of example for the scFv molecule SEQ ID NO: 2, has retained the specificity of the parental antibody 1116-NS-19-9 for the CA19-9 antigen.
  • the molecule SEQ ID NO: 2 binds specifically to CA19-9 antigen (example 1), to CA19-9 antigen-bearing cells (example 2, FIG. 5), but not to other carbohydrate antigens and CA19-9 antigen tested. negative cells (example 1).
  • the molecule SEQ ID NO: 2 shows an unexpectedly good tissue penetration and accumulation on CA19-9 positive cells, preferably tumor cells in vivo. This is shown, for example, after intravenous application of the labeled molecule SEQ ID NO: 1 in mice which carry experimentally generated CA19-9 positive tumors (Example 6). However, there is no accumulation of healthy tissue.
  • the preferred use of the molecule according to the invention is the detection and localization of CA19-9 positive cells in vivo, in particular tumors, for example gastric and pancreatic carcinoma, in imaging diagnostics.
  • the molecule SEQ ID NO: 2 is not intemalized after binding to the antigen-carrying cell. This property is all the more surprising since the bound ligands are internalized in the vast majority of the receptor-ligand systems. This applies all the more to antibodies that bind to a molecule in the outer cell membrane.
  • the lack of internalization of the molecule SEQ ID NO: 2 also ensures a long residence time on the cell surface without degradation due to cytoplasmic proteases. This property proves to be particularly advantageous when using the SEQ ID NO: 2 molecule for drug targeting, pro-drug activation, radioactive / ⁇ -Vo labeling and other methods in which the molecule is used for targeting.
  • the molecule SEQ ID NO: 2 is free of constant proportions of the mouse immunoglobulin, so that no pronounced HAMA immune reaction is to be expected when the molecule is used repeatedly. This property, unlike the previously existing anti-CA19-9 antibodies, the use of the SEQ ID NO: 2 molecule in / ti-wVo methods in diagnostics and therapy.
  • NK cells natural killer cells
  • a preferred use of the molecule SEQ ID NO: 2 after coupling with Ig CH2CH3, CH2 or CH3 domains is the elimination of CA19-9 positive tumor cells in vivo and in vitro.
  • the preferred object of the invention is the covalent or non-covalent coupling of the anti-CA19-9 scFv molecule to a further large number of molecules in accordance with the desired use.
  • the following modifications of the polypeptide according to the invention and their uses should only be mentioned here by way of example.
  • the polypeptide according to the invention accumulates, e.g. B. the molecule SEQ ID NO: 2 at the location of the CA19-9 positive tumor and its metastases.
  • the tumors can be localized in vivo using scintigraphic methods.
  • the z. B. with a radioisotope labeled polypeptide administered to the patient and the binding to the CA19-9 carrying tissue is detected from outside the body.
  • Other indirect or direct methods for immunoscintigraphy can also be used for the detection of CA19-9 positive tumors in vivo, the molecules according to the invention, preferably SEQ ID NO: 2, mediating the specific binding to the tumor cells.
  • polypeptide according to the invention is the radiotherapeutic elimination of CA19-9 positive cells after application in vivo.
  • Direct or indirect radioactive labels are possible. Examples include the direct labeling of the molecule SEQ ID NO: 2 with 188Re or the indirect labeling by sequential or simultaneous application of a tag-carrying fusion molecule SEQ ID NO: 2 -tag and a radioactively labeled anti-tag molecule.
  • tag is understood to mean a protein domain (short peptide sequence) which detects the detection of the protein, eg. B. with the help of antibodies and / or the purification of the protein z. B. with the help of Ni bond.
  • the preferred therapeutic use of the 188Re-labeled molecule SEQ ID NO: 2 is to achieve tumor reduction in vivo to eliminate metastases or to create the prerequisites for successful operative therapy.
  • a preferred accumulation of the 188Re-labeled molecule SEQ ID NO: 2 on the tumor results in an accumulation of the radiation on the tumor while at the same time reducing the haematopoietic toxicity, while when using other carrier molecules or using external radiation sources, there is often a pronounced toxicity for cells of the hematopoietic system (Iznaga-Escobar, Nucl. Med. Biol. 25, 441-447, 1998).
  • polypeptides according to the invention preferably of the molecule SEQ ID NO: 2
  • modified molecules with "designer" effector functions are produced, for example by conjugations to other polypeptides, by known processes (review by Neri et al., Engineering recombinant antibodies for immunotherapy, Cell. Biophys. 27: 47-61, 1995)
  • Sugar molecules, lipids or other synthetic or natural substances, chelators, or also the coupling with pharmacologically active substances for example Molecules with cytolytic activity (e.g. perforin) or pro-drug substances, proteins with signal functions (e.g.
  • cytokines examples 3
  • proteins with enzymatic activity e.g. metalloproteinase, endopeptidases.
  • Coupling with nucleic acids is also possible.
  • the polypeptide according to the invention in particular SEQ ID NO: 2, to achieve the accumulation of a desired substance at the site of CA19-9 expression, preferably at the site of the tumor, in vivo.
  • a further use is the coupling of the polypeptide according to the invention, in particular the molecule SEQ ID NO: 2, to binding domains of other molecules or Antibodies for the generation of bi- and multispecific hybrid molecules, eg bispecific antibodies.
  • a recombinant anti-CA19-9 / anti-CD3 (OKT3) bispecific scFv antibody may be mentioned for use in the immunotherapy of CA19-9 positive tumors.
  • the polypeptide according to the invention in particular the molecule SEQ ID NO: 2, can be fused with an intracellular signal chain and expressed in suitable host cells as a signal-transducing receptor molecule.
  • SEQ ID NO: 2 a binding of the CA19-9 antigen to SEQ ID NO: 2 triggered a biochemical signal on the fused heterologous signal peptide chain (Example 5).
  • Numerous signal chain combinations are available to the person skilled in the art, which cannot be mentioned comprehensively here.
  • polypeptides according to the invention are suitable for the localization and / or elimination of cells carrying CA 19-9 antigen.
  • the invention thus also relates to a method for activating cells carrying CA 19-9, comprising the administration of the (optionally derivatized or modified) polypeptide (1) to humans or animals.
  • Example 1 The single chain molecule SEQ ID NO: 2 has binding specificity for the CA19-9 antigen and binds to the CA19-9 antigen from CA19-9 positive tumor cells.
  • the molecule SEQ ID NO: 1 was generated using the phage display technique (McCafferty et al., Nature 348: 552-554 (1990); Winter and Milstein, Nature 349: 293-299 (1991)) from the hybridoma 1116-NS-19-9 (ATCC HB- 8059), which expresses the 1116-NS-19-9 antibody specific for the CA19-9 antigen (Koprowski et al., Somatic Cell Genet. 5, 957-971, 1979).
  • PolyA + mRNA was obtained from the hybridoma cells by known methods (Sambrook et al., Molecular Clonig, 2nd Edition, Cold Spring Harbor Laboratory Poress, Cold Spring Harbor, NY, pp. 8.11 - 8.13, 1989) and with the aid of the reverse transcriptase and the "primed first-strand reaction mix" oligonucleotides (Pharmacia Biotech, Freiburg, Cat. No. 27-9400-01) were rewritten in cDNA.
  • the cDNA coding for the variable region of the heavy (VH) and light (V ⁇ _) immunoglobulin chain was determined with the help of immunoglobulin-specific oligonucleotides ("heavy primer 1", "heavy primer 2", "light primer mix", Pharmacia, Cat No.
  • scFv fragment is expressed in this phagemid as a fusion protein with the phage protein gp3 on the phage surface.
  • TG1 E. coli bacteria were transformed with the recombinant phagemid DNA and then infected with M13K07 helper phages.
  • the phages that can bind the CA19-9 antigen were selectively enriched.
  • the Recombinant scFv molecule (NS19-9-scFv) binding CA19-9 antigen has the DNA sequence shown in Fig. 1 and SEQ ID NO: 1 and the amino acid sequence shown in SEQ ID NO: 2.
  • the phage antibody SEQ ID NO: 2-scFv-M13 is expressed on the surface of the phage as a fusion protein scFv-gp3 and specifically binds the CA19-9 antigen from CA19-9 positive tumor cells (FIG. 2).
  • the gp3 protein of the M13 phage has no binding specificity.
  • the binding of the SEQ ID NO: 2-scFv fragment is specific for the CA19-9 antigen, since the binding of the SEQ ID NO: 2-scFv to CA19-9 antigen by the monoclonal antibody NS19-9 (FIG. 3a) and by soluble CA19-9 antigen (Fig. 3b) is competed.
  • Example 2 The polypeptide SEQ ID NO: 2 retains the bundling specificity for the CA19-9 antigen even after fusion to the human immunoglobulin IgG1 CH 2 CH 3 domain.
  • the CA19-9 binding scFv molecule SEQ ID NO: 2 was linked to a second, independent protein domain while maintaining the binding specificity for the CA19-9 antigen to form a fusion protein.
  • the DNA SEQ ID NO: 1, which codes for the anti-CA19-9 scFv was ligated with the cDNA which codes for the human IgG1 CH 2 CH 3 domain.
  • the resulting cDNA, which codes for the scFv-CH 2 CH 3 fusion protein is inserted into the pRSV expression vector and, after transfection, is expressed in Ag8.653 cells.
  • the pRSV expression vector (Eshhar et al., Proc. Natl. Acad. Sci.
  • the fusion protein SEQ ID NO: 2-scFv-CH 2 CH 3 is isolated from the serum-free culture supernatant of transfected Ag8.653 cells with the aid of affinity chromatography by binding to agarose coupled to anti-human Fc antibodies.
  • the SEQ ID NO: 2 scFv domain has retained the binding specificity for the CA19-9 antigen in the fusion molecule.
  • the preserved bond specificity was determined by binding the SEQ ID NO: 2-scFv-CH 2 CH 3 fusion protein to CA19-9 antigen (FIG. 4a) and by competition for the binding by the monoclonal antibody NS19-9 (FIG. 4b) and soluble CA19- 9 antigen (Fig. 4c) detected.
  • the fusion molecule SEQ ID NO: 2-scFv CH 2 CH 3 binds specifically to CA19-9 positive tumor cells, but not to CA19-9 negative tumor cells (FIG. 5).
  • CA19-9 binding domain SEQ ID NO: 2 on the one hand has the same binding specificity for the CA19-9 antigen as the monoclonal NS19-9 antibody, but on the other hand maintains the binding specificity even if the SEQ ID NO: 2- scFv domain is part of a fusion protein with other functional domains.
  • Example 3 The fusion of the molecule SEQ ID NO: 2-CH 2 CH 3 to human interleukin-2 (IL-2) generates a bifunctional protein with binding specificity for the CA19-9 antigen.
  • IL-2 human interleukin-2
  • a bifunctional fusion protein was generated by fusion of the molecule SEQ ID NO: 2-CH 2 CH 3 (from Example 2) with interleukin-2 (IL-2), which expresses binding specificity for the CA19-9 antigen and at the same time the IL-2 protein wearing.
  • IL-2 interleukin-2
  • the DNA SEQ ID NO: 1-CH 2 CH 3 (from Example 2) was fused with the cDNA which codes for human IL-2 and inserted into the expression cassette of the expression vector pRSV.
  • the pRSV expression vector (Eshhar et al., Proc. Natl. Acad.
  • Sei USA 90: 720-714 (1993) is characterized by an RSV-LTR-controlled expression cassette with an immunoglobulin kappa light chain leader peptide, so that the ex - Primed fusion protein is secreted into the culture supernatant.
  • the fusion protein SEQ ID NO: 2-CH 2 CH 3 -IL-2 was extracted from the culture supernatant by affinity chromatography by binding to an anti-human Fc antibody which is CH 2 CH 3 - Domain binds, cleaned.
  • the fusion protein SEQ ID NO: 2-CH 2 CH 3 -IL-2 is characterized by a pharmacologically active cytokine (IL-2), fused with the CA19-9 binding domain SEQ ID NO: 2, the binding specificity of the scFv for the CA19-9 antigen too has been preserved in this configuration of the fusion protein.
  • the binding of the fusion protein SEQ ID NO: 2-CH 2 CH 3 -IL-2 to CA19-9 is documented in Fig. 6a, b.
  • Example 4 The molecule SEQ ID NO: 2 preserves the binding specificity for the CA19-9 antigen after denaturation and renaturation.
  • the soluble molecule SEQ ID NO: 2 (without phage fraction) is produced after transfection of the SEQ ID NO: 1 DNA in pCANTAB 5E vector in E. coli HB2151 bacteria and induction with IPTG and accumulates in the periplasm of the bacteria.
  • the SEQ ID NO: 2 protein is isolated from the periplasm of the bacteria by incubating the bacteria in 1/5 x (v / v) TES buffer (1 x TES: 200 mM Tris-HCl, 0.5 mM EDTA, 500 mM sucrose, pH 8.0) for 30 min at 4 ° C and ammonium sulfate precipitation of the bacterial supernatant. The precipitate is renatured in PBS.
  • the renatured SEQ ID NO: 2 molecule was compared with the native SEQ ID NO: 2-gp3 phage molecule in terms of the specificity of binding to CA19-9 antigen. It was found that the SEQ ID NO: 2 molecule after denaturation and renaturation in the soluble form has the same binding specificity to the CA19-9 antigen as the SEQ ID NO: 2-gp3 phage body, the isolation of which does not require any denaturation and renaturation.
  • Example 5 The molecule SEQ ID NO: 2 as a domain is able to activate the fused signal domain through antigen binding.
  • a recombinant transmembrane molecule was generated whose extracellular domain is formed by the CA19-9 binding molecule SEQ ID NO: 2 and whose transmembrane and intracellular domain is formed by the ⁇ chain of the Fc ⁇ RI receptor.
  • This fusion molecule is intended for the specific cellular activation of immunocompetent cells (e.g. T cells) after contact with cells carrying CA19-9 antigen (e.g. tumor cells).
  • the DNA coding for SEQ ID NO: 2-CH 2 CH 3 was inserted into the Snabl and BamHI site of the vector pRSV- ⁇ (Eshhar et al. Proc. Natl. Acad. Sei.
  • Receptor-bearing MD45 cells were tested for their ability to be specifically activated after contact with CA19-9 positive tumor cells.
  • receptor-bearing MD45 cells and, as a control, non-transfected MD45 cells (10 5 cells each) were co-cultivated in parallel batches with CA19-9 + H498 tumor cells and with CA19-9-H716 tumor cells (10 5 cells each). After 48 hours, the amount of IL-2 secreted in the culture supernatant was determined as a measure of the cellular activation. It was found that MD45 cells carrying SEQ ID NO: 2-CH 2 CH 3 - ⁇ receptor are activated by coculture with CA19-9-positive H498 tumor cells, but not by CA19-9-negative H716 tumor cells.
  • Non-transfected MD45 cells are not activated by either H498 or H716 cells. Furthermore, this result shows that the CA19-9 binding extracellular domain SEQ ID NO: 2, after binding of the cell-bound CA19-9 antigen, transmits a signal to the fused intracellular ⁇ signal chain, which in turn initiates cellular activation.
  • Example 6 The molecule SEQ ID NO: 2 accumulates in vivo on cells with CA19-9 expression.
  • the soluble protein SEQ UD NO: 2 (from Example 1) was radioactively labeled with 125 J using the Bolton-Hunter reagent and purified from unbound 125 J.
  • the binding specificity of the 125 J-SEQ ID NO: 2 molecule in vitro is the same as that of the unlabelled SEQ ID NO: 2 molecule.
  • CA19-9 positive H498 cells and, as a control, CA19-9-H716 cells were injected subcutaneously into nude mice (nu / nu) to generate tumors (approx. 10 6 cells per injection and animal). After 5 - 8 days, a subcutaneous tumor with a diameter of 2 - 4 mm developed.
  • 125 J-SEQ ID NO: 2 was administered intravenously into tumor-bearing mice (5 ⁇ g protein / mouse). After 24 hours, the distribution of radioactivity in the mouse was determined by whole body autoradiography. The radioactivity was found to be accumulated on the H498 cell tumor (CA19-9 +), but not on the H716 cell tumor (CA19-9-). The organs of the mice show low radioactivity in the same distribution in the H498 tumor as in the H716 tumor-bearing mouse.

Abstract

L'invention concerne des polypeptides qui présentent une spécificité de liaison à l'antigène CA19-9, leur production et leur utilisation pour détecter et éliminer des cellules réagissant de manière positive à l'antigène CA19-9, c'est-à-dire pour le diagnostic et la thérapie de tumeurs. L'invention concerne en outre des dérivés radiomarqués de ces polypeptides, leur production et leur utilisation dans des dosages radioimmunologiques, en radioscintigraphie et radioimmunothérapie, ainsi que des produits de couplage de ces polypeptides avec des substances indicatrices ou des substances pharmacologiquement actives.
EP00979484A 1999-10-08 2000-10-05 Polypeptides pour la detection et l'elimination de cellules reagissant de maniere positive a l'antigene ca19-9 Withdrawn EP1218414A2 (fr)

Priority Applications (1)

Application Number Priority Date Filing Date Title
EP00979484A EP1218414A2 (fr) 1999-10-08 2000-10-05 Polypeptides pour la detection et l'elimination de cellules reagissant de maniere positive a l'antigene ca19-9

Applications Claiming Priority (4)

Application Number Priority Date Filing Date Title
EP99120119A EP1090927A1 (fr) 1999-10-08 1999-10-08 Polypeptide (scFv) pour la détection et l'élimination de cellules porteuses de l'antigène CA19-9
EP99120119 1999-10-08
EP00979484A EP1218414A2 (fr) 1999-10-08 2000-10-05 Polypeptides pour la detection et l'elimination de cellules reagissant de maniere positive a l'antigene ca19-9
PCT/EP2000/009714 WO2001027159A2 (fr) 1999-10-08 2000-10-05 Polypeptides pour la detection et l'elimination de cellules reagissant de maniere positive a l'antigene ca19-9

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EP00979484A Withdrawn EP1218414A2 (fr) 1999-10-08 2000-10-05 Polypeptides pour la detection et l'elimination de cellules reagissant de maniere positive a l'antigene ca19-9

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US6949629B2 (en) 2002-03-13 2005-09-27 Aspenbio, Inc. Methods for purifying selected CEA family member proteins
CA2650380A1 (fr) * 2006-04-28 2007-11-08 Oregon Health & Science University Anticorps monoclonaux et leur utilisation
EP4065606A4 (fr) * 2019-11-26 2023-08-02 Ramot at Tel-Aviv University Ltd. Récepteur antigénique chimérique pour antigènes glucidiques
EP4065605A4 (fr) * 2019-11-26 2023-07-12 Ramot at Tel-Aviv University Ltd. Anticorps dirigés contre des antigènes glucidiques
WO2022113066A1 (fr) * 2020-11-24 2022-06-02 Ramot At Tel-Aviv University Ltd. Anticorps humanisés et fragments de ceux-ci se liant à des antigènes glucidiques et leurs utilisations
CN114605550B (zh) * 2020-12-08 2023-09-22 东莞市朋志生物科技有限公司 抗ca19-9的抗体、其应用和检测ca19-9的试剂盒
CN114573700B (zh) * 2022-02-18 2023-05-26 河北渤腾医药技术有限公司 糖类抗原ca19-9的检测试剂盒

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WO2001027159A3 (fr) 2001-10-25
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JP2004507205A (ja) 2004-03-11

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