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WO2001081423A1 - Anticorps contre gp96 naissante, fabrication et utilisation de ces anticorps - Google Patents

Anticorps contre gp96 naissante, fabrication et utilisation de ces anticorps Download PDF

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Publication number
WO2001081423A1
WO2001081423A1 PCT/EP2001/004382 EP0104382W WO0181423A1 WO 2001081423 A1 WO2001081423 A1 WO 2001081423A1 EP 0104382 W EP0104382 W EP 0104382W WO 0181423 A1 WO0181423 A1 WO 0181423A1
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Prior art keywords
seq
antibodies
sequence
native
recombinant
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German (de)
English (en)
Inventor
Peter Terness
Christian Kleist
Gerhard Opelz
Martin Welschof
Danièle ARNOLD-SCHILD
Hansjörg Schild
Hans-Georg Rammensee
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DR FENNING BIOMED GmbH
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DR FENNING BIOMED GmbH
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Priority to AU2001273950A priority Critical patent/AU2001273950A1/en
Publication of WO2001081423A1 publication Critical patent/WO2001081423A1/fr
Anticipated expiration legal-status Critical
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    • 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
    • A61K39/385—Haptens or antigens, bound to carriers
    • 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
    • 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
    • A61K2039/6043—Heat shock proteins
    • 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
    • A61K2039/622—Medicinal preparations containing antigens or antibodies characterised by the link between antigen and carrier non-covalent binding
    • 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)

Definitions

  • Antibodies against native gp96 their production and use
  • the invention relates to recombinant antibodies against native gp96, processes for their preparation, their use, separating materials, devices and kits comprising the said antibodies, peptides bound to native gp96 and their isolation, nucleic acid sequences which code for the said recombinant antibodies and host cells which produce them contain the named DNA and enable the production of corresponding antibodies, as well as further aspects described below.
  • gp96 which belongs to the group of heat shock proteins, is a protein which is able to associate with peptides in the endoplasmic reticulum (ER) and thus to form an immunogenic context with them . If such gp96 / peptide complexes are injected into mice, strong immune responses can be induced. These immune responses are specific to the associated peptides, as evidenced by induction of specific responses by cytotoxic T lymphocytes.
  • the high effectiveness of gp96 in particular can possibly be explained by the fact that antigen-presenting cells (e.g. macrophages, dendritic cells) carry receptors that are specific for heat shock proteins and enable them to be absorbed efficiently. Based on these applications, heat shock proteins can be used for cancer immunotherapy in mice (see Tamura et al., Science 271, 117-120 (1994)). They also appear to be ideal adjuvants for immunotherapy protocols tailored to individual patients.
  • the antibodies previously produced by conventional immunization protocols are of very limited suitability because they react only weakly with the native gp96 - possibly because the native gp96 is present intracellularly in the ER in the animals used for immunization, that in the resulting B cell hybridoma
  • the antibody present is also present in the ER and there is interference with the normal gp96 function in the hybridoma cells by reaction of native gp96 with the antibody formed, which does not allow the corresponding hybridoma cells to perform their function.
  • the object of the present invention is to provide antibodies which enable the practically exclusive recognition of native gp96. This task could now be solved by using recombinant antibodies.
  • the present disclosure describes the construction of antigen-binding domains of gp96-specific antibodies starting from a semi-synthetic immunoglobulin gene library.
  • the resulting single chain antibodies (scFvAbs) are used for a rapid one-step chromatographic purification method for the isolation of gp96 molecules. These are still able to trigger specific immune responses, such as native gp96 molecules, which are obtained using more complex conventional protocols, in which ConA-Sepharose column chromatography and further purification steps are used.
  • the ConA-Sepharose treatment leads to contamination with ConA as a result of "column bleeding". Because ConA is a T cell mitogen, it can interfere with a person's immune system or interfere with an animal to be vaccinated.
  • proteases such as cathepsin E
  • gp96 preparations purified in a conventional manner All the potential risks that exist due to the contamination of gp96 can be avoided by using the affinity purification according to the invention by means of recombinant scFv antibodies. In addition, they accelerate the cleaning process and allow the processing of small tumor samples. These features make the recombinant anti-gp96 antibodies an ideal tool for the purification of gp96, which can then also be used in the treatment itself, for example of cancer and infectious diseases.
  • FIG. 1 shows the sequence analysis of the recombinant scFv antibodies.
  • the amino acid sequences of the V H and the V L chain sections are shown.
  • the V H segments of the B10C, G12D and H11B clones belong to the V H 1 gene family and are derivatized from the germline gene DP-3.
  • the V L gene segment sequences are identical in all clones and match the V ⁇ 3 gene segment IGLV3S1 / DPL16.
  • the determined V H and V L sequences were compared to sequences from the V Base (Medical Research Council, Cambrdige, supra), the GenBank and the EMBL sequence database.
  • the interruptions within the sequence representation in each case within the sequence of the heavy and the light chain should not mean missing binding, but only make it easier to recognize different sections (FR1, CDR1 etc.).
  • sections FR1 to FR4 of the heavy chain and FR1 'to FR4' of the light chain form a coherent chain.
  • Figure 2 This figure shows the DNA sequences underlying the amino acid sequences in Figure 1.
  • the start of the scaffolding and complementary deteming regions (FR1, CDR1 etc.) is indicated above the coding triplets.
  • CDR2 CTT GTT GAT CCT GAA GAT GGT GAA ACA ATA TAC GCA GAG AAG
  • TTC CAG GGC SEQ ID NO: 17;
  • CDR2 CTT GTT GAT CCT GAA GAT GGT GAA ACA ATA TAC GCA GAG AAG
  • TTC CAG GGC SEQ ID NO: 17;
  • CDR2 CTT GTT GAT CCT GAA GAT GGT GAA ACA ATA TAC GCA GAG AAG
  • Purified scFv Abs are examined for binding to mouse gp96, using a nitrocellulose membrane (1) under reducing conditions (SDS plus ß-mercaptoethanol), (2) denaturing conditions (SDS) and (3) native (PBS) conditions is examined.
  • the membrane is developed with mouse IgG anti-human c-myc monoclonal, polyclonal peroxidase-conjugated rabbit anti-mouse IgG and substrate.
  • Fig. 4 Precipitation of native gp96 from mouse tumor cell lysates. Hypotonic lsyates of radiolabeled IGELa2 cells are incubated with the antibodies mentioned, followed by protein G-Sepharose precipitation and SDS-polyacrylamide gel electrophoresis. The control lane shows precipitation only with protein G Sepharose alone as a control.
  • (1) control
  • (2) anti-BiP (SPA-847)
  • (3) anti-gp96 (SPA-850)
  • (4) scFv (clone H11B).
  • Fig. 6 Activation of CTL by scFv-purified gp96 molecules.
  • C57BL / 6 mice are i.p. with PBS (A), gp96 bound to anti-gp96-scFv-Sepharose (B), untreated (C), gp96, purified according to standard methods (D), pH 10, 5-eluted gp96 molecules (E) and 1.3 M NaCl eluted gp96 molecules immunized.
  • splenocytes are incubated with irradiated BALB.B cells for 5 days and the CTL activity is measured on C57BL / 6 (filled circles) and BALB.B (filled triangles) ConA blasts.
  • (9) % specific lysis
  • (10) E: T ratio.
  • the invention relates to recombinant antibodies which bind native gp96, in particular those which determine the third complement-determining region (CDR3) from a variable portion of a heavy chain of an antibody according to SEQ ID NO: 1, SEQ ID NO: 2 or SEQ ID NO: 3 (FIG. 1), in particular those which in FIG. 1 (a) contain the sections CDR3 derived from the variable part of a heavy chain of the sequences SEQ ID NO: 1, SEQ ID NO: 2 or SEQ ID NO: 3; CDR2 of the sequence SEQ ID NO: 4; and CDR
  • CDR3 third complement-determining region
  • the invention also relates to the use of the recombinant antibodies mentioned for the purification or labeling of gp96.
  • the invention also relates to the use of the above recombinant antibodies for the (rapid) purification of intact gp96 / peptide complexes from small amounts of tumor material or infected cells, which can then be used for autologous or heterologous immunotherapy.
  • the invention also relates to gp96 with bound peptides which can be obtained or has preferably been obtained by the purification method described above, in particular for use in a method for (preferably autologous) immunization, and pharmaceutical formulations which contain a product which can be obtained by the purification method described above. in particular obtained, gp96 / peptide complex and possibly further carrier materials.
  • the invention also relates to gp96 with bound peptides according to the last paragraph for use in a method for therapeutic treatment of the human or animal body, in particular for heterologous or, above all, autologous immunization; the use of the corresponding gp96 with bound peptides for the production of a medicament for (in particular autologous) immunization in patients suffering from a tumor disease or an infection, and the use of the corresponding gp96 with bound peptides for the immunization of these patients or methods for (in particular autologous ) Immunization of patients by administering gp96 according to the previous paragraph to a patient who has a tumor or an infection and needs such treatment.
  • the invention also relates to the isolation of peptides which are bound to native gp96 from warm-blooded cells, in particular in warm-blooded animals which suffer from a disease, for example ischemia (for example as a result of an infarction or from atherosclerosis), a tumor disease or an infection and the like Peptides as new and their use.
  • a disease for example ischemia (for example as a result of an infarction or from atherosclerosis), a tumor disease or an infection and the like Peptides as new and their use.
  • the invention also relates to separation materials (matrices) and devices for purifying native gp96, which comprise the recombinant antibodies according to the invention.
  • the invention also relates to kits for the purification or detection of native gp96, which comprise the recombinant antibodies according to the invention.
  • the invention also relates to nucleic acid sequences, in particular DNA sequences, which code for recombinant antibodies according to the invention.
  • the invention also relates to host cells, in particular prokaryotes such as E. coli, which comprise sequences coding for recombinant antibodies according to the invention.
  • a recombinant antibody that binds native gp96 is a protein made up of one or more non-covalently or covalently linked peptide chains that define the complementarity-determining regions that bind to native gp96
  • CDR comprises, whereby glycosylations or other derivatizations can also be present; these are in particular antibodies constructed in the usual way from a heavy and a light chain, which are covalently linked to one another, for example via disulfide bridges or in some other way, or preferably a fragment of such an antibody, in particular of the F (ab) 2 type, or in particular single chain antibodies (sc Abs) with all complementarity-determining regions on a polypeptide chain, which also have further sequences, such as framework sequences, linker sequences, marker sequences (for example c-myc) or sequences which allow a specific separation, for example for IMAC (Immobilized Metal Affinity Chromatography), and / or can also be present as an aggregate of two or more such chains, as is frequently found (both the actual single chains and their aggregates are shown in - The following is meant when talking about scFv antibodies (Abs) - The minimum for such single chains consists of the so-called Fv fragments, which include the
  • Recombinant means that at least part of the sequence of the corresponding antibody is modified by methods of recombinant genetic engineering, the corresponding antibody also being able to be produced by enzymatic or chemical means or preferably by expression of suitable nucleic acid sequences in a host organism.
  • CDR Complementarity-determining regions
  • Antibodies are preferred which comprise the third complementarity-determining region (CDR3) of the heavy chain of an antibody according to SEQ ID NO: 1, SEQ ID NO: 2 or SEQ ID NO: 3 (FIG. 1), in particular those which in FIG. 1 (a), the parts derived from variable heavy chain sequences CDR3 (SEQ ID NO: 1, SEQ ID NO: 2 or SEQ ID NO: 3), CDR2 (SEQ ID NO: 4) and CDR 1 (SEQ ID NO: 5 or in particular SEQ ID NO: 6) and (b) those of CD 1 '(SEQ ID NO: 7), CDR2' (SEQ ID NO: 8) and CDR3 '(SEQ ID NO: 9).
  • Antibodies are also possible in which these CDR are separated from one another by framework regions other than those shown in FIG. 1 (FR).
  • the invention also relates to native gp96-binding antibodies which contain variants of the sequences identified as preferred, for example variants which result from a DNA which have been subjected to in vitro mutagenesis, the condition being that they still have the binding capacity mentioned , in particular in the case of conventional measurement methods for affinity determination, have at least twice, preferably at least 10 times stronger affinity for slightly denatured or, in particular, native gp96 than the antibody SPA-850, which recognizes a primary (sequence) structure of gp96, which is native as well is present in denatured gp96 and is therefore not selective for native gp96, see below).
  • modifications may consist in addition, substitution or deletion of amino acids, preferably up to 5, e.g.
  • an amino acid - particularly preferred are those recombinant antibodies which have an optimal affinity high enough for strong gp96 binding but still release again the same from gp96 / antibody complexes allowed to release the bound gp96 again, which is essential for the purification methods described below.
  • conservative exchanges are preferred, for example the replacement of one hydrophobic amino acid by another, one acidic by another, one basic by another, from Ser by Thr, an armatic by another or the like, or further addition a homologous amino acid so that an amino acid is replaced by two homologous amino acids, or deletion of an amino acid.
  • framework regions FR such as, for example, the FR1 to FR4 on the heavy chain, FR1 'to FR4' on the light chain, as shown in FIG. 1
  • framework regions FR such as, for example, the FR1 to FR4 on the heavy chain, FR1 'to FR4' on the light chain, as shown in FIG. 1
  • other amino acid sequences which impair the ability of the antibodies to bind to easily denatured or in particular have native gp96).
  • Particularly preferred recombinant antibodies are those which contain at least one of the sequences for the variable parts of the heavy chains according to FIG. 1 for one of the clones G12D, H11B or B1 OC (SEQ ID NO: 10; SEQ ID NO: 11; or SEQ ID NO 12), in particular the mutant with a CDR 1 of the sequence DYYMY (SEQ ID NO: 6), which could not be expected with the method described in the examples, that is to say the sequence for the variable section of a heavy chain of the clone H11B (SEQ ID NO: 12); and the sequence for the variable section of the light chain according to FIG. 1 (SEQ ID NO: 13), which are separated from one another if necessary by a suitable spacer section.
  • Native gp96 is one that essentially corresponds to the conformation of the cell normally present in the ER and is still able to bind the antigenic peptides bound under physiological conditions so that they can be isolated in complex with gp96 - with others Words that 3D
  • the purification of gp96 is accomplished by conventional methods, for example by precipitation of the gp96 from solutions such as cell lysates, provided that at least two binding sites per recombinant Antibodies for gp96 are present or aggregates of such recombinant antibodies are present, or if further anti-antibodies are used which have more than one binding site for sequences on the recombinant antibodies according to the invention (for example c-myc sequences) and are thus able to crosslink, in particular if the anti-antibodies are polyclonal; cleaning after binding an antibody according to the invention to a suitable particulate carrier material with an adequately large surface area, for example to a gel matrix based on carbohydrates or plastics, such as, in particular, Sepharose, is preferred.
  • a suitable particulate carrier material with an adequately large surface area, for example to a gel matrix based on carbohydrates or plastics, such as, in particular, Sepharose, is preferred.
  • the purification can then be carried out by binding in suspension or by applying the sample to be purified to a column which is filled with a matrix comprising a bound recombinant antibody. It is also possible to bind to membranes, for example nitrocellulose, which are coated with the corresponding recombinant antibody, or corresponding other carrier materials.
  • the gp96 bound in this way is eluted with suitable solutions, in particular with slightly basic (pH preferably between 7, 2 and 12, in particular between 7, 2 and 10.5; adjusted, for example, with the aid of organic bases, in particular nitrogen bases, such as triethanolamine) and / or saline eluents, in particular 0.3 to 4, preferably 0.5 to 1.5 M salt solutions, such as 0.7 to 1.5 M salt solution, preferably with common salt (NaCl) as the salt.
  • suitable solutions in particular with slightly basic (pH preferably between 7, 2 and 12, in particular between 7, 2 and 10.5; adjusted, for example, with the aid of organic bases, in particular nitrogen bases, such as triethanolamine) and / or saline eluents, in particular 0.3 to 4, preferably 0.5 to 1.5 M salt solutions, such as 0.7 to 1.5 M salt solution, preferably with common salt (NaCl) as the salt.
  • a pH in the range between 6 and 8 is preferably chosen and an increased salt concentration, in particular 0.7 to 1.5 M saline in the presence of a buffer such as phosphate buffer.
  • the labeling (ie, qualitative or quantitative determination) of gp96 can be carried out, for example, with conventional assays for the detection of antigens with antibodies. So they can recombinant antibodies according to the invention preferably themselves directly contain sequences which enable labeling (for example sequence sections which have an enzyme function and thus enable detection directly by reaction with appropriate substrates, or can be recognized by antibodies directed against them, which in turn can be detected by standard methods ), for example as phosphatase, e.g. alkaline phosphatase from E. coli or alkaline phosphatase from mammals (e.g. from beef), peroxidases, such as horseradish peroxidase (with e.g.
  • immunoassays in particular radio-immunoassays and especially immunofluorescence labeling, immunoblotting, enzyme immunoassays, in particular EIA or ELISA, or dot assays
  • EIA or ELISA enzyme immunoassays
  • dot assays can be carried out in order to gp96 to prove qualitatively and / or quantitatively.
  • the rapid purification is preferably achieved by separating a cell lysate (if necessary pre-clarified by ultracentrifugation) over a column which is loaded with a matrix which carries bound recombinant antibodies according to the invention (see above under Purification, where suitable elution conditions are also given), especially about appropriately modified Sepharose.
  • the immunotherapy uses the gp96 obtained in this way, which is essentially in its native form if appropriate elution conditions are selected (see above or in the examples), and bound peptides from the tumor cells or the infected cells are present, which are characteristic of disease states such as Ischemia, cancer or infection.
  • heterologous immunotherapy is also possible, for example if several patients are infected by the same pathogen or have the same tumor disease.
  • Tumor disease means a proliferative disease, for example solid tumors or blood cancer, including any metastases that may be present.
  • the cells used to isolate the gp96 should then in particular comprise tumor cells.
  • “Infected” means that the corresponding cells are infected by a microorganism, such as a virus, bacteria, mycoplasmas, a fungus or a parasite, such as protozoa.
  • the immunotherapy is carried out by enteral (eg nasal) or preferably parenteral, in particular intramuscular, subcutaneous or further intracranial or intralumbar administration of a gp96 / peptide complex purified as described above to a patient, preferably one in need of such treatment, in the absence or presence of further adjuvants, such as aluminum hydroxide, lysolecithin, Pluronic polyols, polyanions, peptides, muramyldi or tripeptides, squalene mixtures (SAF-1), saponin derivatives, cell wall preparations of mycobacteria, Quil A, subunit B from cholera emulsions , or combinations of two or more of them.
  • enteral eg nasal
  • parenteral in particular intramuscular, subcutaneous or further intracranial or intralumbar administration of a gp96 / peptide complex purified as described above
  • further adjuvants such as aluminum hydroxide, lysolecithin, Plur
  • the invention thus also relates to the use of the recombinant antibodies according to the invention for the purification of gp96 from cells or tissue samples, in particular from a patient who suffering from a disease, such as ischemia (e.g. as a result of an infarction), a tumor or an infection, and (especially in the case of an infection or a tumor disease) the subsequent administration of the gp96 thus obtained to a patient, in particular one who has such Treatment is required for autologous or further heterologous immunization.
  • a disease such as ischemia (e.g. as a result of an infarction), a tumor or an infection, and (especially in the case of an infection or a tumor disease)
  • ischemia e.g. as a result of an infarction
  • a tumor or an infection e.g. as a result of an infarction
  • the subsequent administration of the gp96 thus obtained to a patient, in particular one who has such Treatment is required for autologous or further heterologous
  • the invention also relates to gp96 with bound peptides, which can be obtained or has preferably been obtained by the purification method described above, in particular for use in a method for (preferably autologous) immunization, and pharmaceutical formulations which can be obtained by the purification method described above. in particular obtained, include gp9 ⁇ / peptide complex and possibly further carrier materials.
  • Particularly suitable carrier materials are physiologically acceptable carrier materials, in particular physiological saline, phosphate-buffered physiological saline, sterile water and optionally adjuvants, as mentioned above. Other buffering and dispersing carrier materials can be used.
  • physiological saline physiological saline
  • phosphate-buffered physiological saline sterile water
  • optionally adjuvants as mentioned above.
  • Other buffering and dispersing carrier materials can be used.
  • the skilled person knows the possible compositions, examples can be found in Remington's Pharmaceutical Sciences (Alfonso R.
  • compositions are sterilized and packaged using conventional methods, e.g. in ampoules or jars, or administered directly.
  • concentration of gp96 / peptide complements can vary, for example from approximately 0.001 to 20% by weight, preferably from 0.01 to 0.5%.
  • a dose is administered during administration which is normally suitable for stimulating an immune response (in particular based on cytotoxic T lymphocytes).
  • the dose depends on the individual patient parameters such as size, age, gender and condition, but will normally preferably be in the range from 0.001 to 20,000 ⁇ g, preferably 0.005 to 4,000 ⁇ g gp96 / peptide complex per kg patient weight.
  • Isolation of peptides bound to native gp96 from warm-blooded cells, particularly warm-blooded animals suffering from a disease, e.g. Ischemia (e.g. as a result of an infarction), a tumor disease or an infection is possible by releasing native gp96, which has bound such peptides, according to the invention from corresponding gp96 / peptide complexes (for example by changing the ionic strength, increasing the basicity, for example by means of Separates nitrogen bases, such as triethanolamine, to a pH between 7.4 and 12, preferably between 9 and 11.5) and, if necessary, for example by gel filtration from the gp96.
  • the peptides can then be further investigated (e.g.
  • corresponding peptides are also the subject of the present invention, as well as their use, for example in immunization protocols.
  • Chromatographic columns which are filled with a matrix which comprises a recombinant antibody according to the invention are to be mentioned in particular as devices for cleaning.
  • Such basic atrices are found in particular for the production of the matrix with functional groups, such as hydroxyl, carboxy, amino, epoxy, diene or mercapto groups, which allow coupling with an antibody according to the invention, in particular those based on polymers, such as those based on polyacrylic or polyacrylic derivatives (in particular oxirane-modified, such as in the case of Eupergit (Röhm Phar a, Darmstadt) or in particular on the basis of activated polymeric carbohydrates, such as cyanogen bromide-activated Sepharose (especially Sepharose 4, 4B or 6MB), and the antibody-modified matrices according to the invention are based on this of polymers, such as polyacrylic or its derivatives (eg Eupergit), or based on polymeric carbohydrates, which carry antibodies according to the invention (preferably covalently bound) .
  • a matrix (carrier material), as described above - in particular Sepharose - which carries an antibody according to the invention, in particular if it is covalently bound, is itself the subject of the invention.
  • a corresponding device preferably also comprises a column which contains a matrix coated with a non-specific material, in particular serum albumin, such as bovine serum albumin (as described above, but with a ligand other than the recombinant antibody), which is suitable for precleaning (as a precolumn) and preferably directly upstream of the actual column occupied by the recombinant antibody.
  • Kits for the purification or detection of native gp9 ⁇ which comprise the recombinant antibodies according to the invention, are in particular those which have a chromatographic column as described in the last paragraph and required further components, such as a precolumn also described above and, if appropriate Buffer salts or buffer solutions, eluents, or the like, and if desired also detection systems for isolated native gp96 or peptides bound to it, such as components of corresponding immunoassays or the like, can contain, for example antibodies against c-myc, anti-antibodies against these antibodies, for example with a Enzyme as a component of an enzymatic detection system, such as a peroxidase, are marked, as well as corresponding reagent solutions and substrates for these enzymes.
  • the invention also relates to nucleic acid sequences, in particular DNA sequences, which code for recombinant antibodies according to the invention. Preference is given to those nucleic acid sequences which, as the nucleic acid sequence coding for the variable section of a heavy chain, are one of those in FIG. 2 and its legend for the CDR 3 from the clones B1 OC (SEQ ID NO: 23), G12D (SEQ ID NO: 24) or H11B (SEQ ID NO: 25), and / or as the nucleic acid sequence coding for the variable portion of a light chain include the one shown for the CDR3 'in Fig. 2 and its legend (SEQ ID NO: 22), as in parentheses each indicated above; as well as any sequences FR1 to FR4 and FR1 'to FR4' coding for framework regions.
  • nucleic acid sequences which, as the nucleic acid sequences coding for the variable section of a heavy chain for CDR1, comprise one of the sequences mentioned in FIG. 2 and its legend with SEQ ID NO: 18 or in particular SEQ ID NO: 19, for which CDR2 Sequence according to SEQ ID NO: 17 and for CDR3 one of the sequences according to SEQ ID NO: 14, SEQ ID NO: 15 and SEQ ID NO: 16, and / or as the nucleic acid sequence coding for the variable portion of a light chain that for CDR1 ' according to SEQ ID NO: 20, for the CDR2 'according to SEQ ID NO: 21 and for the CDR3' according to SEQ ID NO: 22 in FIG. 2 and their sequences; as well as any sequences FR1 coding for framework regions to FR4 and FR1 'to FR4', in particular those mentioned in FIG. 2 and its legend.
  • nucleic acid sequences which, as the coding sequence for the variable part of the heavy chain, that of clone S1 OC (SEQ ID NO: 23), clone G12D (SEQ ID NO: 24) or in particular the clone shown in FIG. 2 H11B (SEQ ID NO: 25) comprise and / or as the sequence coding for the variable part of the light chain that shown in FIG. 2 for all clones (SEQ ID NO: 26).
  • nucleic acid sequences are particularly preferred, in which the sequences coding for the variable sections of the heavy and light chain are contained in one and the same nucleic acid strand (coding for single-chain antibodies).
  • nucleic acid sequences which are also suitable for further sequences, such as Gerüs sequences, linker sequences, marker sequences (e.g. c-myc) or sequences which allow specific separation, e.g. for IMAC (Immobilized Metal Affinity Chromatography) encoding sequence sections.
  • marker sequences e.g. c-myc
  • IMAC Immobilized Metal Affinity Chromatography
  • nucleic acid sequences according to the invention also include those which code for the amino acid deletions, insertions or substitutions mentioned above for antibodies which contain variants of the sequences designated as preferred, thus itself contain corresponding deletions, insertions or substitutions of nucleic acids.
  • nucleic acids are varied (deleted, inserted or substituted).
  • vectors such as plasmids, cosmids or the like, which comprise one of the sequences mentioned and which can be expressed in suitable host cells.
  • a preferred vector is based on the pHEN1 or pHOG 21 vector (see examples).
  • Suitable host cells are those which can be cultivated in vitro.
  • Eukaryotic or in particular prokaryotic cells e.g. Yeasts, such as Saccharomyces cerevisiae, mammalian cells, hybridomas or in particular bacteria, such as E. coli, which are transformed by suitable vectors.
  • Eukaryotic host cells are required for the production of glycosylated antibodies, and bacteria are particularly suitable for the production of single-stranded antibodies. Examples of bacteria are especially those that are low in restriction or modification enzymes, such as E. coli, e.g. E. coli X776, E.coli TG1 or in particular E.coli XL1 -Blue (Stratagene).
  • the antibodies, host cells (including cultivation and transformation), labeled antibodies, devices, etc. are produced by methods known per se, for example analogously to the methods mentioned in the examples.
  • slightly denatured or native GP96-binding antibodies, methods, vectors and devices mentioned in the examples are particularly preferred.
  • mice are from Charles River WIGA
  • mice are from Harlan Winkelmann
  • the monoclonal antibodies against gp96 (anti-Grp94, SPA-850, clone 9G10) and against BiP (SPA-827, clone 10C3) are from StressGen Biotechnologies Corp., Victoria, BC, Canada.
  • Cell culture and CTL assay The mouse cell line IGELa2 and the human C1 cell lines are from ATCC, Rockville, MD, USA; they are cultured in RPMI 1640 containing 10% fetal calf serum and supplemented with 0.3 mg / ml L-glutamine, penicillin / streptomycin (100 units / ml) and 2-mercaptoethanol (2 ⁇ l / l).
  • CTL lines are obtained by weekly stimulation with irradiated spleen cells from BALB.B mice (using a cesium ⁇ radiation source).
  • CTL assays are carried out as described in the literature (Arnold et al., J. Exp. Med. 182, 885-889 (1995)) using ConA blasts (concanavalin A-activated T cells) from spleen cells as target cells. Con A blasts are also produced as described in the last-mentioned publication.
  • the standard method for purifying gp96 is described in PK Srivastava, METHODS: A Co panion to Methods Enzymol. 12, 165-71 (1997) and includes purification using ConA-Sepharose and MonoQ anion exchange chromatography.
  • a semisynthetic phage display library which consists of 50 different human germline genes for heavy chains with CDR3 loops randomized in sequence and a constant human V ⁇ 3 gene of a light chain and has a complexity of more than 10 8 clones (Nissim et al., EMBO J. 13, 692-698 (1997), is used for the selection of single-chain Fv (scFv) antibodies (Abs).
  • synthetic CDR3 loops are used, which randomized sequences of 4-12 amino acids have inserted into 50 variable sections of heavy chains, then the repertoire of sections of heavy chains obtained is inserted into the phagemid vector pHEN1 (see Hoogenboom et al., Nucleic Acids Res.
  • coli TG1 are 5 x 10 12 colony-forming units (cfu) in phosphate-buffered saline (PBS; 150 mM NaCl, 150 mM sodium phosphate, pH 7.2 with 2% skimmed milk powder (MPBS) in Nunc Maxisorb immune tubes (Nunc, Roskilde, Denmark) coated with 80 ⁇ g mouse gp96 (mgp96), for panning. Unspecifically bound phages are removed by intensive washing. Specific phages are eluted with 100 mM triethylamine (pH12) and expanded after neutralization and used for further selection steps until a total of four have been carried out.
  • PBS phosphate-buffered saline
  • MPBS skimmed milk powder
  • the binding specificity is determined by ELISA: microtiter plates are coated with mgp96 (0.5 ⁇ g / well), blocked with 2% skim milk powder solution and then mixed with phage particles (10 10 cfu).
  • Commercially available polyclonal chicken anti-gp96 serum (1: 500) serves as a positive and PBS as a negative control.
  • the gene segments of the three specific scFv-Abs (B10C, G12D and H11B) are obtained by Ncol-Notl digestion into the plasmid pHOG21 (see Kipryanov et al., J. Immunol. Meth. 200, 60-77 (1997)) ligated and expressed in E. coli XL1-Blue (Stratagene).
  • the soluble fraction of the periplasmic extract and the culture supernatant are combined, concentrated and dialyzed against 50 mM Tris-HCl, 1M NaCl, pH 7.0.
  • the samples are applied to a "Chelating Sepharose Fast Flow Column” (Amersham Pharmacia Biotech, Upsala, Sweden), which has been previously loaded with Ni 2+ ions and equilibrated with dialysis buffer. After extensive washing, the bound material is eluted with 250 nM imidazole and dialyzed against PBS. The purity is checked by means of polyacrylamide gel electrophoresis (PAGE) (not shown).
  • PAGE polyacrylamide gel electrophoresis
  • 500 ng mgp96 are applied to nitrocellulose membranes under native (PBS), denaturing (2% SDS) or reducing (100 mM ⁇ -mercaptoethanol) conditions. Both the western and dot blot membranes are blocked with 2% MPBS.
  • the mgp96 is detected using rat IgG anti-Grp94 (SPA-850, StressGen Biotechnologies) and peroxidase conjugated rabbit anti-rat IgG (DAKO, Glostrup, Denmark) or with the scFv antibodies.
  • All scFv Abs carry a c-myc marker, which allows labeling with mouse IgG anti-human myc (Genosys, The Woodlands, USA) and peroxidase-conjugated rabbit anti-mouse antibody (DAKO).
  • the detection of soluble scFv Abs can be determined with a range of concentrations (1/1 to 1/8192). 3 shows the results of the dot blot analysis.
  • scFv Abs show only binding to the native protein when analyzed on the nitrocellulose membranes labeled with reduced, denatured or native mgp96 (FIG. 3). Western blot analysis data (not shown) confirm this finding.
  • Example 4 scFv antibodies precipitate native gp96 molecules If the recombinant scFv Abs are tested in an ELISA, they only recognize gp96 in its native form, while the commercially available monoclonal antibody SPA-850 (StressGen Biotechnologies) hardly does this , which severely limits its usefulness for affinity purification of gp96.
  • Mouse tumor cell lysates from IGELa2 cells are used for this. 10 7 cells are metabolically labeled in the presence of 35 S-methionine (150 ⁇ Ci) in 10 ml of methionine-free RPMI medium for 16 hours. After lysis of the cells in hypotonic buffer (30 mM NaHC0 3 pH 7.1), the lysates are pre-adsorbed overnight on protein G-Sepharose (Amersham Pharmacia Biotech). Specific monoclonal antibodies or scFv-Anti-gp96 Abs and anti-c-myc Abs become clear lysates in an amount of
  • the immunocomplexes are washed repeatedly and washed by SDS polyacrylamide gel electrophoresis.
  • the fixed gels are dried and exposed on a phosphoimager screen.
  • SPA-850 FIG. 4, lane (3)
  • the bands with scFv Abs according to the invention show strong precipitation of the gp96 molecules (FIG. 4, lane (4)).
  • the recombinant antibodies according to the invention thus recognize native gp96- Molecules much better than a monoclonal antibody can do.
  • Clones B1 OC and G12D show comparable results (not shown).
  • the scFv-anti-gp96 Abs are also more efficient in the preparation of gp96 than a monoclonal antibody against the chaperone BiP present in the endoplasmic reticulum, which is known to cross-react with an unknown 40kDa protein and also with gp96 (Fig. 4 , Track (2)).
  • Antibody H11B coupled to Sepharose beads 5 mg thereof or
  • a strongly radioactive labeled band of the protein which runs close to the 94 kDa marker band, shows that the immobilized recombinant ⁇ CFVH11B is able to specifically bind gp96 (Fig. 5, (8)).
  • This band is missing in the control lane (7), in which only BSA-Sepharose beads are used for immunoprecipitation.
  • the recombinant scFv antibody should also be able to recognize human gp96. To test whether this is possible, the immunoprecipitation experiments are carried out with hypotonic lysates from human C1R cells.
  • immobilized scFv-H11B antibody is able to bind human gp96 molecules (6).
  • the difference in the intensities between the mouse and the human band in FIG. 5 can be explained by the higher expression levels (concentrations) of gp96 in IGELa2 cells compared to C1R cells (not shown).
  • Immobilized B1 OC or G12D scFv antibodies also precipitate mouse and human gp96 in the same way.
  • mice Since native gp96 molecules are recognized by the immobilized scFv-Abs, it is examined whether they are still associated with antigenic peptides and are able to induce specific immune responses. 8 to 10 week old C57BL / 6 mice are treated with 30 ⁇ g from IGELa2 cells using gp96 purified in accordance with the standard method or with the scFv-anti-gp96 method in 300 ⁇ l PBS, or with 80 ⁇ l Sepharose beads (based on the volume of beads) coupled to BSA or to scFv, which is complexed to gp96, immunized in 300 ⁇ l PBS.
  • the spleens are removed and the splenocytes are stimulated with irradiated (33Gy) spleen cells from BALB.B mice for 5 days.
  • irradiated (33Gy) spleen cells from BALB.B mice for 5 days.
  • Sepharose columns which are coupled with recombinant scFv antibody, are loaded with the hypotonic lysates from IGELa2 cells (which come from BALB / c mice and express H2 d ) and the gp96 / scFv-Sepharose beads in C57BL / 6 -Mice injected 10 days after immunization, the recipient splenocytes are stimulated with irradiated BALB.B spleen cells (of the H2 b haplotype).
  • SEQ ID NO: 1 Randomized CDR3 loop as part of an antibody heavy chain, see Nissim et al, EMBO J. 11 (3), 692-98 (1994);
  • SEQ ID NO: 2 and 3 Random sequence for CDR3 of an antibody heavy chain, produced according to Nissim et al., 1994.
  • SEQ ID NO: 10 to 12 Recombinant sequence for variable part of the heavy chain of antibodies with a random sequence for CDR3 (Nissim , 1994);
  • SEQ ID NO 14 to 16 random sequence, encoded for CDR3 of a heavy chain of an antibody, production see Nissim et al. , 1994;
  • SEQ ID NO: 23 to 25 recomb. Sequence encoded for variable part of the antibody heavy chain with random sequence for CDR 3 (Nissim, 1994).

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Abstract

L'invention concerne des anticorps recombinants contre la gp96 naissante, des procédés de fabrication de ces anticorps, leur utilisation, des éléments de séparation, des dispositifs et des ensembles contenant lesdits anticorps, des peptides liés à la gp96 naissante, leur isolation, des séquences d'acides nucléiques codant lesdits anticorps recombinants, et des cellules hôtes contenant ledit ADN et permettant la fabrication d'anticorps correspondants.
PCT/EP2001/004382 2000-04-20 2001-04-18 Anticorps contre gp96 naissante, fabrication et utilisation de ces anticorps Ceased WO2001081423A1 (fr)

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DE2000119967 DE10019967A1 (de) 2000-04-20 2000-04-20 Antikörper gegen natives gp96, deren Herstellung und Verwendung
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EP2185579A1 (fr) * 2007-07-24 2010-05-19 Primiero, Paolo Complexes composés de grp94 et d'immunoglobuline g humaine
US7767207B2 (en) 2000-02-10 2010-08-03 Abbott Laboratories Antibodies that bind IL-18 and methods of inhibiting IL-18 activity
WO2010142651A3 (fr) * 2009-06-10 2011-03-17 Fundació Privada Institut D'investigació Biomèdica De Bellvitge (Idibell) Procédé pour déterminer le risque de développement d'une métastase cérébrale, et trousse pour mettre en oeuvre ledit procédé
WO2021251564A1 (fr) * 2020-06-09 2021-12-16 국민대학교산학협력단 Anticorps se liant de manière spécifique à grp94 ou un fragment de liaison à l'antigène de celui-ci, et utilisations associées

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GB0210783D0 (en) * 2002-05-10 2002-06-19 Polonelli Luciano Anti-microbial polypeptides
EP1561107A1 (fr) * 2002-11-13 2005-08-10 Micromet AG Procede d'identification de cellules b specifiques d'un antigene
MX2008001818A (es) * 2005-08-19 2008-04-09 Indivumed Gmbh Uso de un fragmento de endoplasmina y derivados del mismo como un biomarcador para adenoma y/o carcinoma colorrectal, metodo para deteccion y sistema de prueba.

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WO2000026667A1 (fr) * 1998-10-30 2000-05-11 Miller Jonathan L Regions variables de chaine lourde et de chaine legere d'anticorps diriges contre la glycoproteine plaquettaire humaine ib alpha

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FELDWEG ANNA M ET AL: "Molecular heterogeneity of tumor rejection antigen/heat shock protein GP96.", INTERNATIONAL JOURNAL OF CANCER, vol. 63, no. 2, 1995, pages 310 - 314, XP001015894, ISSN: 0020-7136 *

Cited By (8)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US7767207B2 (en) 2000-02-10 2010-08-03 Abbott Laboratories Antibodies that bind IL-18 and methods of inhibiting IL-18 activity
EP2332579A3 (fr) * 2000-02-10 2011-09-21 Abbott Laboratories Anticorps qui se lient à l'interleukine-18 humaine et procédés de fabrication et d'utilisation
EP2338515A3 (fr) * 2000-02-10 2011-11-16 Abbott Laboratories Anticorps qui se lient à l'interleukine-18 humaine et procédés de fabrication et d'utilisation
EP2360184A3 (fr) * 2000-02-10 2011-11-16 Abbott Laboratories Anticorps qui se lient à l'interleukine-18 humaine et procédés de fabrication et d'utilisation
EP2185579A1 (fr) * 2007-07-24 2010-05-19 Primiero, Paolo Complexes composés de grp94 et d'immunoglobuline g humaine
WO2010142651A3 (fr) * 2009-06-10 2011-03-17 Fundació Privada Institut D'investigació Biomèdica De Bellvitge (Idibell) Procédé pour déterminer le risque de développement d'une métastase cérébrale, et trousse pour mettre en oeuvre ledit procédé
WO2021251564A1 (fr) * 2020-06-09 2021-12-16 국민대학교산학협력단 Anticorps se liant de manière spécifique à grp94 ou un fragment de liaison à l'antigène de celui-ci, et utilisations associées
US12448436B2 (en) 2020-06-09 2025-10-21 Kookmin University Industry Academy Cooperation Foundation Antibody specifically binding to GRP94 or antigen-binding fragment thereof, and uses thereof

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