Classifications

• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07K—PEPTIDES
  • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
  • A61P31/04—Antibacterial agents
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
  • A61P31/12—Antivirals
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P37/00—Drugs for immunological or allergic disorders
  • A61P37/02—Immunomodulators
  • A61P37/04—Immunostimulants
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P37/00—Drugs for immunological or allergic disorders
  • A61P37/02—Immunomodulators
  • A61P37/06—Immunosuppressants, e.g. drugs for graft rejection
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P39/00—General protective or antinoxious agents
  • A61P39/02—Antidotes
• • 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
  • C07K16/34—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against blood group antigens
• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07K—PEPTIDES
  • C07K2317/00—Immunoglobulins specific features
  • C07K2317/50—Immunoglobulins specific features characterized by immunoglobulin fragments
  • C07K2317/52—Constant or Fc region; Isotype

Definitions

• the present invention relates to the use of metal cations, in particular divalent or trivalent, and more particularly of Zinc, Copper, Cadmium or Iron, to improve the functional activity of antibodies. More particularly, the subject of the invention is pharmaceutical compositions of antibodies comprising divalent or trivalent metal cations.
• Passive immunotherapy which is very widespread, is based on the administration of antibodies, for example monoclonal antibodies directed against a given cell or substance. Passive immunotherapy using monoclonal antibodies has given encouraging results. However, if the use of monoclonal antibodies has several advantages, such as an assurance of product safety as regards the absence of infectious contamination, it can however prove difficult to obtain an effective monoclonal antibodies.
• one of the objects of the invention is to provide new products or methods making it possible to overcome the drawbacks encountered during the industrial development of antico ⁇ s, namely their low efficiency and their high cost.
• the Fc region of IgG consists of 2 globular domains named CH2 and CH3.
• the 2 heavy chains interact closely at the level of the CH3 domains while at the level of the CH2 domains, the presence, on each of the 2 chains, of an oligosaccharide linked to Asn 297 (Kabat numbering) contributes to the separation of the 2 CH2 domains.
• the CH2 and CH3 domains of the same chain are separated by a flexible region defining an interface between the 2 domains.
• the interface between the CH2-CH3 domains has been described as a common site for the attachment of numerous (glyco) proteins such as FcRn, rheumatoid factors (Co ⁇ er et al., 1997) and certain bacterial and viral proteins, for example the protein A of Staphylococcus aureus (Deisenhofer, 1981), protein G of Streptococcus group G (Sauer-Eriksson et al., 1995), the gE-gl complex of the He ⁇ es simplex 1 virus (Chapman et al., 1999)) and the “core” protein of the hepatitis C virus (Maillard and ⁇ /., 2004).
• Glyco proteins such as FcRn, rheumatoid factors (Co ⁇ er et al., 1997) and certain bacterial and viral proteins, for example the protein A of Staphylococcus aureus (Deisenhofer, 1981), protein G of Streptococcus group G (S
• this zinc atom plays an important role in the general conformation of Fc, and thereby allows the improvement of the binding of Fc to its Fc ⁇ Rs receptors.
• the invention provides an economical and universal solution to solve the problems linked to the low efficiency of the monoclonal antico ⁇ s available or under development by the use of metal cations which improve the functional activity of antico ⁇ s.
• the invention also provides antico ⁇ s of class IgGl having a reduced capacity for activating the receptor Fc ⁇ RI ⁇ , as well as antico ⁇ s of class IgG3 artificially having a binding site for a cation metallic.
• a first object of the invention is the use of divalent or trivalent metal cations to improve the functional activity of antico ⁇ s.
• the metal cation used is zinc.
• this zinc atom plays an important role in the general conformation of the Fc region, and thereby allows the improvement of the binding of the Fc region to its receptors.
• these cations are used to interact with the Fc region of the antico ⁇ s in order to participate in the stabilization of this region.
• these metal cations are used in order to participate in controlling the opening of the Fc region of the antico ⁇ s and thus of promoting the maintenance of the so-called “open” conformation of the antico ⁇ s, that is to say the maintenance of a certain separation between the CH2 domains favoring the attachment of the Fc region to its receptors.
• the presence of the metal cations makes it possible to induce an opening of the Fc region, even if the metal cation does not remain in its site.
• these metal cations promote the attachment of the antico ⁇ s to the FcyR receptors, in particular to the FcvRIII receptor.
• the metal cation can, in another aspect of the invention, promote the bringing together of several Fc regions of antico ⁇ s, via a second binding site involving the histidine residues 268 and 285 (Kabat numbering), facilitating the activation of Fc ⁇ Rs, more particularly Fc ⁇ RI ⁇ and the transduction of signaling via these receptors.
• functional activity is meant, without limitation, the ADCC activity (Antibody-Dependent Cell-mediated Cytotoxicity), CDC activity (Complement Dépendent Cytotoxicity), phagocytosis activity, endocytosis activity or further induction of cytokine secretion.
• ADCC activity Antibody-Dependent Cell-mediated Cytotoxicity
• CDC activity Complement Dépendent Cytotoxicity
• phagocytosis activity endocytosis activity or further induction of cytokine secretion.
• receptors is meant not only the molecules of FcyR., Such as FcyRIII, present on the cells of the immune system such as monocytes, macrophages, B and T lymphocytes, NK cells and dendritic cells but also FcRn, complement molecules such as Cl q and those of bacterial walls such as protein A.
• antico ⁇ s any polyclonal or monoclonal antico ⁇ s. If the antico ⁇ s is a monoclonal antico ⁇ s, it can be chimerical, humanized or human.
• this antico ⁇ s is an IgG, for example an IgGl or an IgG3, in particular human.
• antico ⁇ s also includes any glycoprotein comprising an Fc region, for example human, and one or more fragments, domains or derivatives of antico ⁇ s.
• antico ⁇ s domain is understood to mean any of the domains VL, CL, VH, CH1, CH2, CH3, CH4; by "fragment of antico ⁇ s” any fragment which contains a complete binding site for an antigen, chosen from the fragments Fv, scFv, Fab, Fab ', F (ab') 2, and by "derivative of antico ⁇ s" all antico ⁇ s may include one or more mutations, substitutions, deletions and / or additions of one or more amino acid residues, as well as multi-specific and polyfunctional antico ants.
• divalent or trivalent metal cation or by “metal cation” is meant any metal cation having an oxidation state +2 or +3 and more particularly zinc, iron, copper, cadmium, cobalt, nickel , manganese, gallium, gadolinium, selenium, gold, platinum or palladium or the like.
• these metal cations are zinc, iron, copper or cadmium, and in a particularly advantageous manner it is zinc.
• a second object of the invention relates to a method for potentiating the functional activity of the antico ⁇ s via the Fc region, comprising a step consisting in adding an appropriate amount of at least one metal cation in the biological system producing the antico ⁇ s or in a solution comprising antico ⁇ s before and / or after purification or in the preservation solution or in the final formulation in the form of an injectable solution of the antico ⁇ s.
• these metal cations are zinc, iron, copper or cadmium.
• biological system is understood to mean cell lines, non-human transgenic plants or animals.
• cells it is possible to choose cells originating from cell lines, transfected using a vector comprising the gene coding for said antico ⁇ s, for example eukaryotic or prokaryotic cells, in particular mammalian, insect, plants, bacteria or yeast. More specifically, rat myeloma cells such as YB2 / 0 can be used.
• CHO cells can also be used, in particular CHO-K, CHO-LeclO, CHO-Lecl, CHO Pro-5, CHO dhfr- or other cell lines among Wil-2, Jurkat, Vero, Molt-4, COS- 7, 293-HEK, K6H6, NSO, SP2 / 0-Ag 14 and P3X63Ag8.653, PERC6 or BHK.
• a molar concentration of zinc at least equal to the molar concentration of antico ⁇ s is added.
• a molar zinc concentration at least equal to 2 times is added, and preferably 3 times or 4 times the molar concentration of antico ⁇ s.
• a molar concentration of metal cations is added which makes it possible to improve the functional activity of the antico ⁇ s by at least 25%, preferably 50% or 60%, 70%, 80%, 100%, and preferably 200 or 300 %.
• the metal cations exist in different forms.
• the zinc ions can be in the form of zinc acetate, zinc bromide, zinc hydrochloride, zinc chloride, zinc citrate, zinc gluconate, hydroxycarbonate zinc, zinc iodide, zinc L-lactate, zinc nitrate, zinc stearate, or zinc sulfate.
• Another subject of the invention relates to antico ⁇ s of class IgG3, and more particularly the allotypes G3m (b) and G3m (g), having a binding site for a metal cation comprising the residues His 310 and His 435 on its Fc region created by molecular engineering.
• antico ⁇ s of class IgG3 having an improved fixation of metal cations compared to unmodified antico ⁇ s , by creating a binding site involving a His 435 residue in substitution for the Arg 435 residue.
• these IgG3 antico ⁇ s have a binding site for a metal cation, in particular zinc, iron, copper, cadmium, cobalt, nickel, manganese, gallium, selenium, gold, platinum or palladium, comprising the residues His 310 and His 435, and advantageously also comprising the residue Asn 434 and / or His 433.
• a metal cation in particular zinc, iron, copper, cadmium, cobalt, nickel, manganese, gallium, selenium, gold, platinum or palladium
• the residues His 310 and His 435 and advantageously also comprising the residue Asn 434 and / or His 433.
• at least one of these histidine residues is replaced by at least one of the residues chosen among cysteine, aspartic acid and glutamic acid.
• these residues also have the capacity to fix such metal cations.
• the metal cation is zinc, iron, copper or cadmium, and preferably zinc.
• the antico ⁇ s has a metal cation, - and more particularly a zinc atom, linked to one or more residues of the Fc region.
• this IgG3 antico ⁇ s has a capacity for fixing to Fc ⁇ RI ⁇ and an improved functional activity compared to the native antico ⁇ s.
• An object of the invention is thus the use of the antico ⁇ s IgG3 having a binding site for a metal cation previously described for the preparation of a medicament intended for the treatment of a pathology such as hemolytic disease of the newborn, viral, bacterial or parasitic pathology, pathology linked to pathogenic agents or derived toxins, listed as being particularly dangerous in cases of bioterrorism (classification of the Centers for Disease Control, CDC), in particular anthrax (Bacillus anthracis), botulism (Clostridium botulium), plague (Yersinia pestis), smallpox (Variola major), latularemia (Francisella tularensis), viral hemorrhagic fevers (linked to filoviruses -Ebola, Marburg and arenaviruses -Lassa, Machupo), epsilon toxin Clostridium perfringens, brucellosis (Brucella species), melioidosis (Burkholderia
• Another subject of the invention is a pharmaceutical composition of therapeutic antico ⁇ s comprising divalent or trivalent cations and at least one excipient.
• these metal cations are zinc, iron, copper or cadmium, or a mixture of several of them.
• zinc is chosen, which can be in the form of zinc acetate, zinc bromide, zinc hydrochloride, zinc chloride, zinc citrate, zinc gluconate, hydroxycarbonate zinc, zinc iodide, zinc L-lactate, zinc nitrate, zinc stearate, or zinc sulfate.
• the antico ⁇ s contained in the composition have a metal cation according to the invention linked to the residues His 310 and His 435, the residues His 433 and Asn 434 can also participate in the fixation.
• the antico ⁇ s of the pharmaceutical composition are the antico ⁇ s of class IgG3 created by molecular engineering described above. In another preferred aspect of the invention, these are human IgGs or having a human Fc region.
• the presence of such metal cations in the composition improves the fixation of the therapeutic antico ⁇ s that it contains to its receptors, in particular the Fc RULs, the composition thus having better therapeutic activity.
• Another subject of the invention is a pharmaceutical composition in which at least 50%, 60%, 70%, 80%, 90%, or even 99% of the antico ⁇ s have a divalent or trivalent metal cation, in particular a zinc ion, bound to a site located in the Fc region. It may preferably be the binding site comprising the amino acids His 310 and His 435, the amino acid Asn 434 and / or His 433 being capable of participating in the binding. In another aspect of the invention, it can be the binding site comprising the amino acids His 268 and His 285. In another aspect of the invention, the 2 sites can be occupied by a metal cation as described in the invention.
• the metal cation is preferably one of those already mentioned above, in particular zinc, iron, copper or cadmium, or a mixture of several of them, possibly in the forms already mentioned.
• Another object of the invention is a solution comprising a monoclonal antico ⁇ s or monoclonal antico ⁇ s and an appropriate quantity of divalent or trivalent metal cations, in particular of zinc ions at least equal to the molar concentration in antico ⁇ s, this solution being suitable for injection by intravenous, subcutaneous or intramuscular route.
• the metal cations can be any divalent or trivalent metal cation, in particular zinc, iron, copper, cadmium, cobalt, nickel, manganese, gallium, selenium, gold, platinum or palladium or a similar.
• the cation is a zinc ion or zinc acetate, zinc bromide, zinc hydrochloride, zinc chloride, zinc citrate, zinc gluconate, zinc hydroxycarbonate, zinc iodide, zinc L-lactate, zinc nitrate, zinc stearate, or zinc sulfate.
• Another object of the invention is the use of zinc ions to improve the crystallization of therapeutic antico ⁇ s, and more particularly of monoclonal IgG, the zinc ions stabilizing the Fc region of the antico ⁇ s.
• the addition of divalent cations, and more particularly of zinc, significantly increases the solubility of Fc of IgGs, by promoting crystalline contacts which facilitates the obtaining of the crystals necessary for structural studies.
• the invention also aims to provide a test for evaluating the efficacy of an antico l'mentss comprising the study of the 3D conformation, in particular of the field involving the residues His 310, His 435, His 433 and / or Asn 434 of the Fc region as shown in FIG. 1 or 2 or also a determination of the zinc content of said antico ⁇ s, the presence of zinc being an indication of the effectiveness of the antico ⁇ s.
• Another subject of the invention relates to an antico ⁇ s having at least one of its residues His 310 and His 435 modified.
• the modification of the antico ⁇ s is a mutation, in particular a substitution by an amino acid having a low affinity for divalent or trivalent metal cations.
• the His 310 residue and / or the His 435 residue can be substituted with a residue of lysine, alanine, glycine, valine, leucine, isoleucine, proline, methionine, tryptophan, phenylalanine, serine or threonine.
• the His 310 and His 435 residues are both substituted by lysine residues.
• mutants can be produced from any antico ⁇ s possessing in the “natural” state, that is to say non-mutated, a binding site for metal cations comprising the residues His 310 and His 435. It can be act in particular of IgGl, of IgG3 allotypes G3m (s) or G3m (st), IgG2 or IgG4.
• the modification can be carried out with DEPC (diethyl pyrocarbonate), a histidine modifying agent.
• DEPC diethyl pyrocarbonate
• these antico ⁇ s are IgGl, or in any case antico ⁇ s having in the “natural” state, that is to say not mutated, a binding site for metal cations comprising the residues His 310 and His 435.
• antico ⁇ s have a reduced functional activity compared to the same unmodified antico ⁇ s. However, they retain their ability to fix the antigen and the
• the invention provides antico ⁇ s having a low ADCC activity, which is of particular interest in therapy to replace IgG4, or to prevent transplant rejection.
• the double mutant antico ⁇ s according to the invention can also be used as anti-tetanus, anti-diphtheria or directed against pathogens or toxins derived therefrom, listed as being particularly dangerous in cases of bioterrorism (classification of the Centers for Disease Control, CDC) , including anthrax (Bacillus anthracis), botulism (Clostridium botulium), plague (Yersinia pestis), smallpox (Variola major), tularemia (Francisella tularensis), viral haemorrhagic fevers (linked to filoviruses -Ebola, Marburg and arenaviruses -Lassa, Machupo), epsilon toxin from Clostridium perfringens, brucellosis (Brucella species ), melioidosis (Burk
• Another object of the invention therefore relates to the use of modified antico ⁇ s as described above, and therefore having a weak functional activity, for the preparation of a medicament intended for the prevention of transplant rejection or for the treatment of 'a pathology chosen from tetanus, diphtheria, or caused by a pathogen or derived toxin, listed as being particularly dangerous in cases of bioterrorism " (classification by the Centers for Disease Control, CDC), in particular anthrax (Bacillus anthracis ), botulism (Clostridium botulium), plague (Yersinia pestis), smallpox (Variola major), tularemia (Francisella tularensis), viral haemorrhagic fevers (linked to filoviruses -Ebola, Marburg and arenaviruses -Lassa, Machupo) , epsilon toxin from Clostridium perfringens, brucellosis (Brucella species), melioidosis
• antico ⁇ s having an impaired functional activity as previously described are also used for the preparation of a medicament to replace IgG4.
• the antico ⁇ s described in the invention in particular the antico ⁇ s having an improved functional activity due to metal cations, or the modified antico donts whose functional activity is impaired, or the compositions or solutions of the invention, can be chosen from anti Ep-CAM, anti-KIR3DL2, anti-EGFR, anti-VEGFR, anti HER1, anti HER2, anti GD, anti GD2, anti GD3, anti-CD20, anti CD-23, anti CD-25 , anti-CD30, anti-CD33, anti-CD38, anti-CD44, anti CD52, anti CA125 and anti ProteinC, anti-HLA-DR, anti-virals: HBV, HCV, HIV and RS V, and more particularly among the antico ⁇ s of Table I below: Table I: Name and brand Target company commercial indication of the antibody Edrecolomab Centocor anti Ep-CAM colorectal cancer PANOREX Rituximab Idea anti CD20 B cell lymphoma RITUXAN Licensed to thrombocytopenia pu
• IDEC-114 IDEC inhibition of non-Hodgkin's lymphoma ProteinC For example, the invention relates to a solution in the form of a concentrate at a concentration of antico ⁇ s ranging from 0.1 to 50 mg / ml, or 1 to 25 mg / ml which can be formulated for administration IV.
• the solute may contain as an example: 9.0 mg / mL sodium chloride, 7.35 mg / mL sodium citrate dihydrate, and 0.7 mg / mL polysorbate-80 in sterile water.
• 0.1 to 50 mg / ml, or 1 to 20 mg / ml of a cation are added to this solute or concentrate, the concentration of said cation being 1, 2, 3, 4 or 5 times the antico ants concentration either from 0.1 to 250 mg / mL or from 1100 mg / mL.
• This concentrate can be injected into a pocket of serum or infusion liquid so as to obtain the dose which it is desired to administer, while maintaining the same cation content relative to the antico ⁇ s content.
• the invention also relates to a lyophilized powder, sterile in a container and which can be reconstituted with sterile water just before injection comprising the appropriate amount of antico ⁇ s and the amount of said cation according to the invention 1 ", 2, 3 , 4 or 5 times higher than that of the antico ⁇ s.
• This powder can be reconstituted for IV or subcutaneous injection.
• the powder can comprise from 10 to 500 mg of antico ⁇ s and a quantity of said cation according to the invention 1, 2, 3, 4 or 5 times greater than that of antico ⁇ s, ie for example from 10 to 500 mg.
• excipients such as sucrose, an amino acid, polysorbate:
• the invention preferably relates to the compositions described above in which the cation content is at least equal to the content of antico ⁇ s, but also relates to any composition in which an amount of cation is added (zinc, iron, copper , cadmium, or the like, or a mixture thereof) less than said equimolar amount, for example 0.1 to 0.99 molar (0.1, 0.2, 0.3, 0.4, 0.5, 0 , 6, 0.7, 0.8 or 0.9 molar), but still sufficient to improve by at least 10% or 25%, or 50% or even 100% the effectiveness and / or the functional properties of the antico ⁇ s.
• an amount of cation is added (zinc, iron, copper , cadmium, or the like, or a mixture thereof) less than said equimolar amount, for example 0.1 to 0.99 molar (0.1, 0.2, 0.3, 0.4, 0.5, 0 , 6, 0.7, 0.8 or 0.9 molar), but still sufficient to improve by at least 10% or
• Figure 1 Diagram showing the position of Zn2 + ions in the vicinity of the Fc fragment of the monoclonal antico ⁇ s EMAB5 (2 ⁇ ).
• Figure 2 Detail of the electronic density map in the vicinity of histidine residues 310 and 435.
• Figure 3 Supe ⁇ osition of the structures of the Fc fragment of the antico ⁇ s EMAB5 obtained in the absence (gray) and in the presence (white) of Zn2 + ion.
• Figure 4 Attachment of the antico ⁇ s EMAB5 modified by DEPC to the Fc ⁇ RIH receptor. This figure shows, on the abscissa, the fixation of the antico ⁇ s on the red cells and on the ordinate the fixation of the CD 16 (Fc ⁇ RJH) present on the surface of the Jurkat CD 16 cells.
• f antico ⁇ s witness AD1;
• M EMAB5 control;
• A EMAB5 FNR;
• Figure 5 Activation by antico ⁇ s EMAB5 modified by DEPC of the Fc ⁇ PJII receptor present on Jurkat CD 16 cells.
• This figure represents the quantity, expressed in pg / ml, of IL-2 secreted by Jurkat CD16 cells whose CD16 receptor was activated by the antico EMs EMAB5 control () and the fractions separated on protein A after modification of the antico ⁇ s EMAB5 by DEPC: FNR (•), FR (M).
• Figure 6 Effect of the DEPC modification on the ADCC activity of the EMAB5 monoclonal antibody.
• This figure represents the percentage of lysis of Rh red blood cells (D +) induced by the control monoclonal antico ⁇ s EMAB5 and by the 2 fractions FR and FNR, separated on Sepharose-protein A gel, of the antico ⁇ s modified by DEPC.
• Figure 7 Measurement of the Fc binding of the doubly mutated His310-435Lys antico ⁇ s to the Fc ⁇ R ⁇ I receptor.
• Anticoips 1C7, 2H11, 4G5 and 4H10 are the mutated antico ⁇ s.
• Antico ⁇ s 16D11, 11G5 and 6H11 are not mutated.
• This figure shows on the abscissa, the fixation of the antico ⁇ s on the red cells and on the ordinate the fixation of the CD 16 (Fc ⁇ RIII) present on the surface of the Jurkat CD 16 cells.
• the figure groups together the results obtained with the supernatants containing the doubly mutated antico ⁇ s (curves in solid lines) and those containing non-mutated antico ⁇ s (dotted curves).
• Figure 10 Study by flow cytometry of the binding of antico ⁇ s carrying the double mutation His310-435Lys to the CD 16 receptor
• EMAB5 It is a human IgGl ( ⁇ ), directed against the Rh (D) antigen, produced in the cell line YB2 / 0 (rat myeloma, line ATCC No. CRL 1662) suitable for culture in medium without serum.
• EMAB5 was purified by affinity chromatography on Sepharose- protein A.
• ADCC activity of EMAB5 is at least equal to that of the reference anti-Rh (D) polyclonal antibody, WinRho (Cangene).
• the purified antico ⁇ s EMAB5 is dialyzed overnight against 50 mM Tris buffer, pH 8.0.
• the antico d'ants solution adjusted to 50 mM CaCl 2 and 10 mM cysteine, is incubated for 30 min. at 37 ° C before adding the trypsin solution (1 mg / ml) in an enzyme / substrate ratio of 1/25. After 5 a.m. incubation at 37 ° C, the reaction is stopped by the addition of diisopropyl fluorophosphate (1 mM final).
• the hydrolyzate is dialyzed overnight against 50 mM Imidazole buffer, pH 7.8.
• the dialysis hydrolyzate is brought into contact with Affarose-protein L at the rate of 1 ml of gel for 3.6 mg of antico ⁇ s. After 4 h. incubation at room temperature with shaking, the gel is mounted in a column and washed with 50 mM Imidazole buffer, pH 7.8. The effluent and the washing buffer which contain the Fc fragments are combined, concentrated by centrifugation on Vivaspin 20 using the conditions described by the manufacturer.
• Crystallogenesis After development, the crystallization conditions adopted are as follows: the solution of Fc fragments, at 2 mg / ml in 50 mM imidazole buffer, pH 7.8, is brought to 10% of 5,000, 100 mM monomethyl polyethylene glycol sodium cacodylate, 0.1 mM zinc chloride, pH 5.1 by vapor diffusion in seated drops at 17 ° C. Collection of diffraction data and determination of the structure: The crystal obtained is subjected to X-rays at the ESRF in Grenoble and the information collected is processed by the DENZO and SCALEPACK programs (Otwinowski and Minor, 1997). The structure is solved and refined to 2.3 ⁇ (Fig.l) using the rest of the CCP4 programs.
• the three-dimensional structure (shown diagrammatically in FIG. 1) makes it possible to highlight the presence of zinc ions (in white) near the CH2 and CH3 domains (in gray).
• the electronic density map presented in FIG. 2 shows the zinc ion linked to the histidine residues 310 (CH2) and 435 (CH3). Another zinc ion is linked to histidine 268 from an Fc and to histidine 285 from a symmetrical Fc. The third is near histidine 433.
• the crystals obtained all belong to the space group
• P2 (l) 2 (l) 2 (l) with two chains per asymmetric unit and the opening of the Fc is characterized by the distances between the proline residues 329 of chains A and B and between the mannose residues 4 of chains A and B.
• the Fc fragments of Pantico ⁇ s EMAB5 are characterized by a distance between pralines 329 of 32.53 ⁇ and a distance between mannoses 4 of 17.61 ⁇ . When a metal ion is added to the crystallization solution (see Table II), these characteristic distances increase.
• Table II Main characteristics of the crystals of the Fc fragment of the monoclonal antico ⁇ s EMAB5 in the space group P2 (l) 2 (l) 2 (l).
• FIG. 3 shows the supe ⁇ osition of the main chains of the structures obtained in the absence (in gray) and in the presence of zinc (white) in the crystallization solution.
• the addition of a metal salt to the Fc solution therefore promotes a so-called open conformation, a conformation close to that of Fc linked to the Fc ⁇ RHI receptor.
• Diethylpyrocarbonate is a reagent that has been widely used to modify and study the role of histidine residues in proteins (Miles, 1977). Firan et al. (2001) showed that human IgGl treated with DEPC lost their ability to bind the FcRn receptor which is involved in the transfer of maternal IgG to the fetus. DEPC acts by substitution of a nitrogen group present on the imidazole cycle of histidine, thus transforming the histidine residue into 3-carboethoxy histidine.
• the monoclonal antico ⁇ s EMAB5 dialysis against 0.1 M sodium acetate buffer, pH 6.0, is brought into contact with DEPC at a rate of 70 ⁇ g of DEPC / mg of IgG. After 30 minutes of incubation at room temperature, the reaction is stopped by the addition of imidazole (0.2 mg / ml final).
• the modified monoclonal antibody is fractionated by affinity chromatography on Sepharose-protein A.
• a fraction of the modified monoclonal antibody is not retained on the affinity gel and constitutes the non-retained fraction or FNR.
• the fraction retained on the gel and called FR is eluted with 0.1 M Glycine-HCl buffer, pH 2.8.
• the two fractions thus obtained are dialyzed against 20 mM sodium phosphate buffer, 50 mM NaCl, pH 7.2, concentrated on Vivaspin according to the manufacturer's recommendations and stored at 4 ° C for 15 days maximum.
• the control antico ⁇ s which serves as a reference, underwent the same treatment except for DEPC which was replaced by an identical volume of ethanol.
• the antico ⁇ s are subjected to a test, called CFC, which estimates the capacity of the antico ⁇ s to fix, firstly, the antigen against which they are directed then, in a second, the Fc ⁇ RlJJ (CD 16) receptor expressed on the surface of the Jurkat CD 16 cell line.
• CFC a test
• the wells of a microtiter plate are covered with papain Rb (D +) red cells.
• Anti-Rit (D) antico ⁇ s, diluted to concentrations varying from 7.8 to 500 ng / ml in UVIDM + 2.5% fetal calf serum (SVF) are deposited in parallel on two microtiter plates previously "coated” with red cells. After 90 min. incubation at 37 ° C, the wells are washed.
• One of the plates, used to detect IgG fixed on red cells, is incubated in the presence of an anti-human mouse Fc ⁇ antibody labeled with alkaline phosphatase (Jackson ImmunoResearch Laboratories).
• the Jurkat CD16 cells In the other plate, the Jurkat CD16 cells, diluted to the concentration of 2 ⁇ 10 6 cells / ml in DVLDM + 1% FCS, are added. After 15 min. of contact at 37 ° C., the plate is centrifuged by gradually increasing the speed and the duration of centrifugation until the negative controls are negativized.
• the expression “negative control” means antico ⁇ s which bind to the red cells immobilized in the wells of the microtitration plate but which do not fix the Fc ⁇ RIII receptor present on the surface of the Jurkat CD 16 cells. In the wells containing the negative control, the Jurkat cells CD 16, after centrifugation, form a cluster in the center of the well whereas in wells containing a positive control, Jurkat CD 16 cells line the well.
• the activation test of the Jurkat CD16 cells measures the secretion of interleukin-2 (IL-2) induced by the binding of the Fc of the antico ⁇ s to the Fc ⁇ RIJI receptor (CD 16) after binding of Fab to its antigen, present on the target cell.
• IL-2 interleukin-2
• the level of IL-2 secreted by Jurkat CD 16 cells is proportional to the activation of the CD 16 receptor.
• a 96-well microtiter plate 50 ⁇ l of dilutions of antico ⁇ s, 50 ⁇ l of a red cell suspension at 6.10 5 / ml, 50 ⁇ l of a suspension of Jurkat CD 16 cells at 1.10 6 / ml and 50 ⁇ l of a PMA solution at 40 ng / ml. All dilutions were carried out in EVIDM culture medium containing 5% FCS. After 16 hours of incubation at 37 ° C and 7% CO 2, the microtiter plate is centrifuged and the amount of IL-2 contained in the supernatant is assayed by a commercial kit (Duoset, R&D). The levels of secreted IL-2 are expressed in pg / ml. The results are expressed in% of CD16 activation, the level of dTL-2 secreted in the presence of the control monoclonal antibody being considered equal to 100%.
• ADCC Antibody-Dependent Cell-mediated Cytotoxicity
• the red cells of a globular concentrate RbD (+) are treated with papain (1 mg / ml, 10 min at 37 ° C.) then washed in 0.9% NaCl.
• the effector cells are isolated from a pool of at least 3 buffy coats, by centrifugation on Ficoll (Amersham Biosciences), followed by an adhesion step in the presence of 25% of FCS, so as to obtain a lymphocyte / monocyte ratio of the order of 9.
• each well is deposited: 100 ⁇ l of a dilution of purified anti-Rh (D) antico ⁇ s (from 9.3 to 150 ng / ml), 25 ⁇ l of Rh (Dr-) papain red cells at 4 x 10 7 , 25 ⁇ l of effector cells at 8 x .10 7 and 50 ⁇ l of polyvalent IgG (Tegeline, LFB) at the usual concentrations of 2 and 10 mg / ml.
• the dilutions are made in EVIDM containing 0.25% of SVF.
• the plates are centrifuged, then the hemoglobin released in the supernatant is measured via its peroxidase activity in the presence of a chromogenic substrate, 2,7-diaminofluorene (DAF).
• DAF 2,7-diaminofluorene
• the results are expressed as a percentage of lysis, 100% corresponding to the total lysis of the red cells in NH 4 C1 (control 100%) and 0% to the reaction mixture without antico ⁇ s (control 0%).
• the specific lysis is calculated as a percentage according to the following formula:
• the F ⁇ R fraction After treatment with DEPC according to the conditions described above, approximately 20% of the molecules of the monoclonal antico ⁇ s EMAB5, constituting the FNR fraction, lose their capacity to bind to the gel of Sepharose-protein A. Knowing that histidine 435 is an essential amino acid for the attachment of IgG to protein A, it seems likely that the IgG of the FNR fraction differs from the FR fraction, retained on the Sepharose-protein A gel, by the modification of the residue His435. In the test for measuring the binding to the CD 16 receptor in the presence of antigen, the F ⁇ R fraction has the same capacities as the FR fraction, capacities which are identical to those of the control antico ⁇ s (Fig. 4).
• the binding of the different fractions of the monoclonal antico ⁇ s EMAB5, modified or not by DEPC, is much higher than that of the monoclonal antico ⁇ s AD1, used in this test as a negative control.
• the modification of histidine residues by DEPC does not induce a change in the ability of the monoclonal antibody EMAB5 to bind to Rh (D +) red blood cells or to the CD 16 receptor present on the surface of the Jurkat cell line. CD 16.
• the functional activity of the monoclonal antico ⁇ s EMAB5 modified by DEPC is very slight.
• the secretion of IL-2 of the Jurkat CD16 line induced by the FR and FNR fractions of the monoclonal antibody EMAB5 modified by DEPC represents, respectively, 42.8% and 19.5% of the secretion induced by the antibody witness (Fig. 5).
• the results of the ADCC activity, expressed as% of actual lysis, show that the activity of the FNR fraction is lower than that of the FR fraction (FIG. 6).
• the FR fraction shows a decrease in ADCC activity compared to the control antico ⁇ s, when the amount of antico ⁇ s added to the well is lower ( ⁇ 20 ng / ml) and that the amount of Multipurpose IgG is 2.5 mg / ml.
• ADCC induction of cytokine secretion
• histidine residues by a chemical agent (DEPC or other) does not make it possible to control either the degree or the location of the modifications.
• a chemical agent DEPC or other
• His310 and His435 which play an essential role in the binding of the zinc cation at the CH2-CH3 interface of IgGl, are replaced by lysine residues, by site-directed mutagenesis.
• the YB2 / 0 cells co-transfected by electroporation with the mutated vector EMAB5- H-K338-K463-1 and the vector EMAB5- dhfr-K- Spel encoding the light chain of the antico EMs EMAB5, are cultured in RPMI medium supplemented with 5% of SVF dialysis, 0.5% of G418 and 25 nM of Methotrexate (MTX).
• the clones secreting the highest levels of human IgG are cultured in 24-well plates in medium without MTX. The supernatants, harvested after 7 days of culture, are used to carry out the tests described below. 2.
• CFC Fc ⁇ RIII receptor
• This test is carried out on culture supernatants, the level of human IgG contained in the supernatants being determined by ELISA assay.
• the wells of a microtiter plate are covered by papainized Rh (D +) red cells.
• Culture supernatants containing native or mutated anti-Rh (D) antico dils and diluted to concentrations varying from 7.8 to 500 ng / ml in IMDM + 2.5% FCS are deposited in parallel on two microtiter plates previously “coated” by the red cells. After 90 min. incubation at 37 ° C, the wells are washed.
• One of the plates, used to detect the IgGs fixed on the red cells is incubated in the presence of an anti-human mouse anti-Fc ⁇ labeled with alkaline phosphatase (Jackson LnmunoReaserch Laboratories).
• Jurkat CD 16 cells are added after 15 min. contact at 37 ° C., the plate is centrifuged by gradually increasing the speed and the duration of centrifugation until the negative controls are negativized. After centrifugation, the wells are read and a score is given as a function of the spreading of Jurkat CD 16 cells in the well.
• the microtiter plate After 16 hours of incubation at 37 ° C and 7% CO 2, the microtiter plate is centrifuged and the amount of IL-2 contained in the supernatant is assayed by a commercial kit (Duoset, R&D). The levels of secreted IL-2 are expressed in pg / ml.
• the various tests were carried out on the culture supernatants containing the anti-Rh (D) monoclonal antibodies that were mutated or not.
• the results of the CFC test show that the double mutation His310-435Lys does not induce any modification in the binding of the antico ⁇ s to the Fc ⁇ RIII receptor, carried by the Jurkat CD16 cell line (Fig. 7). While the non mutated clone 6H11 (negative control) exhibits binding to the receptor Fc ⁇ RUI decreased, the mutated clones 1C7, 2H11, 4G5 and 4H10 bind the Fc ⁇ RJJJ receptor similarly to the non-mutated clones 16D11 and 11G5 (positive controls).
• CD 16 activation was carried out on the culture supernatants of 4 mutated clones mentioned above and of 3 non-mutated clones (16D11, 11G5 and 24G9).
• the results represent the mean (+/- standard deviation) of the levels of IL-2 secreted by the Jurkat CD 16 cells in the presence of the non-mutated clones (Native) and of the mutated clones (His310-435Lys).
• the mutated antico ⁇ s induce a secretion of_L-2 very reduced compared to that induced by the native antico ⁇ s (Fig. 8).
• the mutated antico ⁇ s have a decrease in capacity to activate the Fc ⁇ RI ⁇ receptor by 50%.
• the addition of 50 mM of imidazole in the Pantico ⁇ s incubation buffer with the cells causes a decrease in fixation of the antico ⁇ s which results in a significant decrease in the percentage of labeled cells; at the antico ants concentration of 1.5 ⁇ g / ml, the presence of imidazole induces a 40% decrease in the number of labeled cells.
• Imidazole is a reagent that has the property of fixing cations.
• the fixation of the antico ⁇ s on the CD 16 receptor is reduced.
• the results expressed as a percentage of cells labeled as a function of the amount of antico ⁇ s added show that the antico ⁇ s 4G5 and 4H10, which have the double mutation His310-435Lys, bind significantly less well to Jurkat CD 16 cells than the clone 24G9, which is the non-mutated control antico ⁇ s.

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