EP3174904A1 - Verfahren zur herstellung von varianten mit einem fc mit verbesserter sialylierung - Google Patents

Verfahren zur herstellung von varianten mit einem fc mit verbesserter sialylierung

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Publication number
EP3174904A1
EP3174904A1 EP15759880.6A EP15759880A EP3174904A1 EP 3174904 A1 EP3174904 A1 EP 3174904A1 EP 15759880 A EP15759880 A EP 15759880A EP 3174904 A1 EP3174904 A1 EP 3174904A1
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EP
European Patent Office
Prior art keywords
fragment
parent polypeptide
variant
amino acid
mutation
Prior art date
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EP15759880.6A
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English (en)
French (fr)
Inventor
Céline MONNET
Alexandre Fontayne
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LFB SA
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LFB SA
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    • CCHEMISTRY; METALLURGY
    • C07ORGANIC CHEMISTRY
    • C07KPEPTIDES
    • C07K16/00Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • CCHEMISTRY; METALLURGY
    • C07ORGANIC CHEMISTRY
    • C07KPEPTIDES
    • C07K16/00Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/18Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
    • C07K16/28Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants
    • C07K16/2887Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants against CD20
    • CCHEMISTRY; METALLURGY
    • C07ORGANIC CHEMISTRY
    • C07KPEPTIDES
    • C07K16/00Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/18Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
    • C07K16/34Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against blood group antigens
    • CCHEMISTRY; METALLURGY
    • C07ORGANIC CHEMISTRY
    • C07KPEPTIDES
    • C07K2317/00Immunoglobulins specific features
    • C07K2317/40Immunoglobulins specific features characterized by post-translational modification
    • C07K2317/41Glycosylation, sialylation, or fucosylation
    • CCHEMISTRY; METALLURGY
    • C07ORGANIC CHEMISTRY
    • C07KPEPTIDES
    • C07K2317/00Immunoglobulins specific features
    • C07K2317/50Immunoglobulins specific features characterized by immunoglobulin fragments
    • C07K2317/52Constant or Fc region; Isotype
    • CCHEMISTRY; METALLURGY
    • C07ORGANIC CHEMISTRY
    • C07KPEPTIDES
    • C07K2317/00Immunoglobulins specific features
    • C07K2317/70Immunoglobulins specific features characterized by effect upon binding to a cell or to an antigen
    • C07K2317/71Decreased effector function due to an Fc-modification
    • CCHEMISTRY; METALLURGY
    • C07ORGANIC CHEMISTRY
    • C07KPEPTIDES
    • C07K2317/00Immunoglobulins specific features
    • C07K2317/90Immunoglobulins specific features characterized by (pharmaco)kinetic aspects or by stability of the immunoglobulin
    • CCHEMISTRY; METALLURGY
    • C07ORGANIC CHEMISTRY
    • C07KPEPTIDES
    • C07K2317/00Immunoglobulins specific features
    • C07K2317/90Immunoglobulins specific features characterized by (pharmaco)kinetic aspects or by stability of the immunoglobulin
    • C07K2317/94Stability, e.g. half-life, pH, temperature or enzyme-resistance
    • CCHEMISTRY; METALLURGY
    • C07ORGANIC CHEMISTRY
    • C07KPEPTIDES
    • C07K2319/00Fusion polypeptide
    • C07K2319/30Non-immunoglobulin-derived peptide or protein having an immunoglobulin constant or Fc region, or a fragment thereof, attached thereto
    • CCHEMISTRY; METALLURGY
    • C07ORGANIC CHEMISTRY
    • C07KPEPTIDES
    • C07K2319/00Fusion polypeptide
    • C07K2319/90Fusion polypeptide containing a motif for post-translational modification

Definitions

  • the present invention relates to a method for increasing the sialylation of an Fc fragment, comprising a step of mutating at least one amino acid selected from amino acids in position 240 to 243, 258 to 267 and 290 to 305 of said fragment.
  • Fc the numbering being that of the EU index or equivalent in Kabat.
  • Monoclonal antibodies are used today as therapeutics to treat a variety of conditions, including cancers, autoimmune diseases, chronic inflammatory diseases, transplant rejection, infectious diseases, and cardiac diseases. vascular. They are therefore a major therapeutic issue. Many of them are already on the market, and an ever increasing proportion is under development or clinical trials. However, there is an important need to optimize the structural and functional properties of the antibodies, in order to control the side effects.
  • Immunoglobulin isotype G (IgG) is the class of immunoglobulin most common in humans and also the most used in therapy.
  • Various experiments of specific mutagenesis in the constant region (Fc) of mouse IgG have identified certain critical amino acid residues involved, for some, in the interaction between IgG and FcRn (Kim et al, 1994, Eur J Immunol; 24: 2429-34;
  • the present invention provides means for obtaining a variant of a parent polypeptide comprising an Fc fragment having optimized sialylation.
  • This optimized sialylation i.e. improved, in particular gives the variant an increased half-life, as well as optimized anti-inflammatory properties compared to a parent polypeptide.
  • half-life refers to a biological half-life of a polypeptide of interest in the circulation of a given animal, and is represented by the time required for half the amount present in the circulation of a given animal. animal to be removed from the circulation and / or other tissues of the animal.
  • the subject of the present invention is therefore a process for increasing the sialylation of an Fc fragment, comprising a step of mutation of at least one amino acid chosen from amino acids in position 240 to 243, 258 to 267 and 290 to 305 of said fragment Fc, the numbering being that of the EU index or equivalent in Kabat.
  • said method for increasing the sialylation of an Fc fragment comprises:
  • the present invention also relates to a method for producing a variant of a parent polypeptide comprising an Fc fragment, said variant having a improved sialylation of said Fc fragment with respect to sialylation of the Fc fragment of the parent polypeptide, which comprises a step of mutating at least one amino acid selected from amino acids at position 240 to 243, 258 to 267 and 290 to 305 of said fragment Fc, the numbering being that of the EU index or equivalent in Kabat.
  • said method of producing a variant of a parent polypeptide comprising an Fc fragment comprises:
  • said method of producing a variant of a parent polypeptide comprising an Fc fragment is such that the variant has at least one effector activity mediated by said Fc fragment decreased relative to the effector activity of the parent polypeptide.
  • sialylation or “improved sialylation” it is meant that the sialylation of the obtained protein is increased by at least 10%, preferably at least 15%, preferably at least 20%, preferably at least 25%. %, preferably at least 30%, preferably at least 35%, preferably at least 40%, preferably at least 45%, preferably at least 50%, preferably at least 55%, preferably at least 60% preferably at least 65%, preferably at least 70%, preferably at least 75%, preferably at least 80%, preferably at least 85%, preferably at least 90%, preferably at least 95%, with respect to the sialylation of said Fc fragment prior to the mutation step or said Fc fragment of the parent polypeptide.
  • Sialylation of a protein is a well known glycosylation mechanism (see especially Essentials of Glycobiology, 2 nd edition, Varki et al, 2009). It corresponds to a covalent addition of at least one sialic acid (ie N-acetylneuraminic acid and its derivatives, such as N-glycosylneuraminic acid, N-acetylglycoylneuraminic acid) in the glycosylated chain of the protein.
  • sialic acid ie N-acetylneuraminic acid and its derivatives, such as N-glycosylneuraminic acid, N-acetylglycoylneuraminic acid
  • protein and “polypeptide” are used interchangeably herein and refer to a sequence of at least two covalently linked amino acids, including proteins, polypeptides, oligopeptides, and peptides. .
  • protein and polypeptide include especially antibodies or immunoglobulins, in particular whole, monoclonal, multi-specific, bi-specific, dual-specific, synthetic, chimeric, humanized, human, fusion proteins with immunoglobulins, conjugated antibodies, and their fragments.
  • protein and polypeptide also include Fc polypeptides defined by a polypeptide comprising all or part of an Fc region, including isolated Fc, conjugated Fc, multimeric Fc fragments and Fc fusion proteins.
  • Fc fragment or “Fc region” is meant the constant region of a full length immunoglobulin excluding the first immunoglobulin constant region domain (ie CH1-CL).
  • the Fc fragment refers to a homodimer, each monomer comprising the last two constant domains of IgA, IgD, IgG (ie CH2 and CH3), or the last three constant domains of IgE and IgM (ie CH2, CH3 and CH4), and the flexible N-terminal hinge region of these domains.
  • the Fc fragment when it is derived from IgA or IgM, can comprise the J chain.
  • an Fc fragment of an IgG1, which consists of the flexible N-terminal hinge is used in the present invention.
  • an Fc fragment of a human IgG1 ie amino acids 226 to 447 according to the EU index or equivalent in Kabat
  • the lower hinge refers to positions 226 to 230
  • the CH2 domain refers to positions 231 to 340
  • the CH3 domain refers to positions 341-447 according to the EU index or equivalent in Kabat.
  • the Fc fragment used according to the invention may also comprise part of the upper hinge region, upstream of the position 226.
  • a Fc fragment of a human IgG1 comprising part of the region is used. located between positions 216 to 226 (according to the EU index).
  • the Fc fragment human IgG1 refers to the portion from amino acid 216, 217, 218, 219, 220, 221, 222, 223, 224 or 225 to the C-terminus.
  • Fc fragment includes a scFc fragment for "single chain Fc".
  • scFc fragment is meant a single chain Fc fragment, obtained by genetic fusion of two Fc monomers linked by a polypeptide linker. The scFc folds naturally into a functional dimeric Fc region.
  • the Fc fragment used in the context of the invention is chosen from the Fc fragment of an IgG1 or IgG2. More preferably, the Fc fragment used is the Fc fragment of an IgG1.
  • the numbering of the residues of the Fc fragment is that of the EU index or equivalent in Kabat (Sequences of Proteins of Immunological Interest, 5th Ed. Public Health Service, National Institutes of Health, Bethesda, Md. (1991) ).
  • the parent polypeptide comprises an Fc fragment, referred to as a "parent Fc fragment”.
  • amino acid mutation is meant here a change in the amino acid sequence of a polypeptide.
  • a mutation is chosen in particular from a substitution, an insertion and a deletion.
  • substitution is meant the replacement of one or more amino acids at a particular position in a parent polypeptide sequence by the same number of other amino acids.
  • the substitution is punctual, ie it concerns only one amino acid.
  • the N434S substitution refers to a variant of a parent polypeptide, wherein the asparagine at position 434 of the Fc fragment according to the EU index or equivalent in Kabat is replaced by serine.
  • insertion is meant the addition of at least one amino acid at a particular position in a parent polypeptide sequence.
  • insertion G> 235-236 refers to a glycine insertion between positions 235 and 236.
  • deletion is meant the removal of at least one amino acid at a particular position in a parent polypeptide sequence .
  • E294del refers to the removal of glutamic acid at position 294; such a deletion is called Del294.
  • parent polypeptide is meant an unmodified polypeptide which is then modified to generate a variant.
  • the parent polypeptide may be a naturally occurring polypeptide, a variant of a naturally occurring polypeptide, a modified version of a natural polypeptide, or a synthetic polypeptide.
  • the parent polypeptide comprises an Fc fragment selected from wild-type Fc fragments, fragments and mutants thereof. Therefore, the parent polypeptide may optionally include pre-existing amino acid modifications in the Fc fragment relative to wild-type Fc fragments.
  • the Fc fragment of the parent polypeptide already comprises at least one additional mutation (ie pre-existing modification), preferably selected from P230S, T256N, V259I, N315D, A330V, N361D, A378V, S383N, M428L, N434Y.
  • the Fc fragment of the parent polypeptide comprises at least one combination of additional mutations chosen from P230S / N315D / M428L / N434Y, T256N / A378V / S383N / N434Y, V259I / N315D / N434Y and N315D / A330V / N361D / A378V / N434Y .
  • the parent polypeptide consists of an Fc fragment, and preferably an entire Fc fragment.
  • the parent polypeptide consists of an amino acid sequence fused to N- or C-terminal to an Fc fragment.
  • the parent polypeptide is an antibody, an Fc fusion polypeptide or an Fc conjugate.
  • the Fc fragment of the parent polypeptide is chosen from the sequences SEQ ID NO: 1, 2, 3, 4 and 5.
  • the Fc fragment of the parent polypeptide has the sequence SEQ ID NO: 1.
  • sequences shown in SEQ ID Nos: 1, 2, 3, 4 and 5 are free of an N-terminal hinge region.
  • sequences represented in SEQ ID NO: 6, 7, 8, 9 and 10 respectively correspond to the sequences represented in SEQ ID NOs: 1, 2, 3, 4 and 5 with their hinge regions at the N-terminal.
  • the Fc fragment of the parent polypeptide is chosen from the sequences SEQ ID NO: 6, 7, 8, 9 and 10.
  • the Fc fragment of the parent polypeptide has a sequence corresponding to positions 1-232, 2-232, 3-232, 4-232, 5-232, 6-232, 7-232, 8-232, 9-232. , 10-232 or 11-232 of the sequence SEQ ID NO: 6.
  • the parent polypeptide consists of an immunoglobulin, an antibody or an amino acid sequence fused to an N- or C-terminal to an antibody or an immunoglobulin.
  • variant is meant a polypeptide sequence which is different from the parent polypeptide sequence by at least one amino acid modification.
  • the sequence of the variant has at least 80% identity with the sequence of the parent polypeptide, and more preferably at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%. , 98%, 99% or 99.5% identity.
  • percent identity between two amino acid sequences in the sense of the present invention, it is meant to designate a percentage of identical amino acid residues between the two sequences to be compared, obtained after the best alignment, this percentage being purely statistics and the differences between the two sequences being randomly distributed over their entire length.
  • Best alignment or “optimal alignment” is meant the alignment for which the percentage of identity determined as hereinafter is the highest.
  • Sequence comparisons between two amino acid sequences are traditionally performed by comparing these sequences after optimally aligning them, said comparison being performed by segment or by "comparison window" to identify and compare the local regions of sequence similarity. .
  • the optimal alignment of the sequences for comparison can be realized, besides manually, by means of the local homology algorithm of Smith and Waterman (1981, J.
  • the parent polypeptide is an immunoglobulin or an antibody, preferably an IgG, and the variant according to the invention is then selected from IgG variants. More preferentially, the variant according to the invention is chosen from human IgG 1, IgG 2, IgG 3 and IgG 4 variants.
  • the process for producing a variant according to the invention or the process for increasing the sialylation according to the invention comprises a mutation carried out on at least one amino acid of the Fc fragment located at position 240, 241, 242, 243, 258, 259, 260, 261, 262, 263, 264, 265, 266, 267, 290, 291, 292, 293, 294, 295, 296, 297, 298, 299, 300, 301, 302, 303, 304 or 305, the numbering being that of the EU index or equivalent in Kabat.
  • the process for producing the variant or the method for increasing the sialylation according to the invention comprises at least one mutation on the Fc fragment chosen from V262del, V263F, V263K, V263W, V264K, V264P, D265A, D265E, D265G.
  • the invention relates to a method for increasing the sialylation of an Fc fragment, comprising a step of mutating at least one amino acid selected from amino acids in position 240 to 243, 258 to 267 and 290 to 305 of said Fc fragment, the numbering being that of the EU index or equivalent in Kabat, excluding the amino acids at position 262 and 264.
  • the invention also preferably relates to a method for producing a variant of a parent polypeptide comprising an Fc fragment, said variant having a sialylation improved of said Fc fragment relative to the parent polypeptide, which comprises a step of mutating at least one amino acid selected from amino acids in position 240 to 243, 258 to 267 and 290 to 305 of said Fc fragment, the numbering being that of the EU index or equivalent in Kabat, and wherein said mutating step does not relate to any of the amino acids at position 262 or 264.
  • the mutation is carried out on the amino acid of the Fc fragment located in position 240, 241, 242, 243, 258, 259, 260, 261, 263, 265, 266, 267, 290, 291, 292, 293, 294, 295, 296, 298, 299, 300, 301, 302, 303, 304 or 305. More preferably, the mutation is carried out on the amino acid of the Fc fragment located in position 293 or 294, the numbering being that of the EU index or equivalent in Kabat. According to a particular embodiment, the mutation is carried out on the two amino acids of the Fc fragment located at position 293 and at position 294, the numbering being that of the EU index or equivalent in Kabat.
  • the subject of the present invention is also a process for producing a variant of a parent polypeptide comprising an Fc fragment, said variant having at least one effector activity mediated by said Fc fragment, decreased with respect to the effector activity of the parent polypeptide , said method comprising a step of mutating at least one amino acid selected from amino acids in position 240 to 243, 258 to 267 and 290 to 305 of said Fc fragment, the numbering being that of the EU index or equivalent in Kabat .
  • Fc fragment-mediated effector activity is meant, in particular, antibody-dependent cellular cytotoxicity (ADCC or Antibody-Dependent Cell- mediated Cytotoxicity), complement-dependent cytotoxicity (CDC or Complement Depends Cytotoxicity), cell-dependent cellular phagocytosis. antibodies (ADCP), endocytosis activity or secretion of cytokines.
  • the effector activity mediated by the Fc fragment considered in the invention is selected from antibody-dependent cellular cytotoxicity (ADCC), complement-dependent cytotoxicity (CDC), and antibody-dependent cellular phagocytosis (ADCP).
  • DCC antibody-dependent cellular cytotoxicity
  • CDC complement-dependent cytotoxicity
  • ADCP antibody-dependent cellular phagocytosis
  • diminished effector activity is meant a diminished or abolished effector activity.
  • a variant of a parent polypeptide produced by a method according to the invention may have at least one effector activity mediated by the abolished Fc fragment.
  • a variant of a parent polypeptide produced by a method according to the invention has an effector activity mediated by the decreased Fc region, relative to that of the parent polypeptide, of at least 10%, preferably at least 20%, 30%, 40%, 50%, 60%, 70%, 80%, or 90%.
  • the invention provides a method for producing a variant of a parent polypeptide comprising an Fc fragment, said variant lacking any effector activity mediated by said Fc fragment, comprising a mutation step of at least one amino acid selected from amino acids in position 240 to 243, 258 to 267 and 290 to 305 of said Fc fragment, the numbering being that of the EU index or equivalent in Kabat.
  • the invention provides a method for producing a variant of a parent polypeptide comprising an Fc fragment, said variant exhibiting at least one effector activity mediated by said Fc fragment, decreased in relation to the effector activity of the parent polypeptide, comprising a step of mutating at least one amino acid selected from amino acids in position 240 to 243, 258 to 267 and 290 to 305 of said Fc fragment, the numbering being that of the EU index or equivalent in Kabat, and wherein the mutation step is not directed to any of the amino acids at position 262, 264, 293 or 294.
  • the present invention provides a method for producing a variant of a parent polypeptide comprising an Fc fragment, said variant having an affinity mediated by said Fc fragment, decreased relative to the affinity of the parent polypeptide for at least one of the Fc region receptors (FcR) comprising a step of mutating at least one amino acid selected from amino acids at position 240 to 243, 258 to 267 and 290 to 305 of said Fc fragment, numbering being that of the EU index or equivalent in Kabat.
  • Fc region receptor or “FcR” is meant in particular Clq and Fcy receptors (FcyR).
  • Fc ⁇ receptors or “Fc ⁇ R” refer to the IgG-type immunoglobulin receptors, called CD64 (Fc ⁇ RIII), CD32 (Fc ⁇ RII), and CD16 (FCYRIII), in particular to the five expressed Fc ⁇ RIa, Fc ⁇ RIIa, Fc ⁇ RIIb, Fc ⁇ RIIIa receptors. and PcyRIIIb. All are effector cell activating receptors, except for human Fc ⁇ RIIb which is an inhibitory receptor for immune cell activation (Muta T et al., Nature, 1994, 368: 70-73).
  • the Clq complement is involved in the CDC activity.
  • the FcgRIIIa receptor (CD 16a) is involved in the ADCC; it has a V / F polymorphism at position 158.
  • the FcgRIIa receptor (CD32a) is involved in platelet activation and phagocytosis; it has an H / R polymorphism at position 131.
  • FcgRIIb receptor (CD32b) is involved in the inhibition of cellular activity.
  • the affinity is reduced, relative to that of the parent polypeptide comprising the Fc fragment, by at least 10%, preferably by at least 20%, 30%, 40%, 50%, 60%, 70%. , 80%, or 90%.
  • the invention provides a method for producing a variant of a parent polypeptide comprising an Fc fragment, said variant having an affinity mediated by said decreased Fc fragment relative to the affinity of the parent polypeptide, for at least one receptors of the Fc (FcR) region chosen from Clq complement and FcgRIIIa (CD16a), FcgRIIa (CD32a) and FcgRIIb (CD32b) receptors, comprising a step of mutating at least one amino acid selected from amino acids in position 240 to 243, 258 to 267 and 290 to 305 of said Fc fragment, the numbering being that of the EU index or equivalent in Kabat.
  • FcR Fc receptor region chosen from Clq complement and FcgRIIIa
  • CD32a FcgRIIa
  • CD32b FcgRIIb
  • the variant produced according to the invention has an affinity mediated by the decreased Fc fragment relative to that of the parent polypeptide, by at least 10%, of preferably at least 20%, 30%, 40%, 50%, 60%, 70%, 80%, or 90%.
  • the affinity of the mutated Fc fragment for a FcR is less than that of the parent polypeptide.
  • the ratio of the affinity mediated by the Fc fragment of a variant according to the invention relative to that of the parent polypeptide is less than 0.9, preferably less than 0.8, preferably less than 0.7, preferably less than 0.7. 0.6, preferably less than 0.5, preferably less than 0.4, preferably less than 0.3, preferably less than 0.2, preferably less than 0.1.
  • the ratio of Fc fragment-mediated affinity of a variant according to the invention to that of the parent polypeptide is less than 0.7. More preferably, the ratio of the affinity mediated by the Fc fragment of a variant according to the invention relative to that of the parent polypeptide is between 0.9 and 0.1, preferably between 0.8 and 0.2, between 0.7 and 0.3, or between 0.6 and 0.4.
  • the affinity of a polypeptide comprising an Fc fragment for a FcR can be evaluated by methods well known in the art. For example, one skilled in the art can determine affinity (Kd) using surface plasmon resonance (SPR).
  • Binding affinity can be indifferently determined by evaluating whole polypeptides or evaluating isolated Fc regions thereof.
  • a variant produced according to a method which is the subject of the invention has an affinity mediated by said Fc fragment, decreased with respect to the affinity of the parent polypeptide, for the FcgRIIIa receptor (CD 16a), and the FcgRIIa receptor ( CD32A).
  • the invention provides a method for producing a variant of a parent polypeptide comprising an Fc fragment, said variant having an affinity mediated by said Fc fragment, decreased relative to the affinity of the parent polypeptide for at least one of the Fc region receptors (FcR), preferably selected from complement C lq and FcgRIIIa receptors (CD 16a), FcgRIIa (CD32a), and FcgRIIb (CD32b), and comprising a step of mutating at least one amino acid selected from amino acids in position 240 to 243, 258 to 267 and 290 to 305 of said Fc fragment, the numbering being that of the EU index or equivalent in Kabatet wherein the mutation step is not on any of the amino acids at position 262, 264, 293 or 294.
  • FcR Fc region receptors
  • the mutation is chosen from an insertion, a substitution, preferably a single substitution, and a deletion, and is carried out on at least one amino acid located at position 240, 241, 242, 243, 258, 259, 260, 261, 263, 265, 266, 267, 290, 291, 292, 295, 296, 298, 299, 300, 301, 302, 303, 304 or 305, the numbering being that of the EU index or equivalent in Kabat.
  • a variant of a parent polypeptide comprising an Fc fragment produced according to the invention may have an affinity for the FcRn receptor mediated by the Fc fragment, conserved or increased, with respect to said affinity of the parent polypeptide.
  • the mutation or mutations that comprise the variant according to the invention does not affect the affinity for the FcRn receptor mediated by the Fc fragment.
  • the variant of a parent polypeptide comprising an Fc fragment produced according to the invention comprises one or more mutations that do not affect the affinity for the FcRn receptor mediated by the Fc fragment relative to the affinity of the parent polypeptide.
  • FcRn or “neonatal Fc receptor” as used herein is meant a protein that binds to the Fc region of IgG and is encoded at least in part by an FcRn gene.
  • FcRn can be from any organism including, but not limited to, humans, mice, rats, rabbits and monkeys.
  • the functional FcRn protein comprises two polypeptides, often referred to as heavy chain and light chain. The light chain is beta-2-microglobulin and the heavy chain is encoded by the FcRn gene.
  • FcRn or FcRn protein refers to the ⁇ -chain complex with beta-2-microglobulin.
  • the gene encoding FcRn is called FCGRT.
  • the mutation step of the method for preparing the variant according to the invention is obtained as follows:
  • ii) modifying the nucleic sequence provided in i) to obtain a nucleic sequence coding for the variant; and iii) expressing the nucleic sequence obtained in ii) in a host cell, and recovering the variant.
  • Such a mutation step is thus performed using a nucleic sequence (polynucleotide or nucleotide sequence) encoding said parent polypeptide (step i)).
  • the nucleic acid sequence encoding the parent polypeptide may be synthesized chemically (Young L and Dong, 2004, -Nucleic Acids Res., Apr. 15; 32 (7), Hoover, DM and Lubkowski, J. 2002, Nucleic Acids Res., 30, Villalobos A, et al., 2006. BMC Bioinformatics, Jun 6; 7: 285).
  • the nucleotide sequence encoding the parent polypeptide may also be amplified by PCR using suitable primers.
  • the nucleotide sequence encoding the parent polypeptide may also be cloned into an expression vector.
  • the DNA coding for such a parent polypeptide is inserted into an expression plasmid and inserted into an ad hoc cell line for its production (for example the HEK-293 FreeStyle line, the YB2 / 0 line, or the CHO line).
  • the protein thus produced is then purified by chromatography.
  • nucleic sequence provided in i) (polynucleotide), which encodes the parent polypeptide, is then modified to obtain a nucleic sequence encoding the variant. This is step ii).
  • This step is the actual mutation stage. It can be performed by any known method of the prior art, in particular by site-directed mutagenesis or by random mutagenesis.
  • the random mutagenesis as described in the application WO02 / 038756 is used: it is the Mutagen technique. This technique uses a human DNA mutase, in particular chosen from DNA polymerases ⁇ , ⁇ and ⁇ . A step of selecting mutants having retained FcRn binding is necessary to retain the mutants of interest.
  • amino acid substitutions are preferably carried out by site-directed mutagenesis, by the use of P CR for the use of seeds.
  • degenerate oligonucleotides see, e.g., Zoller and Smith, 1982, Nucl Acids Res 10: 6487-6500, Kunkel, 1985, Proc Natl, Acad Sci USA 82: 488).
  • step iii) the nucleic sequence obtained in ii) is expressed in a host cell, and the variant thus obtained is recovered.
  • the cellular host may be chosen from prokaryotic or eukaryotic systems, for example bacterial cells, but also yeast cells or animal cells, in particular mammalian cells. Insect cells or plant cells can also be used.
  • the preferred host cells are the YB2 / 0 rat line, the CHO hamster line, in particular the CHO dhfr- and CHO Lecl3, PER.C6 TM (Crucell), HEK293, T1080, EB66, K562, NS0, SP2 / lines. 0, BHK or COS. More preferably, the YB2 / 0 rat line is used.
  • the host cells may be modified transgenic animal cells to produce the polypeptide in the milk.
  • the expression of a DNA sequence coding for the polypeptide according to the invention is controlled by a mammalian casein promoter or a mammalian whey promoter, said promoter not naturally controlling the transcription of said gene, and the DNA sequence further containing a secretion sequence of the protein.
  • the secretion sequence comprises a secretion signal interposed between the coding sequence and the promoter.
  • the animal can thus be chosen from sheep, goat, rabbit, sheep or cow.
  • the polynucleotide encoding the variant obtained in step ii) may also comprise optimized codons, in particular for its expression in certain cells (step iii)).
  • said cells include COS cells, CHO cells, HEK cells, BHK cells, PER.C6 cells, HeLa cells, NIH / 3T3 cells, 293 (ATCC # CRL1573), T2 cells, dendritic cells or monocytes.
  • the purpose of codon optimization is to replace natural codons with codons whose amino acid transfer RNAs (tRNAs) are the most sensitive. frequent in the cell type considered.
  • Codon optimization also plays on the prediction of mRNA secondary structures that could slow down reading by the ribosomal complex. Codon optimization also has an impact on the percentage of G / C that is directly related to the half-life of the mRNAs and therefore to their translation potential (Chechetkin, J. of Theoretical Biology 242, 2006 922-934). Codon optimization can be done by substitution of natural codons using codon frequency (Codon Usage Table) tables for mammals and more specifically for Homo sapiens. There are algorithms available on the internet and made available by the suppliers of synthetic genes (DNA2.0, GeneArt, MWG, Genscript) that make this sequence optimization possible.
  • the polynucleotide comprises codons optimized for expression in HEK cells, such as HEK293 cells, CHO cells, or YB2 / 0 cells. More preferably, the polynucleotide comprises codons optimized for its expression in YB2 / 0 cells. Alternatively, preferably, the polynucleotide comprises codons optimized for its expression in the cells of transgenic animals, preferably the goat, the rabbit, the ewe or the cow.
  • the variant obtained according to the invention may be combined with pharmaceutically acceptable excipients, and optionally extended release matrices, such as biodegradable polymers, to form a therapeutic composition.
  • the pharmaceutical composition can be administered orally, sublingually, subcutaneously, intramuscularly, intravenously, intraarterially, intrathecally, intraocularly, intracerebrally, transdermally, pulmonally, locally or rectally.
  • the active ingredient alone or in combination with another active ingredient, can then be administered in unit dosage form, in admixture with conventional pharmaceutical carriers.
  • Unit dosage forms include oral forms such as tablets, capsules, powders, granules and oral solutions or suspensions, sublingual and oral forms of administration, aerosols, subcutaneous implants cutaneous, transdermal, topical, intraperitoneal, intramuscular, intravenous, subcutaneous, intrathecal, intranasal administration forms and rectal administration forms.
  • the pharmaceutical composition contains a pharmaceutically acceptable carrier for a formulation that can be injected.
  • a pharmaceutically acceptable carrier for a formulation that can be injected.
  • a pharmaceutically acceptable carrier for a formulation that can be injected.
  • It may be in particular sterile iso-tonic formulas, saline solutions (with monosodium or disodium phosphate, sodium chloride, potassium chloride, calcium or magnesium chloride and the like, or mixtures of such salts), or compositions freeze-dried, which, during the addition of sterilized water or physiological saline as appropriate, allow the constitution of injectable solutions.
  • Dosage forms suitable for injectable use include sterile aqueous solutions or dispersions, oily formulations, including sesame oil, peanut oil, and sterile powders for the extemporaneous preparation of sterile injectable solutions or dispersions.
  • the form must be sterile and must be fluid to the extent that it must be injected by syringe. It must be stable under the conditions of manufacture and storage and must be preserved against the contaminating action of microorganisms, such as bacteria and fungi.
  • the dispersions according to the invention can be prepared in glycerol, liquid polyethylene glycols or mixtures thereof, or in oils. Under normal conditions of storage and use, these preparations contain a preservative to prevent the growth of micro-organisms.
  • the pharmaceutically acceptable carrier may be a solvent or dispersion medium containing, for example, water, ethanol, a polyol (eg, glycerine, propylene glycol, polyethylene glycol, and the like), suitable mixtures of these, and / or vegetable oils.
  • a surfactant such as lecithin.
  • Prevention of the action of microorganisms can be caused by various antibacterial and antifungal agents, for example, parabens, chlorobutanol, phenol, sorbic acid or thimerosal. In many cases, it will be preferable to include isotonic agents, for example, sugars or sodium chloride.
  • Prolonged absorption injectable compositions can be caused by the use in the compositions of agents delaying absorption, for example, aluminum monostearate or gelatin.
  • Sterile injectable solutions are prepared by incorporating the active ingredients in the required amount in the appropriate solvent with several of the other ingredients listed above, if appropriate, followed by sterilization by filtration.
  • the dispersions are prepared by incorporating the various sterilized active ingredients into a sterile vehicle that contains the basic dispersion medium and the other required ingredients from those enumerated above.
  • the preferred methods of preparation are vacuum drying and lyophilization.
  • the solutions will be administered in a manner compatible with the dosage formulation and in a therapeutically effective amount.
  • the formulations are easily administered in a variety of dosage forms, such as the injectable solutions described above, but drug release capsules and the like can also be used.
  • parenteral administration in an aqueous solution for example, the solution should be suitably buffered and the liquid diluent rendered isotonic with sufficient saline or glucose.
  • aqueous solutions are particularly suitable for intravenous, intramuscular, subcutaneous and intraperitoneal administration.
  • sterile aqueous media that can be used are known to those skilled in the art.
  • a dose may be dissolved in 1 ml of isotonic NaCl solution and then added to 1000 ml of appropriate liquid, or injected at the proposed site of the infusion.
  • the pharmaceutical composition of the invention can be formulated in a therapeutic mixture comprising about 0.0001 to 1.0 milligrams, about 0.001 to 0.1 milligrams, about 0.1 to 1.0 milligrams, or about 10 milligrams per dose or more. Multiple doses may also be administered.
  • the level of therapeutically effective dose specific for a particular patient will depend on a variety of factors, including the disorder being treated and the severity of the disease, the activity of the specific compound employed, the specific composition used, the age, the body weight, general health, sex and diet of the patient, the moment administration, the route of administration, the rate of excretion of the specific compound used, the duration of treatment, or the drugs used in parallel.
  • FIG. 1 shows alignments of native human IgG1 sequences referring to positions 216 to 447 (according to the index UE) with the corresponding sequences of human IgG2 (SEQ ID NO: 2 and 7), human IgG3 (SEQ ID NO: 3 and 8) and human IgG4 (SEQ ID NO: 4 and 9).
  • the IgG1 sequences refer to the Glml allotype, 17 (SEQ ID NO: 1 and 6) and the Glm3 allotype (SEQ ID NOs: 5 and 10).
  • the "lower hinge CH2-CH3" domain of IgG1 begins at position 226 (see arrow). The CH2 domain is highlighted in gray and the CH3 domain is italicized.
  • A) Represents the evolution over time of the concentration of plasma IgG
  • Example 1 Production of deleted variants at position 294
  • the inventors have analyzed the sialylation of several variants in accordance with the invention, in particular deleted at position 294 (EU number or equivalent in Kabat).
  • several variants comprise a combination of additional mutations among the combinations described to confer an optimized binding to FcRn in the patent application EP 0 233 500.
  • this method includes the following steps:
  • the human Fc gene coding for residues 226 to 447 (according to the EU index of Kabat and represented in FIG. 1) derived from the heavy chain of a human IgG1 is cloned in a suitable vector, such as the phagemid vector pMG58 according to standard protocols well known to those skilled in the art.
  • a suitable vector such as the phagemid vector pMG58 according to standard protocols well known to those skilled in the art.
  • the Fc libraries are expressed using the Phage-display technique according to standard protocols, for use in the selection of Fc fragments.
  • the selection can be done according to the protocol detailed in the European patent application EP 2 233 500, in particular by selection on FcRn in solid or liquid phase, then determination of the binding characteristics of FcRn fragments by ELISA.
  • the Fc SEQ ID NO: 1 fragment sequence was cloned into a generic eukaryotic expression vector derived from pCEP4 (Invitrogen) and containing the heavy chain of a chimeric anti-CD20 antibody according to standard PCR protocols.
  • the light chain of this antibody was inserted into a similar pCEP4 derived vector.
  • All mutations of interest in the Fc fragment were inserted into the expression vector containing the anti-CD20 heavy chain by overlap PCR.
  • the 294Del variant was obtained using two sets of primers adapted to integrate the 294 deletion on the heavy chain contained in the expression vector.
  • the fragments thus obtained by PCR were combined and the resulting fragment was amplified by PCR using standard protocols.
  • the PCR product was purified on 1% (w / v) agarose gels, digested with the appropriate restriction enzymes and cloned into the anti-CD20 heavy chain expression vector.
  • HEK 293 cells were cotransfected with the light chain and heavy chain anti-CD20 IgG expression vectors in equimolar amounts according to standard protocols (Invitrogen). The cells were cultured to produce the antibodies transiently. The antibodies produced could be isolated and purified according to standard techniques of the art, with a view to their characterization. 2- Production of IgG variants in YB2 / 0 cells
  • Fc variants were prepared in full IgG format in the YB2 / 0 cell line (ATCC, CRL-1662) with anti-CD20 and anti-RhD specificity.
  • the IgG heavy and light chain was cloned into a bicistronic vector HKCD20 optimized for production in YB2 / 0.
  • the production was carried out in stable pools of YB2 / 0 cells.
  • the cell culture production and antibody purification steps were carried out according to standard techniques of the art, with a view to their characterization.
  • the inventors have verified that the deionization at position 294 did not have a significant impact on FcRn binding.
  • the variants deleted at position 294 retain their binding to FcRn relative to the parent IgG (IgG WT or IgG comprising mutations. "Optimized FcRn").
  • the sample to be analyzed was desalinated according to standard protocols so as to eliminate all the free reducing glucides potentially present as well as substances that could interfere during the subsequent steps (salts and excipients).
  • the sample was dried and the glycols were released by the enzymatic action of N-Glycannase under denaturing and reducing conditions, in order to maximize the yield of N-deglycosylation.
  • the dry sample was taken up in 45 of the PNGase F digestion solution diluted 1/5. 1.5 of a solution of ⁇ -mercaptoethanol 10% (v / v) in ultrapure water was added before stirring and incubation for 15 minutes at room temperature.
  • the isolation of the exoglycosidic degradation products was carried out by cold alcohol extraction by adding 60 (3 volumes) of absolute ethanol equilibrated at -20 ° C., before stirring and then incubation at -20 ° C. for 15 minutes. Centrifugation at 10,000 rpm was performed for 10 minutes at + 4 ° C, and the supernatant was immediately transferred to a 0.5 mL microtube before being dried under vacuum.
  • oligosaccharides obtained were then labeled with a fluorochrome, the APTS, then separated and quantified in HPCE-LIF.
  • the identification of the N-glycanse peaks is carried out using a standard glycoprotein standard whose N-glycosylation is perfectly known, by comparing the migration times of its N-glycans with those of the species observed on the profiles. electrophoretic samples to be analyzed. In addition, the migration times of the standard oligosaccharides are converted into units of glucose (GUs) after analysis of a heterogeneous mixture of a glucose homopolymer (Glc ladder). These values of GUs will then be compared with those of some standard oligosaccharides of known GUs, and will make it possible to increase the confidence index of the identifications. Results
  • the electrophoregrams obtained show biantennian glycan structures. These structures are mainly sialylated.
  • the electrophoregrams obtained show biantennian glycan structures. These structures are mainly sialylated.
  • the electrophoregrams obtained show biantennian glycan structures. These structures are mainly sialylated.
  • the electrophoregrams obtained show biantennary glycan structures mainly consisting of non-fucosylated agalactosylated short structures (GO: 52.06%). Fucosylated structures are a minority. Some structures possessing a GlcNac in bisecting position (G0B, G0FB) are observed.
  • the predominant oligosaccharide structure is: G0 (52.06%).
  • the calculated fucosylation rate is 17.05%
  • the fucosylation rate obtained with the run DSial + DGal + DhexNAc (*) is 13.07%.
  • the rate of forms having a GlcNac bisector is 2.87%.
  • the calculated galactosylation rate is 40.5%.
  • the electrophoregrams obtained show biantennary glycan structures and some triantennial structures. These structures are mainly sialylated.
  • the predominant oligosaccharide structure is: GO (55.20%).
  • the calculated fucosylation rate is 12.37%), the fucosylation rate obtained with the run DSial + DGal + DhexNAc (*) is 10.63%.
  • the rate of forms having a GlcNac bisector is 2.27%.
  • the calculated galactosylation rate is 39.13%.
  • the electrophoregrams obtained show biantennary glycan structures and some triantennial structures. These structures are mainly sialylated.
  • the variant T5A-74H differs from the parent variant T5A-74 by the V264E mutation.
  • the mutant T5A-74Del294 differs from the parent variant T5A-74 by the deletion of the amino acid at position 294.
  • mice hFcRn which are homozygous for a murine FcRn KO allele and heterozygous for a human FcRn transgene (mFcRn - / - hFcRnTg).
  • each animal received a single intravenous injection of 5 mg / kg IgG at the retro-orbital sinus in a protocol similar to that previously described (Petkova SB, et al., Enhanced half-life of genetically engineered human IgGl antibodies in a humanized FcRn mouse model: potential application in humorally mediated autoimmune disease (Int Immunol 2006). Blood samples were taken from the retro-orbital sinus at multiple time points and IgGs titrated by ELI SA. Results:
  • AUCO-t Area under the time / plasma concentration curve (from time T0 to the last time t where the antibody is still quantifiable)
  • Each mutation of interest in the Fc fragment was inserted independently into an expression vector containing the anti-CD20 heavy chain by overlap PCR using two sets of primers adapted to integrate a deletion or a degenerate codon (NNN or NNK ) in the targeted position (240 to 243, 258 to 267, 290 to 305).
  • the fragments thus obtained by PCR were combined and the resulting fragment was amplified by PCR using standard protocols.
  • the PCR product was purified on gels agarose 1% (w / v), digested with the appropriate restriction enzymes and cloned into the eukaryotic expression vector pMGM05-CD20 (pCEP4 InvitroGen), which contains cloning sites for the Fc fragment (BamHI and NotI) and the VH variable chain of the anti-CD20 antibody.
  • pMGM05-CD20 pCEP4 InvitroGen
  • This construction leads to the mutation of two amino acids in Fc (aa224 and 225, HT changed to GS) and the addition of the EFAAA sequence to the C-terminal of Fc, but makes it possible to very rapidly test a very large number of clones. Initially, it was verified that these mutations did not modify the binding of IgG-WT to the different receptors. Subsequently, the positive controls were cloned into this system in order to validate it:
  • IgG1-G236A from Xencor (C4): positive control for CD32aH / R;
  • the DNA of isolated clones was sequenced after colony PCR. After bioinformatic analyzes, clones with new mutations were frozen at -80 ° C with XL1-Blue bacteria and the sequences included in our database. Thus, 268 variants were constructed.
  • the anti-CD20 light chain was inserted into a pCEP4 vector identical to the vector used for the heavy chain, denoted pMGM01-CDC20 (pCEP4 InvitroGen).
  • HEK293-F Freestyle TM cells (Invitrogen), grown in 24-well plates, were co-transfected with the pMGM01-CD20 and pMGM05-CD20 vectors (Fc-WT and variants) in equimolar amounts (250 ng / ml). ) with a transfection reagent (1 ⁇ / ml) using standard protocols (Invitrogen).
  • the cells were cultured in suspension in serum-free medium for 7-9 days post transfection and the IgG-containing supernatants (1 ml) were harvested after centrifugation of the cells at 100 g for 10 min. IgG secreted in the supernatants were quantified using an ELISA test (FastELISA, R & D biotech). 3. Recombinant Fc Receptors Used:
  • CD16a is an activator receptor that has a V / F polymorphism at position 158 at the Fc binding site. The affinity is better for CD16aV.
  • CD16aV is commercially available (R & D System).
  • CD32a is an activating receptor that has an H / R polymorphism at position 131 at the Fc binding site. The affinity is better for CD32aH.
  • CD32aH was produced by PX'Therapeutics.
  • CD32aR and CD32b are commercially available (R & D System).
  • IgG variants were tested for binding to several human FcRs and FcRn by ELISA.
  • Maxisorp immunoplates were coated with 0.1 ⁇ g CD32 ⁇ H / well, or 0.2 ⁇ g CD16 ⁇ / well in PBS or 0.25 ⁇ g FcRn in P6 (100mM sodium phosphate, 50mM sodium chloride pH6.0).
  • NiNTA plates HisGrab Pierce
  • the supernatants were diluted in PBS (or P6 for the FcRn test) to a final concentration of 0.5 ⁇ g of IgG / ml and mixed with F (ab ') 2 IgG HRP of goat anti-human at the same concentration for 2 hours at room temperature.
  • IgG aggregated with F (ab ') 2 are then incubated with gentle shaking for 1 hour at 30 ° C on saturated ELISA plates without dilution for CD16aV, CD32aR and CD32b (ie IgG at 0.5 ⁇ g / ml), diluted in of PBS at 0.25 ⁇ g / ml for CD32 ⁇ H and diluted in P6 at 0.035 ⁇ g / ml for FcRn.
  • the plates are then revealed with TMB (Pierce) and the absorbance is read at 450 nm.

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