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    • 专利摘要:
    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. The present invention also relates to a method for producing a variant of a parent polypeptide comprising an Fc fragment, said variant having an improved sialylation of said Fc fragment relative to the parent polypeptide, which comprises a step of mutating at least one acid amine 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.
    公开号:FR3024453A1
    申请号:FR1457504
    申请日:2014-08-01
    公开日:2016-02-05
    发明作者:Celine Monnet；Alexandre Fontayne
    申请人:LFB SA；
    IPC主号:
    专利说明:

    [0001] 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.
    [0002] Monoclonal antibodies are used today as therapeutic agents 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.
    [0003] One of the critical questions in the use of monoclonal antibodies in therapy is their persistence in the bloodstream. The clearance of the antibody directly affects the effectiveness of the treatment, and therefore the frequency and amount of drug delivery, which can cause adverse effects in the patient.
    [0004] Immunoglobulin isotype G (IgG) is the class of immunoglobulin most common in humans and also the most used in therapy. Various specific mutagenesis experiments 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; Kim et al., 1994, Eur J Immunol., 24: 542-8; Medesan et al., 1996, Eur J Immunol., 26: 2533-6; Medesan et al. al, 1997, Immunol 158: 2211-7). Studies have more recently been conducted in humans (Shields et al., 2001, J. Biol Chem, 276: 65916604). [0009] However, there is still a need to find antibodies, or fragments of antibodies, having an improved half-life, and having interesting biological properties.
    [0005] 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. The term "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. Indeed, surprisingly, the inventors have discovered that a Fc fragment mutated at a specific position, close to the N-glycosylation site, has a highly increased sialylation compared to the non-mutated Fc fragment. This thus makes it possible to increase the properties of interest of the Fc fragment, and in particular its half-life. This may also allow to increase its anti-inflammatory activity. 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 at 305 of said Fc fragment, the numbering being that of the EU index or equivalent in Kabat. The present invention also relates to a method for producing a variant of a parent polypeptide comprising an Fc fragment, said variant having an improved sialylation of said Fc fragment relative to the parent polypeptide, which comprises 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. By "increased sialylation" or "improved sialylation" 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% , relative to the protein before the mutation step. Sialylation of a protein is a well-known glycosylation mechanism (see, in particular, Essentials of Glycobiology, 2nd edition, Varki et al, 2009). It corresponds to a covalent addition of at least one sialic acid (i.e. N-acetylneuraminic acid and its derivatives, such as N-glycosylneuraminic acid, N-acetylglycoylneuraminic acid) in the glycosylated chain of the protein. As used herein, the terms "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. . The terms "protein" and "polypeptide" include especially antibodies or immunoglobulins, in particular whole, monoclonal, multi-specific, bi-specific, dual-specific, synthetic, chimeric, humanized, human, immunoglobulin fusion proteins, conjugated antibodies, and their fragments. The terms "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.
    [0006] By "Fc fragment" or "Fc region" is meant the constant region of a full length immunoglobulin excluding the first immunoglobulin constant region domain (i.e. CH1-CL). Thus 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. Preferably, an Fc fragment of an IgG1, which consists of the flexible N-terminal hinge, is used in the present invention. and CH2-CH3 domains, i.e. the portion from amino acid C226 to the C-terminus, the numbering being indicated according to the EU index or equivalent in Kabat. Preferably, an Fc fragment of a human IgG1 (i.e. amino acids 226 to 447 is used according to the EU index or equivalent in Kabat). In this case, the lower hinge refers to positions 226 to 230, the CH2 domain refers to positions 231 to 340 and 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 a part of the upper hinge region, upstream of the position 226. In this case, preferably, 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). In this case, the Fc fragment of a human IgG1 refers to the portion from amino acid 216, 217, 218, 219, 220, 221, 222, 223, 224 or 225 to the C-terminus -terminal.
    [0007] The definition of "Fc fragment" includes a scFc fragment for "single chain Fc". By "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. Preferably, 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. In the present application, the numbering of Fc residues 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) ). By "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. By "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. Preferably, the substitution is punctual, i.e. it concerns only one amino acid. For example, 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. By "insertion" is meant the addition of at least one amino acid at a particular position in a parent polypeptide sequence. For example, insertion G> 235-236 refers to a glycine insertion between positions 235 and 236. By "deletion" is meant the removal of at least one amino acid at a particular position in a parent polypeptide sequence . For example, E294de1 refers to the suppression of glutamic acid at position 294; such a deletion is called De1294.
    [0008] By "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. Preferably, 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. Thus, preferably, the Fc fragment of the parent polypeptide already comprises at least one additional mutation (i.e. pre-existing modification), preferably selected from P230S, T256N, V259I, N315D, A330V, N361D, A378V, S383N, M428L, N434Y. Preferably, 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 .
    [0009] Preferably, according to a first alternative, the parent polypeptide consists of an Fc fragment, and preferably an entire Fc fragment. Preferably, according to a second alternative, the parent polypeptide consists of an amino acid sequence fused to N- or C-terminal to an Fc fragment. In this case, advantageously, the parent polypeptide is an antibody, an Fc fusion polypeptide or an Fc conjugate.
    [0010] Preferably, the Fc fragment of the parent polypeptide is chosen from the sequences SEQ ID NO: 1, 2, 3, 4 and 5. Preferably, the Fc fragment of the parent polypeptide has for sequence SEQ ID NO: 1. The sequences represented in FIG. SEQ ID NO: 1, 2, 3, 4 and 5 are free from an N-terminal hinge region. The 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. Also, in a particular embodiment, the Fc fragment of the parent polypeptide is chosen from the sequences SEQ ID NO: 6, 7, 8, 9 and 10. Preferably, the Fc fragment of the parent polypeptide has a sequence corresponding to the 1-position. -232, 2-232, 3-232, 4-232, 5-232, 6-232, 7-232, 8-232, 9-232, 10-232 or 11232 of the sequence SEQ ID NO: 6.
    [0011] Alternatively, the parent polypeptide consists of an immunoglobulin, an antibody or an amino acid sequence fused to N- or C-terminal to an antibody or immunoglobulin. By "variant" is meant a polypeptide sequence which is different from the sequence of the parent polypeptide by at least one amino acid modification. Preferably, 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. By "percent identity" between two amino acid sequences in the sense of the present invention, it is meant a percentage of identical amino acid residues between the two sequences to be compared, obtained after the best alignment, this percentage being purely statistical and the differences between the two sequences are randomly distributed over their entire length. By "best alignment" or "optimal alignment" is meant the alignment for which the percentage of identity determined as hereinafter is the highest. The sequence comparisons between two amino acid sequences are traditionally performed by comparing these sequences after optimally aligning them, said comparison being made by segment or by "comparison window" to identify and compare the local regions of similarity of the amino acid sequence. sequence. 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. Mol Evol., 18: 38-46), by means of the the local homology algorithm of Neddleman and Wunsch (1970), using the similarity search method of Pearson and Lipman (1988, PNAS, 85: 2444-2448), using computer programs using these algorithms (GAP, BESTFIT, BLAST P, BLAST N, FASTA and TFASTA in the Wisconsin Genetics Software Package, Genetics Computer Group, 575 Science Dr., Madison, WI).
    [0012] In preferred embodiments, 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 preferably, the variant according to the invention is chosen from human IgG1, IgG2, IgG3 and IgG4 variants.
    [0013] Preferably, the method for producing the variant or the method 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, numbering being that of the EU index or equivalent in Kabat. Preferably, the mutation is carried out on the amino acid of the Fc fragment located at 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 fragment Fc located in position 293 or 294, the numbering being that of the EU index or equivalent in Kabat.25 The sequences described in the present application can be summarized as follows: SEQ ID NO: Protein 1 Fragment Fc of human IgG1 Glm1,17 (residues 226-447 according to the EU index or equivalent in Kabat) without an N-terminal hinge region. 2 Fragment Fc of human IgG2 without N-terminal hinge region. 3 Fragment Fc of human IgG3 without N-terminal hinge region. 4 Fragment Fc of human IgG4 without N-terminal hinge region.
    [0014] Fc fragment of human IgG1 G1m3 without N-terminal hinge region. 6 Fragment Fc of human IgG1 Glm1,17 (residues 226-447 according to the EU index or equivalent in Kabat) with N-terminal hinge region. 7 Fragment Fc of human IgG2 with N-terminal hinge region. 8 Fragment Fc of human IgG3 with N-terminal hinge region. 9 Fragment Fc of human IgG4 with N-terminal hinge region.
    [0015] Glm3 human IgG1 Fc fragment with N-terminal hinge region. More preferably, the mutation step of the method for preparing the variant according to the invention is obtained as follows: i) a nucleic sequence encoding the parent polypeptide comprising the Fc fragment is provided; 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 / O line, or the CHO line). the protein thus produced is then purified by chromatography. These techniques are described in detail in the reference manuals: Molecular cloning: a laboratory manual, 3rd edition-Sambrook and Russel eds. (2001) and Current Protocols in Molecular Biology - Ausubel et al. eds (2007).
    [0016] The 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. Preferably, the random mutagenesis as described in the application W002 / 038756 is used: it is the Mutagen technique. This technique uses a human DNA mutase, in particular chosen from DNA polymerases (3, ri and t). A step of selecting mutants having retained FcRn binding is necessary to retain the mutants of interest.
    [0017] Alternatively, the amino acid substitutions are preferably performed by site-directed mutagenesis, using the assembly PCR technique using degenerate oligonucleotides (see, for example, Zoller and Smith, 1982, Nucl Acids Res., 10: 6487-6500. Kunkel, 1985, Proc Natl Acad Sci USA 82: 488). Finally, in 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.
    [0018] The preferred host cells are the YB2 / 0 rat line, the CHO hamster line, in particular the CHO dhfr- and CHO Lec13 lines, PER.C6TM (Crucell), HEK293, T1080, EB66, K562, NSO, SP2 / 0. , BHK or COS. More preferably, the YB2 / 0 rat line is used.
    [0019] Alternatively, the host cells may be modified transgenic animal cells to produce the polypeptide in the milk. In this case, 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.
    [0020] The polynucleotide encoding the variant obtained in step ii) may also comprise optimized codons, in particular for its expression in certain cells (step iii)). For example, 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. Codon optimization aims to replace natural codons by codons whose transfer RNA (tRNA) carrying the amino acids are most common in the cell type considered. The mobilization of frequently encountered tRNAs has the major advantage of increasing the translation speed of the messenger RNAs (mRNA) and therefore of increasing the final titre (JM Carton et al., Protein Expr Purif, 2007). 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).
    [0021] 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.
    [0022] Preferably, 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.
    [0023] 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, transdermal, topical, intraperitoneal, intramuscular, intravenous, subcutaneous, intrathecal, intranasal administration forms and rectal administration forms. Preferably, the pharmaceutical composition contains a pharmaceutically acceptable carrier for a formulation that can be injected. It may be in particular isotonic, sterile, saline solutions (with monosodium or disodium phosphate, sodium chloride, potassium chloride, calcium or magnesium chloride and the like, or mixtures of such salts), or freeze-dried compositions which, when adding 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. In all cases, 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 microorganisms. 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. The proper fluidity can be maintained, for example, by the use of 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 of the injectable compositions may 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. In general, 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. In the case of sterile powders for the preparation of sterile injectable solutions, the preferred methods of preparation are vacuum drying and lyophilization. During formulation, 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. For 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. These particular aqueous solutions are particularly suitable for intravenous, intramuscular, subcutaneous and intraperitoneal administration. In this regard, sterile aqueous media that can be used are known to those skilled in the art. For example, 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 infusion. Certain dosage variations will necessarily occur depending on the condition of the subject being treated.
    [0024] 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 time of administration, the route of administration, the rate of excretion of the specific compound used, the duration of treatment, or the drugs used in parallel. FIGURES FIG. 1: Alignments of native human IgG1 sequences referring to positions 216 to 447 according to the index UE FIG. 1 shows alignments of native human IgG1 sequences referring to positions 216 to 447 (according to the EU index ) 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 Glm1,17 allotype (SEQ ID NO: 1 and 6) and the G1m3 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.
    [0025] Figure 2: Half-life of anti-CD20 and anti-Rhesus D antibodies produced in YB2 / 0: The persistence of immunoglobulins in the serum of transgenic mice for human FcRn was evaluated. Two antigenic specificities were tested; antiCD20 IgG and anti-RhD IgG deleted at position 294 were tested in comparison with the corresponding WT IgGs. A) Represents the evolution over time of the concentration of plasma IgG B) Represents the half-life observed for the two IgGs deleted at position 294 and IgG WT. EXAMPLES The following examples are given to illustrate various embodiments of the invention. Example 1: Production of the variants deleted at position 294 The inventors analyzed the sialylation of several variants in accordance with the invention, in particular deleted at position 294 (EU number or equivalent in Kabat). Among the De1294 variants analyzed, several variants comprise a combination of additional mutations from the combinations described to confer an optimized binding to FcRn in patent application EP 0 233 500. The identification and obtaining of such "optimized FcRn" variants can according to the methods described in the prior art, in particular the European patent application EP 0 233 500, which describes the production of such mutants according to the so-called MutaGenTM technique technique. Typically, this method comprises the following steps: A / Construction of an Fc library 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 human IgG1 is cloned into a suitable vector, such as the phagemid vector pMG58 according to standard protocols well known to those skilled in the art. B / Mutagenesis Several libraries are then generated, according to the procedure described in WO 02/038756, which uses low fidelity human DNA polymerases in order to introduce random mutations homogeneously on the entire target sequence. Specifically, three distinct mutases (pol (3, ri, and t) were used under different conditions to create complementary mutational patterns C / Expression of Fc Banks by Phage-display and Selection of Enhanced Binding Variants 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 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.
    [0026] D / Production of Variants as Integral Ig and Deletion in Position 294 Several combinations of "optimized FcRn" mutations were selected to serve as a basis for the production of mutants deleted at position 294. The following combinations were selected: N315D / A330V / N361D / A378V / N434Y (T5A-74) T256N / A378V / S383N / N434Y (C6A-78) V259I / N315D / N434Y (C6A-74) 1- Production of IgG variants in HEK cells Seq Fc fragment sequence ID NO: 1 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. For example, 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.
    [0027] 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.
    [0028] 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 in the art, for their characterization. 2- Generation of IgG Variants in YB2 / 0 Cells The Fc variants were prepared in a full IgG format in the YB2 / O cell line (ATCC, CRL-1662) with anti-CD20 and anti-RhD specificity. For this, the IgG heavy and light chain was cloned in 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.
    [0029] The inventors verified that the deletion at position 294 did not have a significant impact on FcRn binding. Variants deleted at position 294 retain their binding to FcRn relative to the parent IgG (IgG WT or IgG comprising "optimized FcRn" mutations).
    [0030] EXAMPLE 2 Analysis of the Sialylation of Different Proteins Procedure: Preparation of the Sample 1 Desalting and N-Deglycosylation In a first step, 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 may interfere during later stages (salts and excipients). After desalting, 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. For N-deglycosylation of Ig, the dry sample was taken up in 45 μl of the PNGase F digestion solution diluted 1/5. 1.5 μl of a 10% (v / v) 13-mercaptoethanol solution in ultrapure water was added before stirring and incubation for 15 minutes at room temperature. Then, 1 μL of the PNGase F solution (2.5 mUffl) was added before shaking and incubating in a 37 ° C water bath for 12-18 hours. The glycans were then separated from the deglycosylated proteins by precipitation with cold EtOH.
    [0031] The glycan extract obtained was then divided into 4 fractions before being treated with exoglycosidases. Quantification of Fucosylation and GlcNAc Rates Between Amounts, and N-Glycan Number of Galactosylation Each N dried alcoholic subfraction, containing the equivalent of 100 μg glycoprotein, was respectively digested (1 ) by α-sialidase, 13-galactosidase and Nacetyl-13-hexosaminidase, to determine the fucosylation rate; (2) asialidase, 13-galactosidase and -Fucosidase, for calculating the level of GlcNAc 10 between; and (3) u-sialidase and -fucosidase, to determine the galactosylation index. These deglycosylations were carried out at 37 ° C for 12 to 18 hours. The isolation of exoglycosidic degradation products was carried out by cold alcohol extraction by adding 60 μl (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. The oligosaccharides obtained were then labeled with a fluorochrome, APTS, and then separated and quantified in HPCE-LIF. 3 Exploitation of the results The identification of the N-glycans peaks is carried out using a reference glycoprotein standard whose N-glycosylation is perfectly known, by comparison of the migration times of its N-glycans with those of the N-glycans. species observed on the electrophoretic profiles of the 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 30 standard oligosaccharides of known GUs, and will make it possible to increase the confidence index of the identifications.
    [0032] A-Variant Results Produced in YB2 / 0 The following polypeptides were analyzed: De1294 De1294 Anti-CD2O-C6A 78-De1294 T256N / A378V / S383N / N434Y / De1294 Anti-CD2O-C6A De1294 Anti-CD20 74-De1294 V2591 / Anti-CD20 Mutations N315D / N434Y / De1294 Anti-RhD Anti-RhD De1294 De1294 Anti-RhD -C6A 78 T256N / A378V / S383N / N434Y Anti-RhD -C6A 78-De1294 T256N / A378V / S383N / N434Y / De1294 Anti-CD20 De1294: Electropherograms obtained show biantennian glycan structures. These structures are mainly sialylated. 87.98% of the structures seem sialylated. The calculated fucosylation rate is 48.24%. 42 11.9 A2F 19.8 A1 5.5 A1F 1..6 C.:.:- 0.S4 0.5 "G :: i_. I. F 0.19 2.44 GI_: 1: 0.14 G - + .1,3) F 2.C5 G212 0.51 G2F12.3, S4 Unidentified sialyl structures 49.18 - - - :: f, ic._., 1 -_, --..- ..: Anti-CD2O-C6A 78-De1294: The electrophoregrams obtained show glycan structures biantennées.
    [0033] These structures are mainly sialylated. 88.69% of the structures seem sialylated. The calculated fucosylation rate is 51.87%.
    [0034] A ") A 2 F! Jh Al 7.2 AlF j..12 C: _: 1.17` ._. '. 31, - GI: 1:' 3F 0 _ r - G2 + C-1. ## STR1 ## 1, ## STR1 ## 1, ## STR1 ##, and Anti-CD2O-C6A 74-De1294: Electrophoregrams Obtained. glycannic structures biantennées.
    [0035] These structures are mainly sialylated. 93.48% of the structures seem sialylated. The calculated fucosylation rate is 51.24%.
    [0036] Al. 2: 1, 77. 1.92 G = '! -.--: G '- {'. 1. L-α: 1 0.43 (: L3 :, GC:, 3, F 0.1Q: 2 G = 1.6: 0.11 + G i: 1.2: 2.05 G2L 0.15 G2F i ## EQU1 ## Anti-RhD: The electrophoregrams obtained show biantennary glycan structures mainly consisting of non-fucosylated agalactosylated short structures (GO: 52.06). The fucosylated structures are in the minority.Some structures with a GlcNac in bisecting position (GOB, GOFB) are observed.The predominant oligosaccharide structure is: GO (52.06%) .The calculated fucosylation rate is 17.05%. Fucosylation obtained with DSial + DGal + DhexNAc (*) min is 13.07% The rate of forms having a GlcNac bisector is 2.87% The calculated galactosylation rate is 40.5%.
    [0037] Structure re ,, HPCE-LIF Sia'ylated 0.00 --- 2%,: - a.d ::; n Bissecing 2.87 17 sylated * 13, D7 F ..; cosylated 1. .5 A2 0.00 A2F 0.00 VI3N2 0.00 M3N2F 0: CO AL 0.00. H 0.00 G2F6 0.00 0.47 G2F G28 0.00 G2 456 G1FB MO G1F 5.65 - _.3. : 2 GiB 0.00 G1 24.59 -I D .: :: GDFB: 1.24 9.59 F n g 1.63. , 52.06 7., IAN-5 0.00 deni9s) .5-0 Anti-RhD De1294: The electrophoregrams obtained show biantennary glycan structures and some triantennial structures. These structures are mainly sialylated. 92.25% of the structures seem sialylated. The calculated fucosylation rate is 37.08%. -. F: 3:, 1 r_. ## EQU1 ## 1 ## EQU1 ## EJ: E: 37: Anti-RhD -C6A 78: The predominant oligosaccharide structure is: GO (55.20%) The calculated fucosylation rate is 12.37%, the fucosylation rate obtained with DSial + min. DGal + DhexNAc (*) is 10.63%, the rate of forms having a bisecting GlcNac is 2.27%, and the calculated galactosylation rate is 39.13%.
    [0038] St-uctre: ':: - 0 HPCE-L1F If ylatec 0.00 Bissec.inR 2.27 F ..: cosylated * 11-2. ,, -. 1.3 F, cosvlated 1 ,: AZ 0.00 A.2F 0.00 M3N2 0.00 5113N2F 0.00 AI '0.00 AF 0.00 G2FB 0.00 G2F 0.00 GZB 0.00 G2 4.00 G1FB 0.00 G1F 4.20 ^ - - _ G1B 0.83 G1 26.10 GDFB 0.66 G CiF 7.51 1.50. 55.20 MAN-5 0.00 Identified ... _ Anti-RhD -C6A 78-De1294: The electrophoregrams obtained show biantennary glycan structures and some triantennial structures. These structures are mainly sialylated. 91.83% of the structures seem sialylated. The calculated fucosylation rate is 57.81%. .. ". ## EQU1 ## Variants produced in HEK lines are described in US Pat. following polypeptides were analyzed (Anti-CD20 IgG variants): Name Mutations T5A-74 N315D / A330V / N361D / A378V / N434Y T5A-74H V264E / N315D / A330V / N361D / A378V / N434Y T5A-74De1294 E294de1 / N315D / A330V / N361D / A378V / N434Y WT / The variant T5A-74H differs from the parent variant T5A-74 by the mutation V264E The mutant T5A-74De1294 differs from the parent variant T5A-74 by the deletion of the amino acid at position 294. They have The results are summarized in the table below (in percentage): Example 3: Analysis of the half-life of the IgGs deleted in position 294 The persistence of the immunoglobulins in the serum of Transgenic mice for human FcRn were evaluated, two antigenic specificities were tested, and anti-CD20 IgG and anti-RhD IgG deleted at position 294 were tested. compared with the corresponding WT IgGs. Pharmacokinetic experiments were thus performed in hFcRn mice that are homozygous for a murine and heterozygous FcRn KO allele for a human FcRn transgene (mFcRn - / - hFcRnTg). For these pharmacokinetic studies, each animal received a single intravenous injection of IgG at 5 mg / kg at the retroorbital sinus, in a protocol similar to that previously described (Petkova SB, et al., Enhanced half-life of genetically engineered human IgG1 antibodies in a humanized FcRn mouse model: Immunol 2006). Blood samples were taken from the retro-orbital sinus at multiple time points and IgGs titrated by ELISA. Results: T5A-74De1294 T5A-74H T5A-74 27 0 No 2.13 GI (GOF 81.44 G1 60FB 1 G1 (.6 0.51 10.08 5.64 9.18 19.56 3.34 1, 7 / 10.27 28.76 79.47 1.21 oo 4.92 0.88 0.71 9.23 0 3.68 0 2 5.94 0.91 1.57 0> 51.11 15.44 > 34.38> 67.64 94.17 Al AlF G1 (1.3) F G1 (1.6) F8 G28 Gatactasyta, SiatyfatiQn 2 G F Fucosylatior 1.8 6.41 0.84 5.57 0 1.65 16, 27 - + -> 58.36 0> 37.8 97.36 740.88 6.72 29.28 2.37 0.65 In this test, the two IgG deleted in position 294 showed an increase of half life with a ratio (half-life variant / WT) of 1.7 (Figure 2).
    [0039] The parameters analyzed are grouped in the table below: CO AUCO-t ._! ::: 11nf e Vd CI Uh Anti-CD20 WT 29.2 4.07 0.097 Anti-CD20 De1294 48.8 2. 0.037 Anti-RhD WT O 7491 80 0 65.7 2 0.017 Anti-RhD De1294 141 11142 13354 111 1.66 0.010 The parameters analyzed are defined below: CO: Maximum concentration extrapolated to TO AUCO-t: Area under the time / plasma concentration curve (from time TO to last time t where the antibody is still quantifiable) AUCinf: Area under the time / plasma concentration curve of TO at infinity (= AUCO-t + extrapolation to infinity) T1 / 2: Half-life Vd: Volume of distribution Cl: Clearance
    权利要求:
    Claims (14)
    [0001]
    REVENDICATIONS1. A method of 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.
    [0002]
    A method of producing a variant of a parent polypeptide comprising an Fc fragment, said variant having enhanced sialylation 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.
    [0003]
    3. Method according to claim 1 or 2, characterized in that the sialylation of said Fc fragment or said obtained variant 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 said Fc fragment or said variant before the mutation step.
    [0004]
    4. Method according to any one of claims 1 to 3, characterized in that the mutation is selected from an insertion, a substitution, preferably punctual, and a deletion.
    [0005]
    5. Method according to any one of claims 2 to 4, characterized in that the parent polypeptide consists of an Fc fragment.
    [0006]
    6. Method according to any one of claims 2 to 4, characterized in that the parent polypeptide consists of an amino acid sequence fused in N- or C-terminal to an Fc fragment.
    [0007]
    7. Method according to any one of claims 2 to 4, characterized in that said parent polypeptide is an immunoglobulin or an antibody.
    [0008]
    The method according to any one of claims 2 to 4 or claim 7, characterized in that the parent polypeptide consists of an amino acid sequence fused to N- or C-terminal to an antibody or immunoglobulin.
    [0009]
    9. Method according to one of claims 2 to 8, characterized in that the Fc fragment of the parent polypeptide is selected from the sequences SEQ ID NO: 1, 2, 3, 4 and 5, preferably corresponds to the sequence SEQ ID NO: 1. 15
    [0010]
    10. Method according to one of the preceding claims, characterized in that the mutation is carried out on at least one amino acid located in 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 index 20 EU or equivalent in Kabat.
    [0011]
    11. Method according to one of the preceding claims, characterized in that the mutation is carried out on at least one amino acid 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, the numbering being that of the EU index or equivalent in Kabat.
    [0012]
    12. Method according to one of the preceding claims, characterized in that the mutation is carried out on the amino acid in position 293 or 294, the numbering being that of the EU index or equivalent in Kabat.
    [0013]
    13. Method according to one of claims 2 to 12, characterized in that the Fc fragment of the parent polypeptide already comprises at least one additional mutation, preferably selected from P230S, T256N, V2591, N315D, A330V, N361D, A378V, S383N , M428L, N434Y, preferably a combination of additional mutations selected from P230S / N315D / M428L / N434Y, T256N / A378V / S383N / N434Y, V2591 / N315D / N434Y and N315D / A330V / N361D / A378V / N434Y.
    [0014]
    14. Method according to one of claims 2 to 13, characterized in that the mutation step is obtained as follows: i) provides a nucleic sequence encoding the parent polypeptide comprising the Fc fragment; 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.
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    CA2834589A1|2011-05-25|2012-11-29|Merck Sharp & Dohme Corp.|Method for preparing fc-containing polypeptides having improved properties|FR3058159B1|2016-10-28|2022-02-25|Lab Francais Du Fractionnement|POLYPEPTIDE FC VARIANTS WITH AN INCREASED HALF-LIFE|
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    WO2021057726A1|2019-09-23|2021-04-01|南开大学|SCREENING OF FC SPECIFICALLY BINDING TO FCγR BY USING MAMMALIAN DISPLAY|
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    优先权:
    申请号 | 申请日 | 专利标题
    FR1457504A|FR3024453B1|2014-08-01|2014-08-01|PROCESS FOR PRODUCING VARIANTS HAVING FC HAVING ENHANCED SIALYLATION|FR1457504A| FR3024453B1|2014-08-01|2014-08-01|PROCESS FOR PRODUCING VARIANTS HAVING FC HAVING ENHANCED SIALYLATION|
    AU2015295090A| AU2015295090A1|2014-08-01|2015-07-31|Method for producing variants having an Fc with improved sialylation|
    US15/500,105| US20170260254A1|2014-08-01|2015-07-31|Method for producing variants having an fc with improved sialylation|
    KR1020177002628A| KR20170035923A|2014-08-01|2015-07-31|Method for producing variants having an fc with improved sialylation|
    JP2017505476A| JP2017522040A|2014-08-01|2015-07-31|Methods for making variants with Fc having improved sialylation|
    CA2956822A| CA2956822A1|2014-08-01|2015-07-31|Method for producing variants having an fc with improved sialylation|
    MX2017001516A| MX2017001516A|2014-08-01|2015-07-31|Method for producing variants having an fc with improved sialylation.|
    PCT/FR2015/052123| WO2016016586A1|2014-08-01|2015-07-31|Method for producing variants having an fc with improved sialylation|
    BR112017001966A| BR112017001966A2|2014-08-01|2015-07-31|"Method for producing variants with an improved sialylated fc"|
    CN201580041852.8A| CN106573978A|2014-08-01|2015-07-31|Method for producing variants having an Fc with improved sialylation|
    EP15759880.6A| EP3174904A1|2014-08-01|2015-07-31|Method for producing variants having an fc with improved sialylation|
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