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    • 专利摘要:
    The present invention relates to the use of antibody Fc fragments in the treatment of autoimmune and / or inflammatory diseases, said Fc fragments being isolated recombinant Fc fragments having a modified affinity for at least one of the Fc receptors ( FcR), in particular increased affinity to FcRn.
    公开号:FR3038517A1
    申请号:FR1556399
    申请日:2015-07-06
    公开日:2017-01-13
    发明作者:Philippe Mondon；Mars Celine Monnet；Alexandre Fontayne；Romeuf Christophe De；Abdessatar Chtourou
    申请人:LFB SA；
    IPC主号:
    专利说明:

    The present invention relates to immunotherapy of autoimmune and / or inflammatory diseases.
    BACKGROUND OF THE INVENTION Immunotherapy, the therapy of administering exogenous antibodies to patients, is nowadays widely used for the treatment of various pathologies, particularly for autoimmune diseases and inflammatory diseases. Immunoglobulin-based therapy is typically proposed for the treatment of these diseases: i) Intravenous immunoglobulin (IVgV) -based therapies, which consist in administering to patients intravenously immunoglobulins (IgG most often) from pools human plasma donations; and (ii) therapies that rely on the use of recombinant antibodies (ie, genetically engineered antibodies). These have led to real advances in the management of patients with inflammatory diseases and autoimmune diseases, particularly because they offer the possibility of overcoming the disadvantages of using immunoglobulins of origin the risk of supply disruption, and the risk of transmission of pathogens possibly present in the plasma.
    The presence of Fab fragments in immunoglobulins may be responsible for the occurrence of significant adverse reactions in treated patients. To avoid these side effects, the patent application WO 2004/099374 discloses the use of isolated recombinant Fc fragments for the treatment of patients, in particular for patients suffering from an autoimmune disease.
    However, there is still a need to optimize these Fc fragments, in particular to increase their half-life time, and / or their therapeutic efficacy. Summary of the invention
    The inventors now propose to use Fc fragments having a modified affinity for at least one of the Fc receptors (FcR) relative to a parent Fc fragment. According to the invention, the Fc fragments are isolated, namely that they are not associated with Fab fragments, or conjugated or fused to any other protein, polypeptide or peptide. In particular, it is not a question of complete immunoglobulins. The invention thus provides a composition comprising antibody Fc fragments for use in the treatment of an autoimmune and / or inflammatory disease, said Fc fragments being isolated Fc fragments having a modified affinity for at least one of the Fc receptors (FcR) compared to a parent Fc fragment.
    In a particular embodiment, the composition comprises antibody Fc fragments, comprising at least one mutation of one or more amino acids and / or having on their glycosylation site (Asn 297) N-glycans, said N- glycan Fc fragments with a fucosylation level of less than 65%, Legend of figures:
    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: 7), human IgG3 (SEQ ID NO: 8) and human IgG4 (SEQ ID NO: 9). The IgG1 sequences refer to the Glml allotype, 17 (SEQ ID NO: 6) and the Glm3 allotype (SEQ ID NO: 10). The "lower hinge CH2-CH3" domain of IgG1 begins at position 226 (see arrow). Domain 0142 is highlighted in gray and the CH3 domain is italicized.
    Figure 2 shows schematic representations of Fc IgG1 (A) and scFc (B). The CH2 are represented in gray and the CH3 in white. The disulfide bridges are represented in fine gray dots. In scFc, the two CH2-CH3 polypeptides are linked by a peptide linker represented in bold dotted lines.
    FIG. 3 shows the GO, GOF, G1 and GIF forms of the glycan structures likely to be present on the Fc fragments used in the invention.
    FIG. 4 is a graph showing the percentage of red blood cell lysis D + mediated by effector cells in the presence of an anti-Rhesus D (RhD) monoclonal antibody, observed following the addition of different amounts of polyvalent immunoglobulins ( IglV) or recombinant, non-mutated or mutated Fc fragments (Fc fragment carrying the 315D / 330V / 361D / 378V / 434Y mutations).
    Detailed description of the invention: Definitions
    Throughout the present description, the numbering of the residues in the Fc fragment is that of the immunoglobulin heavy chain according to the EU index or as described in Kabat et al., Protein sequences of immunological interest, 5th ed. . Public Health Service, National
    Institutes of Health, Bethesda, Md. (1991), expressly incorporated herein by reference. The "EU index" or "EU index or equivalent in Kabat" refers here to the numbering of the residues of the human IgG1 antibody.
    The term "immunoglobulin" refers to the structure that constitutes the naturally occurring biological form of an antibody, including constant and variable regions (also called fragments). An immunoglobulin molecule is a molecule whose base unit is a heterotetramer consisting of two heavy chains of about 50-70 kDa each (called H chains for Heavy) and two light chains of about 25 kDa each ( say the L chains for Light), linked together by disulfide bridges intra and intercatenaries.
    Each chain consists, in the N-terminal position, of a region or variable domain, called VL for the light chain, VH for the heavy chain, and in the C-terminal position, of a constant region, consisting of a single domain called CL for the light chain and three or four domains named CH1, CH2, CH3, CH4, for the heavy chain.
    Only IgM and IgE have the CH4 domain.
    Each domain comprises about 110 amino acids and is structurally comparable. The 2 heavy chains are linked by disulfide bridges at CH2 and each heavy chain is linked to a light chain by a disulphide bridge between CH1 and CL. The region that determines the specificity of the antibody for the antigen is carried by the variable parts, whereas the constant parts can interact with the Fc receptors (RFc) of effector cells or molecules such as complement proteins to induce different properties. functional. The assembly of the chains that make up an antibody makes it possible to define a characteristic three-dimensional structure in Y, where the base of the Y corresponds to the constant region Fc; or Fc fragment; which is recognized by the complement and the Fc receptors to mediate the effector functions of the molecule, and the ends of the Y arms correspond to the respective assembly of the variable region of a light chain and the variable region of a heavy chain, said ends constitute the Fab fragment and determine the specificity of the antibody for the antigen.
    More precisely, there are five isotypes of heavy chains (gamma, alpha, mu, delta and epsilon) and two isotypes of light chains (kappa and lambda, the lambda chains being themselves divided into two types: lambda 1 and lambda 2). . It is the heavy chain that determines the class of immunoglobulin. There are five Ig classes: IgG for Gamma isotype, IgA for Alpha isotype, IgM for Mu isotype, IgD for Delta isotype and IgE for Epsilon isotype.
    The kappa and lambda light chains are shared by all classes and subclasses. In humans, the proportion of kappa and lambda produced is in a ratio of 2 to 1.
    Immunoglobulin G (IgG) is the most abundant immunoglobulin in the serum (75 to 80% circulating antibodies). Present as monomers, they have the longest serum half-life of all immunoglobulins (about 21 days).
    There are four types of heavy gamma chains, which determines four IgG subclasses (IgG1 for gamma, IgG2 for gamma2, IgG3 for gamma3, and IgG4 for gamma4). These four subclasses differ in numbers and varying positions of disulfide bridges {Basic and Clinical Immunology, 8th Edition, Daniel P. Stites, Abba I. Terr and Tristram G. Parslow (eds.), Appleton & Lange, Norwalk, Conn., 1994, p. 71 and Chapter 6).
    The four subclasses of human IgG also differ in their biological activities, despite very homologous structures (more than 95% sequence homology for Fc regions).
    By biological activity, reference is made in particular to the capacity of the IgG constant region to bind: in particular the complement proteins (the Clq protein for example [Basic and Clinical Immunology, 8th edition, Daniel P. Stites, Abba I. Terr and Tristram G. Parslow (eds.)]) And / or IgG receptors: RFcy (RFcyl, RFcylI, RFcylII, Ravetch and Kinet, AnnualReview of Immunology, Vol. 9: 457-492 (1991)).
    Depending on the type of binding, various mechanisms of action may be activated: opsonization, phagocytosis, ADCC (Antibody Dependent Cellular Cytotoxicity) or CDC (Complement Dependent Cytotoxicity) for example. See Uananue and Benacerraf, Textbook of Immunology, 2nd Edition, Williams & Wilkins, p. 218 (1984)) for more details.
    By "Fc fragment" is meant the constant region of a full length immunoglobulin excluding the first immunoglobulin constant region domain. Thus, the Fc fragment refers to the last two constant domains (CH2-CH3) of IgA, IgD, IgG and the last three constant domains (CH2-CH3-CH4) of IgE and IgM, and the flexible N-terminal hinge of these domains. . For IgA and IgM, the Fc fragment may comprise the J chain. For IgGs, the Fc fragment comprises the CH2, CH3 and lower hinge region regions between CH1 and CH2. In other words, the Fc region of an IgG1 consists of the lower hinge CH2-CH3, ie the domain, that is to say the portion from the amino acid C226 up to the carboxy-terminal end, the numbering being indicated according to the EU index or equivalent in Kabat. Analogous domains for other IgG subclasses can be determined from the alignment of the heavy chain or heavy chain amino acid sequences of the IgG subclasses with that of an IgG1 human (see Figure 1). The fragment Fc used according to the invention may also comprise part of the upper hinge region, upstream of the position 226 (according to the index EU). In this case, preferably, a Fc fragment of a human IgG1 comprising a portion of the region between positions 216 to 226 is used. 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 carboxy terminus. By "Fc fragment" is also meant 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.
    By "parent Fc fragment" or "parent polypeptide" as used herein is meant a reference polypeptide among wild-type Fc regions or variants possibly carrying other mutations than that considered. By "wild type or WT" is meant here an amino acid sequence or a nucleotide sequence found in nature, ie which is of natural origin, including allelic variations, and which is not It has not been intentionally modified by molecular biology techniques such as mutagenesis. For example, the Fc "wild-type" regions refer in particular to the Fc region of IgG1 having the sequence SEQ ID No. 1 (Glml allotype, 17), the Fc region of IgG2 having the sequence SEQ ID No. 2, the Fc region of IgG3 having the sequence SEQ ID No. 3, the Fc region of IgG4 having the sequence SEQ ID No. 4, and the Fc region of IgG1 having the sequence SEQ ID No. 5 (Glm3 allotype ).
    By "modified affinity" is meant a diminished or increased affinity to a parent Fc fragment.
    By "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 a FcRn gene. FcRn can be from any organism including, but not limited to, humans, mice, rats, rabbits and monkeys. As is known in the art, 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. Unless otherwise noted herein, FcRn or FcRn protein refers to the α-chain complex with beta-2-microglobulin. In humans, the gene encoding FcRn is called FCGRT.
    By "increased FcRn binding" as used herein is meant the increase in the binding affinity, in vivo or in vitro, of the mutated Fc fragment of the invention for FcRn, relative to parent polypeptide. The ability of the mutated Fc fragment of the invention to bind to a FcRn receptor can be evaluated in vitro by an ELISA test, as described for example in the patent application WO2010 / 106180.
    The term "half-life" in this invention refers to the time taken by the Fc fragment to be eliminated for one-half of the circulation or other tissues, once present in the serum of the patient to whom it has been administered.
    The term "Fcγ receptor" or "FcγR" refers to the IgG-type immunoglobulin receptors, called CD64 (FcγRI), CD32 (FcγRII), and CD16 (FcγRIII), in particular five expressed receptors (FcγRIa, FcγRIIa, FcγRIIb, FcγRIIIa). , FcγRIIIb). All are effector cell activating receptors, except for human FcγRIIb which is an inhibitory receptor for the activation of immune cells (Muta T et al., Nature, 1994, 368: 70-73).
    By "effector cell" is meant any cell carrying an Fc receptor, such as lymphocytes, monocytes, neutrophils, Natural Killer (NK) cells, eosinophils, basophils, mast cells, dendritic cells, Langerhans cells and platelets.
    The term "glycosylation" in the context of the invention refers to the addition, by enzymatic reaction, of one or more carbohydrates to the sequence of a recombinant Fc fragment.
    The term "hypersialysation" in the context of the invention refers to the addition of one or more sialic acid groups to the sequence of an Fc fragment. The addition of one or more sialic acid groups can be achieved by enzymatic reaction, cellular reaction or by site-directed mutagenesis of Fc targeting one or more amino acids involved in sialylation of Fc.
    The term "patient" refers to any human or animal subject, preferably mammalian. In a preferred embodiment, the patient is a human being, regardless of age and gender.
    The term "treatment" or "treating" means an improvement, prophylaxis, or reversal of a disease or disorder, or at least one symptom that can be discerned from it, or an improvement, the prophylaxis, or inversion of at least one measurable physical parameter associated with the disease or disorder being treated, which is not necessarily discernible in or by the subject being treated. The term "treatment" or "treating" further includes the inhibition or slowing down of the progression of a disease or disorder, physically, for example, the stabilization of a discernible symptom, physiologically, for example, the stabilization of a physical parameter, or both.
    Compositions of Fc fragments according to the invention
    The antibody Fc fragments used in the invention are preferably Fc fragments of an IgG1, IgG2, IgG3, or IgG4 immunoglobulin, having a modified affinity for at least one of the Fc receptors (FcR) relative to an Fc fragment. parent.
    The Fc fragments according to the invention have a decreased affinity to at least one Fc receptor and / or an increased affinity to at least one Fc receptor, relative to a parent Fc fragment.
    Preferably, the affinity is increased, relative to that of the parent Fc, by a ratio at least equal to 2, preferably greater than 5, preferably greater than 10, preferably greater than 15, preferably greater than 20, preferably, greater than 25, and preferably greater than 30. In other words, the affinity of the mutated Fc region for a FcR is greater than that of the parent polypeptide. Alternatively, said mutated Fc region has a decreased affinity for at least one of the FcRs. Preferably, the affinity is reduced, relative to that of the parent Fc, by a ratio of at least 2, preferably greater than 5, preferably greater than 10, preferably greater than 15, preferably greater than 20, preferably, greater than 25, and preferably greater than 30. In other words, the affinity of the mutated Fc region for a FcR is lower than that of the parent polypeptide. The affinity of a polypeptide comprising an Fc region 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). Alternatively, one skilled in the art can perform an appropriate ELISA test. An appropriate ELISA assay compares the binding forces of the parent Fc and the mutated Fc. The detected signals specific for the mutated Fc and the parent Fc are compared. Binding affinity can be indifferently determined by evaluating whole polypeptides or evaluating isolated Fc regions thereof.
    In particular, the Fc fragments used according to the invention have a decreased affinity to at least one Fc receptor chosen from FcRn, a Fcγ receptor, and the Clq complement, and / or an increased affinity to at least one Fc receptor selected from the group consisting of FcRn, a Fcγ receptor, and the complement Clq, with respect to a parent Fc fragment.
    According to a particular embodiment, the Fc fragments used according to the invention have a modified affinity, advantageously increased, to the FcRn receptor. This increase in FcRn binding results in an improvement in serum retention in vivo and, therefore, an increase in half-life.
    The Fc fragments can be used alone or in a mixture, for example several different Fc fragments can be administered in a mixture or co-administered.
    According to one aspect of the invention, Fc fragments having a modified affinity for at least one Fc receptor comprise a mutation of at least one amino acid with respect to a parent Fc fragment. The mutations concerned are not the natural variations that define the isotype of the immunoglobulin, but artificial mutations, the production process generating the Fc fragment carrying the desired mutation (s).
    Preferably, the invention implements a composition comprising a single type of mutated Fc fragment. In other words, the composition then comprises molecules of Fc fragments of identical sequence.
    Preferably, the mutation is a substitution, deletion or insertion of one or more amino acids. The mutated Fc fragments may have several mutations, affecting several amino acids, preferably from two to ten.
    In a preferred embodiment, the Fc fragments carry a mutation selected from the group consisting of a mutation at the amino acid 226, 227, 228, 230, 231, 233, 234, 239, 241, 243, 246, 250, 252, 256, 259, 264, 265, 267, 269, 270, 276, 284, 285, 288, 289, 290, 291, 292, 293, 294, 297, 298, 299, 301, 302, 303, 305, 307, 308, 309, 311, 315, 317, 320, 322, 325, 327, 330, 332, 334, 335, 338, 340, 342, 343, 345, 347, 350, 352, 354, 355, 356, 359, 360, 361, 362, 369, 370, 371, 375, 378, 380, 382, 383, 384, 385, 386, 387, 389, 390, 392, 393, 394, 395, 396, 397, 398, 399, 400, 401, 403, 404, 408, 411, 412, 414, 415, 416, 418, 419, 420, 421, 422, 424, 426, 428, 433, 434, 438, 439, 440, 443, 444, 445, 446 or 447, the amino acid numbering of the Fc fragment referring to that of the EU index, or the equivalent in Kabat.
    Certain amino acid positions of the above list - namely, 226, 230, 241, 256, 259, 264, 307, 315, 330, 342, 361, 362, 378, 382, 383, 389, 396, 397 , 421, 428 and 434 are preferred. In particular, mutated Fc which have a high binding affinity for FcRn may comprise at least one amino acid change at said amino acid positions. Of these, positions 230, 264, 307, 315, 330, 378 and 434 are preferred, more preferably positions 264, 315, 378 and 434.
    In a particular embodiment, at least two, even three, four, or five amino acid mutations can substantially enhance the binding affinity for FcRn compared to the parent Fc.
    In a particular embodiment, the mutations are: (i) one or two mutations selected from the group consisting of positions 226, 230, 241, 264, 307, 315, 330, 342, 362, 378, 382, 389, 396 , 397, 421, and 434; preferably 230, 264, 307, 315, 330, 378 and 434, more preferably 264, 315, 378 and 434 and (ii) at least one other, different, position 226, 227, 228, 230, 231, 233,234,239,241,243,246,250,252,256,259,264,265,267,269,270,276,284,285,288,289,290,291,292,293,294,297 298, 299, 301, 302, 303, 305, 307, 308, 309, 311, 315, 317, 320, 322, 325, 327, 330, 332, 334, 335, 338, 340, 342, 343, 345, 347, 350, 352, 354, 355, 356, 359, 360, 361, 362, 369, 370, 371, 375, 378, 380, 382, 383, 384, 385, 386, 387, 389, 390, 392, 393. , 394, 395, 396, 397, 398, 399, 400, 401, 403, 404, 408, 411, 412, 414, 415, 416, 418, 419, 420, 421, 422, 424, 426, 428, 433 , 434, 438, 439, 440, 443, 444, 445, 446 and 447, preferably 226, 230, 241, 264, 307, 315, 330, 342, 362, 378, 382, 389, 396, 397, 421 and 434.
    In a preferred embodiment, at least one of positions 378 and 434 is mutated; and optionally at least one other selected from the group consisting of 226, 230, 241, 264, 307, 315, 330, 342, 362, 378, 382, 389, 396, 397, 421 and 434.
    Preferably, the mutations are 226G, 226Y, 227S, 227L, 228R, 228L, 230S, 230T, 230L, 230A, 230Q, 231T, 231V, 233D, 234R, 239A, 241L, 241Y, 241R, 243L, 246R, 250A. , 252L, 256N, 2591, 264A, 264E, 264M, 265G, 265N, 267N, 267R, 269D, 269G, 270N, 270E, 276S, 284L, 285Y, 288R, 2891, 290R, 290E, 291S, 291Q 292W, 293del , 294del, 297D, 298G, 298N, 299M, 299A, 299K, 301C, 302A, 303A, 3031, 305A, 307P, 307A, 307N, 3081, 309P, 311R, 315D, 317R, 320T, 320E, 322R, 325S, 327V , 327T, 330V, 330T, 332V, 334E, 334R, 335A, 338R, 340E, 342R, 342E, 342K, 343S, 345Q, 345G, 347R, 350A, 352S, 354P, 355Q, 355G, 356N, 359A, 360N, 360R , 361D, 361S, 362R, 362E, 369A, 370R, 371D, 375A, 375G, 378V, 378T, 378S, 380Q, 382V, 382G, 383R, 383N, 3841, 384T, 385R, 386R, 386K, 387S, 387T, 389T, 389K, 389R, 390S, 392E, 392R, 393N, 394A, 395A, 395S, 396S, 396L, 397A, 397M, 398P, 399N, 400P, 401A, 401G, 403T, 404L, 408T, 411A, 412A , 414R, 415D, 415N, 416K, 416G, 418R, 418K, 418E, 419H, 420R, 421T, 421S, 421D, 422 A, 424L, 426T, 428L, 433R, 433P, 434Y, 434S, 434H, 438R, 439R, 440R, 440N, 443R, 444F, 444P, 445S, 446A, 447Net 447E, and are also described in WO2010 / 106180.
    In another embodiment, the Fc fragments comprise at least one mutation selected from the group consisting of 226G, 227L, 230S, 230T, 230L, 231T, 241L, 243L, 250A, 256N, 2591, 264E, 265G, 267R, 290E. , 293del, 294del, 303A, 305A, 307P, 307A, 3081, 315D, 322R, 325S, 327V, 330V, 342R, 347R, 352S, 361D, 362R, 362E 370R, 378V, 378T, 382V, 383N, 386R, 386K, 387T, 389T, 389K, 392R, 395A, 396L, 397M, 403T, 404L, 415N, 416K, 421T, 426T, 428L, 433R, 434Y, 434S and 439R, preferably 226G, 230S, 230T, 230L, 241L, 264E , 307P, 315D, 330V, 342R, 362R, 362E, 378V, 378T, 382V, 389T, 389K, 396L, 397M, 421T, 434Y, and 434S.
    Examples of combinations of particular mutations are shown below: 226G / 330V, 230L / 264E, 230L / 378V, 230S / 315D, 230S / 434Y, 230T / 378V, 241L / 434S, 250A / 434Y, 264E / 378T, 305A / 315D, 305A / 330V, 305A / 434Y, 307P / 434Y, 315D / 389T, 330V / 382V, 330V / 389T, 378V / 421T, 389K / 434Y, 389T / 434Y, 396L / 434S, 230T / 264E, 230T / 315D , 230T / 434S, 230T / 434Y, 241L / 307P, 264E / 307P, 264E / 396L, 315D / 362R, 315D / 382V, 362R / 434Y, 378V / 434Y, 382V / 434Y, 226G / 315D, 226G / 434Y, 241L / 378V, 307P / 378V, 241L / 264E, 378V / 434S, 264E / 378V, 264E / 434S, 315D / 330V, 330V / 434Y and 315D / 434Y; or 226G / 315D / 330V, 226G / 315D / 434Y, 226G / 330V / 434Y, 230L / 264E / 378V, 230T / 264E / 378V, 230T / 264E / 434S, 230S / 315D / 434Y, 230T / 315D / 434Y, 230T / 389T / 434S, 241L / 264E / 434S, 241L / 264E / 378V, 241L / 264E / 307P, 241L / 307P / 378V, 250A / 389K / 434Y, 256N / 378V / 434Y, 2591 / 315D / 434Y, 264E / 378T / 396L, 264E / 378V / 416K, 294del / 307P / 434Y, 264E / 307P / 378V, 264E / 396L / 434S, 264E / 378V / 434S, 305A / 315D / 330V, 305A / 315D / 434Y, 305A / 330V / 434Y, 307P / 378V / 434Y, 315D / 330V / 382V, 315D / 330V / 389T, 315D / 378V / 434Y, 315D / 389T / 434Y, 315D / 362R / 434Y, 315D / 382V / 434Y, 315D / 330V / 434Y, 330V / 382V / 434Y, 330V / 389T / 434Y and 378V / 383N / 434Y.
    In a particularly advantageous embodiment, the Fc fragments carry a combination of mutations selected from 315D / 330V / 361D / 378V / 434Y, 230S / 315D / 428L / 434Y, 307A / 315D / 330V / 382V / 389T / 434Y, 2591 / 315D / 434Y, 256N / 378V / 383N / 434Y.
    In another embodiment, the Fc fragments carry at least one mutation chosen from: G316D, K326E, N315D, N361H, P396L, T350A, V284L, V3231, P352S, A378V, Y436H, V266M, N421T, G385R, K326T, H435R, K447N, N434K, K334N, V397M, E283G, A378T, F423L, A431V, F423S, N325S, P343S, K290E, S375R, F405V, K322E, K340E, N389S, F243I, T307P, N389T, S442F, K248E, Y349H, N286I, T359A, ## EQU1 ## R255W, A287T, N286Y, A231V, K274R, V308G, K414R, M428T, E345G, F243L, P247T, Q362R, S440N, Y278H, D312G, V262A, V305A, K246R, V308I, E380G, N276S, K439Q, S267G, F423Y, A231T, K320R, L410R, K320M, V412M, T307N, T366A, P230S, Y349S, A339T, K246E, K274E, A231P, I336T, S298N, L234P, S267N, V263A, E333G, V308A, K439R, K392R, S440G, V397I, I336V, Y373D, K288E, L309P, P227S, V379A, K288R, K320T, V282A, I377T, N421S and C261R, numbering being that of the EU index or equivalent in Kabat.
    In a particular embodiment, the Fc fragments carry at least one combination of 2 mutations, said combination being chosen from: (i) a mutation chosen from 307N, 326E, 326T, 334N, 334R, 352L, 378V, 378T, 394P, 396L, 397M and 421T and; (ii) at least one mutation selected from 226Y, 227S, 230S, 231V, 234P, 2431, 243L, 246R, 246E, 247T, 248E, 253F, 254F, 255W, 259A, 261R, 262A, 263A, 266M, 267N, 267G; , 274E, 274R, 276S, 278H, 282A, 283G, 284L, 2861, 286Y, 287T, 288E, 288R, 290E, 298N, 302A, 305A, 307P, 308A, 3081, 308G, 309P, 312G, 315D, 316D, 319H , 320T, 320R, 320M, 322E, 3231, 325S, 333G, 334N, 334R, 336T, 339T, 340E, 343S, 345G, 349S, 349H, 350A 352S, 359A, 361H, 362R, 3631, 366A, 373D, 375R, 377T, 378V, 378T, 379A, 380G, 383R, 385R, 389S, 389T, 392R, 393A, 3931, 394P, 396L, 3971, 397M, 398P, 405V, 405L, 41OR, 412M, 414R, 421T, 421S, 423L. , 423Y, 423S, 423P, 428T, 431V, 431T, 434K, 434S, 435R, 436H, 439R, 440G, 440N, 442F, 442P, and 447N, the numbering being that of the EU index or equivalent in Kabat and with the condition that mutation (i) does not occur on the same amino acid as mutation (ii).
    Preferably, the mutated Fc fragments have an increased affinity for the Clq complement, and comprise at least one combination of 2 mutations, said combination comprising: i) a mutation selected from 378V, 378T, 396L, 421T, 334R and 326E; and ii) at least one mutation selected from 361H, 290E, 316D, 248E, 410R, 421T, 334R, 394P, 307P, 447N, 378V, 284L, 421T, 396L, 2861, 315D and 397M, the numbering being that of EU index or equivalent in Kabat and with the proviso that mutation (i) does not occur on the same amino acid as mutation (ii).
    Preferably, the mutated Fc fragments have an increased affinity for the FcgRIIIa receptor (CD16a), and comprises at least one combination of 2 mutations, said combination comprising: i) a mutation selected from 378V, 326E, 397M, 334N and 396L; and ii) at least one mutation selected from 316D, 397M, 334N, 248E, 231V, 246R, 336T, 421T, 361H, 366A, 439R, 290E, 394P, 307P, 378V, 378T, 2861, 286Y and 298N, the numbering being that of the EU index or equivalent in Kabat and with the proviso that mutation (i) does not take place on the same amino acid as mutation (ii).
    Preferably, the mutated Fc fragments have an increased affinity for the FcgRIIa receptor (CD32a), and comprises at least one combination of 2 mutations, said combination comprising: i) a mutation selected from 378V, 326E, 397M, 307N, 394P, 326T , 396L and 334N; and ii) at least one mutation selected from: 316D, 334R, 334N, 3231, 231V, 246R, 336T, 378T, 286Y, 2861, 352S, 383R, 359A, 421T, 361H, 315D, 366A, 290E, 307P and 43R , the numbering being that of the index EU or equivalent in Kabat and with the proviso that the mutation (i) does not take place on the same amino acid as the mutation (ii).
    Preferably, the mutated Fc fragments have an increased affinity for the FcgRIIb receptor (CD32b), and comprises at least one combination of 2 mutations, said combination comprising: i) a mutation selected from 326E, 326T, 378V, 397M, 352L, 394P , 396L and 421T; and ii) at least one mutation selected from 316D, 334R, 248E, 334N, 418P, 231V, 320E, 402D, 359A, 383R, 421T and 361H, the numbering being that of the EU index or equivalent in Kabat and with the condition that the mutation (i) does not take place on the same amino acid as the mutation (ii).
    Preferably, the mutated Fc fragments comprise at least one combination of 3 mutations, said combination comprising: (i) a mutation selected from 326E, 326T, 352L, 378V, 378T, 396L, 397M, 421T, 334N, 334R, 307N and 394P; and (ii) at least 2 mutations selected from 226Y, 227S, 230S, 231V, 234P, 2431, 243L, 246R, 246E, 247T, 248E, 253F, 254F, 255W, 259A, 261R, 262A, 263A, 266M, 267N, 267G, 274E, 274R, 276S, 278H, 282A, 283G, 284L, 2861, 286Y, 287T, 288E, 288R, 290E, 298N, 302A, 305A, 307P, 308A, 3081, 308G, 309P, 312G, 315D, 316D, 319H, 320T, 320R, 320M, 322E, 3231, 325S, 333G, 334N, 334R, 336T, 339T, 340E, 343S, 345G, 349S, 349H, 350A 352S, 359A, 361H, 362R, 3631, 366A, 373D, 375R , 377T, 378V, 378T, 379A, 380G, 383R, 385R, 389S, 389T, 392R, 393A, 3931, 394P, 396L, 3971, 397M, 398P, 405V, 405L, 41OR, 412M, 414R, 421T, 421S, 423L, 423Y, 423S, 423P, 428T, 431V, 431T, 434K, 434S, 435R, 436H, 439R, 440G, 440N, 442F, 442P and 447N, the numbering being that of the EU index or equivalent in Kabat and with the condition that mutation (i) does not occur on the same amino acid as mutation (ii).
    According to another particular embodiment, the mutated Fc fragments used in the invention carry a deletion of an amino acid at position 293 or 294 (DEL293 or DEL294), the amino acid numbering of the Fc fragment referring to that of the amino acid. EU index, or the equivalent in Kabat. This deletion may be the only mutation of the Fc fragment, or be accompanied by other mutations, in particular among those listed above.
    This single mutation leads to a particular glycosylation of the fragment, namely a hypersialylation, particularly advantageous vis-à-vis the half-life of glycoproteins and inflammatory processes.
    According to another more particular embodiment, the mutated Fc fragments used in the invention carry the deletion of the amino acid at position 293 or 294, and also carry one or more mutations at selected positions in the list below. 226, 230, 241, 256, 259, 264, 307, 315, 330, 342, 361, 362, 378, 382, 383, 389, 396, 397, 421, 428 and 434.
    For example, a preferred Fc fragment carries the combination of the 307P, 434Y mutations in association with the DEL294 deletion.
    According to another aspect of the invention, there is used a composition comprising a plurality of Fc fragments which all have substantially the same sequence, and which, taken as a whole, have a particular glycosylation profile. According to one particular aspect, a composition used in the context of the invention comprises Fc fragments having on their glycosylation site (Asn 297) N-glycans, characterized in that said N-glycans of Fc fragments have a fucosylation rate. less than 65%, preferably less than 60%, preferably less than 55%, preferably less than 50%, more preferably less than 45%, preferably less than 40%, preferably less than 35%, preferably less than 30%, preferably less than 25%, preferably less than 20%.
    According to another aspect, a composition used in the context of the invention comprises Fc fragments, said Fc fragments having on their glycosylation site (Asn 297) N-glycans, characterized in that said N-glycans of Fc fragments have a glycan structure of biantenné type, with short chains, weak sialylation, having terminal mannoses and / or non-intermediate terminal N-acetylglucosamines.
    According to a more particular aspect, a composition used in the context of the invention comprises Fc fragments, said Fc fragments having on their glycosylation site (Asn 297) N-glycans, characterized in that said N-glycans of Fc fragments have a content greater than 60% for the forms G0 + G1 + G0F + G1F, the G0F + G1F forms being less than 50%.
    According to another more particular aspect, a composition used in the context of the invention comprises Fc fragments, said Fc fragments having on their glycosylation site (Asn 297) N-glycans, characterized in that said N-glycans of Fc fragments have a content greater than 60% for the forms G0 + G1 + G0F + G1F, the fucose content being less than 65%.
    According to another even more particular aspect, a composition used in the context of the invention comprises Fc fragments, said Fc fragments having on their glycosylation site (Asn 297) N-glycans, characterized in that said N-glycans of the fragments Fc have a content of less than 40% for G1F + GOF forms.
    In a more particular aspect, a composition used in the context of the invention comprises Fc fragments which have on their glycosylation site (Asn 297) N-glycans, said N-glycans of Fc fragments having a fucosylation rate equal to 0%. The invention thus provides a composition comprising Fc fragments which have on the Asn297 glycosylation site N-glycans characterized in that said N-glycans of the Fc fragments are free of fucose.
    Also, according to one particular aspect, a composition used in the context of the invention comprises Fc fragments which have on the Asn297 glycosylation site N-glycans, characterized in that said N-glycans of the Fc fragments have a fucosylation rate. between 20% and 55%. In particular, the invention provides a composition comprising Fc fragments which have, on the Asn297 glycosylation site, N-glycans, characterized in that said N-glycans of the Fc fragments have a fucosylation level of between 20% and 50%, between 25% and 55%, between 25% and 50%, between 20% and 45% or between 25 and 45%.
    According to a more particular aspect, a composition that is useful according to the invention comprises Fc fragments which have N-glycans on the Asn297 glycosylation site, characterized in that said N-glycans of the Fc fragments have a content greater than 60%, of preferably greater than 80%, for the forms GO + G1 + GOF + G1F, the forms G0F + G1F being less than 50%, preferably less than 40%, or 30%.
    According to another more particular aspect, the N-glycans of the Fc fragments within the composition have a content greater than 60% for the forms G0 + G1 + G0F + G1F, the fucose content being less than 65%.
    According to yet another more particular aspect, the N-glycans of the Fc fragments within the composition have a content of less than 50% for the G1F + GOF forms, preferably less than 40%, or 30%.
    The forms G0, G0F, G1 and GIF are selected from the shapes shown in Figure 3.
    Advantageously, the N-glycans of the Fc fragments within the composition have an average sialic acid content of less than 25%, 20%, 15%, or 10%, preferably 5%, 4% 3% or 2%. .
    A composition which can be used in the context of the invention comprises Fc fragments having, at their glycosylation site (Asn 297), N-glycans, said N-glycans of Fc fragments having a glycan structure of biantenné type, with chains short, low sialylation, having terminal mannoses and / or non-intermediate terminal N-acetylglucosamines, the N-glycans having for example a content greater than 60% for the forms G0 + G1 + G0F + GIF, and low fucocylation, the N-glycans having, for example, a content of less than 50% of G0F + GIF forms.
    In a particular embodiment, the Fc fragments according to the invention have glycanic structures as described in Patent Application WO01 / 77181.
    According to an advantageous embodiment, the Fc fragments used in the invention comprise at least one mutation of an amino acid with respect to a parent Fc fragment, and have on their glycosylation site (Asn 297) N-glycans, said N-glycans of Fc fragments having a degree of fucosylation of less than 65%, preferably less than 60%, preferably less than 55%, preferably less than 50%, more preferably less than 45%, preferably less than 40%. %, preferably less than 35%, preferably less than 30%, preferably less than 25%, preferably less than 20%. Preferably, the Fc fragments carry one or more mutations at positions selected from those listed above, in particular at positions selected from the following list: 226, 230, 241, 256, 259, 264, 307, 315, 330 , 342, 361, 362, 378, 382, 383, 389, 396, 397, 421, 428 and 434, and further possess at their glycosylation site (Asn 297) N-glycans having a fucosylation rate of less than 55%, preferably less than 50%, more preferably less than 45%, preferably less than 40%, preferably less than 35%, preferably less than 30%, preferably less than 25%, preferably less than 20%.
    More preferably, the Fc fragments of the composition according to the invention carry a combination of mutations selected from 315D / 330V / 361D / 378V / 434Y, 230S / 315D / 428L / 434Y, 307A / 315D / 330V / 382V / 389T / 434Y , 2591 / 315D / 434Y, 256N / 378V / 383N / 434Y, and DEL294 / 307P / 434Y and further possess at their glycosylation site (Asn 297) N-glycans having a fucosylation level of less than 55%, preferably less than 50%, more preferably less than 45%, preferably less than 40%, preferably less than 35%, preferably less than 30%, preferably less than 25%, preferably less than 20%.
    Advantageously, the Fc fragments having a modified glycosylation at the position 297 glycosylation site, in particular a low fucosylation, has an increase in the binding of said fragment to Fcgamma receptors (FcγR), in particular the FeyRIIIa receptor (CD 16a ).
    Preferably, said Fc fragments have an affinity for CD 16a of at least 2x106 M -1, at least equal to 2x107 M -1, 2 × 10 8 M -1 or 2 × 10 9 M -1, as determined by Scatchard analysis or BIAcore technology (Label-free surface plasmon resonance based technology).
    Production of Fc Fragments According to the Invention
    The Fc fragments used in the invention may be produced by any method known to those skilled in the art, for example by chemical synthesis, or by recombination.
    In a preferred embodiment, the Fc fragments used in the invention are said to be "recombinant", ie they are obtained by recombination.
    When the Fc fragments according to the invention comprise a mutation of one or more amino acids, the mutation (s) may optionally be introduced by known techniques such as gene synthesis, site-directed mutagenesis, in particular obtained by PCR with primers. that introduce the desired mutations, followed by random mutagenesis. Preferably, 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 β, η and i. Conventional recombinant techniques implement a recombination in a host cell, transformed with one or more vectors that allow the expression accompanied or not by the secretion of the Fc fragment sequence in the extracellular medium. The vector generally comprises a promoter, translation initiation and termination signals, as well as appropriate transcriptional regulatory regions. It can be stably maintained in the host cell and may optionally have particular signals that specify the secretion of the translated protein. These different elements are chosen and optimized by those skilled in the art depending on the cellular host used.
    Such vectors are prepared by methods commonly used by those skilled in the art, and the resulting clones can be introduced into a suitable host by standard methods, such as lipofection, electroporation, the use of polycationic agents, thermal shock, or chemical methods. 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 Fc fragments used in the invention can be produced by culturing in a suitable medium and culture conditions of a host cell expressing said Fc fragments; and recovering the fragments thus produced from the culture medium or said cultured cells.
    The mammalian cells preferred for the expression of Fc fragments are the YB2 / 0 rat line, the vero line, the CHO hamster line, in particular the CHO dhfr- and CHO Lecl3 lines, CHO-lec10, CHO-lecl, CHOK1SV Potelligent® (Lonza, Switzerland), CHOGnTIII (Glycart, Switzerland), PER.C6 ™ (Crucell), HEK293, T1080, EB66, K562, NSO, SP2 / 0, BHK or COS.
    Another mode of production is the expression of Fc fragments in transgenic organisms, for example in plants (Ayala M, Gavilondo J, Rodriguez M, Fuentes A, Enriquez G, Perez L, Cremata J, Pujol M. Production of plantibodies in Nicotiana plants, Methods Mol Biol 2009; 483: 103-34.) or in the milk of transgenic animals such as rabbit, goat, rat or pig (Pollock, D P., JP Kutzko, E. Birck-Wilson, JL Williams, Y. Echelard and HM Meade (1999), Transgenic milk as a method for the production of recombinant antibodies, Journal of Immunological Methods, 231: 147-157). See also WO200748077 in this regard.
    In an alternative embodiment, the Fc fragments are obtained by proteolytic processing of mutated immunoglobulins.
    Modification of the glycosylation of Fc fragments can be achieved by known techniques. The Fc fragments possessing a glycosylation according to the invention may in particular be produced from the cleavage of antibodies produced according to the technique described in WO 01/77181, in particular by the papain enzyme. The weakly fucosylated Fc fragments may also be obtained by production in cells cultured in the presence of kifunensine, as described, for example, in document US Pat. No. 3,700,321, or in cells for which the GDP-fucose production route is inhibited, for example by inhibiting at least one of the enzymes of the fucose production cycle (see in particular US 2010291628 or US 20090228994, EP 1 500 698, EP 1 792 987 or US 7 846 725). It is also possible to use interfering RNA (RNAi) inhibiting 1,6-fucosyltransferase as described in US 7,393,683 or WO2006133148. It may also be methods of preparation in yeasts, as described for example in WO 0200879.
    In the case where the Fc fragments have 100% nonfucosylated oligosaccharides, that is to say when the Fc fragments are completely free of fucose, it is possible to use preparation methods known to those skilled in the art. , as for example those described in the documents EPI 176195, US 7,214,775, US 6,994,292, US 7,425,449, US2010223686, WO2007099988, EP 1 705 251, this list not being limiting. It may be for example a method using a host cell expressing at least one nucleic acid encoding an Fc fragment, and whose glycosylation is modified by deletion of the gene coding for α, β-fucosyltransferase or by addition of mutation of this gene to remove al. 6-fucosyltransferase activity, and thereby expressing an antibody fragment lacking fucose.
    Therapeutic applications
    Because of their numerous advantages, in terms of efficacy, optimized effector functions or reduced side effects, the Fc fragments described herein are useful in the treatment of autoimmune and / or inflammatory disease.
    Here is described a method for treating an autoimmune and / or inflammatory disease in a patient comprising administering to said patient a therapeutically effective amount of Fc fragments having altered affinity for at least one Fc receptor as described herein.
    In the present invention, an autoimmune and / or inflammatory disease means a primitive or secondary organ-specific or systemic autoimmune and / or inflammatory disease, associated or not with pathogenic autoantibodies.
    For example, the disease may be selected from thrombotic thrombocytopenic purpura (ITP), transplant or organ rejection, graft-versus-host disease, rheumatoid arthritis, systemic lupus erythematosus, various types of sclerosis, primary Sjögren's syndrome (or Sjögren's syndrome), autoimmune polyneuropathies such as multiple sclerosis, type I diabetes, autoimmune hepatitis, ankylosing spondylitis, Reiter's syndrome, arthritis of gout, celiac disease, Crohn's disease, chronic Hashimoto's thyroiditis (hypothyroidism), Adisson's disease, autoimmune hepatitis, Graves' disease (hyperthyroidism), ulcerative colitis, vasculitis and vasculitis ANCA (anti-cytoplasmic antibodies to neutrophils), autoimmune cytopenia and other hematological complications in adults and children, such as throm Acute or chronic autoimmune bopenias, autoimmune haemolytic anemias, haemolytic newborn disease (HHN), cold agglutinin disease, thrombotic thrombocytopenic purpura, and autoimmune acquired haemophilia; Goodpasture's syndrome, extra-membranous nephropathies, autoimmune bullous skin disorders, refractory myasthenia gravis, mixed cryoglobulinemia, psoriasis, juvenile chronic arthritis, inflammatory myositis, dermatomyositis and autoimmune systemic disorders of the skin. including antiphospholipid syndrome, connective tissue disease, various types of sclerosis, pulmonary autoimmune inflammation, Guillain-Barre Syndrome, chronic demyelinating inflammatory polyradiculoneuropathy (PDCI), autoimmune thyroiditis, mellitis , myasthenia gravis, inflammatory autoimmune disease of the eye, optic neuromyelitis (Deviation Disease), scleroderma, pemphigus, insulin resistance diabetes, polymyositis, Biermer's anemia, glomerulonephritis, Wegener's disease , Horton's disease, periarthritis nodosa and Churg and Strauss syndrome, St. atrophic polychondritis, Behçet's disease, monoclonal gammopathy, Wegener's granulomatosis, lupus, ulcerative colitis, psoriatic arthritis, sarcoidosis, collagenous colitis, dermatitis herpetiformis, familial Mediterranean fever, glomerulonephritis IgA deposits, myasthenic Lambert-Eaton syndrome, sympathetic ophthalmia, Fiessinger-Leroy-Reiter syndrome and uveo-meningoencephalic syndrome. Other inflammatory diseases are also understood, such as acute respiratory distress syndrome (ARDS), acute septic arthritis, adjuvant arthritis, allergic encephalomyelitis, allergic rhinitis, allergic vasculitis, allergy, asthma, atherosclerosis, chronic inflammation due to chronic bacterial or viral infections, chronic obstructive pulmonary disease chronic disease (COPD), coronary heart disease, encephalitis, inflammatory bowel disease, osteolysis inflammatory, inflammation associated with acute and delayed hypersensitivity reactions, inflammation associated with tumors, peripheral nerve injury or demyelinating diseases, inflammation associated with tissue trauma such as burns and ischemia, inflammation due to meningitis, multiorgan organ failure syndrome (multiple organ dysfunction syndrome, MODS), fibrosis pulmonary dystonia, septicemia and septic shock, Stevens-Johnson syndrome, undifferentiated arthritis, and undifferentiated spondyloarthropathies.
    In a particular embodiment of the invention, the autoimmune disease is idiopathic thrombotic purpura (ITP) and chronic inflammatory demyelinating polyradiculoneuropathy (PDCI).
    One of the effects observed is in particular a limitation or a decrease in the platelet destruction observed in the ITP pathology and a limitation or decrease in the loss of myelin sheath of the peripheral nerves in the PDCI.
    In another particular embodiment, the disease is an inflammatory disease, such as graft-versus-host disease. In this case, Fc fragments bearing a hypersialylation-inducing mutation, such as a deletion of an amino acid at position 294 (DEL294) or 293 (DEL293), are particularly advantageous.
    Any route of administration is contemplated, including parenteral routes, such as the intravenous, intramuscular, subcutaneous, intradermal, topical, or mucosal route, for example by inhalation. Enteral (oral, rectal) and intrathecal routes are also possible. Preferably, the intravenous route is used.
    The Fc fragments according to the invention are generally formulated within pharmaceutical compositions comprising pharmaceutically acceptable excipients.
    The pharmaceutical compositions may be in any suitable dosage form depending on the chosen route of administration.
    The pharmaceutical compositions useful according to the invention advantageously comprise one or more excipients or vehicles, which are pharmaceutically acceptable. For example, saline, physiological, isotonic, buffered, etc., solutions compatible with a pharmaceutical use and known to those skilled in the art may be mentioned. The compositions may contain one or more agents or vehicles selected from dispersants, solubilizers, stabilizers, preservatives, etc. Agents or vehicles that can be used in formulations (liquid and / or injectable and / or solid) include methylcellulose, hydroxymethylcellulose, carboxymethylcellulose, polysorbate 80, mannitol, gelatin, lactose, vegetable oils, and the like. acacia, etc. The compositions may optionally be formulated using dosage forms or devices providing sustained and / or delayed release. For this type of formulation, an agent such as cellulose, carbonates or starches is advantageously used.
    The doses administered may vary, inter alia, according to the weight and age of the patient, and the severity of the disease, appreciated by those skilled in the art.
    In a preferred embodiment, the dosage of the Fc fragments according to the invention is in the range of about 0.05mg / kg to about 1g / kg of body weight, or about 20mg to about 100g per day for an adult. Preferably, the dosage is from about 330mg / kg to about 660mg / kg per day.
    Example 1 Efficacy Test (Inhibition of Lysis of Red Blood Cells)
    To mimic the lysis of red blood cells observed in idiopathic thrombocytopenic purpura (ITP), involving the autoantibodies of the patient with ITP, an effector cell-mediated red cell lysis in the presence of an anti-human monoclonal antibody. Rhesus D (RJiD) was conducted, and the ability of different amounts of polyvalent immunoglobulins (IglV) or recombinant, mutated and unmutated Fc fragments to inhibit this lysis, for example by competition with anti-RhD for binding Fc receptors on the surface of the effector cells were evaluated.
    For this, as effector cells, PBMCs (peripheral blood mononuclear cells) were purified from the peripheral blood by Ficoll gradient. Rh-D + RBCs obtained from Rh-D + healthy blood donors were mixed with an anti-RhD monoclonal antibody. The PBMCs were incubated with opsonized Rh-D + red blood cells (effector / target ratio of 2/1).
    To evaluate the ability of candidates (IgM and recombinant Fc, mutated and non-mutated) to inhibit anti-RhD antibody-induced cytotoxicity by competition and / or saturation of Fc receptors, various concentrations of candidates (0 to 9.75 μΜ) were added to each well. After 16 hours, the percentage of lysed red blood cells was estimated chromogenically by measuring the amount of hemoglobin released in the supernatants (optical density measurement or OD). The specific lysis is calculated as a percentage according to the following formula: Sample OD - Control OD 0% / Control OD 100% - Control OD 0% * 100 =% Lysis in which: "100% control OD" corresponds to total lysis of red blood cells (eg NH4Cl); "0% control OD" corresponds to the lysis observed with a reaction mixture without antibodies. The results are expressed as% specific lysis (see Figure 4).
    权利要求:
    Claims (18)
    [1" id="c-fr-0001]
    A composition comprising antibody Fc fragments for use in the treatment of an autoimmune and / or inflammatory disease, said Fc fragments being isolated Fc fragments having a modified affinity for at least one of the Fc receptors (FcR ) with respect to a parent Fc fragment.
    [2" id="c-fr-0002]
    A composition comprising Fc fragments for use according to claim 1, wherein the Fc receptor is selected from FcRn, a Fcγ receptor, and the complement Clq.
    [3" id="c-fr-0003]
    A composition comprising Fc fragments for use according to claim 1 or 2, wherein said Fc fragments comprise a mutation of one or more amino acids.
    [4" id="c-fr-0004]
    A composition comprising Fc fragments for use according to claim 3, wherein the mutation is a substitution, deletion or insertion of one or more amino acids.
    [5" id="c-fr-0005]
    5. A composition comprising Fc fragments for use according to one of claims 1 to 4, wherein the Fc fragments are Fc fragments of an immunoglobulin IgG1, IgG2, IgG3, or IgG4, comprising at least one mutation of a or more amino acids compared to a parent Fc fragment.
    [6" id="c-fr-0006]
    6. A composition comprising Fc fragments for use according to one of claims 1 to 5, wherein the Fc fragments are obtained recombinantly.
    [7" id="c-fr-0007]
    A composition comprising Fc fragments for use according to one of claims 3 to 6, wherein the mutation is involved in increasing the half-life compared to that of the parent Fc fragment.
    [8" id="c-fr-0008]
    A composition comprising Fc fragments for use according to one of claims 3 to 7, wherein the mutation is selected from the group consisting of a mutation at amino acid 226, 227, 228, 230, 231, 233, 234,239,241,243,246,250,252,256,259,264,265,267,269,270,276,284,285,288,289,290,291,292,293,294,297 298, 299, 301, 302, 303, 305, 307, 308, 309, 311, 315, 317, 320, 322, 325, 327, 330, 332, 334, 335, 338, 340, 342, 343, 345, 347, 350, 352, 354, 355, 356, 359, 360, 361, 362, 369, 370, 371, 375, 378, 380, 382, 383, 384, 385, 386, 387, 389, 390, 392, 393, 394, 395, 396, 397, 398, 399, 400, 401, 403, 404, 408, 411, 412, 414, 415, 416, 418, 419, 420, 421, 422, 424, 426, 428, 433, 434, 438, 439, 440, 443, 444, 445, 446 or 447, the amino acid numbering of the Fc fragment referring to that of the EU index, or the equivalent in Kabat.
    [9" id="c-fr-0009]
    A composition comprising Fc fragments for use according to one of claims 3 to 7, wherein the mutation is selected from the group consisting of a mutation at amino acid 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 amino acid numbering of the Fc fragment referring to that of the EU index, or the equivalent in Kabat.
    [10" id="c-fr-0010]
    A composition comprising Fc fragments for use according to claim 9, wherein the mutation is a deletion of an amino acid at position 293 or 294 (DEL293 or DEL294), the amino acid numbering of the Fc fragment referring to that of the EU index, or the equivalent in Kabat.
    [11" id="c-fr-0011]
    A composition comprising Fc fragments for use according to claim 9 or 10, wherein the mutation is involved in glycosylation of the Fc fragment, preferably hypersialylation.
    [12" id="c-fr-0012]
    12. A composition comprising Fc fragments for use according to one of claims 1 to 11, said Fc fragments having at their glycosylation site (Asn 297) N-glycans, characterized in that said N-glycans Fc fragments present a degree of fucosylation of less than 65%, preferably less than 60%, preferably less than 55%, preferably less than 50%, more preferably less than 45%, preferably less than 40%, preferably less than 35%; %, preferably less than 30%, preferably less than 25%, preferably less than 20%.
    [13" id="c-fr-0013]
    13. A composition comprising Fc fragments for use according to one of claims 1 to 12, said Fc fragments having at their glycosylation site (Asn 297) N-glycans, characterized in that said N-glycans Fc fragments have a glycan structure of biantenné type, with short chains, weak sialylation, having terminal mannoses and / or non-intermediate terminal N-acetylglucosamines.
    [14" id="c-fr-0014]
    14. A composition comprising Fc fragments for use according to one of claims 1 to 13, said Fc fragments having at their glycosylation site (Asn 297) N-glycans, characterized in that said N-glycans Fc fragments present a content greater than 60% for the forms G0 + G1 + G0F + G1F, the G0F + G1F forms being less than 50%.
    [15" id="c-fr-0015]
    15. A composition comprising Fc fragments for use according to one of claims 1 to 13, said Fc fragments having at their glycosylation site (Asn 297) N-glycans, characterized in that said N-glycans Fc fragments present a content greater than 60% for the forms G0 + G1 + G0F + G1F, the fucose content being less than 65%.
    [16" id="c-fr-0016]
    16. A composition comprising Fc fragments for use according to claim 14 or 15, said Fc fragments having, at their glycosylation site (Asn 297), N-glycans, characterized in that said N-glycans of the Fc fragments have a lower content. 40% for G1F + GOF forms.
    [17" id="c-fr-0017]
    17. Composition comprising Fcfragments for use according to one of claims 1 to 16, the disease being selected from idiopatic thrombotic purpura, chronic inflammatory demyelinating polyradiculoneuropathy (PDCI), rejection of transplants or organs, the disease of graft versus host, rheumatoid arthritis, systemic lupus erythematosus, various types of sclerosis, primary Sjögren's syndrome (or Sjögren's syndrome), autoimmune polyneuropathies such as multiple sclerosis, diabetes mellitus I, autoimmune hepatitis, ankylosing spondylitis, Reiter's syndrome, gout arthritis, celiac disease, Crohn's disease, chronic Hashimoto's thyroiditis (hypothyroidism), Adisson's disease, hepatitis autoimmune disease, Graves' disease (hyperthyroidism), ulcerative colitis, vasculitis and systemic vasculitis associated with ANCA (anti-cytoplasmic antibodies) neutrophils), autoimmune cytopenias and other hematologic complications in adults and children, such as acute or chronic autoimmune thrombocytopenia, autoimmune haemolytic anemias, haemolytic disease of the newborn ( MHN), cold agglutinin disease, thrombotic thrombocytopenic purpura and autoimmune acquired haemophilia; Goodpasture's syndrome, extramembranous nephropathies, autoimmune bullous skin disorders, refractory myasthenia gravis, mixed cryoglobulinemia, psoriasis, juvenile chronic arthritis, inflammatory myositis, dermatomyositis and autoimmune systemic disorders of child including antiphospholipid syndrome, connective tissue disease, various types of sclerosis, pulmonary autoimmune inflammation, Guillain-Barre syndrome, autoimmune thyroiditis, mellitis, myasthenia gravis, inflammatory autoimmune disease pemphigus, insulin resistance diabetes, polymyositis, Biermer anemia, glomerulonephritis, Wegener's disease, Horton's disease, periarthritis nodosa and Churg and Strauss syndrome, Still's disease , atrophic polychondritis, Behçet's disease, monoclonal gammopathy, Wegener's granulomatosis, lupus, rectocolitis hemorrhagic itch, psoriatic arthritis, sarcoidosis, collagenous colitis, dermatitis herpetiformis, familial Mediterranean fever, glomerulonephritis with IgA deposits, myasthenic Lambert-Eaton syndrome, sympathetic ophthalmia, Fiessinger-Leroy-Reiter syndrome and uveo-meningoencephalic syndrome, optic neuromyelitis (Devia's disease) and scleroderma.
    [18" id="c-fr-0018]
    A composition comprising Fc fragments for use according to claim 17, wherein said Fc fragments comprise a deletion of an amino acid at position 293 or 294 (Del293 or DEL294), and the disease is graft disease against the host.
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    同族专利:
    公开号 | 公开日
    CN108026163A|2018-05-11|
    CA2990354A1|2017-01-12|
    WO2017006052A2|2017-01-12|
    EP3319989A2|2018-05-16|
    US20180355034A1|2018-12-13|
    JP2018524355A|2018-08-30|
    FR3038517B1|2020-02-28|
    CN108026163B|2021-10-08|
    JP2021050235A|2021-04-01|
    WO2017006052A3|2017-04-20|
    CN113817049A|2021-12-21|
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    法律状态:
    2016-06-22| PLFP| Fee payment|Year of fee payment: 2 |
    2017-01-13| PLSC| Publication of the preliminary search report|Effective date: 20170113 |
    2017-06-21| PLFP| Fee payment|Year of fee payment: 3 |
    2018-06-21| PLFP| Fee payment|Year of fee payment: 4 |
    2019-06-21| PLFP| Fee payment|Year of fee payment: 5 |
    2020-06-23| PLFP| Fee payment|Year of fee payment: 6 |
    2021-06-23| PLFP| Fee payment|Year of fee payment: 7 |
    优先权:
    申请号 | 申请日 | 专利标题
    FR1556399A|FR3038517B1|2015-07-06|2015-07-06|USE OF MODIFIED FC FRAGMENTS IN IMMUNOTHERAPY|
    FR1556399|2015-07-06|FR1556399A| FR3038517B1|2015-07-06|2015-07-06|USE OF MODIFIED FC FRAGMENTS IN IMMUNOTHERAPY|
    JP2018500412A| JP2018524355A|2015-07-06|2016-07-06|Use of modified Fc fragments in immunotherapy|
    CN201680051282.5A| CN108026163B|2015-07-06|2016-07-06|Use of modified Fc fragments in immunotherapy|
    CA2990354A| CA2990354A1|2015-07-06|2016-07-06|Use of modified fc fragments in immunotherapy|
    US15/742,268| US20180355034A1|2015-07-06|2016-07-06|Use of modified fc fragments in immunotherapy|
    PCT/FR2016/051708| WO2017006052A2|2015-07-06|2016-07-06|Use of modified fc fragments in immunotherapy|
    EP16747826.2A| EP3319989A2|2015-07-06|2016-07-06|Use of modified fc fragments in immunotherapy|
    CN202111105793.XA| CN113817049A|2015-07-06|2016-07-06|Use of modified Fc fragments in immunotherapy|
    JP2020212333A| JP2021050235A|2015-07-06|2020-12-22|Use of modified Fc fragments in immunotherapy|
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