USE OF MODIFIED FRAGMENTS IN IMMUNE THERAPY

Description

[0001] The present invention relates to the immunotherapy of autoimmune and / or inflammatory diseases. Technological background of the invention :

[0002] Immunotherapy, a therapy consisting of administering exogenous antibodies to patients, is now widely used for the treatment of various pathologies, particularly autoimmune diseases and inflammatory diseases.

[0003] Two types of immunoglobulin therapy are typically proposed for the treatment of these diseases: i) intravenous immunoglobulin (IVIG) therapies, which consist of administering immunoglobulins (most often IgG) to patients intravenously from pooled human plasma donations, and ii) therapies based on the use of recombinant antibodies (i.e. antibodies obtained by genetic engineering). The latter have enabled real advances in the treatment of patients with inflammatory and autoimmune diseases, in particular because they offer the possibility of overcoming the disadvantages associated with the use of plasma-derived immunoglobulins, in particular the risk of supply disruptions and the risk of transmission of pathogens potentially present in the plasma.

[0004] The presence of Fab fragments in immunoglobulins can cause significant adverse reactions in treated patients. To avoid these side effects, patent application WO 2004 / 099374 discloses the use of isolated recombinant Fc fragments for the treatment of patients, particularly those with autoimmune diseases.

[0005] Application EP 2 233 500 describes Fc fragments having improved affinity for FcRn compared to a parent Fc fragment, and comprising at least two mutations.

[0006] However, there is still a need to optimize these Fc fragments, in particular to increase their half-life and / or their therapeutic efficacy. Summary of the invention :

[0007] The inventors now propose to use Fc fragments having a modified affinity for at least one of the Fc receptors (FcR) compared to a parent Fc fragment. According to the invention, the Fc fragments are isolated, i.e. they are not associated with Fab fragments, nor conjugated or fused to any other protein, polypeptide or peptide. In particular, they are not complete immunoglobulins.

[0008] The invention relates to a composition comprising Fc fragments of antibodies, for use in the treatment of a specific disease as defined in claim 1.

[0009] The invention also relates to compositions for their use as defined in one of claims 2 to 8.

[0010] According to preferred aspects, it is about Fc fragments have a combination of K334N / P352S / A378V / V397M mutations relative to a parent Fc fragment of wild-type IgG1, preferably wherein the Fc fragments consist of the sequence SEQ ID NO:19.

[0011] The invention also provides more generally a composition comprising Fc fragments of antibodies, for use in the treatment of a specific disease according to claim 1.

[0012] In a particular embodiment, the composition comprises Fc fragments of antibodies, comprising at least one mutation of one or more amino acids and / or having N-glycans on their glycosylation site (Asn 297), said N-glycans of the Fc fragments having a fucosylation level of less than 65%, Legend of the figures :

[0013] There figure 1shows sequence alignments of native human IgG1 referring to positions 216 to 447 (according to the EU index) 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 G1m1,17 allotype (SEQ ID NO: 6) and the G1m3 allotype (SEQ ID NO: 10). The "lower hinge CH2-CH3" domain of IgG1 starts at position 226 (see arrow). The CH2 domain is highlighted in gray and the CH3 domain is in italics. figure 2 shows schematic representations of the IgG1 Fc (A) and an scFc (B). CH2s are shown in gray and CH3s in white. Disulfide bonds are shown in thin gray dotted lines. In the scFc, the two CH2-CH3 polypeptides are connected by a peptide linker shown in bold dotted lines. figure 3 shows the G0, G0F, G1 and G1F forms of the glycan structures likely to be present on the Fc fragments of the invention. The figure 4is a graph showing the percentage of effector cell-mediated lysis of Rhesus D+ red blood cells in the presence of an anti-Rhesus D (RhD) monoclonal antibody, observed following the addition of different amounts of polyvalent immunoglobulins (IVIg) or recombinant Fc fragments, unmutated or mutated (Fc fragment carrying the mutations 315D / 330V / 361D / 378V / 434Y). Figure 5 is a graph showing the percentage of effector cell-mediated lysis of Rhesus D+ red blood cells in the presence of an anti-Rhesus D (RhD) monoclonal antibody, observed following the addition of different amounts of polyvalent immunoglobulins (IVIg) or recombinant Fc fragments, unmutated or mutated (Fc fragment carrying the mutations according to the Table 1).rFc WT means the wild-type recombinant Fc fragment. Positive controls are represented by: IVIG (i.e. mixture of plasma antibodies extracted from human plasma - IVIG is the standard treatment for idiopathic thrombocytopenic purpura (ITP)) and "IgG Mabs starting" which corresponds to the Fc fragment of herceptin (antibody produced transgenically in a mammal, then digested with papain to generate the Fc fragment). figure 6 represents an in vitro binding assay measuring the affinity (Kd) of WT (wild-type) and mutated Fc fragments for human FcRn (A) and for CD16aV (V polymorphism at position 158 of CD16) (B), assessed by surface plasmon resonance (SPR) using Biacore. Figure 7 shows the map of the pCEP4 vector used for the expression of the T5A-74 variant, defined below, and the map of the OptiCHO vector for the expression of the T5A-74 variant. Detailed description of the invention : Definitions

[0014] Throughout this description, the numbering of residues in the Fc fragment is that of the immunoglobulin heavy chain according to the EU index or as described in Kabat et al., Sequences of Proteins 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" herein refers to the numbering of residues of the human IgG1 antibody.

[0015] The term "immunoglobulin" refers to the structure that constitutes the naturally occurring biological form of an antibody, including both constant and variable regions (also called fragments). An immunoglobulin molecule is a molecule whose basic unit is a heterotetramer consisting of two heavy chains of approximately 50-70 kDa each (called the H chains) and two light chains of approximately 25 kDa each (called the L chains), linked together by intra- and inter-chain disulfide bonds.

[0016] Each chain is made up, in the N-terminal position, of a variable region or domain, called VL for the light chain, VH for the heavy chain, and in the C-terminal position, of a constant region, made up of a single domain called CL for the light chain and three or four domains called CH1, CH2, CH3, CH4, for the heavy chain.

[0017] Only IgM and IgE have the CH4 domain.

[0018] Each domain comprises approximately 110 amino acids and is structured in a comparable manner. The two heavy chains are linked by disulfide bonds at the CH2 and each heavy chain is linked to a light chain by a disulfide bond between the CH1 and the CL. The region that determines the specificity of the antibody for the antigen is carried by the variable parts, while the constant parts can interact with the Fc receptors (FcRs) of effector cells or molecules such as complement proteins to induce different functional properties. The assembly of the chains that make up an antibody allows the definition of a characteristic three-dimensional Y-shaped structure, where the base of the Y corresponds to the Fc constant region; or Fc fragment; which is recognized by complement and Fc receptors in order to mediate the effector functions of the molecule, and the ends of the arms of the Y 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.

[0019] Specifically, there are five heavy chain isotypes (gamma, alpha, mu, delta, and epsilon) and two light chain isotypes (kappa and lambda, with lambda chains themselves divided into two types: lambda 1 and lambda 2). The heavy chain determines the immunoglobulin class. There are five classes of Ig: IgG for the Gamma isotype, IgA for the Alpha isotype, IgM for the Mu isotype, IgD for the Delta isotype, and IgE for the Epsilon isotype.

[0020] 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.

[0021] Immunoglobulin G (IgG) is the most abundant immunoglobulin in serum (75-80% of circulating antibodies). Present as monomers, it has the longest serum half-life of all immunoglobulins (approximately 21 days).

[0022] There are four types of gamma heavy chains, which determine four subclasses of IgG (IgG1 for gamma1, IgG2 for gamma2, IgG3 for gamma3, and IgG4 for gamma4). These four subclasses differ in varying numbers and positions of disulfide bonds (Basic and Clinical Immunology, 8th ed., Daniel P. Stites, Abba I. Terr, and Tristram G. Parslow (eds.), Appleton & Lange, Norwalk, Conn., 1994, page 71 and Chapter 6).

[0023] The four subclasses of human IgG are also distinguished in terms of their biological activities, despite highly homologous structures (more than 95% sequence homology for the Fc regions).

[0024] Biological activity refers in particular to the ability of the IgG constant region to bind: in particular complement proteins (the C1q 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 (RFcyI, RFcyII, RFcyIII, Ravetch and Kinet, AnnualReview of Immunology, vol 9:457-492 (1991)).

[0025] 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.

[0026] 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 N-terminal flexible hinge of these domains. For IgA and IgM, the Fc fragment may comprise the J chain. For IgG, the Fc fragment comprises the CH2, CH3 domains and the lower hinge region between CH1 and CH2. In other words, the Fc region of an IgG1 consists of the lower CH2-CH3 hinge, i.e., the portion from amino acid C226 to the carboxy-terminus, with numbering indicated according to the EU index or equivalent in Kabat.Analogous domains for other IgG subclasses can be determined from the alignment of the amino acid sequences of the heavy chains or heavy chain fragments of the IgG subclasses with that of a human IgG1 (cf. Figure 1). The Fc fragment used according to the invention may further comprise a portion of the upper hinge region, upstream of position 226 (according to the EU index). In this case, preferably, an Fc fragment of a human IgG1 is used comprising a portion of the region located between positions 216 to 226. 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-terminal end. By "Fc fragment", we also mean an scFc fragment for "single chain Fc". By "scFc fragment", we mean a single chain Fc fragment, obtained by genetic fusion of two Fc monomers linked by a polypeptide linker. The scFc naturally folds into a functional dimeric Fc region.

[0027] By "parent Fc fragment" or "parent polypeptide" as used herein is meant a reference polypeptide among the wild-type Fc regions or variants possibly carrying other mutations than the one considered. By "wild-type or WT" is meant herein an amino acid sequence or a nucleotide sequence that is found in nature, i.e., which is of natural origin, including allelic variations, and which has not been intentionally modified by molecular biology techniques such as mutagenesis. For example, "wild-type" Fc regions refer, in particular, to the Fc region of IgG1 having the sequence SEQ ID NO: 1 (allotype G1m1,17), 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 (allotype G1m3).

[0028] The Fc fragments used in the invention are in monomeric form, i.e. they are not fused or conjugated to each other.

[0029] By "altered affinity" we mean a decreased or increased affinity compared to a parent Fc fragment.

[0030] 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 an FcRn gene. The 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 the heavy chain and the light chain. The light chain is beta-2-microglobulin and the heavy chain is encoded by the FcRn gene. Unless otherwise specified herein, FcRn or FcRn protein refers to the complex of the α-chain with beta-2-microglobulin. In humans, the gene encoding FcRn is referred to as FCGRT.

[0031] By "increased FcRn binding" as used herein is meant increased binding affinity, in vivo Or in vitro,of the mutated Fc fragment for FcRn, relative to the parent polypeptide. The ability of the mutated Fc fragment to bind to an FcRn receptor can be assessed in vitro by an ELISA test, as described for example in patent application WO2010 / 106180.

[0032] The term "half-life" refers, as used herein, to the time taken for the Fc fragment to be half eliminated from the circulation or other tissues, once present in the serum of the patient to whom it has been administered.

[0033] The term "Fcy receptor" or "FcyR" refers to the receptors for IgG-type immunoglobulins, called CD64 (FcyRI), CD32 (FcyRII), and CD16 (FcyRIII), specifically five expressed receptors (FcyRIa, FcyRIIa, FcyRIIb, FcyRIIIa, FcyRIIIb). All are activating receptors for effector cells, except for human FcyRIIb, which is an inhibitory receptor for immune cell activation (Muta T et al., Nature, 1994, 368:70-73).

[0034] An "effector cell" means any cell that carries an Fc receptor, such as lymphocytes, monocytes, neutrophils, natural killer (NK) cells, eosinophils, basophils, mast cells, dendritic cells, Langerhans cells, and platelets.

[0035] 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.

[0036] The term "hypersialyzation", 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 carried out by enzymatic reaction, cellular reaction or by site-directed mutagenesis of the Fc targeting one or more amino acids involved in the sialylation of the Fc.

[0037] The term “patient” refers to any human or animal subject, preferably a mammal. In a preferred embodiment, the patient is a human being, regardless of age or gender.

[0038] The term "treatment" or "treat" means an amelioration, prophylaxis, or reversal of a disease or disorder, or at least one discernible symptom thereof, or an amelioration, prophylaxis, or reversal 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 "treat" further includes the inhibition or slowing of the progression of a disease or disorder, physically, e.g., stabilization of a discernible symptom, physiologically, e.g., stabilization of a physical parameter, or both. Compositions of Fc fragments according to the invention

[0039] The antibody Fc fragments used in the invention are Fc fragments of an IgG1 immunoglobulin, having an altered affinity for at least one of the Fc receptors (FcR) compared to a parent Fc fragment.

[0040] The Fc fragments according to the invention have an increased affinity to at least one Fc receptor, compared to a parent Fc fragment.

[0041] 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 an FcR is greater than that of the parent polypeptide.

[0042] The affinity of a polypeptide comprising an Fc region for an FcR can be assessed by methods well known in the prior art. For example, one skilled in the art can determine the affinity (Kd) using surface plasmon resonance (SPR) which can be measured by the Biacore apparatus. Alternatively, one skilled in the art can perform a suitable ELISA test. A suitable ELISA assay allows for the comparison of the binding strengths of the parent Fc and the mutated Fc. The detected signals specific to the mutated Fc and the parent Fc are compared. The binding affinity can be determined either by assessing the whole polypeptides or by assessing the isolated Fc regions thereof.

[0043] According to a particular embodiment of the disclosure, the Fc fragments used according to the invention have a modified, advantageously increased, affinity to the FcRn receptor. This increase in binding to FcRn results in an improvement in serum retention. in vivoand, consequently, an increase in half-life.

[0044] Fc fragments can be used alone or in mixtures, for example several Fc fragments, having different mutations can be administered in mixture or co-administered.

[0045] The Fc fragments of the invention can also be implemented in a composition comprising a single type of mutated Fc fragment. In other words, the composition then comprises Fc fragment molecules of identical sequence.

[0046] In one embodiment of the disclosure, the Fc fragments carry a mutation combination selected from 315D / 330V / 361D / 378V / 434Y (mutation combination also referred to as “T5A-74”. Fc fragments carrying this mutation combination are therefore also referred to as “rFc T5A-74), 230S / 315D / 428L / 434Y, 307A / 315D / 330V / 382V / 389T / 434Y, 259I / 315D / 434Y, 256N / 378V / 383N / 434Y, E294del / T307P / N434Y (mutation combination also referred to as “C6A_66”. Fc fragments carrying this mutation combination are therefore also referred to as “rFc C6A_66).

[0047] In one embodiment of the disclosure, the Fc fragments carry a combination of mutations selected from: (i) T260A and 315D / 330V / 361D / 378V / 434Y (mutation combination also referred to as “T5A-74”)- The fragment designated “T5A-74I” is an Fc fragment that is distinguished from the T5A-74 fragment only by the addition of the T260A mutation. Or (ii) E258I and 315D / 330V / 361D / 378V / 434Y (mutation combination also referred to as “T5A-74”)- The fragment designated “T5A-74J” is an Fc fragment that is distinguished from the T5A-74 fragment only by the addition of the E258I mutation. Or (iii) K290Y and 315D / 330V / 361D / 378V / 434Y (mutation combination also referred to as “T5A-74”) - The fragment designated “T5A-74K” is an Fc fragment that is distinguished from the T5A-74 fragment only by the addition of the K290Y mutation. Or (iv) E294A and 315D / 330V / 361D / 378V / 434Y (mutation combination also referred to as “T5A-74”) - The fragment designated “T5A-74L” is an Fc fragment that is distinguished from the T5A-74 fragment only by the addition of the E294A mutation.Or (v) Y296W and 315D / 330V / 361D / 378V / 434Y (combination of mutations also referred to as “T5A-74”) - The fragment designated “T5A-74M” is an Fc fragment that is distinguished from the T5A-74 fragment only by the addition of the Y296W mutation.

[0048] The sequences of Fc fragments T5A-74, T5A-74I, T5A-74J, T5A-74K, T5A-74L, T5A-74M, are presented in the attached sequence listing. T5A-74 (N315D / A330V / N361D / A378V / N434Y): T5A-74I (T260A / N315D / A330V / N361D / A378V / N434Y) T5A-74J (E258I / N315D / A330VIN361D / A378VIN434Y) T5A-74K (K290Y / N315D / A330V / N361D / A378V / N434Y) T5A-74L (E294A / N315D / A330V / N361D / A378V / N434Y) T5A-74M (Y296W / N315D / A330V / N361D / A378V / N434Y)

[0049] The disclosure also describes mutated Fc fragments which have increased affinity for the FcγRIIIa receptor (CD16a), and comprise 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, 286I, 286Y and 298N, 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).

[0050] The disclosure also describes mutated Fc fragments comprising at least one combination of 3 mutations, said combination comprising: (i) une mutation choisie parmi 326E, 326T, 352L, 378V, 378T, 396L, 397M, 421T, 334N, 334R, 307N et 394P ; et (ii) au moins 2 mutations choisies parmi 226Y, 227S, 230S, 231V, 234P, 243I, 243L, 246R, 246E, 247T, 248E, 253F, 254F, 255W, 259A, 261R, 262A, 263A, 266M, 267N, 267G, 274E, 274R, 276S, 278H, 282A, 283G, 284L, 286I, 286Y, 287T, 288E, 288R, 290E, 298N, 302A, 305A, 307P, 308A, 308I, 308G, 309P, 312G, 315D, 316D, 319H, 320T, 320R, 320M, 322E, 323I, 325S, 333G, 334N, 334R, 336T, 339T, 340E, 343S, 345G, 349S, 349H, 350A 352S, 359A, 361H, 362R, 363I, 366A, 373D, 375R, 377T, 378V, 378T, 379A, 380G, 383R, 385R, 389S, 389T, 392R, 393A, 393I, 394P, 396L, 3971, 397M, 398P, 405V, 405L, 410R, 412M, 414R, 421T, 421S, 423L, 423Y, 423S, 423P, 428T, 431V, 431T, 434K, 434S, 435R, 436H, 439R, 440G, 440N, 442F, 442P et 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).

[0051] The Fc fragments of the invention carry the mutation combination K334N / P352S / V397M / A378V (mutation combination also referred to as “A3A-184A”. Fc fragments carrying this mutation combination are therefore also referred to as “A3A-184A”).

[0052] The sequence of fragment A3A-184A (K334N / P352S / A378V / V397M) is shown in the sequence listing, as SEQ ID NO: 19:

[0053] According to another embodiment of the disclosure, the mutated Fc fragments 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 EU index, or the equivalent in Kabat.

[0054] This single mutation leads to a particular glycosylation of the fragment, namely hypersialylation, which is particularly advantageous with regard to the half-life of glycoproteins and inflammatory processes.

[0055] According to another aspect of the invention, a composition is used comprising a plurality of Fc fragments which all have substantially the same sequence, and which, taken as a whole, have a particular glycosylation profile.

[0056] According to a particular aspect, a composition used in the context of the invention comprises Fc fragments having N-glycans on their glycosylation site (Asn 297), characterized in that said N-glycans of the Fc fragments have a fucosylation level 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%.

[0057] According to yet another aspect, a composition used in the context of the invention comprises Fc fragments, said Fc fragments having N-glycans on their glycosylation site (Asn 297), characterized in that said N-glycans of the Fc fragments have a biantennary type glycan structure, with short chains, low sialylation, having terminal mannoses and / or non-intercalated terminal N-acetylglucosamines.

[0058] According to a more particular aspect, a composition used in the context of the invention comprises Fc fragments, said Fc fragments having N-glycans on their glycosylation site (Asn 297), characterized in that said N-glycans of the Fc fragments have a content greater than 60% for the forms G0+G1+G0F+G1F, the forms G0F+G1F being less than 50%.

[0059] According to another more particular aspect, a composition used in the context of the invention comprises Fc fragments, said Fc fragments having N-glycans on their glycosylation site (Asn 297), characterized in that said N-glycans of the Fc fragments have a content greater than 60% for the forms G0+G1+G0F+G1F, the fucose content being less than 65%.

[0060] According to another even more particular aspect, a composition used in the context of the invention comprises Fc fragments, said Fc fragments having N-glycans on their glycosylation site (Asn 297), characterized in that said N-glycans of the Fc fragments have a content of less than 40% for the G1F+GOF forms.

[0061] According to a more particular aspect, a composition used in the context of the invention comprises Fc fragments which have N-glycans on their glycosylation site (Asn 297), said N-glycans of the Fc fragments having a fucosylation level equal to 0%. The invention thus provides a composition comprising Fc fragments which have N-glycans on the glycosylation site Asn297, characterized in that said N-glycans of the Fc fragments are devoid of fucose.

[0062] Also, according to a particular aspect, a composition used in the context of 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 fucosylation level of between 20% and 55%. In particular, the invention provides a composition comprising Fc fragments which have N-glycans on the Asn297 glycosylation site, 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%.

[0063] According to a more particular aspect, a useful composition 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%, preferably greater than 80%, for the forms G0+G1+G0F+G1F, the forms G0F+G1F being less than 50%, preferably less than 40%, or 30%.

[0064] According to another more particular aspect, the N-glycans of the Fc fragments within the composition have a content greater than 60% for the G0+G1+G0F+G1F forms, the fucose content being less than 65%.

[0065] 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%.

[0066] The shapes G0, G0F, G1 and G1F are selected from the shapes indicated on the Figure 3 .

[0067] 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%.

[0068] A composition which can be used in the context of the invention comprises Fc fragments having on their glycosylation site (Asn 297) N-glycans, said N-glycans of the Fc fragments having a biantennary type glycan structure, with short chains, low sialylation, having terminal mannoses and / or non-intercalated terminal N-acetylglucosamines, the N-glycans having for example a content greater than 60% for the forms G0 + G1 + G0F + G1F, and low fucosylation, the N-glycans having for example a content less than 50% of forms G0F + G1F.

[0069] In a particular embodiment, the Fc fragments according to the invention have glycan structures as described in patent application WO01 / 77181.

[0070] According to an advantageous embodiment, the Fc fragments used in the invention comprise at least the sequence SEQ ID NO: 19, and have N-glycans on their glycosylation site (Asn 297), said N-glycans of the Fc fragments having a fucosylation level 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%.

[0071] Advantageously, Fc fragments having modified glycosylation at the glycosylation site at position 297, in particular low fucosylation, exhibit increased binding of said fragment to Fcgamma receptors (FcγR), in particular the FcγRIIIa receptor (CD16a).

[0072] Preferably, said Fc fragments have an affinity for CD16a at least equal to 2x10 6< M -1< at least equal to 2x10 7< M -1< , 2x10 8< M -1< or 2x10 9< M -1< , as determined by Scatchard analysis or BIAcore technology (Label-free surface plasmon resonance based technology).

[0073] Particularly advantageously, the mutated Fc fragments of the invention can be used in combination with different mutated Fc fragments carrying different mutations. For example, a mixture of mutated Fcs Fc-De1294, mutated Fcs T5A-74 (to also target FcRn), mutated Fcs improved for binding to FcyRs (types A3A-184A) can be advantageously used. Production of Fc fragments according to the invention

[0074] The Fc fragments used in the invention can be produced by any method known to those skilled in the art, for example by chemical synthesis, or by recombination.

[0075] In a preferred embodiment, the Fc fragments used in the invention are said to be “recombinant”, i.e. they are obtained by recombination.

[0076] 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 specific primers which introduce the desired mutations, random mutagenesis. Preferably, random mutagenesis as described in application WO02 / 038756 is used: this is the MutaGen technique. This technique uses a human DNA mutase, in particular chosen from DNA polymerases β, η and . Conventional recombinant techniques involve recombination in a host cell, transformed with one or more vectors that allow expression, with or without secretion, of the Fc fragment sequence into the extracellular medium. The vector generally comprises a promoter, translation initiation and termination signals, as well as appropriate transcription regulation regions. It can be maintained stably in the host cell and may optionally possess specific 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.

[0077] 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, heat shock, or chemical methods.

[0078] The cellular host can 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.

[0079] The Fc fragments used in the invention can be produced by culturing in an appropriate medium and culture conditions a host cell expressing said Fc fragments; and recovering the fragments thus produced from the culture medium or said cultured cells.

[0080] Preferred mammalian cells for expression of Fc fragments are the YB2 / 0 rat line, the vero line, the CHO hamster line, in particular the CHO dhfr- and CHO Lec13, CHO-lec10, CHO-lec1, CHOK1SV Potelligent ®< (Lonza, Switzerland), CHOGnTIII (Glycart, Switzerland), PER.C6 ™< (Crucell), HEK293, T1080, EB66, K562, NS0, SP2 / 0, BHK or COS lines.

[0081] 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, Enríquez G, Pérez 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 rabbits, goats, rats or pigs (Pollock, DP, 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.

[0082] In an alternative embodiment, the Fc fragments are obtained by proteolytic treatment of immunoglobulins which are themselves mutated.

[0083] The modification of the glycosylation of the Fc fragments can be obtained by known techniques. The Fc fragments having glycosylation according to the invention can in particular be produced from the cleavage of antibodies produced according to the technique described in WO 01 / 77181, in particular by the enzyme papain. The weakly fucosylated Fc fragments can also be obtained by production in cells cultured in the presence of kifunensine, as described for example in document US7700321, or in cells for which the GDP-fucose production pathway is inhibited, for example by inhibition of at least one of the enzymes of the fucose production cycle (see in particular documents 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.These may also include methods of preparation in yeasts, as described for example in document WO 0200879.

[0084] In the case where the Fc fragments have 100% non-fucosylated oligosaccharides, that is to say when the Fc fragments are completely devoid of fucose, it is possible to use preparation methods known to those skilled in the art, such as for example those described in documents EP1176195, US 7,214,775, US 6,994,292, US 7,425,449, US2010223686, WO2007099988, EP 1,705,251, this list not being limiting. This may be, for example, a method using a host cell expressing at least one nucleic acid coding for an Fc fragment, and whose glycosylation is modified by deletion of the gene coding for α1,6-fucosyltransferase or by addition of a mutation of this gene to eliminate the α1,6-fucosyltransferase activity, and expressing as such an antibody fragment devoid of fucose. Therapeutic applications

[0085] Due to their numerous advantages, both in terms of efficacy, as well as optimized effector functions or reduced side effects, the Fc fragments of the disclosure are useful for their use in the treatment of an autoimmune and / or inflammatory disease.

[0086] According to the disclosure, Fc fragments having altered affinity for at least one Fc receptor, in a therapeutically effective amount, can be used to treat an autoimmune and / or inflammatory disease in a patient.

[0087] An autoimmune and / or inflammatory disease according to the disclosure means a primary or secondary, organ-specific or systemic autoimmune and / or inflammatory disease, whether or not associated with pathogenic autoantibodies.

[0088] For example, the disease according to the disclosure may be selected from Thrombotic Thrombocytopenic Purpura (TTP), Idiopathic Thrombotic Purpura (ITP), transplant or organ rejection, graft versus host disease, rheumatoid arthritis, systemic lupus erythematosus, various types of sclerosis, primary Sjögren's syndrome (or Gougerot-Sjögren syndrome), autoimmune polyneuropathies such as multiple sclerosis, type 1 diabetes, autoimmune hepatitis, ankylosing spondylitis, Reiter's syndrome, gouty arthritis, celiac disease, Crohn's disease, Hashimoto's chronic thyroiditis (hypothyroidism), Adisson's disease, autoimmune hepatitis, Graves' disease (hyperthyroidism), ulcerative colitis, vasculitis such as systemic vasculitis associated with ANCA (anti-neutrophil cytoplasmic antibodies),autoimmune cytopenias and other hematological complications in adults and children, such as acute or chronic autoimmune thrombocytopenias, autoimmune hemolytic anemias, hemolytic disease of the newborn (HDN), cold agglutinin disease, thrombotic thrombocytopenic purpura and acquired autoimmune hemophilia; Goodpasture syndrome, extramembranous nephropathies, autoimmune bullous skin disorders, refractory myasthenia gravis, mixed cryoglobulinemia, psoriasis, juvenile chronic arthritis, inflammatory myositis, dermatomyositis and systemic autoimmune disorders in children including antiphospholipid syndrome, connective tissue disease, various types of sclerosis, autoimmune pulmonary inflammation, Guillain-Barre syndrome, Kawasaki disease, MMN (Multifocal Motor Neuropathy), chronic inflammatory demyelinating polyradiculoneuropathy (CIDP),autoimmune thyroiditis, mellitis, myasthenia gravis, inflammatory autoimmune eye disease, neuromyelitis optica (Devic's disease), scleroderma, pemphigus, insulin-resistant diabetes, polymyositis, Biermer's anemia, glomerulonephritis, Wegener's disease, giant cell arteritis, periarthritis nodosa and Churg-Strauss syndrome, Still's disease, relapsing polychondritis, Behçet's disease, monoclonal gammopathy, Wegener's granulomatosis, lupus, ulcerative colitis, psoriatic arthritis, sarcoidosis, collagenous colitis, dermatitis herpetiformis, familial Mediterranean fever, IgA-deposited glomerulonephritis, myasthenic syndrome Lambert-Eaton syndrome, sympathetic ophthalmia, Fiessinger-Leroy-Reiter syndrome and uveomeningoencephalic syndrome.

[0089] Other inflammatory diseases are also listed, 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 (COPD), coronary artery disease, encephalitis, inflammatory bowel disease, inflammatory osteolysis, 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, multiple organ dysfunction syndrome (MODS), pulmonary fibrosis,sepsis and septic shock, Stevens-Johnson syndrome, undifferentiated arthritis, and undifferentiated spondyloarthropathies.

[0090] According to the invention, the autoimmune disease is idiopathic thrombotic purpura (ITP).

[0091] One of the observed effects is notably a limitation or reduction of platelet destruction observed in ITP pathology and a limitation or reduction of the loss of the myelin sheath of peripheral nerves in CIDP.

[0092] According to the disclosure, the disease is an inflammatory disease, such as graft-versus-host disease. In this case, the Fc fragments according to the disclosure carry a mutation inducing hypersialylation, such as a deletion of an amino acid at position 294 (DEL294) or 293 (DEL293).

[0093] Any route of administration is considered, including parenteral routes, such as intravenous, intramuscular, subcutaneous, intradermal, topical, or mucosal, for example, by inhalation. Enteral (oral, rectal) and intrathecal routes are also possible. Preferably, the intravenous route is used.

[0094] The Fc fragments according to the invention are generally formulated within pharmaceutical compositions comprising pharmaceutically acceptable excipients.

[0095] Pharmaceutical compositions may be presented in any suitable galenic form depending on the chosen route of administration.

[0096] The pharmaceutical compositions useful according to the invention advantageously comprise one or more pharmaceutically acceptable excipients or vehicles. Examples that may be mentioned are saline, physiological, isotonic, buffered solutions, etc., compatible with pharmaceutical use and known to those skilled in the art. The compositions may contain one or more agents or vehicles chosen 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, acacia, etc. The compositions may optionally be formulated using dosage forms or devices ensuring prolonged and / or delayed release.For this type of formulation, an agent such as cellulose, carbonates or starches is advantageously used.

[0097] The doses administered may vary, among other things, depending on the weight and age of the patient, and the severity of the disease, as assessed by those skilled in the art.

[0098] In a preferred embodiment, the dosage of the Fc fragments according to the invention is in the range of about 0.05 mg / kg to about 1 g / kg of body weight, or about 20 mg to about 100 g per day for an adult. Preferably, the dosage is about 330 mg / kg to about 660 mg / kg per day.

[0099] The following examples illustrate the invention without limiting its scope. Construction of recombinant Fc expression vectors (WT and mutated):

[0100] The recombinant Fc sequence (aa 221-447) was cloned into a generic eukaryotic expression vector derived from pCEP4 (Invitrogen) for expression in HEK cells and the optiCHO vector for expression in YB2 / 0 cells using standard PCR protocols.

[0101] All mutations of interest in the Fc fragment were inserted into the expression vector by overlap PCR using two primers containing the mutation. The fragments thus obtained by PCR were then 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 recombinant Fc expression vector.

[0102] There Figure 7shows the map of the pCEP4 vector used for the expression of the T5A-74 variant in HEK cells, and the map of the OptiCHO vector used for the expression of the T5A-74 variant in YB2 / 0 cells. Production of recombinant Fc in HEK cells

[0103] HEK 293 cells were transfected with the recombinant Fc expression vector (WT or mutated) pCEP4 according to standard protocols (Invitrogen). The cells were cultured to produce antibodies transiently. The antibodies produced were isolated and purified using standard techniques for characterization. The production rates obtained were in the order of 150 to 500 µg / ml. The purity and quality of the products were verified by SDS-PAGE and SEC. Production of recombinant Fc in YB2 cells / 0

[0104] YB2 / 0 cells were stably transfected by electroporation with the recombinant Fc expression vector (WT or mutated) optiCHO according to standard protocols. Production was performed in stable pools or after cloning of YB2 / 0 cells.

[0105] The cell culture production and antibody purification steps were performed using standard state-of-the-art techniques for their characterization. The production rates obtained were in the order of 3 to 30µg / ml. The purity and quality of the products were verified by SDS-PAGE and SEC. Example 1: Efficacy test (inhibition of red blood cell lysis):

[0106] To mimic the situation of red blood cell lysis observed in idiopathic thrombocytopenic purpura (ITP), involving the autoantibodies of the ITP patient, effector cell-mediated red blood cell lysis in the presence of an anti-Rhesus D (RhD) monoclonal antibody was conducted, and the ability of different amounts of polyvalent immunoglobulins (IVIg) or recombinant, mutated Fc fragments (recombinant Fc fragments carrying the mutations according to the Table 1 below) and non-mutated, to inhibit this lysis, for example by competition with anti-RhD for the fixation of Fc receptors on the surface of effector cells, was evaluated. Table 1: Preferred mutated Fc variant constructs from disclosure

[0107] The table below represents the different variants of mutated Fc fragments that were tested in the experiments of efficacy testing (inhibition of red blood cell lysis) and binding test to human FcRn and CD16aV by surface plasmon resonance (SPR). Name Variant Mutation(s) added T5A-74I T5A-74 T260A T5A-74J T5A-74 E258I T5A-74K T5A-74 K290Y T5A-74L T5A-74 E294A T5A-74M T5A-74 Y296W ZAC2-85 WT T260A ZAC3-172 WT K290Y A3A-184A WT K334N, P352S, V397M, A378V A3A-105D WT G316D, K326E, A378V J3B-118A WT P396L, N421T, A378V

[0108] For this purpose, as effector cells, PBMCs (peripheral blood mononuclear cells) were purified from peripheral blood by Ficoll gradient. Rh-D+ red blood cells obtained from healthy Rh-D+ blood donors were mixed with an anti-RhD monoclonal antibody. PBMCs were incubated with opsonized Rh-D+ red blood cells (effector / target ratio of 2 / 1).

[0109] To evaluate the ability of the candidates (IVIg and recombinant Fc fragments, mutated as defined in Table 1 and non-mutated) to inhibit anti-RhD antibody-induced cytotoxicity by competition and / or saturation of Fc receptors, various concentrations of the candidates (0 to 9.75µM) 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 into the supernatants (optical density measurement or OD). Specific lysis is calculated as a percentage according to the following formula: DO échantillon − DO témoin 0 % / DO témoin 100 % − DO témoin 0 % * 100 = % Lyse in which: “DO control 100%” corresponds to the total lysis of red blood cells (for example NH 4 Cl) “DO control 0%” corresponds to the lysis observed with a reaction mixture without antibodies The results are expressed in % of specific lysis (cf Figures 4 and 5 ). Example 2: Human FcRn and CD16aV binding assay by surface plasmon resonance (SPR) on Biacore X100 : Proteins used:

[0110] Recombinant human FcRn (FcRn a and b2 microglobulin) was produced in baculovirus cells by GTP Technology (Labège, France) as previously described (Popov et al., Mol. Immunol. 33:521-530 (1996)). Recombinant human CD16aV is commercially available (R&D system). Test on FcRn:

[0111] FC1 and FC2 cells on a CM5 chip (Biacore, GE Healthcare) were activated for 3 minutes with a 1:1 mixture of 0.1 M N-hydroxysuccinimide and 0.1 M 3-(N,N-dimethylamino)propyl-N-ethylcarbodiimide at 30 µl / min. Recombinant FcRn was then immobilized on the FC2 cell at 32.6 µg / mL in 10 mM Na Acetate buffer pH 5 (immobilization for 100 seconds, final immobilization rate of 350 RU). The FC1 cell was used as a negative control, prepared in the same way as FC2 but in the absence of recombinant FcRn. Recombinant Fc to be tested were injected at 6 different concentrations (300nM, 150nM, 75nM, 30nM, 15nM and 0) in 50mM Na phosphate buffer, 150 mM NaCl, 0.05% tween20 pH6 for 8 minutes at 10µl / min on FC1 and FC2 cells. FC1 and FC2 cells were regenerated between each sample concentration by a one-minute injection of 50mM Na phosphate buffer, 150 mM NaCl, 0.05% tween20 pH7.8.The generated data were analyzed with BIAevaluation software version 3.1 (Biacore), by subtracting the control signal obtained on FC1 from the test signal obtained on FC2. Test on CD16aV:

[0112] FC1 and FC2 cells of a CM5 chip (Biacore, GE Healthcare) were prepared with the GE Healthcare His Capture kit reference 28-9950-56, in order to immobilize the anti-Histidine antibody at 50µg / ml (flow rate of 5 µl / min, activation in EDC / NHS for 7 min, immobilization for 7 minutes, final immobilization rate of 12000 RU on FC1 and FC2). Recombinant human CD16aV was immobilized on the FC2 cell (1 µg / ml in HBS-EP+, Biacore, GE Healthcare) for 60 seconds at 5 µl / min. The recombinant Fc to be tested were injected into FC1 and FC2 cells at 5 different concentrations (1000nM, 500nM, 250nM, 125nM and 25nM) in HBS-EP+ buffer under SCK (Single Cycle Kinetics) conditions, with a 60-second contact, a 300-second dissociation and a flow rate of 30 µl / min without regeneration between each concentration. The final regeneration, between each recombinant Fc, was carried out in 10mM Glycine buffer pH 1.5 for 60 seconds at 30 µl / min on FC1 and FC2 cells.The generated data were analyzed with BIAevaluation software version 3.1 (Biacore), by subtracting the control signal obtained on FC1 from the test signal obtained on FC2.

Claims

1. Composition comprising Fc antibody fragments for use in the treatment of immune thrombocytopenic purpura, said Fc fragments being isolated Fc fragments having improved affinity, by a ratio of at least 2, for the FcRγIII(CD16a) receptor compared with a parent Fc fragment of wild-type IgGI of sequence SEQ ID NO: 1, and comprising at least the sequence SEQ ID NO: 19.

2. Composition for use according to claim 1, wherein the Fc fragments are composed of the sequence SEQ ID NO:19.

3. Composition comprising Fc fragments for use according to one of claims 1 or 2, wherein the Fc fragments are obtained via recombinant route.

4. Composition comprising Fc fragments for use according to one of claims 1 to 3, said Fc fragments having N-glycans on their glycosylation site (Asn 297), characterized in that said N-glycans of the Fc fragments have a fucosylation rate lower than 65 %, preferably lower than 60 %, preferably lower than 55 %, preferably lower than 50 %, more preferably lower than 45 %, preferably lower than 40 %, preferably lower than 35 % , preferably lower than 30 %, preferably lower than 25 %, preferably lower than 20 %.

5. Composition comprising Fc fragments for use according to one of claims 1 to 4, said Fc fragments having N-glycans on their glycosylation site (Asn 297), characterized in that said N-glycans of the Fc fragments have a glycan structure of biantennary type with short chains, weak sialylation, having non-intercalating terminal mannoses and / or terminal N-acetylglucosamines.

6. Composition comprising Fc fragments for use according to one of claims 1 to 4, said Fc fragments having N-glycans on their glycosylation site (Asn 297), characterized in that the content of said N-glycans in the Fc fragments is higher than 60 % for the forms G0+G1+G0D+G1D, and lower than 50 % for the forms G0F+G1F.

7. Composition comprising Fc fragments for use according to one of claims 1 to 5, said Fc fragments having N-glycans on their glycosylation site (Asn 297), characterized in that the content of said N-glycans in the Fc fragments is higher than 60 % for the forms G0+G1+G0F+G1F, the fucose content being lower than 65 %.

8. Composition comprising Fc fragments for use according to claim 6 or 7, said Fc fragments having N-glycans on their glycosylation site (Asn 297), characterized in that the content of said N-glycans in the Fc fragments is lower than 40 % for the forms G1 F+G0F.