Patent Grant
12247076
This application is a national stage filing under 35 U.S.C. § 371 of International Application No. PCT/FR2016/051708, filed Jul. 6, 2016, which claims priority to French Application No. 1556399, filed Jul. 6, 2015, the entire contents of each of which is incorporated herein by reference in its entirety.
The present invention relates to immunotherapy of autoimmune and/or inflammatory diseases.
Immunotherapy, which consists of administering exogenous antibodies to patients, is widely used today for the treatment of various pathologies, particularly for that of autoimmune diseases and inflammatory diseases.
Two types of immunoglobulin-based therapy are typically proposed for the treatment of these diseases: i) therapies based on intravenous immunoglobulins (IVIg), consisting of intravenous administration of immunoglobulins (most often IgG) derived from human plasma pools to patients and ii) therapies based on the use of recombinant antibodies (namely antibodies obtained by genetic engineering). The later have enabled genuine advances in the management of patients with inflammatory diseases and autoimmune diseases, notably because they offer the possibility of avoiding the disadvantages associated with the use of immunoglobulins from plasma, especially the risk of supply shortages, and the risk of transmission of pathogens potentially present in plasma.
The presence of Fab fragments in immunoglobulins may be responsible for 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, in particular for patients with autoimmune disease.
There remains a need to optimize these Fc fragments, however, so as in particular to increase their half-life and/or therapeutic efficacy.
The inventors now propose to use Fc fragments having a modified affinity for at least one Fc receptor (FcR) in comparison with a parent Fc fragment. According to the invention, the Fc fragments are isolated, i.e., they are not associated with Fab fragments or conjugated or fused to any other protein, polypeptide or peptide. In particular, they are not complete immunoglobulins.
The invention relates to 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 an improved affinity for Fc-gamma receptor III (FcγRIII) in comparison with a parent Fc fragment.
In preferred aspects, they are
The invention also more generally 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 modified affinity for at least one Fc receptor (FcR) in comparison with a parent Fc fragment.
In a particular embodiment, the composition comprises antibody Fc fragments, having at least one mutation of one or more amino acids and/or having N-glycans on the glycosylation site (Asn 297) thereof, said N-glycans of the Fc fragments having a degree of fucosylation lower than 65%.
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., Sequences of proteins of immunological interest, 5th Ed. Public Health Service, National Institutes of Health, Bethesda, Md. (1991), expressly incorporated by reference herein. “EU index” or “EU index or the Kabat equivalent” refers herein to the numbering of the residues of human IgG1 antibody.
The term “immunoglobulin” refers to the structure constituting the natural-state biological form of an antibody, including the constant and variable regions (also called fragments). An immunoglobulin molecule is a molecule whose basic unit is a heterotetramer consisting of two heavy (H) chains of roughly 50-70 kDa each and two light (L) chains of roughly 25 kDa each, bound together by intra- and intercatenary disulfide bridges.
Each chain is composed, in the N-terminal position, of a variable domain or region, called VL in the case of the light chain and VH in the case of the heavy chain; and in the C-terminal position, of a constant region consisting of a single domain called CL in the case of the light chain and of three or four domains called CH1, CH2, CH3 and CH4 in the case of the heavy chain.
Only IgM and IgE have the CH4 domain.
Each domain comprises about 110 amino acids and is structured in a comparable manner. The two heavy chains are linked by disulfide bridges at the CH2 domains and each heavy chain is linked to a light chain by a disulfide bridge between CH1 and CL. The region that determines the antibody's specificity for the antigen is carried by the variable portions, whereas the constant portions can interact with the Fc receptors (FcR) of effector cells or molecules such as complement proteins in order to induce various functional properties. The assembly of the chains composing an antibody makes it possible to define a characteristic Y-shaped three-dimensional structure, where
More precisely, there are five heavy chain isotypes (gamma, alpha, mu, delta and epsilon) and two light chain isotypes (kappa and lambda, the lambda chains themselves being divided into two types: lambda 1 and lambda 2). It is the heavy chain which determines the immunoglobulin class. There are thus five classes of Ig: IgG (gamma isotype), IgA (alpha isotype), IgM (mu isotype), IgD (delta isotype) and IgE (epsilon isotype).
The kappa and lambda light chains are shared by all the classes and subclasses. In humans, the proportion of kappa and lambda produced is in a ratio of 2 to 1.
IgG are the most abundant immunoglobulins in serum (75% to 80% of circulating antibodies). Present in monomer form, they have the longest serum half-life of all the immunoglobulins (about 21 days).
There are four types of gamma heavy chains, which determine the four IgG subclasses (IgG1 for gamma 1, IgG2 for gamma 2, IgG3 for gamma 3 and IgG4 for gamma 4). These four subclasses differ in terms of variable numbers and 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, page 71 and Chapter 6).
The four human IgG subclasses are also distinguished by their biological activities, despite highly homologous structures (more than 95% sequence homology for the Fc regions).
The term “biological activity” notably refers to the capacity of the IgG constant region to bind to complement proteins in particular (protein C1q for example [Basic and Clinical Immunology, 8th Edition, Daniel P. Stites, Abba I. Terr and Tristram G. Parslow (eds.)]) and/or to IgG receptors: FcγR (FcγRI, FcγRII, FcγRIII; Ravetch and Kinet, Annual Review of Immunology, Vol. 9:457-492 (1991)).
Depending on the type of binding, various action mechanisms may be activated: opsonization, phagocytosis, antibody-dependent cellular cytotoxicity (ADCC) or complement-dependent cytotoxicity (CDC), for example. See Uananue and Benacerraf, Textbook of Immunology, 2nd Edition, Williams & Wilkins, p. 218 (1984)) for further details.
The term “Fc fragment” refers to the constant region of a full-length immunoglobulin excluding the first immunoglobulin constant region domain. Thus, “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 is composed of the CH2-CH3 lower hinge, i.e., the portion from amino acid C226 to the carboxy-terminal end, the numbering being indicated according to the EU index or the Kabat equivalent. The analogous domains for other IgG subclasses can be determined from the alignment of the amino acid sequences of the heavy chains or the heavy chain fragments of the IgG subclasses with that of human IgG1 (see
The term “parent Fc fragment” or “parent polypeptide” as used herein refers to a reference polypeptide among the wild-type Fc regions or variants optionally containing mutations other than that considered. The term “wild-type” or “WY” refers herein to an amino acid sequence or a nucleotide sequence which is found in nature i.e., which is of natural origin, including allelic variations, and which has not been modified intentionally by molecular biology techniques such as mutagenesis. For example, “wild-type” Fc regions notably refer to the IgG1 Fc region with sequence SEQ ID NO: 1 (G1m1,17 allotype), the IgG2 Fc region with sequence SEQ ID NO: 2, the IgG3 Fc region with sequence SEQ ID NO: 3, the IgG4 Fc region with sequence SEQ ID NO: 4, and the IgG1 Fc region with sequence SEQ ID NO: 5 (G1m3 allotype).
The Fc fragments used in the invention are in monomeric form, i.e., they are not fused or conjugated to each other.
The term “modified affinity” refers to a decreased or increased affinity in comparison with a parent Fc fragment.
The term “neonatal Fc receptor” or “FcRn” as used herein refers to a protein which binds to the IgG Fc region and is encoded at least in part by an FcRn gene. The FcRn may be from any organism, including but not limited to humans, mice, rats, rabbits and monkeys. As known in the state of the art, the functional FcRn protein comprises two polypeptides, often designated by the name of the heavy chain and of the light chain. The light chain is β2-microglobulin and the heavy chain is encoded by the FcRn gene. Unless otherwise specified herein, the term “FcRn” or “FcRn protein” refers to the complex of the α chain with β2-microglobulin. In humans, the gene encoding FcRn is called FCGRT.
The term “increase in FcRn binding” as used herein refers to the increase in the binding affinity, in vivo or in vitro, of the mutated Fc fragment of the invention for FcRn, in comparison with the parent polypeptide. The capacity of the mutated Fc fragment of the invention to bind to an FcRn can be evaluated in vitro by ELISA, as described for example in patent application WO2010/106180.
In the present invention, the term “half-life” refers to the amount of time for the Fc fragment to be eliminated by half from the circulation or from other tissues, once present in the serum of the patient to which it has been administered.
The term “Fcγ receptor” or “FcγR” refers to 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 a receptor that inhibits immune cell activation (Muta T et al., Nature, 1994, 368:70-73).
The term “effector cell” refers to any cell bearing an Fc receptor, such as lymphocytes, monocytes, neutrophils, natural killer (NK) cells, eosinophils, basophils, mastocytes, dendritic cells, Langerhans cells and platelets.
In the context of the invention, the term “glycosylation” refers to the addition, by enzymatic reaction, of one or more carbohydrates to the sequence of a recombinant Fc fragment.
In the context of the invention, the term “hypersialylation” 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 may be carried out by enzymatic reaction, by cellular reaction or by directed mutagenesis of the Fc targeting one or more amino acids involved in Fc sialylation.
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 sex.
The term “treatment” or “treat” refers to an improvement in or the prophylaxis or the reversal of a disease or a disorder, or at least a symptom which can be distinguished therefrom, or an improvement in or the prophylaxis or reversal of at least one measurable physical parameter associated with the disease or the disorder being treated, which is not necessarily distinguishable in or by the treated subject. The term “treatment” or “treat” further includes the inhibition or slowing down of the progression of a disease or a disorder, physically, for example, the stabilization of a distinguishable 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 Fc receptor (FcR) in comparison with a parent Fc fragment.
The Fc fragments according to the invention have a decreased affinity for at least one Fc receptor and/or an increased affinity for at least one Fc receptor, in comparison with a parent Fc fragment.
Preferentially, the affinity is increased, in comparison with that of the parent Fc, by a ratio at least equal to 2, preferably higher than 5, preferably higher than 10, preferably higher than 15, preferably higher than 20, preferably higher than 25, and preferably higher than 30. In other words, the affinity of the mutated Fc region for an FcR is higher than that of the parent polypeptide. Alternatively, said mutated Fc region has a decreased affinity for at least one FcR. Preferentially, the affinity is decreased, in comparison with that of the parent Fc, by a ratio at least equal to 2, preferably higher than 5, preferably higher than 10, preferably higher than 15, preferably higher than 20, preferably higher than 25, and preferably higher than 30. In other words, the affinity of the mutated Fc region for an FcR is lower than that of the parent polypeptide.
The affinity of a polypeptide comprising an Fc region for an FcR may be evaluated by methods well-known in the prior art. For example, the person skilled in the art may determine the affinity (Kd) by using surface plasmon resonance (SPR), which can be measured by the Biacore system. Alternatively, the person skilled in the art may perform a suitable ELISA. A suitable ELISA can be used to compare the binding forces of the parent Fc and of the mutated Fc. The specific signals detected from the mutated Fc and from the parent Fc are compared. The binding affinity may be equally determined by evaluating the entire polypeptides or by evaluating the Fc regions isolated therefrom.
In particular, the Fc fragments used according to the invention have a decreased affinity for at least one Fc receptor selected from FcRn, an Fcγ receptor, and complement C1q, and/or an increased affinity for at least one Fc receptor selected from FcRn, an Fcγ receptor, and complement C1q, in comparison with a parent Fc fragment.
According to a particular embodiment, the Fc fragments used according to the invention have a modified affinity, advantageously an increased affinity, for FcRn. This increase in FcRn binding results in an improvement in serum retention in vivo and, consequently, an increase in half-life.
The Fc fragments may be used alone or in mixture; for example, several Fc fragments having different mutations may be administered in a mixture or co-administered.
The Fc fragments of the invention may also be used in a composition comprising a single type of mutated Fc fragment. In other words, the composition comprises molecules of Fc fragments of identical sequence.
According to an aspect of the invention, the Fc fragments having a modified affinity for at least one Fc receptor contain a mutation of at least one amino acid in comparison with a parent Fc fragment. The mutations concerned are not the natural variations that define the immunoglobulin isotype, but artificial mutations, the production process generating the Fc fragment containing the desired mutation(s).
Preferably, the mutation is a substitution, a deletion or an 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 have a mutation 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 numbering of the Fc fragment amino acids referring to that of the EU index or the Kabat equivalent.
Certain amino acid positions from the list above—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, the mutated Fc fragments which have a high binding affinity for FcRn may comprise at least one amino acid change at said amino acid positions. Among 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, indeed three, four or five amino acid mutations may substantially improve the binding affinity for FcRn in comparison with the parent Fc.
In a particular embodiment, the mutations are:
In a preferred embodiment, at least one of positions 378 and 434 is mutated; and optionally also 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, 259I, 264A, 264E, 264M, 265G, 265N, 267N, 267R, 269D, 269G, 270N, 270E, 276S, 284L, 285Y, 288R, 289I, 290R, 290E, 291S, 291Q, 292W, 293del, 294del, 297D, 298G, 298N, 299M, 299A, 299K, 301C, 302A, 303A, 303I, 305A, 307P, 307A, 307N, 308I, 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, 384I, 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, 422A, 424L, 426T, 428L, 433R, 433P, 434Y, 434S, 434H, 438R, 439R, 440R, 440N, 443R, 444F, 444P, 445S, 446A, 447N and 447E, and are also described in patent application 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, 259I, 264E, 265G, 267R, 290E, 293del, 294del, 303A, 305A, 307P, 307A, 308I, 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 particular combinations of mutations are presented below:
In a particularly advantageous embodiment, the Fc fragments have a combination of mutations selected from 315D/330V/361D/378V/434Y (combination of mutations also called “T5A-74”; Fc fragments with this combination of mutations are thus also called “rFc T5A-74”), 230S/315D/428L/434Y, 307A/315D/330V/382V/389T/434Y, 259I/315D/434Y, 256N/378V/383N/434Y, E294del/T307P/N434Y (combination of mutations also called “C6A_66”; Fc fragments with this combination of mutations are thus also called “rFc C6A_66”).
In another embodiment, the Fc fragments have at least one mutation selected from V240H, V240I, V240M, V240N, V240S, F241H, F241Y, L242A, L242F, L242G, L242H, L242I, L242K, L242P, L242S, L242T, L242V, F243L, F243S, E258G, E258I, E258R, E258M, E258Q, E258Y, V259C, V259I, V259L, T260A, T260H, T260I, T260M, T260N, T260R, T260S, T260W, V262A, V262S, V263T, V264L, V264S, V264T, V266L, V266M, S267A, S267Q, S267V, K290D, K290E, K290G, K290H, K290L, K290N, K290Q, K290R, K290S, K290Y, P291G, P291Q, P291R, R292I, R292L, E293A, E293D, E293G, E293M, E293Q, E293S, E293T, E294A, E294G, E294P, E294Q, E294R, E294T, E294V, Q295I, Q295M, Y296H, Y296W, S298A, S298R, Y300I, Y300V, Y300W, R301A, R301M, R301P, R301S, V302A, V302F, V302L, V302M, V302R, V302S, V303S, V303Y, S304T, V305A, V305F, V305I, V305L, V305R and V305S of said Fc fragment; the numbering being that of the EU index or the Kabat equivalent.
In an embodiment, the variant according to the invention has an increased affinity for FcγRIIIa (CD16a). In this particular embodiment, said variant comprises at least one mutation i) selected from S298A, S298R, F243S, F243L, L242A, L242F, L242G, L242I, L242K, L242S, L242V, V240I, V240M, V240N, V240S, E258I, T260A, K290D, K290E, K290G, K290H, K290Q, K290S, K290Y, Y296H, Y296W of said Fc fragment;
the numbering being that of the EU index or the Kabat equivalent.
Particular Fc fragments are fragments ZAC2-85 (T260A), of sequence SEQ ID NO: 17:
Or ZAC3-172 (K290Y), of sequence SEQ ID NO: 18:
In another embodiment, the variant according to the invention has an increased affinity for FcγRIIa (CD32a). In this particular embodiment, said variant comprises at least one mutation i) selected from F241H, F241Y, F243L, L242A, L242F, L242G, L242H, L242I, L242K, L242P, L242S, L242T, L242V, V240H, V240I, V240M, V240S, E258G, E258I, E258R, E258M, E258Q, E258Y, S267A, S267Q, S267V, T260A, T260H, T260I, T260M, T260N, T260R, T260S, T260W, V259C, V259I, V259L, V262A, V262S, V263T, V264L, V264S, V264T, V266L, V266M, E293A, E293D, E293G, E293M, E293Q, E293S, E293T, E294A, E294G, E294P, E294Q, E294R, E294T, E294V, K290D, K290E, K290G, K290H, K290L, K290N, K290Q, K290R, K290S, K290Y, P291G, P291Q, P291R, Q295I, Q295M, R292I, R292L, R301A, R301P, R301S, S304T, V302A, V302F, V302L, V302M, V302R, V302S, V303Y, V305A, V305F, V305L, V305R, V305S, Y300I, Y300V or Y300W; the numbering being that of the EU index or the Kabat equivalent.
In another embodiment, the variant according to the invention has an increased affinity for FcγRIIb (CD32b). In this particular embodiment, said variant comprises at least one mutation i) selected from E258R, E258Y, V262A, S267A, S267Q, S267V, V264S, V266L, V266M, K290R, R301A, R301M, S304T, V302A, V302L, V302R, V303S, V305A, V305F, V305I, V305R, Y300V of said Fc fragment; the numbering being that of the EU index or the Kabat equivalent.
Preferably, the variant according to the invention is characterized in that the Fc fragment of the parent polypeptide comprises at least:
In a particularly advantageous embodiment, the Fc fragments have a combination of mutations selected from:
The sequences of Fc fragments T5A-74, T5A-74I, T5A-74J, T5A-74K, T5A-74L, T5A-74M are presented in the attached sequence listing.
Preferably, the variant according to the invention is characterized in that the Fc fragment of the parent polypeptide comprises at least:
In another embodiment, the Fc fragments have at least one mutation selected from:
In a particular embodiment, the Fc fragments have at least a combination of 2 mutations, said combination being selected from:
Preferably, the mutated Fc fragments have an increased affinity for complement C1q, and comprise at least a combination of 2 mutations, said combination comprising:
Preferably, the mutated Fc fragments have an increased affinity for FcγRIIIa (CD16a), and comprise at least a combination of 2 mutations, said combination comprising:
Preferably, the mutated Fc fragments have an increased affinity for FcγRIIa (CD32a), and comprise at least a combination of 2 mutations, said combination comprising:
Preferably, the mutated Fc fragments have an increased affinity for FcγRIIb (CD32b), and comprise at least a combination of 2 mutations, said combination comprising:
Preferably, the mutated Fc fragments comprise at least a combination of 3 mutations, said combination comprising:
In a particularly advantageous embodiment, the Fc fragments have a combination of mutations selected from K334N/P352S/V397M/A378V (combination of mutations also called “A3A-184A”; Fc fragments with this combination of mutations are thus also called “A3A-184A”), G316D/K326E/A378V (combination of mutations also called “A3A-105D”; Fc fragments with this combination of mutations are thus also called “A3A-105D”), P396L/N421T/A378V (combination of mutations also called “J3B-118A”; Fc fragments with this combination of mutations are also called “J3B-118A”).
The sequence of fragment A3A-184A (K334N/P352S/A378V/V397M) is presented in the sequence listing, as SEQ ID NO: 19:
The sequence of fragment A3A-105D (G316D/K326E/A378V) is presented in the sequence listing, as SEQ ID NO: 20:
The sequence of fragment J3B-118A (A378V/P396L/N421T) is presented in the sequence listing, as SEQ ID NO: 21:
According to another particular embodiment, the mutated Fc fragments used in the invention have a deletion of an amino acid at position 293 or 294 (DEL293 or DEL294), the numbering of the Fc fragment amino acids referring to that of the EU index or the Kabat equivalent. This deletion may be the only mutation of the Fc fragment, or may be accompanied by other mutations, in particular among those listed above.
This single mutation leads to a specific glycosylation of the fragment, namely a hypersialylation, which is particularly advantageous with respect to glycoprotein half-life and to inflammatory processes.
According to another more particular embodiment, the mutated Fc fragments used in the invention have the deletion of the amino acid at position 293 or 294, and have in addition one or more mutations 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.
For example, a preferred Fc fragment has the combination of mutations 307P, 434Y, in combination with deletion DEL294 or DEL293.
According to another aspect of the invention, use is made of a composition comprising a plurality of Fc fragments which all have substantially the same sequence, and which, taken as a whole, have a specific glycosylation profile.
According to a particular aspect, a composition used in the context of the invention comprises Fc fragments having N-glycans on the glycosylation site (Asn 297) thereof, characterized in that said N-glycans of the Fc fragments have a degree of fucosylation 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%.
According to still another aspect, a composition used in the context of the invention comprises Fc fragments, said Fc fragments having N-glycans on the glycosylation site (Asn 297) thereof, characterized in that said N-glycans of the Fc fragments have a biantennary-type glycan structure, with short chains, a low sialylation, with terminal mannoses and/or non-intercalated 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 N-glycans on the glycosylation site (Asn 297) thereof, characterized in that greater than 60% of said N-glycans of the Fc fragments are the G0+G1+G0F+G1F forms, 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 N-glycans on the glycosylation site (Asn 297) thereof, characterized in that greater than 60% of said N-glycans of the Fc fragments are the G0+G1+G0F+G1F forms, the fucose content being lower 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 N-glycans on the glycosylation site (Asn 297) thereof, characterized in that less than 40% of said N-glycans of the Fc fragments are the G1F+G0F forms.
According to a more particular aspect, a composition used in the context of the invention comprises Fc fragments having N-glycans on the glycosylation site (Asn 297) thereof, said N-glycans of the Fc fragments having a degree of fucosylation equal to 0%. The invention thus provides a composition comprising Fc fragments having N-glycans on the glycosylation site Asn297 thereof, characterized in that said N-glycans of the Fc fragments are fucose-free.
Also, according to a particular aspect, a composition used in the context of the invention comprises Fc fragments having N-glycans on the glycosylation site Asn297 thereof, characterized in that said N-glycans of the Fc fragments have a degree of fucosylation in the range between 20% and 55%. In particular, the invention provides a composition comprising Fc fragments having N-glycans on the glycosylation site Asn297 thereof, characterized in that said N-glycans of the Fc fragments have a degree of fucosylation in the range 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 useful composition according to the invention comprises Fc fragments having N-glycans on the glycosylation site Asn297 thereof, characterized in that greater than 60%, preferably greater than 80%, of said N-glycans of the Fc fragments are the G0+G1+G0F+G1F forms, the G0F+G1F forms being less than 50%, preferably less than 40%, or 30%.
According to another more particular aspect, greater than 60% of the N-glycans of the Fc fragments within the composition are the G0+G1+G0F+G1F forms, the fucose content being lower than 65%.
According to still another more particular aspect, less than 50%, preferably less than 40%, or 30% of the N-glycans of the Fc fragments within the composition are the G1F+G0F forms.
The G0, G0F, G1 and G1F forms are selected from the forms indicated in
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 that may be used in the context of the invention comprises Fc fragments having N-glycans on the glycosylation site (Asn 297) thereof, said N-glycans of the Fc fragments having a biantennary-type glycan structure, with short chains, a low sialylation, with terminal mannoses and/or non-intercalated terminal N-acetylglucosamines, greater than 60% of the N-glycans consisting of the G0+G1+G0F+G1F forms, and a low fucosylation, less than 50% of the N-glycans consisting of the G0F+G1F forms, for example.
In a particular embodiment, the Fc fragments according to the invention have glycan 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 in comparison with a parent Fc fragment and have N-glycans on the glycosylation site (Asn 297) thereof, said N-glycans of the Fc fragments having a degree of fucosylation 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%. Preferably, the Fc fragments have one or more mutations at positions selected from those listed above, notably 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, in addition, on the glycosylation site (Asn 297) thereof, have N-glycans with a degree of fucosylation 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%.
More preferably, the Fc fragments of the composition according to the invention have a combination of mutations selected from 315D/330V/361D/378V/434Y, 230S/315D/428L/434Y, 307A/315D/330V/382V/389T/434Y, 259I/315D/434Y, 256N/378V/383N/434Y and DEL294/307P/434Y; and, in addition, on the glycosylation site (Asn 297) thereof, have N-glycans with a degree of fucosylation 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%.
Advantageously, the Fc fragments having a modified glycosylation at the glycosylation site at position 297, in particular a low fucosylation, have an increased binding to Fc-gamma receptors (FcγR), in particular FcγRIIIa (CD16a).
Preferably, said Fc fragments have an affinity for CD16a at least equal to 2×106 M−1, at least equal to 2×107 M−1, 2×108 M−1 or 2×109 M−1, as determined by Scatchard analysis or BIAcore technology (label-free surface plasmon resonance-based technology).
Particularly advantageously, the mutated Fc fragments of the invention may be used in combination with different mutated Fc fragments having different mutations. For example, use may advantageously be made of a mixture of mutated Fc Fc-Del294, mutated Fc T5A-74 (to also target FcRn), mutated Fc improved for binding to FcγRs (types A3A-184A).
Production of the Fc Fragments According to the Invention
The Fc fragments used in the invention may be produced by any method known to persons skilled in the art, for example by chemical synthesis or by recombination.
In a preferred embodiment, the Fc fragments used in the invention are referred to as “recombinant,” i.e., they are obtained by recombination.
When the Fc fragments according to the invention have a mutation of one or more amino acids, the mutation(s) may optionally be introduced by known techniques such as gene synthesis, directed mutagenesis, notably obtained by PCR with specific primers that introduce the desired mutations, or random mutagenesis. Preferably, random mutagenesis as described in application WO02/038756 is used, namely the MutaGen technique. This technique uses a human DNA mutase, notably selected from DNA polymerases β, η and ι. Conventional recombinant techniques involve recombination in a host cell, transformed with one or more vectors that enable expression with or without secretion of the Fc fragment sequence into the extracellular medium. The vector generally comprises a promoter, signals for initiation and termination of translation, as well as appropriate regions for regulation of transcription. It may be maintained stably in the host cell and may optionally have particular signals that specify secretion of the translated protein. These various elements are selected and optimized by persons skilled in the art according to the host cell used.
Such vectors are prepared by methods commonly used by persons skilled in the art, and the resulting clones may be introduced into a suitable host by standard methods, such as lipofection, electroporation, use of polycationic agents, heat shock, or chemical methods.
The host cell may be selected 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 may also be used.
The Fc fragments used in the invention may be produced by culturing, in suitable medium and culture conditions, a host cell expressing said Fc fragments; and recovering the fragments thus produced from the culture medium or from said cultured cells.
The preferred mammalian cells for expressing the Fc fragments are the rat cell line YB2/0, the cell line Vero, the hamster cell line CHO, in particular the cell lines 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.
Another production mode is expression of the Fc fragments in transgenic organisms, for example in plants (Ayala M, Gavilondo J, Rodríguez M, Fuentes A, Enríquez 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 rabbits, goats, rats or pigs (Pollock, D. P., J. P. Kutzko, E. Birck-Wilson, J. L. Williams, Y. Echelard and H. M. Meade. (1999). Transgenic milk as a method for the production of recombinant antibodies. Journal of Immunological Methods. 231:147-157). Also see document WO200748077 in this respect.
In an alternative embodiment, the Fc fragments are obtained by proteolytic treatment of immunoglobulins that are themselves mutated.
The glycosylation of the Fc fragments may be modified by known techniques. The Fc fragments having glycosylation according to the invention may notably be produced from the cleavage of the antibodies produced according to the technique described in WO01/77181, notably by the enzyme papain. Slightly fucosylated Fc fragments may also be obtained by production in cells cultured in the presence of kifunensine, as described for example in document U.S. Pat. No. 7,700,321, or in cells for which the GDP-fucose production pathway is inhibited, for example by inhibition of at least one enzyme of the fucose production cycle (see notably documents US 2010291628 or US 20090228994, EP 1500698, EP 1792987 or U.S. Pat. No. 7,846,725). It is also possible to use interfering RNA (RNAi) that inhibit 1,6-fucosyltransferase as described in document U.S. Pat. No. 7,393,683 or document WO2006133148. It may be a matter of preparation methods in yeasts, as described for example in document WO 0200879.
If the Fc fragments have 100% non-fucosylated oligosaccharides, i.e., when the Fc fragments are completely fucose-free, it is possible to use preparation methods known to persons skilled in the art, such as for example those disclosed in documents EP1176195, U.S. Pat. Nos. 7,214,775, 6,994,292, 7,425,449, US2010223686, WO2007099988, EP 1705251, this list being non-limiting. It may be a matter for example of a method using a host cell expressing at least one nucleic acid encoding an Fc fragment, and of which the glycosylation is modified by deletion of the gene encoding α1,6-fucosyltransferase or by addition of a mutation of said gene to eliminate α1,6-fucosyltransferase activity, and consequently expressing a fucose-free antibody fragment.
Therapeutic Applications
Because of their many advantages, in terms of both efficacy and optimized effector functions or reduced side effects, the Fc fragments described herein are useful in the treatment of an autoimmune and/or inflammatory disease.
A method is described herein for treating an autoimmune and/or inflammatory disease in a patient, comprising administering to said patient a therapeutically effective amount of Fc fragments with an altered affinity for at least one Fc receptor as described herein.
In the present invention, the expression “an autoimmune and/or inflammatory disease” refers to an organ-specific or systemic, primary or secondary autoimmune and/or inflammatory disease, optionally associated with pathogenic autoantibodies.
For example, the disease may be selected from thrombocytopenic thrombotic purpura (TTP), idiopathic thrombotic purpura (ITP), organ or graft rejection, graft-versus-host disease, rheumatoid arthritis, systemic lupus erythematosus, the different types of sclerosis, primary Sjögren syndrome (or Gougerot-Sjögren syndrome), autoimmune polyneuropathies such as multiple sclerosis, type 1 diabetes, autoimmune hepatitis, ankylosing spondylitis, Reiter syndrome, gouty arthritis, celiac disease, Crohn's disease, Hashimoto's thyroiditis (hypothyroidism), Addison disease, autoimmune hepatitis, Basedow disease (hyperthyroidism), ulcerative colitis, vasculitis such as antineutrophil cytoplasmic antibody (ANCA)-associated systemic vasculitis, autoimmune cytopenias and other hematological complications in adults and children, such as acute or chronic autoimmune thrombopenia, autoimmune hemolytic anemia, hemolytic disease of the newborn (HDN), cold agglutinin disease, thrombocytopenic thrombotic purpura and acquired autoimmune hemophilia; Goodpasture syndrome, extra-membranous nephropathy, autoimmune bullous skin disorders, refractory myasthenia, mixed cryoglobulinemia, psoriasis, juvenile chronic arthritis, inflammatory myositis, dermatomyositis and systemic autoimmune disease in children including antiphospholipid syndrome, connective tissue disease, the different types of sclerosis, pulmonary autoimmune inflammation, Guillain-Barré syndrome, Kawasaki disease, multifocal motor neuropathy (MMN), chronic demyelinating inflammatory polyradiculoneuropathy (CDIP), autoimmune thyroiditis, mellitus, myasthenia gravis, inflammatory autoimmune disease of the eye, neuromyelitis optica (Devic disease), scleroderma, pemphigus, diabetes due to insulin resistance, polymyositis, Biermer anemia, glomerulonephritis, Wegener disease, giant cell arteritis, periarteritis nodosa and Churg-Strauss syndrome, Still's disease, atrophic polychondritis, Behçet's disease, monoclonal gammopathy, Wegener's granulomatosis, lupus, ulcerative colitis, psoriatic rheumatism, sarcoidosis, collagenous colitis, dermatitis herpetiformis, familial Mediterranean fever, glomerulonephritis with deposits of IgA, Lambert-Eaton myasthenic syndrome, sympathetic ophthalmia, Fiessinger-Leroy-Reiter syndrome and uveomeningoencephalitic syndrome.
Other inflammatory diseases are also included, such as for example 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 heart disease, encephalitis, inflammatory bowel disease, inflammatory osteolysis, inflammation associated with acute and delayed hypersensitivity reactions, inflammation associated with tumors, peripheral nerve damage or demyelinating diseases, inflammation associated with tissue trauma such as burns and ischemia, inflammation due to meningitis, multiple organ dysfunction syndrome (MODS), pulmonary fibrosis, septicemia and septic shock, Stevens-Johnson syndrome, undifferentiated arthritis, and undifferentiated spondyloarthropathy.
In a particular embodiment of the invention, the autoimmune disease is idiopathic thrombotic purpura (ITP) and chronic demyelinating inflammatory polyradiculoneuropathy (CDIP).
One of the effects observed is notably a limitation or a reduction of the destruction of platelets observed in ITP pathology and a limitation or a reduction of the loss of the myelin sheath of peripheral nerves in CDIP.
In another particular embodiment, the disease is an inflammatory disease, such as in particular graft-versus-host disease. In this case, Fc fragments with a mutation that induces hypersialylation, such as a deletion of an amino acid at position 294 (DEL294) or 293 (DEL293), are particularly advantageous.
Any route of administration is envisaged, notably parenteral routes, such as the intravenous, intramuscular, subcutaneous, intradermal or topical routes, or via the mucosal route, for example by inhalation. The 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 pharmaceutical form suited to the selected route of administration.
The useful pharmaceutical compositions according to the invention advantageously comprise one or more pharmaceutically acceptable excipients or carriers. For example, mention may be made of saline, physiological, isotonic or buffered solutions, etc., compatible with pharmaceutical use and known to persons skilled in the art. The compositions may contain one or more agents or carriers selected from dispersants, solubilizers, stabilizers, preservatives, etc. Agents or carriers usable in formulations (liquid and/or injectable and/or solid) are notably methylcellulose, hydroxymethylcellulose, carboxymethylcellulose, polysorbate 80, mannitol, gelatin, lactose, plant oils, acacia, etc. The compositions may optionally be formulated by means of pharmaceutical forms or devices providing extended and/or delayed release. For this type of formulation, an agent such as cellulose, carbonate or starch is advantageously used.
The administered doses may vary, inter alia, according to the patient's weight and age, and the severity of the disease, assessed by the person skilled in the art.
In a preferred embodiment, the dosage of the Fc fragments according to the invention is in the range from about 0.05 mg/kg to about 1 g/kg of body weight, i.e., about 20 mg to about 100 g per day for an adult. Preferably, the dosage is from about 330 mg/kg to about 660 mg/kg per day.
The following examples illustrate the invention without limiting the scope thereof.
Construction of Expression Vectors for the Recombinant Fc (WT and Mutated):
The sequence of the recombinant Fc (aa 221-447) was cloned into a generic eukaryotic expression vector derived from pCEP4 (Invitrogen) for expression in HEK cells and into the OptiCHO vector for expression in YB2/0 cells using standard PCR protocols.
All the mutations of interest in the Fc fragment were inserted into the expression vector by overlapping 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 gel, digested with adequate restriction enzymes and cloned into the expression vector for the recombinant Fc.
Production of the Recombinant Fc in HEK Cells
HEK 293 cells were transfected with the pCEP4 expression vector for the recombinant Fc (WT or mutated) according to standard protocols (Invitrogen). The cells were cultured so as to produce antibodies transiently. The antibodies produced were able to be isolated and purified according to standard techniques of the art, with a view to the characterization thereof. The production rates obtained are on the order of 150 to 500 μg/mL. The purity and the quality of the products were verified by SDS-PAGE and SEC.
Production of Recombinant Fc in YB2/0 Cells
YB2/0 cells were stably transfected by electroporation with the OptiCHO expression vector for the recombinant Fc (WT or mutated) according to standard protocols. Production was carried out in stable pools or after cloning of YB2/0 cells.
The steps of producing the antibodies by cell culture and of purifying same were carried out according to standard techniques of the art, with a view to the characterization thereof. The production rates obtained are on the order of 3 to 30 μg/mL. The purity and the quality of the products were verified by SDS-PAGE and SEC.
To mimic the situation of red blood cell lysis observed in idiopathic thrombocytopenic purpura (ITP), involving the ITP patient's autoantibodies, effector-cell mediated red blood cell lysis in the presence of a monoclonal anti-Rhesus D (RhD) antibody was carried out, and the capacity of various amounts of polyvalent immunoglobulins (IVIg) or of recombinant Fc fragments, mutated (recombinant Fc fragments containing the mutations according to Table 1 below) and non-mutated, to inhibit said lysis, for example by competition with anti-RhD for Fc receptor binding to the surface of effector cells, was evaluated.
To that end, as effector cells, peripheral blood mononuclear cells (PBMC) 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. The PBMC were incubated with opsonized Rh-D+ red blood cells (effector/target ratio of 2:1).
To evaluate the capacity of the candidates (IVIg and recombinant Fc fragments, mutated as defined according to Table 1 and non-mutated) to inhibit cytotoxicity induced by an anti-RhD antibody by competition and/or saturation of Fc receptors, various concentrations of the candidates (0 to 9.75 μM) 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 (measurement of optical density (OD)). Specific lysis is calculated in percentage according to the following formula:
OD sample−OD control 0%/OD control 100%−OD control 0%×100=% Lysis
wherein:
The results are expressed as % specific lysis (see
Proteins Used:
Recombinant human FcRn (FcRn α and β2-microglobulin) was produced in baculovirus cells by GTP Technology (Labege, France) as previously described (Popov et al., Mol. Immunol. 33:521-530 (1996)). Recombinant human CD16aV is available commercially (R&D Systems).
Test on FcRn:
The FC1 and FC2 cells of 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 sodium acetate buffer (pH 5) (immobilization for 100 seconds, final immobilization level of 350 RU). The FC1 cell was used as negative control, prepared in the same manner as FC2 but in the absence of recombinant FcRn. The recombinant Fc to be tested were injected at 6 different concentrations (300 nM, 150 nM, 75 nM, 30 nM, 15 nM and 0) in 50 mM Na phosphate, 150 mM NaCl, 0.05% Tween 20 buffer (pH 6) for 8 minutes at 10 μL/min on the FC1 and FC2 cells. The FC1 and FC2 cells were regenerated between each sample concentration by an injection for 1 minute of 50 mM Na phosphate, 150 mM NaCl, 0.05% Tween 20 buffer (pH 7.8). The data generated were analyzed with the BIAevaluation version 3.1 software (Biacore), by subtracting the control signal obtained on FC1 from the test signal obtained on FC2.
Test on CD16aV:
The FC1 and FC2 cells of a CM5 chip (Biacore, GE Healthcare) were prepared with the His Capture Kit (GE Healthcare, item 28-9950-56), so as to immobilize the anti-histidine antibody at 50 μg/mL (flow rate of 5 μL/min, EDC/NHS activation for 7 minutes, immobilization for 7 minutes, final immobilization level of 12,000 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 on the FC1 and FC2 cells at 5 different concentrations (1000 nM, 500 nM, 250 nM, 125 nM and 25 nM) in HBS-EP+ buffer in single cycle kinetics (SCK) conditions, with a contact time of 60 seconds, a dissociation of 300 seconds and a flow rate of 30 μL/min without regeneration between each concentration. The final regeneration, between each recombinant Fc, was carried out in 10 mM glycine buffer (pH 1.5) for 60 seconds at 30 μL/min on the FC1 and FC2 cells. The data generated were analyzed with the BIAevaluation version 3.1 software (Biacore), by subtracting the control signal obtained on FC1 from the test signal obtained on FC2.
| Number | Date | Country | Kind |
|---|---|---|---|
| 1556399 | Jul 2015 | FR | national |
| Filing Document | Filing Date | Country | Kind |
|---|---|---|---|
| PCT/FR2016/051708 | 7/6/2016 | WO |
| Publishing Document | Publishing Date | Country | Kind |
|---|---|---|---|
| WO2017/006052 | 1/12/2017 | WO | A |
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| Number | Date | Country | |
|---|---|---|---|
| 20180355034 A1 | Dec 2018 | US |
