Compositions and methods related to engineered fc constructs

Abstract

To provide engineered Fc constructs.SOLUTION: The present invention provides an Fc construct comprising: a first polypeptide having a formula A-L-B (where A comprises a first Fc domain monomer; L is a linker; and B comprises a second Fc domain monomer); a second polypeptide comprising a third Fc domain monomer; and a third polypeptide comprising a fourth Fc domain monomer. The first Fc domain monomer and the third Fc domain monomer form a first Fc domain, and the second Fc domain monomer and the fourth Fc domain monomer form a second Fc domain. At least one of the Fc domains includes at least one amino acid modification that alters one or more of binding affinity to one or more Fc receptors, effector functions, a level of Fc domain sulfation, half-life, protease resistance, Fc domain stability, and / or susceptibility to degradation.SELECTED DRAWING: Figure 16

Description

[Technical field]

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS This application is a joint venture of co-pending U.S. Provisional Patent Application No. 62 / 340,3 filed May 23, 2016. No. 22, filed January 6, 2017, and / or copending U.S. Provisional Patent Application No. 62 / This application claims the benefit of priority to US Pat. No. 4,443,451, the disclosure of which is incorporated herein by reference in its entirety. This is hereby incorporated by reference. [Background technology]

[0002] background Therapeutic proteins, such as therapeutic antibodies and Fc fusion proteins, can rapidly improve immune disease outcomes. and has become a clinically important drug class for patients with inflammatory diseases. Summary of the Invention

[0003] The present disclosure features therapeutic constructs that contain a biologically active Fc domain. Such constructs may be used to provide desired serum half-life and / or binding to Fc receptors. These constructs can have binding affinity and / or avidity. reducing inflammation in a subject, promoting clearance of autoantibodies in a subject, promoting antigen presentation in a subject Inhibition of immune responses, e.g., prevention of activation of immune complex-based immune responses in a subject. Prevention of responses and immune and inflammatory diseases (e.g., autoimmune diseases) in a subject. The Fc constructs described herein are useful for the treatment of It can be used to treat patients with immune and inflammatory diseases without the need for any therapeutic agent.

[0004] Generally, this disclosure relates to Fc constructs having 2-10 Fc domains, e.g. Fc constructs having 2, 3, 4, 5, 6, 7, 8, 9, or 10 Fc domains wherein at least one of the Fc domains comprises: (i) one or more (ii) binding affinity to multiple Fc receptors; (iii) effector function; and (iv) Fc domain structure. (iv) half-life, (v) protease resistance, (vi) Fc domain structure, and / or (vii) susceptibility to degradation. and at least one amino acid modification. In some embodiments, the Fc construct comprises: 2-10 Fc domains, 2-5 Fc domains, 2-4 Fc domains, 2-3 Fc domains, 3 to 5 Fc domains, 2 to 8 Fc domains, or 2 to 6 In some embodiments, the Fc construct comprises 5-10 Fc domains. The construct comprises 2 to 6 (e.g., 2, 3, 4, 5, or 6) domains. Each polypeptide may comprise at least one Fc domain monomer. Each Fc domain monomer of the construct is identical or and other monomers, for example, 20 or less, 15 or less, 10 or less, 8 or less, 7 or less, 6 or less 20 or less amino acids, which may be 1 or less, 5 or less, 4 or less, 3 or less, or 2 or less For example, 15 or fewer, 10 or fewer amino acids differ. In some embodiments, the Fc polypeptide does not contain an antigen-binding domain of an immunoglobulin. The construct (or the Fc domain in the Fc construct) may be contained within a different polypeptide. It is formed in whole or in part by the association of the Fc domain monomers present. In this case, the Fc construct contains an additional domain (e.g., For example, the antibody does not contain an IgM tailpiece or an IgA tailpiece. In another embodiment, there is a linker between two Fc domain monomers that link to form an Fc domain. In other embodiments, the covalent bond is present only in the Fc construct. In yet another embodiment, the Fc construct does not contain a covalent bond between the Fc domains. The construct comprises an Fc construct in which all or substantially all of the Fc domains in the Fc construct are expressed on the cell surface. It has sufficient flexibility to be able to simultaneously interact with multiple Fc receptors. In embodiments, the Fc construct comprises a linker (e.g., a flexible amino acid spacer) In one embodiment, the domain monomer comprises at least two Fc domains linked via The variants are those whose primary sequence differs from the wild type or from each other and which are capable of dimerization. Dimerization selectivity module ).

[0005] The Fc construct of this disclosure can be in a pharmaceutical composition, For example, 2, 3, 4, 5, 6, 7, 8, 9, or 10 A construct having an Fc domain, 2 to 10 Fc domains (e.g., 2 to 8 Fc domains, 2-6 Fc domains, 2-4 Fc domains, 2-3 F Fc domain, 3 to 5 Fc domains, or 5 to 10 Fc domains A substantially homogenous population of constructs (e.g., at least 85%, 90%, 95%, 98% %, or 99% homogeneity). As a result, there is no substantial aggregation or undesirable aggregation of the Fc construct. It is possible to prepare pharmaceutical compositions that are free of excessive multimerization.

[0006] In one embodiment, the Fc construct comprises three polypeptides forming two Fc domains. The first polypeptide has the formula ALB, where A is a first Fc domain. The second polynucleotide comprises a monomer, L is a linker, and B comprises a second Fc domain monomer. The peptide comprises a third Fc domain monomer and the third polypeptide comprises a fourth Fc domain monomer. In this embodiment, the first Fc domain monomer and the third Fc domain monomer are Similarly, a second Fc domain monomer and a third Fc domain monomer are combined to form a first Fc domain. The four Fc domain monomers combine to form a second Fc domain. Exemplary Fc constructs are shown in Figures 4 and 6.

[0007] In some embodiments, the first Fc domain monomer and the third Fc domain monomer are Complementary dimerization selectivity modules promote dimerization between Fc domain monomers In another embodiment, the second Fc domain monomer and the fourth Fc domain monomer comprise These Fc domain monomers are associated with complementary dimerization selectivity modules that promote dimerization. Includes rules.

[0008] In some embodiments, one of A, B, the second polypeptide, and the third polypeptide. In one embodiment, one or more of the A, B, and second polypeptides are comprised of an Fc domain monomer. The first polypeptide, the second polypeptide, and the third polypeptide each consist of an Fc domain monomer.

[0009] In some embodiments, the Fc construct is coupled to, e.g., a peptide, e.g., a serum protein. The heterologous moiety may further comprise a binding peptide, e.g., an albumin binding peptide. The moiety may be attached, for example via a linker, to the N-terminus or N-terminus of the B or third polypeptide. can be linked to the carboxy terminus.

[0010] In some embodiments, the Fc construct comprises IgG C L Antibody constant domains and IgG C H IgG C H 1. Antibody constant domains include, for example, It can be linked to the N-terminus of A or the second polypeptide via a linker.

[0011] In other embodiments, the second and third polypeptides of the Fc construct have the same amino acid sequence. It has the following acid sequence:

[0012] In another embodiment, the disclosure provides a method for the preparation of a fusion protein comprising the steps of: The present invention features an Fc construct comprising a first polypeptide having the formula ALB, , A comprises a first Fc domain monomer, L is a linker, and B is a second Fc domain. The second polypeptide has the formula A'-L'-B', where A' is a third Fc domain. It comprises the main monomer, L' is a linker and B' comprises the fourth Fc domain monomer. The third polypeptide comprises a fifth Fc domain monomer and the fourth polypeptide comprises a sixth Fc domain monomer. In this embodiment, A and A' are linked to form a first Fc domain monomer. and B and the fifth Fc domain monomer combine to form a second Fc domain, and B The third Fc domain monomer and the sixth Fc domain monomer combine to form a third Fc domain. An exemplary Fc construct of this embodiment is shown in FIG.

[0013] In some embodiments, A and A' each represent a dimeric form between these Fc domain monomers. In another embodiment, the B and fifth Fc Each of the Fc domain monomers has a dimer that promotes dimer formation between these Fc domain monomers. In yet another embodiment, the B' and sixth Fc domains comprise a somatotopic selectivity module. Each monomer has a dimerization selection group that promotes dimerization between these Fc domain monomers. Includes a selectivity module.

[0014] In some embodiments, A, B, A', B', a third polypeptide, and a fourth polypeptide. In one embodiment, one or more of the A, B, A Each of the Fc domain alone is a polypeptide B', a third polypeptide, and a fourth polypeptide. It consists of monomers.

[0015] In some embodiments, the Fc construct comprises IgG C L Antibody constant domains and IgG C H IgG C L The antibody constant domain is Via IgG C H 1 Binds to the N-terminus of the antibody constant domain and binds to IgG C H 1. Antibody constant domain In one embodiment, the Fc component is linked to the N-terminus of A, e.g., via a linker. The struc- ture is the second IgG C L Antibody constant domains and second IgG C H 1. Antibody Stationary The second IgG C L The antibody constant domain can be, for example, Second IgG C H 1. Binds to the N-terminus of the antibody constant domain and the second IgG C H 1 antibody The constant domain is attached to the N-terminus of A', for example via a linker.

[0016] In some embodiments, the Fc construct can be linked, e.g., via a linker, to a peptide A heterologous portion, e.g., an albumin-binding peptide linked to the N-terminus or C-terminus of B or B'. minutes.

[0017] In other embodiments, the first and second polypeptides of the Fc construct have the same amino acid sequence. and the third and fourth polypeptides of the Fc construct have the same amino acid sequence. It has an array.

[0018] In another aspect, the disclosure features an Fc construct comprising two polypeptides. The first polypeptide has the formula ALB, where A is a first Fc domain monomer. wherein L is a linker and B is a serum protein binding moiety, e.g., an albumin binding peptide. The second polypeptide comprises a second Fc domain monomer. The first Fc domain monomer and the second Fc domain monomer combine to form an Fc domain. Complete.

[0019] In some embodiments, the first Fc domain monomer and the second Fc domain monomer are and a complementary Fc domain monomer that promotes dimer formation between the first Fc domain monomer and a second Fc domain monomer. The dimerization selectivity module is

[0020] In some embodiments, A and the second polypeptide each comprise an Fc domain monomer. do.

[0021] In yet another aspect, the disclosure features an Fc construct comprising two polypeptides. The first polypeptide has the formula A-L1-B-L2-C, where A is IgG C L It contains an antibody constant domain, L1 and L2 are linkers, and B is IgG C H C comprises a first antibody constant domain monomer, and C comprises a first Fc domain monomer. The peptide has the formula A'-L1'-B'-L2'-C', where A' is IgG C L Antibody determination L1' and L2' are linkers, and B' is an IgG C H 1 C comprises a second Fc domain monomer, and C' comprises a second Fc domain monomer. The first Fc domain monomer and the second Fc domain monomer bind to form an Fc domain. An exemplary Fc construct according to this embodiment of the disclosure is shown in Figure 7A.

[0022] In some embodiments, the first Fc domain monomer and the second Fc domain monomer are Dimers that promote dimer formation between a first Fc domain monomer and a second Fc domain monomer. It includes a formation selectivity module.

[0023] In some embodiments, C and C' each consist of an Fc domain monomer.

[0024] In some embodiments, the Fc construct can be linked via a linker, e.g., N of C or C'. The serum protein binding moiety is an albumin-binding peptide linked to the terminal or C-terminus of the Including etc.

[0025] In yet another aspect, the disclosure provides an Fc construct comprising four or more polypeptides. The first polypeptide has the formula A-L1-B-L2-C, where A is I gG C LIt contains an antibody constant domain, L1 and L2 are linkers, and B is Ig GC H C comprises a first antibody constant domain monomer, and C comprises a first Fc domain monomer. The peptide has the formula A'-L1'-B'-L2'-C', where A' is IgG C L anti L1' and L2' are linkers, and B' is an IgG C H C' comprises one antibody constant domain monomer and C' comprises a second Fc domain monomer. The first Fc domain monomer binds to the third Fc domain monomer to form the first Fc domain. The second Fc domain monomer binds to a fourth Fc domain monomer to form a second Fc domain. Further, the first polypeptide IgG C H 1. an antibody constant domain, Second Polypeptide IgG C L A polypeptide that is linked to an antibody constant domain and IgG C H 1. An antibody constant domain is an IgG C L Antibody constant The antibody can be combined with a polypeptide to form an Fc construct containing two or more Fc domains. An exemplary Fc construct according to this embodiment is shown in FIG. 7B.

[0026] In another aspect, the disclosure features an Fc construct comprising two polypeptides. The first polypeptide comprises a first Fc domain monomer and the second polypeptide comprises In this embodiment, the first Fc domain monomer and the second Fc domain monomer are and c domain monomers combine to form the Fc domain. The Fc construct is shown in Figure 1. Further in this embodiment, a first Fc domain monomer The first Fc domain monomer and the second Fc domain monomer are, respectively, a first Fc domain monomer and a second Fc domain monomer. The dimerization selectivity module promotes dimerization between domain monomers. Exemplary Fc constructs of the embodiments are shown in FIGS.

[0027] In some embodiments, the first polypeptide and the second polypeptide each comprise an Fc domain. It is composed of yne monomers.

[0028] In some embodiments, the Fc construct comprises a first polypeptide, e.g., a polypeptide linked to the first polypeptide, e.g., via a linker. an albumin-binding peptide linked to the N-terminus or C-terminus of a second polypeptide; The antibody further comprises a serum protein binding moiety which is a cytochrome P450 antibody.

[0029] In another aspect, the disclosure features an Fc construct comprising two polypeptides. The first polypeptide has the formula ALB, where A is a first Fc domain monomer. wherein L is a linker and B comprises a second Fc domain monomer. The Fc domain has the formula A'-L'-B', where A' comprises a third Fc domain monomer and L' is a phosphoryl group. and B' comprises a fourth Fc domain monomer. In this embodiment, the first and second F Each of the c domain monomers is H 3. Engineered cavities in antibody constant domains and each of the second and fourth Fc domain monomers comprises their respective C H 3. Antibody constant domain The device has a manipulated protrusion in the cavity, and the manipulated cavity and the manipulated protrusion The projections are positioned to form pairs with the projections inserted into the cavities. The first Fc domain monomer and the third Fc domain monomer are combined to form the first Fc domain. and the second Fc domain monomer binds to the fourth Fc domain monomer to form a second Fc domain monomer. It forms the c domain.

[0030] In some embodiments, one or more of A, B, A', and B' is an Fc domain alone. In one embodiment, each of A, B, A', and B' consists of a single Fc domain. It consists of monomers.

[0031] In some embodiments, the Fc construct can be linked, e.g., via a linker, to, e.g., B or B' A serum protein-binding moiety that is an albumin-binding peptide linked to the N-terminus or C-terminus of , and further includes.

[0032] In some embodiments, the Fc construct comprises IgG C L Antibody constant domains and IgG C H IgG C L Antibody constant domains include, for example, IgG C via Car H 1 Binds to the N-terminus of the antibody constant domain and binds to IgG C H 1 antibody determination The common domain is linked to the N-terminus of A via a linker. In one embodiment, the Fc domain is The struc- ture is the second IgG C L Antibody constant domains and second IgG C H 1. Antibody Stationary The second IgG C L The antibody constant domain is connected to the second IgG C H 1. Binds to the N-terminus of the antibody constant domain and the second IgG C H 1. Antibody Constant The main is bound to the N-terminus of A' via a linker.

[0033] In another aspect, the disclosure provides a method for the detection of a polypeptide comprising: a) a first polypeptide having the formula ALB, wherein A comprises or consists of a first Fc domain monomer, L is a linker, and B is a second Fc domain monomer. a) a first polypeptide comprising or consisting of two Fc domain monomers; and b) a polypeptide of the formula A'-L' - a second polypeptide having a structure of formula B', wherein A' is a third Fc domain monomer; wherein L' is a linker and B' comprises a fourth Fc domain monomer or and c) a third polypeptide comprising or consisting of a fifth Fc domain monomer. and d) a fourth polypeptide comprising or consisting of a sixth Fc domain monomer. The present invention is characterized by an Fc construct comprising a first polypeptide A and a second polypeptide B. A' of the peptide binds to form a first Fc domain, and B of the first polypeptide binds to a second Fc domain. and five Fc domain monomers are combined to form a second Fc domain, The B' and the sixth Fc domain monomer combine to form the third Fc domain. Each of the Fc domain monomer and the third Fc domain monomer is a first Fc domain monomer. Dimerization Selection Promotes Dimerization between a First and a Third Fc Domain Monomer Each of the second Fc domain monomer and the fifth Fc domain monomer comprises a functional module. This promotes dimer formation between the second Fc domain monomer and the fifth Fc domain monomer. a fourth Fc domain monomer and a sixth Fc domain monomer, Each of the Fc domain monomers is linked to a fourth Fc domain monomer and a sixth Fc domain monomer. The Fc component comprises complementary dimerization selectivity modules that promote dimerization between Fc components. The construct contains no more than three Fc domains.

[0034] In some embodiments of this aspect, the first Fc domain monomer or the third Fc domain monomer One of the Fc domain monomers contains a negatively charged amino acid substitution, while the other contains a positively charged a second Fc domain monomer and a fourth Fc domain monomer, each of which comprises an amino acid substitution having a different amino acid sequence. or the fifth Fc domain monomer and the sixth Fc domain monomer include an engineered protrusion. In some embodiments, the linker comprises only one Fc domain monomer and the other Fc domain monomer comprises an engineered cavity. L1, L2, L1', and / or L2' are 3 to 200 amino acids in length. In an embodiment, the linker L and / or L' is any one of SEQ ID NOs: 1 to 27 and 51 to 55. In some embodiments, the nucleic acid comprises, consists of, or consists essentially of one of the sequences. The linkers L and / or L' may be up to 10 (e.g., up to 9, 8, 7, 6, 5, 4, 3, 2, or 1) single amino acid modification (e.g., substitution, e.g., conservative substitution), Nos. 1 to 27 and 51 to 55, or substantially any one of the sequences. The nucleic acid sequence essentially consists of the sequence.

[0035] In another aspect, the disclosure provides a method for detecting a polypeptide comprising: a) a first polypeptide having the formula A-L1-B-L2-C; wherein A comprises or consists of a first Fc domain monomer and L1 is a linker B comprises or consists of a second Fc domain monomer, L2 is a linker, and C and b) a first polypeptide comprising or consisting of a third Fc domain monomer of formula A'- A second polypeptide having L1'-B'-L2'-C', wherein A' is a fourth and L1' is a linker and B' is a fifth Fc domain monomer. comprising or consisting of a main monomer, L2' is a linker, and C' is a sixth Fc domain and c) a second polypeptide comprising or consisting of a seventh Fc domain monomer. and d) a third polypeptide comprising or consisting of an eighth Fc domain monomer. and e) a fifth polypeptide comprising or consisting of a ninth Fc domain monomer. a peptide; and f) a sixth polypeptide comprising or consisting of a tenth Fc domain monomer. The present invention features an Fc construct comprising a first polypeptide A and a seventh Fc domain. The first polypeptide and the second polypeptide are linked to form a first Fc domain, and the B of the first polypeptide and the B of the second polypeptide are linked to form a first Fc domain. and B' of the first polypeptide to form a second Fc domain, and C of the first polypeptide to and an eighth Fc domain monomer to form a third Fc domain, The A' of the second Fc domain monomer binds to the ninth Fc domain monomer to form the fourth Fc domain, and the second C' of the polypeptide and the tenth Fc domain monomer combine to form the fifth Fc domain The first Fc domain monomer and the seventh Fc domain monomer each have a first Fc domain monomer. Complementary dimers that promote dimer formation between the c domain monomer and the seventh Fc domain monomer. and a second Fc domain monomer and a fifth Fc domain monomer. Each of the dimers is a dimer between a second Fc domain monomer and a fifth Fc domain monomer. and a third Fc domain monomer, which comprises a complementary dimerization selectivity module that promotes dimerization. and the eighth Fc domain monomer are each a third Fc domain monomer and an eighth Fc domain monomer. It contains complementary dimerization selectivity modules that promote dimerization between c domain monomers. and each of the fourth Fc domain monomer and the ninth Fc domain monomer is a fourth Fc Complementary dimers that promote dimer formation between the Fc domain monomer and the ninth Fc domain monomer. a sixth Fc domain monomer and a tenth Fc domain monomer comprising a somatic selectivity module; Each of the dimers is a dimer between a sixth Fc domain monomer and a tenth Fc domain monomer. and a complementary dimerization selectivity module that promotes dimerization, and the Fc construct comprises It contains no more than one Fc domain.

[0036] In some embodiments of this aspect, the first, third, fourth, and sixth Fc domain monomers Each includes an engineered protuberance, and the second Fc domain monomer includes a negatively charged amino acid. the fifth Fc domain monomer comprises a positively charged amino acid substitution; the seventh Fc domain monomer comprises a positively charged amino acid substitution; Each of the eighth, ninth, and tenth Fc domain monomers contains an engineered cavity. In this embodiment, the linkers L1, L2, L1', and / or L2' are 3 to 200 amino acids in length. In some embodiments, the linkers L1, L2, L1', and / or L2' are , comprising, consisting of, or essentially consisting of any one of SEQ ID NOs: 1, 2, and 3. The sequence consists of:

[0037] In another aspect, the disclosure provides a method for detecting a polypeptide comprising: a) a first polypeptide having the formula A-L1-B-L2-C; wherein A comprises or consists of a first Fc domain monomer and L1 is a linker B comprises or consists of a second Fc domain monomer, L2 is a linker, and C and b) a first polypeptide comprising or consisting of a third Fc domain monomer of formula A'- A second polypeptide having L1'-B'-L2'-C', wherein A' is a fourth and L1' is a linker and B' is a fifth Fc domain monomer. comprising or consisting of a main monomer, L2' is a linker, and C' is a sixth Fc domain and c) a second polypeptide comprising or consisting of a seventh Fc domain monomer. and d) a third polypeptide comprising or consisting of an eighth Fc domain monomer. and e) a fifth polypeptide comprising or consisting of a ninth Fc domain monomer. a peptide; and f) a sixth polypeptide comprising or consisting of a tenth Fc domain monomer. The present invention is characterized in that the Fc construct comprises a first polypeptide A and a second polypeptide B. The A' of the first polypeptide binds to form a first Fc domain, and the B of the first polypeptide binds to a seventh F and a c domain monomer combine to form a second Fc domain, and and an eighth Fc domain monomer to form a third Fc domain, The B' of the second Fc domain monomer binds to the ninth Fc domain monomer to form the fourth Fc domain, and the second C' of the polypeptide and the tenth Fc domain monomer combine to form the fifth Fc domain Each of the first Fc domain monomer and the fourth Fc domain monomer is a first Fc domain monomer. A complementary dimer that promotes dimer formation between a c domain monomer and a fourth Fc domain monomer. a second Fc domain monomer and a seventh Fc domain monomer comprising a merization selectivity module; Each of the dimers is a dimer between a second Fc domain monomer and a seventh Fc domain monomer. and a third Fc domain monomer, which comprises a complementary dimerization selectivity module that promotes dimerization. and the eighth Fc domain monomer are each a third Fc domain monomer and an eighth Fc domain monomer. It contains complementary dimerization selectivity modules that promote dimerization between c domain monomers. and the fifth Fc domain monomer and the ninth Fc domain monomer each have a fifth Fc Complementary dimers that promote dimer formation between the Fc domain monomer and the ninth Fc domain monomer. a sixth Fc domain monomer and a tenth Fc domain monomer comprising a somatic selectivity module; Each of the dimers is a dimer between a sixth Fc domain monomer and a tenth Fc domain monomer. and a complementary dimerization selectivity module that promotes dimerization, and the Fc construct comprises It contains no more than one Fc domain.

[0038] In some embodiments of this aspect, the first Fc domain monomer comprises a negatively charged amino acid the fourth Fc domain monomer comprises a positively charged amino acid substitution; the second, The third, fifth, and sixth Fc domain monomers each contain an engineered protrusion, and the seventh, Each of the eighth, ninth, and tenth Fc domain monomers contains an engineered cavity. In this embodiment, the linkers L1, L2, L1', and / or L2' are 3 to 200 amino acids in length. In some embodiments, the linkers L1, L2, L1', and / or L2' are , comprising, consisting of, or essentially consisting of any one of SEQ ID NOs: 1, 2, and 3. The sequence consists of:

[0039] In another aspect, the disclosure provides an Fc construct comprising one or more Fc domains. The Fc construct is constructed from a single polypeptide sequence. The polypeptide has the formula ALB, where A comprises a first Fc domain monomer. and L is a linker (optionally a cleavage site, e.g., with one, two or more cleavage sites). A comprises a second Fc domain monomer, and B comprises a cleavable linker. length (e.g., at least 15 amino acids in length, preferably at least about 20 amino acids in length) a first F of the polypeptide, e.g., a first F of the polypeptide having a length of 15 to 200 amino acids The c domain monomer and the second Fc domain monomer are allowed to combine to form an Fc domain. In one embodiment, the first F The c domain monomer and the second Fc domain monomer are a first Fc domain monomer and a second Complementary dimerization selectivity modules that promote dimerization between Fc domain monomers of Such constructs include those that are capable of producing a single polypeptide sequence in a host cell. In one embodiment, the polypeptide can be formed according to the formula A-L1-B-L2 -C, wherein A comprises a first Fc domain monomer and L1 is a linker (optionally (e.g., a linker cleavable by one, two, or more cleavage sites), B is a second L2 is a linker, and C is a third Fc domain monomer. The linker is of sufficient length (e.g., at least 15 amino acids in length, preferably is at least about 20 amino acid residues, e.g., 15 to 200 amino acids in length), and The first Fc domain monomer and the second Fc domain monomer of the polypeptide are bound to each other to form an Fc domain. It is possible to use a flexible amino acid spacer to form a c domain. In one embodiment, the first Fc domain monomer and the second Fc domain monomer are Complementary dimers that promote dimer formation between a c domain monomer and a second Fc domain monomer. In this embodiment, the Fc domain comprises a merization selectivity module. An example of a struct is shown in FIG.

[0040] In any of the Fc constructs described herein, at least one of the Fc domains At least one of the antibodies has (i) binding affinity to one or more Fc receptors, (ii) an effector (iii) the level of sulfation of the Fc domain; (iv) half-life; and (v) proteasome function. (vi) Fc domain stability; and / or (vii) susceptibility to degradation. The Fc constructs described herein include amino acid modifications that change one or more of the following: In one embodiment, the amino acid sequence of the amino acid sequence alters the binding affinity to one or more Fc receptors. The amino acid modification can be any one of the amino acid modifications in Table 2. In one or more of the constructs, the binding affinity for one or more Fc receptors is altered. The amino acid modification is S267E / L328F. In some embodiments, the Fc receptor is In some cases, the modifications described herein are directed to the FcγRIIb receptor. In some cases, the S267E / L328F modification increases affinity for FcγRII. b. In the present invention, the amino acid modification that changes the effector function is any one of the amino acid modifications in Table 6. In any of the Fc constructs described herein, the Fc domain is The amino acid modifications that change the sulfation level of the ribonuclease are 241F, 243F, 246K, and 260T. In any of the Fc constructs described herein, The amino acid modification that alters the reduction life is any one of the amino acid modifications in Table 4. In any of the Fc constructs described herein, amino acids that alter protease resistance may be included. The acid modifications were the following set: 233P, 234V, 235A, and 236del; 237A , 239D, and 332E; 237D, 239D, and 332E; 237P, 239D, and 332E;237Q, 239D, and 332E;237S, 239D, and 332E ;239D, 268F, 324T, and 332E;239D, 326A, and 333A; 239D and 332E; 243L, 292P, and 300L; 267E, 268F, 32 4T, and 332E;267E and 332E;268F, 324T, and 332E;32 6A, 332E, and 333A; or 326A and 333A. In any of the Fc constructs described herein, an amino acid that alters Fc domain stability may be used. The amino acid modification is any one of the amino acid modifications in Table 8. In either construct, an amino acid that alters the susceptibility of the Fc domain to degradation is The acid modifications are C233X, D234X, K235X, S236X, T236X, and H237X. , C239X, S241X, and G249X, where X is any amino acid.

[0041] In another aspect, the disclosure provides a first polypeptide having the formula: (a) ALB, wherein A comprises or consists of a first Fc domain monomer, L is a linker, and B is (b) a first polypeptide comprising or consisting of a second Fc domain monomer; and A second polypeptide having L'-B', where A' is a third Fc domain monomer. wherein L' is a linker and B' comprises a fourth Fc domain monomer. or (c) a second polypeptide comprising or consisting of a fifth Fc domain monomer. and d) a fourth polypeptide comprising or consisting of a sixth Fc domain monomer. In some cases, the first polypeptide comprises a Fc construct comprising a first polypeptide and a second polypeptide. a of the first polypeptide binds to A' of the second polypeptide to form a first Fc domain, The polypeptide B and the fifth Fc domain monomer combine to form a second Fc domain. and B' of the second polypeptide binds to the sixth Fc domain monomer to form a third Fc domain. In some cases, the first polypeptide and the second polypeptide form a polypeptide having the sequence comprising, consisting of or consisting essentially of the sequence of No. 50; The third polypeptide and the fourth polypeptide comprise the sequence of SEQ ID NO: 48. In some embodiments of the disclosure, the first Each of the polypeptide and the second polypeptide may have up to 10 (e.g., up to 9, 8, 7, 6, 5, 4, 3, 2, or 1) single amino acid modifications (e.g., substitutions, e.g., conservative substitutions) The present invention relates to a method for producing a nucleic acid sequence comprising the sequence of SEQ ID NO: 50, the nucleic acid sequence comprising the sequence of SEQ ID NO: 50, or the nucleic acid sequence comprising the sequence of SEQ ID NO: 50. and the third polypeptide and the fourth polypeptide each have a maximum of 10 (e.g. , up to 9, 8, 7, 6, 5, 4, 3, 2, or 1 single amino acid modification (e.g., substitution, A nucleic acid sequence comprising, consisting of, or essentially consisting of the sequence of SEQ ID NO: 48, with, for example, conservative substitutions. The sequence is as follows:

[0042] The Fc domain monomers of the construct have the same primary amino acid sequence. For example, both Fc domain monomers of the Fc domain may be of the wild type conformation. Alternatively, both Fc domain monomers of the Fc domain may be identical dimer-forming A selectivity module may be present, for example, where both Fc domain monomers of the Fc domain are C H At least two positions within the ring of charged residues at the interface between the three domains In the above, the amino acid sequence can have the same opposite charge mutation.

[0043] In any of the Fc constructs described herein, the F of a construct The Fc domain monomer of the c domain is between two Fc monomers (i.e., Fc construct between the Fc domain monomer of the same monomer and other monomers, 15 or less, 10 or less, 8 or less, 7 or less, 6 or less, 5 or less, 4 or less, 3 or less, or 2 or less differ by 20 or fewer amino acids (e.g., 15 or fewer, 10 or fewer amino acids) that are For example, the constructs described herein may have a sequence The Fc monomer sequences may be different, but it is possible to use either of the Fc constructs. A complementary dimerization selectivity module is located in one of the C domain monomers. H 3. Antibody Constant Domains The cavity engineered in the other C of the Fc domain monomer H 3. Engineered into Antibody Constant Domains and an engineered protrusion, the engineered cavity and the engineered protrusion being an Fc domain. The protrusions are positioned to form pairs inserted into the cavities of the main monomers. Exemplary engineered cavities and protrusions are shown in Table 9. In other embodiments, The complementary dimerization selectivity module is located at the C H 3. Antibody Constant Domain A negatively charged amino acid was engineered (substituted) into the Fc domain monomer on the other side of the Fc domain monomer. C H 3. Positively charged amino acids engineered (substituted) into the antibody constant domain; Negatively and positively charged amino acids are complementary to each other in the domain monomers. Exemplary complementary amino acid changes are positioned to facilitate formation of the Fc domain. As shown in Table 10.

[0044] In some embodiments, in addition to the dimer formation selectivity module (e.g., an engineered cavity and protuberances, or engineered positively and negatively charged amino acids (e.g., exemplary amino acids See Tables 1 and 2 for modifications), and the Fc constructs described herein also include, e.g., F c) F, which serves to stabilize the construct or prevent protein aggregation; c may contain further amino acid substitutions from the wild-type sequence in the monomer sequence.

[0045] In some embodiments, the Fc constructs described herein comprise between 2 and 10 Fc domains. In (e.g., 2-8 Fc domains, 2-6 Fc domains, 2-4 Fc domains 2-3 Fc domains, 3-5 Fc domains, or 5-10 Fc domains domains, e.g., 2, 3, 4, 5, 6, 7, 8, 9, 10 domains) and constructs At least two of the Fc domains of the target have different dimer selectivity modules. In some embodiments, the Fc constructs described herein comprise 5 to 10 Fc domains. Containing the main (e.g., 5, 6, 7, 8, 9, 10 domains) and the F At least two of the c domains have different dimer selectivity modules. For example, constructs 5, 8, 9, and 10 comprise at least one engineered cavity and protrusion. one Fc domain and at least one Fc domain containing complementary opposite charge mutations. Yes.

[0046] In other embodiments, one or more linkers in the Fc constructs described herein is a bond.

[0047] In other embodiments, one or more linkers in the Fc constructs described herein The spacer is, for example, 2 to 200 amino acids (for example, 2 to 100, 3 to 20 0, 3~150, 3~100, 3~60, 3~50, 3~40, 3~30, 3~20, 3 ~10, 3~8, 3~5, 4~30, 5~30, 6~30, 8~30, 10~20, 10 ~30, 12~30, 14~30, 20~30, 15~25, 15~30, 18~22, and an amino acid spacer of 20 to 30 amino acids.

[0048] In some embodiments, the amino acid spacer is a glycine-rich spacer and / or a selenium-rich spacer. A phosphorus-rich spacer, for example the spacer has the sequence GS, GGS, GGGG S (SEQ ID NO: 1), GGSG (SEQ ID NO: 2), or SGGG (SEQ ID NO: 3) The present invention comprises, consists of, or consists essentially of two or more of the motifs. In some cases, the amino acid spacer includes only glycines, includes only serines, or In some cases, the amino acid spacer contains only serine and glycine. In some cases, the amino acid sequence may contain only glycine. The spacer contains 3 to 20 amino acids (e.g., 20 amino acids) and may contain glycine and selenium. Contains only phosphorus.

[0049] In one embodiment, when the Fc construct comprises an albumin binding peptide, Albumin-binding peptides In one embodiment, the Fc Where the construct comprises an albumin binding peptide, the albumin binding peptide may be up to 10 (e.g. up to 9, 8, 7, 6, 5, 4, 3, 2, or 1) single amino acids having modifications (e.g., substitutions, e.g., conservative substitutions); It comprises, consists of, or consists essentially of the sequence of TIFF2025026880000003.tif4128.

[0050] In other embodiments, the vesicles of Fc domain monomers within the Fc constructs described herein One or more of these may be an IgG hinge domain, an IgG C H 2 antibody constant domains, and I gG C H 3 antibody constant domains.

[0051] In some embodiments, each of the Fc domain monomers in the Fc construct comprises: IgG hinge domain, IgG C H 2 antibody constant domains, and IgG C H 3. Antibody Stationary Includes the domain.

[0052] In some embodiments, the IgG is selected from the group consisting of IgG1, IgG2a, IgG2b, IgG3, and I. gG4.

[0053] In some embodiments, each of the Fc domain monomers has 10 or fewer (e.g., 1 or fewer , 2 or less, 3 or less, 4 or less, 5 or less, 6 or less, 7 or less, 8 or less, 9 or less , 10 or less) amino acid modifications. In some embodiments, the Fc domain monomer One or more of the following may be selected from the group consisting of human IgG Fc (e.g., IgG1, IgG2a, IgG2 In some embodiments, the Fc domain monomer is One or more of the following may occur up to 10 times (e.g., up to 1, 2, 3, 4, 5, 6, 7, 8, 9, or is a human IgG Fc domain monomer having 10 amino acid modifications.

[0054] In yet another aspect, the disclosure provides a method for the production of a medicament for the treatment of a cancer, comprising administering to the patient a therapeutically effective amount of any of the Fc constructs described herein. homogeneous (e.g., at least 85%, 90%, 95%, 97%, 98%, 99% homogeneous) In one embodiment, the pharmaceutical composition is delivered in a sterile syringe or syringes suitable for pharmaceutical use. The vial contains a pharmaceutical composition, the sole or primary active ingredient being any of the active ingredients described herein. Substantially homogeneous (e.g., at least 85%, 90%, 95%, 9 The pharmaceutical composition is a homogeneous (8%, or 99% homogeneous) population. tergents, surfactants, fillers, polymers, preservatives, and other pharmaceutical excipients In another embodiment, the composition may include one or more inactive ingredients selected from the group consisting of A substantially homogenous pharmaceutical composition has less than 10% aggregates or unwanted multimers of the Fc construct; Contains less than 5%, less than 4%, less than 3%, less than 2%, less than 1%, or less than 0.5%.

[0055] In another aspect, the disclosure provides a method for preparing any one of the foregoing Fc constructs. The method includes the steps of: providing a host cell comprising a polynucleotide encoding an Fc construct; expressing the polypeptide in the host cell under conditions that allow for the formation of an Fc component; and recovering (e.g., purifying) the construct.

[0056] In some embodiments, the Fc construct comprises Fc domains present in different polypeptides. In one embodiment, the Fc constellation is formed at least in part by the association of the main monomers. Tracts are formed by the association of Fc domain monomers present in different polypeptides. In these embodiments, the Fc construct promotes the association of the two polypeptides. It does not contain additional domains (e.g., IgM tailpiece or IgA tailpiece) that are In this embodiment, the Fc domain is only between two Fc domain monomers that are linked to form an Fc domain. In another embodiment, the Fc construct is a covalent bond (e.g., a disulfide bridge). Lactates do not contain covalent bonds (e.g., disulfide bridges) between Fc domains. In other embodiments, the Fc construct comprises all of the Fc domains in the Fc construct. or substantially all of the Fc receptors on the cell surface simultaneously. In some of these embodiments, the Fc component is The construct may comprise a small number of amino acids linked via a linker (e.g., a flexible amino acid spacer). It contains at least two Fc domains.

[0057] In one embodiment, the Fc domain monomers of the Fc domain associate to form an Fc domain. For example, the constructs shown in Figures 4 and 6 , and has two Fc domains comprising three associated polypeptides. one of the polypeptides contains two Fc domain monomers, and the other two of the polypeptides Each contains one Fc domain monomer. The constructs shown in FIG. It has three Fc domains containing four polypeptides, two of which are , two Fc domain monomers, and the other two of the four polypeptides each have one The Fc construct shown in FIG. 7B has 2n polypeptides. Each polypeptide may have n Fc domains (n ​​is 2 to 10) each including a peptide. The peptides are Fc domain monomers, IgG C L Antibody constant domains, and IgG C H 1 The constructs shown in Figures 8 and 9 each contain six associated antibody constant domains. It has five Fc domains containing polypeptides. Two of the six polypeptides are It has three Fc domain monomers, and the other four of the six polypeptides each have one The construct shown in FIG. 10 has two Fc domain monomers. Each of the two polypeptides is linked in tandem. It contains three linked Fc domain monomers.

[0058] In another aspect, the disclosure provides a composition that promotes selective dimerization of Fc domain monomers. The disclosure features products and methods comprising an Fc domain, and two Fs of the Fc domain. The c domain monomer is C H The classification of rings of charged residues at the interface between three antibody constant domains. The disclosure also includes mutations with identical opposite charges at at least two positions within this range. The present invention includes a method for producing such an Fc domain. H 3. Charges at the interface between antibody constant domains At least two positions within the ring of residues having two Fc domain monomer sequences This involves introducing a complementary dimerization selectivity module having an identical mutation within the C H The interface between three antibody constant domains is a hydrophobic patch surrounded by a ring of charged residues. (hydrophobic patch). H 3. Antibody constant domains When combined with , these charged residues pair with residues of the opposite charge. The mutated Fc is characterized by the inversion of charges on both members of two or more complementary pairs of residues. The domain monomers maintain complementarity to Fc domain monomers of the same mutated sequence, but The complementarity to an Fc domain monomer without such mutations is less. The same dimerization selectivity module promotes homodimerization. The double mutants K409D / D339K, K392D / D399K, and E357K / K370E, D356K / K439D, K409E / D339K, K392E / D39 9K, E357K / K370D, or D356K / K439E-containing Fc monomers. In another embodiment, the Fc domain is, for example, K409D / D399K / E357K / The Fc monomers include those containing the quadruple mutant combining any pair of double mutants, one of which is K370E. In another embodiment, in addition to the same dimerization selectivity module, the Fc domain of the Fc domain The main monomers contain non-identical mutations that promote specific association (e.g., engineered cavities and protrusions). As a result, two Fc domains have complementary dimerization selectivity modules. The dimer monomer contains two dimer-forming selectivity modules and maintains complementarity with each other. However, it has little complementarity to other Fc domain monomers. This embodiment is similar to the cavity-containing Fc domain. Promotes heterodimer formation between the main and protuberance-containing Fc domain monomers. In this study, we investigated the effect of the same mutations in the charged pair residues of both Fc domain monomers on the The protrusion on the Fc domain monomer combines with a cavity on the other Fc domain monomer.

[0059] In another aspect, this disclosure features a method of reducing inflammation in a subject in need thereof. In another aspect, the disclosure relates to a method for clearing autoantibodies in a subject in need thereof. In another aspect, the disclosure features a method for providing a subject in need thereof with In another aspect, the disclosure features a method for inhibiting antigen presentation in a subject in need thereof. Methods for reducing an immune response in a subject, e.g., in a subject in need thereof The present invention features methods for reducing activation of an immune response based on immune complexes. These methods include administering to a subject The method includes administering to the subject an Fc construct or pharmaceutical composition described herein.

[0060] In another aspect, the disclosure provides a method for administering to a subject an Fc construct or a pharmaceutical agent described herein. Pharmaceutical compositions (e.g., Constructs 1-10 and 5 * (one of the following) Thus, a method of treating an inflammatory disease or an autoimmune disease or an immune disease in a subject is provided. Exemplary diseases include rheumatoid arthritis (RA); systemic lupus erythematosus (SLE); ANCA-associated vasculitis; antiphospholipid syndrome; autoimmune hemolytic anemia; chronic inflammatory demyelinating anemia Neuropathies; anti-allo in transplants, anti-autologous in GVHD (anti-self), anti-substitution, IgG therapy, clearance of IgG paraprotein; Dermatomyositis; Goodpasture's syndrome; Organogenesis mediated by antibody-dependent cell-mediated cytotoxicity Type II hypersensitivity syndromes targeting the nervous system, e.g. Guillain-Barré syndrome, CIDP, dermatomyositis inflammation, Felty's syndrome, antibody-mediated rejection, autoimmune thyroid disease, ulcerative colitis, autoimmune Autoimmune liver disease; idiopathic thrombocytopenic purpura; myasthenia gravis, neuromyelitis optica; pemphigus and Other autoimmune blistering diseases; Sjogren's syndrome; Antibody-dependent phagocytosis Mediated autoimmune cytopenias and other disorders; e.g. synovitis, dermatomyositis, systemic vasculitis Other FcR-dependent inflammatory syndromes include glomerulitis, and vasculitis.

[0061] In another aspect, the disclosure provides a method for the treatment of inflammation in a subject in need thereof, comprising administering to said subject a therapeutic agent comprising the method of the present invention. The Fc construct or pharmaceutical composition described in the specification (e.g., Constructs 1 to 10 and 5 * In another aspect, the present disclosure provides a method for treating a subject in need thereof, comprising: Fc constructs as described herein for use in promoting clearance of autoantibodies in or pharmaceutical compositions (e.g., Constructs 1-10 and 5 * (one of the following) In another aspect, the disclosure provides a method for the inhibition of antigen presentation in a subject in need thereof. , the Fc construct or pharmaceutical composition described herein (e.g., Constructs 1-1 0 and 5 * In another aspect, the disclosure provides a method for the preparation of a medicament for use in a medical device, comprising: Reducing an immune response in a subject in need thereof, e.g., reducing immune complex responses in a subject in need thereof. The Fc construct or medicament described herein for use in reducing activation of an immune response based on Compositions (e.g., Constructs 1-10 and 5 * The present invention is characterized by the following:

[0062] In another aspect, the disclosure provides a method for treating an inflammatory disease or an autoimmune disease or an immune disorder in a subject. The Fc constructs or pharmaceutical compositions (e.g., constructs) described herein for use in therapy. Lacto 1-10 and 5 * Exemplary diseases include rheumatoid arthritis, Systemic lupus erythematosus (SLE); ANCA-associated vasculitis; antiphospholipid antibodies syndrome; autoimmune hemolytic anemia; chronic inflammatory demyelinating neuropathy; anti-allo (anti-allo), anti-self in GVHD, anti-substitution, I gG treatment, clearance of IgG paraprotein; dermatomyositis; Goodpasture's syndrome; Type II hypersensitivity syndrome targeting organ systems mediated by antibody-dependent cell-mediated cytotoxicity Guillain-Barré syndrome, CIDP, dermatomyositis, Felty syndrome, antibody-mediated rejection Refractory disease, autoimmune thyroid disease, ulcerative colitis, autoimmune liver disease; idiopathic thrombocytopenic Purpura; Myasthenia gravis, Neuromyelitis optica; Pemphigus and other autoimmune blistering diseases; Sjoegren's disease Renn's syndrome; an autoimmune cytopenia mediated by antibody-dependent phagocytosis Other disorders; e.g., synovitis, dermatomyositis, systemic vasculitis, glomerulitis, and other Fs that are vasculitis. Includes cR-dependent inflammatory syndromes.

[0063] In any of the Fc constructs described herein, the order of the Fc domain monomers is It is understood that the order can be interchanged. For example, a polypeptide having the formula ALB In the peptide, the carboxy terminus of A can be linked to the amino terminus of L, and L can be The carboxy terminus of B is linked to the amino terminus of B. Alternatively, the carboxy terminus of B is linked to the amino terminus of L. L can be linked to the amino terminus of C, and its carboxy terminus can be linked to the amino terminus of C. Both of these configurations are included in the formula ALB.

[0064] In a related aspect, the disclosure provides a method for the production of a medicament for the treatment of a pulmonary fibrosis, comprising: The host cell is characterized in that it contains the polypeptides necessary to construct the Fc construct. The polynucleotide comprises a polynucleotide encoding a polypeptide, the polynucleotide being expressed in a host cell. can be.

[0065] definition As used herein, the term "Fc domain monomer" refers to a monomer that contains at least one hinge domain and a A second antibody constant domain and a third antibody constant domain (C H 2 and C H 3) Poly A peptide chain or a functional fragment thereof (e.g., (i) a peptide chain that dimerizes with another Fc domain monomer) (ii) a fragment capable of binding to an Fc receptor to form an Fc domain; Fc domain monomers include IgG, IgE, IgM, IgA, or IgD. The antibody may be of any immunoglobulin antibody isotype, including those with an Fc domain. In monomers are IgG subtypes (e.g., IgG1, IgG2a, IgG2b, IgG The Fc domain monomer can be an antigen recognition region, e.g., IgG1, IgG2, or IgG3. For example, immunoglobulins capable of functioning as a variable region or a complementarity determining region (CDR). The Fc domain monomer does not contain any portion of the wild-type Fc domain monomer sequence. Approximately 10 modifications (e.g., 1 to 10, 1 to 8, 1 to 6, 1 to 4 amino acid substitutions, additions, or The alteration may include a deletion (a deletion) of the Fc domain, which alters the interaction between the Fc domain and the Fc receptor. Examples of suitable modifications are known in the art.

[0066] As used herein, the term "Fc domain" refers to two domains capable of binding to an Fc receptor. A dimer of Fc domain monomers. In the wild-type Fc domain, there are two Fc domains Two monomers C H 3. Not only do dimers form through interactions between antibody constant domains, Between the hinge domains of the two dimerized Fc domain monomers, one or more Forms disulfide bonds.

[0067] In the present disclosure, the term "Fc construct" refers to a construct that comprises 2 to 10 amino acids, as described herein. Fc domains (e.g., 2, 3, 4, 5, 6, 7, 8, 9, or 10 Fc domains) 2-8 Fc domains, 2-6 Fc domains, 2-4 Fc domains, 2-3 Fc domains, 5 to 10 Fc domains, 5 to 8 Fc domains, or 5 to 6 The term "antibody" refers to the associated polypeptide chain formed between the Fc domain of an antibody or antibody complex as described herein. The Fc constructs may contain Fc domain monomers having the same or different sequences. For example, the Fc construct can have two Fc domains. one of which contains an IgG1 or IgG1-derived Fc domain monomer, and the other In another embodiment, the Fc construct comprises an IgG2 or an Fc domain monomer derived from an IgG2. A peptide can have two Fc domains, one of which has a protuberance inserted into a cavity. Protuberance-into-cavity pairs, The other does not contain a "pair of protrusions inserted into a cavity." In the present disclosure, the Fc domain is, for example, For example, V H , V L The Fc domain does not include the variable regions of an antibody that are CDRs, HVRs, or CDRs. , e.g., FcγRI, FcγRIIa, FcγRIIb, FcγRIIIa, FcγRI IIb, forms the minimal structure that binds to Fc receptors, FcγRIV.

[0068] As used herein, the term "antibody constant domain" refers to the constant region domain of an antibody (e.g., L anti C H 1. Antibody constant domain, C H 2 antibody constant domain, or C H 3 antibody determination It refers to a polypeptide corresponding to the common domain.

[0069] As used herein, the term "promotes" refers to, for example, a more specific Fc domain monomer than other separate Fc domain monomers. The Fc domain is formed by two Fc domain monomers that have high binding affinity to each other. "encourages" means to encourage and support, such as to support the Thus, two Fc domain monomers that combine to form an Fc domain are C H 3. Compatible amino acid modifications (e.g., engineered protuberances) at the antibody constant domain interface and engineered cavities). from other Fc domain monomers that have no amino acid modifications or that have incompatible amino acid modifications. These interactions between Fc domain monomers also promote or favor selective interactions between these Fc domain monomers. This is a set of two interacting C H 3. Amino acid modifications at the antibody constant domain interface Therefore, the Fc domain monomers have a higher affinity for other Fc domain monomers that do not have the amino acid modifications. This occurs because the two molecules have a higher affinity for each other than the other molecules.

[0070] As used herein, the term "dimerization selectivity module" refers to a selective selectivity module that selectively selectively binds two Fc domain monomers. Refers to sequences of Fc domain monomers that promote favorable pairing. "Complementary" dimerization selection The sex module promotes the selective interaction of two Fc domain monomers with each other. or a supporting dimerization selectivity module. Complementary dimerization selectivity modules can have the same sequence or different sequences. Selectivity modules are described herein.

[0071] Here, the term "engineered cavity" refers to H 3. At least one of the original amino acid residues in the antibody constant domain is the original amino acid By replacing the C residue with a different amino acid residue that has a lower molecular weight side chain than the C residue, H 3 Refers to the three-dimensional cavity formed in the antibody constant domain. The term refers to the wild-type C H 3 Natural amines encoded by the genetic code of antibody constant domains It refers to an acid residue.

[0072] Here, "engineered protuberance" means The word is C H 3 At least one of the original amino acid residues in the antibody constant domain is By replacing the original amino acid residue with a different amino acid residue having a higher molecular weight side chain, Therefore, C H 3 Refers to the three-dimensional protrusion formed in the antibody constant domain. The term "acid residue" refers to the wild-type C H 3. Antibody constant domains are encoded by the genetic code It refers to a naturally occurring amino acid residue.

[0073] The term "pair with a protuberance inserted into the cavity" refers to a pair in which the first Fc domain monomer has a protuberance inserted into its C H 3 The antibody constant domain contains an engineered cavity, while the second Fc domain monomer contains C H 3. Two Fc domains, each of which contains engineered protuberances within the antibody constant domain This illustrates an Fc domain containing monomers. In a pair of protuberances inserted into the cavity. is the C of the first Fc domain monomer H 3. Engineered protrusions in antibody constant domains but the C of the second Fc domain monomer H 3. Engineered Cavities in Antibody Constant Domains and H 3 The antibody constant domains interact without significantly interfering with the normal association of the dimer at the interface. The devices are arranged for use.

[0074] As used herein, the term "linking" includes chemical linkages, recombinant means, as well as, for example, disulfide linkages. The bonding of two or more components, elements or constituents by means of chemical bonds including amide bonds and amide bonds. By combining or linking protein domains, e.g., polypeptides, For example, two single polypeptides can be joined together to form a chemical linkage. , a chemical bond, a peptide linker, or other covalent bonding means, In some embodiments, the first Fc domain is capable of forming a protein structure. The monomer is linked to a second Fc domain monomer via a peptide linker, The N-terminus of the peptide linker is linked to the first Fc domain via a chemical bond, e.g., a peptide bond. The C-terminus of the peptide linker is linked to the C-terminus of the main monomer, and the C-terminus of the peptide linker is, for example, a peptide bond. In another embodiment, the Fc domain monomer is linked to the N-terminus of the second Fc domain monomer via a chemical bond. The N-terminus of the albumin-binding peptide is linked to a linker in the same manner as described above. C of Fc domain monomer via H 3 linked to the C-terminus of an antibody constant domain.

[0075] As used herein, the term "associated" refers to a plurality of polypeptides (or a single polypeptide). (a plurality of sequences in each of the above) form an Fc construct having at least one Fc domain. such that the junction is positioned such that the junction is oriented ... To describe interactions between polypeptides (or sequences within one single polypeptide) that are For example, two polypeptides each containing one Fc domain monomer may be combined. It is possible to combine the two to form an Fc construct (see, for example, the diagrams shown in Figures 1 to 3). In some embodiments, for example, a polypeptide comprising two Fc domain monomers. and two polypeptides each containing one Fc domain monomer. The peptides associate to form Fc constructs with two Fc domains (e.g. For example, as shown in Figures 4 and 6. In some embodiments, for example, two Fc domain monomers Two polypeptides each containing one Fc domain monomer and two polypeptides each containing one Fc domain monomer. The four polypeptides are associated with each other to form three Fc domains. In another embodiment, the Fc construct is formed (e.g., as depicted in FIG. 5). For example, Fc domain monomer, IgG C L Antibody constant domains, and IgG C H 1 antibody determination The assembly of 2n polypeptides, each of which contains a common domain, results in n Fc domains. The Fc construct is formed with two polypeptides (as shown in FIG. 7B). The Fc domains of the peptides may be linked together via their respective Fc domain monomers or via other elements of the polypeptide. For example, in FIG. 7B, polypeptide 708 is capable of associating with its F Associated with polypeptide 706 via a c domain monomer and with polypeptide 710 C H 1 domain and its associated C L Associated with polypeptide 710 via domain association The association between the polypeptides does not involve covalent interactions. For example, in FIG. Fc monomer sequences 1014 and 1012 in the polypeptide associate to form an Fc domain; This is also the case for Fc monomer sequences 1004 and 1006.

[0076] The term "linker" as used herein refers to a bond between two elements, e.g., protein domains. The linker can be a covalent bond or a spacer. is a chemical bond, such as an amide bond or a disulfide bond, or a chemical linkage, such as The term "spacer" refers to any type of bond formed by a chemical reaction in which between two polypeptides or polypeptide domains (e.g., Fc domain monomers). to provide space and / or flexibility between the two polypeptides or polypeptide domains. Such moieties (e.g., polyethylene glycol (PEG) polymers) or amino acid sequences (e.g., For example, 3 to 200 amino acids, 3 to 150 amino acids, 3 to 100 amino acids, 3 to 60 amino acids 3-50 amino acids, 3-40 amino acids, 3-30 amino acids, 3-20 amino acids, 3 ~10 amino acids, 3~8 amino acids, 3~5 amino acids, 4~30 amino acids, 5~30 amino acids Acid, 6-30 amino acids, 8-30 amino acids, 10-20 amino acids, 10-30 amino acids, 12-30 amino acids, 14-30 amino acids, 20-30 amino acids, 15-25 amino acids, The term refers to a sequence of 15 to 30 amino acids, 18 to 22 amino acids, and 20 to 30 amino acids. The amino acid spacer is part of the primary sequence of the polypeptide (e.g., through the polypeptide backbone). such that the polypeptide or polypeptide domain is linked to a separate polypeptide or polypeptide domain. For example, between two hinge regions forming an Fc domain or between two Fc domain monomers. Disulfide bond formation is not considered a linker.

[0077] As used herein, the term "cleavable linker" refers to a linker that can be selectively removed after the construct is formed. "Linker" refers to a linker that contains one or more elements that can be cleaved into a single molecule, e.g., a cleavable linker. Possible linkers include polypeptides that can be selectively cleaved by a protease. It contains a nucleotide sequence.

[0078] Herein, the term "albumin-binding peptide" refers to a peptide that has an affinity for serum albumin and It refers to an amino acid sequence of 12 to 16 amino acids that has the function of binding to serum albumin. The ubiquinone-binding peptides may be of various origins, e.g., human, mouse, or rat. In some embodiments of the present disclosure, the albumin binding peptide has an Fc domain. The albumin is fused to the C-terminus of the Fc monomer to increase the serum half-life of the Fc construct. The peptide is attached to the N-terminus or C-terminus of the Fc domain monomer, either directly or via a linker. It is possible to combine with

[0079] The term "multimer" herein refers to at least two associated Fc constellation complexes as described herein. Refers to a molecule that contains a tract.

[0080] As used herein, the term "polynucleotide" refers to an oligonucleotide or nucleotide, and It refers to any fragment or portion thereof, and to DNA or RNA of genomic or synthetic origin, It may be single-stranded or double-stranded and may represent the sense or antisense strand. The nucleotides are translated into a single polypeptide.

[0081] As used herein, the term "polypeptide" refers to an amide molecule in which monomers are linked together through amide bonds. The term "polypeptide" describes a single polymer of amino acid residues. or any amino acid sequence that is synthetically produced.

[0082] As used herein, the term "amino acid position" refers to an amino acid within a protein or protein domain. The amino acid positions of an antibody or Fc construct are indicated by the Kabat numbering system. Sequences of Protein s of Immunological Interest,National Ins. titutes of Health,Bethesda,Md.,ed 5,1991 ) were used to number the nuclei.

[0083] As used herein, the term "amino acid modification" refers to a modification of a reference sequence (e.g., a wild-type, unmutated, or modified amino acid). The pharmacokinetic (PK) and / or pharmacokinetic (AAC) profiles of the Fc constructs were compared to those of the unmodified Fc sequences. or pharmacodynamic (PD) properties, blood half-life, effector functions (e.g., cytolysis (e.g., antibody-dependent cell-mediated toxicity (ADCC) and / or complement-dependent cytotoxic activity (CDC)); Phagocytosis (e.g., antibody-dependent cellular phagocytosis (ADCP) and / or complement-dependent cytotoxicity (CDCC), immune activation and T-cell activation, and Fc receptor binding. Affinity for (e.g., Fc-gamma receptors (FcγR) (e.g., FcγRI (CD6 4), FcγRIIA(CD32), FcγRIIB(CD32), FcγRIIIA( CD16a), and / or FcγRIIIB (CD16b), Fc-alpha receptor ( FcαR), Fc-epsilon receptor (FcεR), and / or fetal Fc receptor (Fc Rn), affinity for proteins involved in the complement cascade (e.g., C1q), translation Post-translational modifications (e.g., glycosylation, sialylation), aggregation (e.g., dimerization (e.g., phosphatase), ability to form monodimers and / or heterodimers and / or multimers; and biophysical properties. and / or or altering sensitivity to temperature and / or pH) and may have an effect on immunity. The present invention provides a method for the preparation of polypeptides having Fc domains that may facilitate improved efficacy in the treatment of pharmacological and inflammatory diseases. An amino acid modification refers to an alteration of the amino acid sequence. An amino acid modification includes an amino acid substitution, deletion, and / or insertion. In some embodiments, the amino acid modification is a single amino acid modification. An amino acid modification is a modification of multiple (e.g., more than one) amino acids. The amino acid modifications may include a combination of amino acid substitutions, deletions, and / or insertions. The present invention relates to point mutations (e.g., single mutations) in the nucleotide sequence encoding the Fc polypeptide. exchange of one nucleotide for another), insertions, and deletions (e.g., one or more nucleotides) Genetic (i.e., DNA and RNA) modifications, such as the addition and / or removal of amino acids do.

[0084] In some embodiments, at least one of the Fc constructs (e.g., at least one 2, 3, 4, 5, 6, 7, 8, 9, 10) of the Fc domains contain amino acid modifications. In some examples, at least one Fc domain may be one or more (e.g., 2, 3, 4, 5, 6, In some examples, the amino acid sequence includes at least one amino acid modification, such as at least one amino acid sequence having at least one amino acid modification, such as at least one amino acid sequence having at least one amino acid modification, At least one Fc domain has 16 or fewer amino acid modifications (e.g., no more than 1, 2 Below, 3 or less, 4 or less, 5 or less, 6 or less, 7 or less, 8 or less, 9 or less, 10 11 or less, 12 or less, 13 or less, 14 or less, 15 or less, or 16 or less In some cases, the Fc domain monomer comprises 10 or fewer amino acids. In some cases, the Fc domain monomer may have 12 or fewer amino acid modifications. In some cases, the Fc domain monomer comprises no more than 14 amino acid modifications.

[0085] As used herein, the term "host cell" refers to a cell that is capable of expressing proteins from their corresponding nucleic acids. It refers to a medium that contains the necessary cellular components, e.g., organelles, as needed. Typically, the vector is produced by conventional techniques known in the art (transformation, transfection, electroporation, etc.). They can be introduced into host cells by various methods (e.g., pores, calcium phosphate precipitation, direct microinjection, etc.). The host cell may be a prokaryotic cell, e.g., a bacterial cell, or The cell can be a eukaryotic cell, for example a mammalian cell (e.g., a CHO cell). As described herein, host cells can be used to subsequently bind and express the desired Fc construct. The method includes the steps of: generating one or more polypeptides encoding desired domains capable of forming Make it happen.

[0086] The term "pharmaceutical composition" as used herein means a pharmaceutical composition that contains an active ingredient and a suitable formulation for the method of administration of the active ingredient. and one or more excipients and diluents capable of The pharmaceutical compositions of the present disclosure comprise pharma- ceutical acceptable components that are compatible with the Fc construct. The pharmaceutical compositions are typically in aqueous form for intravenous or subcutaneous administration.

[0087] Herein, a "substantially homogenous population" of polypeptides or Fc constructs refers to a composition (e.g. For example, at least 85% of the polypeptide or Fc construct in the pharmaceutical composition. However, they have the same number of Fc domains and the same Fc domain structure. In embodiments, at least 90% of the polypeptides or Fc constructs in the composition %, 92%, 95%, 97%, 98%, 99%, or 99.5% identical. Therefore, a pharmaceutical composition comprising a substantially homogenous population of Fc constructs can be prepared by selectively isolating the Fc constructs in the composition. At least 85% of the constructs have the same number of Fc domains and identical structure. A substantially homogenous population of Fc constructs is Contains no more than 10% dimers or aggregates (e.g., no more than 8%, 5%, 2%, or 1%) stomach).

[0088] As used herein, the term "pharmaceutical acceptable carrier" refers to an excipient or diluent in a pharmaceutical composition. A pharma- ceutically acceptable carrier is one that is compatible with the other ingredients of a formulation and is easy for the recipient to use. In this disclosure, a pharma- ceutically acceptable carrier is any carrier that is not harmful to the body. The nature of the carrier must provide sufficient pharmaceutical stability to the construct. It depends on the mode of administration. For example, for oral administration, solid carriers are preferred, and for intravenous administration, Generally, an aqueous carrier (eg, WFI and / or a buffer) is used.

[0089] As used herein, a "therapeutically effective amount" refers to a desired therapeutic effect in a subject or patient, e.g., a drug dosage. Useful for inducing a biological effect or for treating a patient having a condition or disorder described herein. As used herein, a "therapeutically effective amount" refers to an amount that is effective in a single dose or any administration. Either by any route or by any other method, alone or in combination with other therapeutic agents, It is also to be understood that the term "therapeutic effect" can be interpreted as an amount that produces a therapeutic effect. [Brief description of the drawings]

[0090] [Figure 1] FIG. 1 is an illustration of an Fc construct (construct 1) containing a dimer of two wild-type (wt) Fc domain monomers (102 and 104). [Diagram 2] Figure 2 is an illustration of an Fc construct (Construct 2) containing a dimer of two Fc domain monomers. The first Fc domain monomer (202) contains a protuberance in its CH3 antibody constant domain, while in close proximity, the second Fc domain monomer (204) contains a cavity in its CH3 antibody constant domain. [Diagram 3] Figure 3 is an illustration of another Fc construct (construct 3), which contains a dimer of two Fc domain monomers (302 and 304) that contain differently charged amino acids at their CH3-CH3 interfaces compared to the wt sequence, promoting favorable electrostatic interactions between the two Fc domain monomers. [Figure 4]Figure 4 is an illustration of an Fc construct (Construct 4) containing two Fc domains. This construct is formed of three polypeptides. The first polypeptide (402) contains two wt Fc domain monomers (404 and 406) linked in tandem. The second and third polypeptides (408 and 410, respectively) each contain a wt Fc domain monomer. [Diagram 5] FIG. 5 is an illustration of an Fc construct (Construct 5 or Construct 5*) containing three Fc domains formed by four polypeptides. The first polypeptide (502) contains one Fc domain monomer (506) containing an amino acid with a different charge at the CH3-CH3 interface compared to the wt sequence linked in tandem with a protuberance-containing Fc domain monomer (504). The second polypeptide (508) contains an Fc domain monomer (512) containing an amino acid with a different charge at the CH3-CH3 interface compared to the wt sequence linked in tandem with another protuberance-containing Fc domain monomer (510). The third and fourth polypeptides (514 and 516, respectively) each contain a cavity-containing Fc domain monomer. [Figure 6] 6 is an illustration of an Fc construct (Construct 6) containing two Fc domains formed from three polypeptides. The first polypeptide (602) contains two protuberance-containing Fc domain monomers (604 and 606) linked in tandem, while the second and third polypeptides (608 and 610, respectively) each contain an Fc domain monomer engineered to contain a corresponding cavity. [Figure 7]FIG. 7A is an illustration of another Fc construct (Construct 7). This Fc construct contains a dimer of two CL-CH1-Fc domain monomers (702 and 704). In this embodiment, a CL antibody constant domain is linked to an adjacent CH1 antibody constant domain. FIG. 7B is an illustration of an Fc construct (Construct 8) containing a multimer of CL-CH1-Fc domain monomers (e.g., 706, 708, and 710) that contains multiple Fc domains. In this Fc construct, the constituent polypeptides can be the same as those of Construct 7. The CL antibody constant domain of one Fc construct (e.g., 712) interacts with the adjacent CH1 antibody constant domain of a second Fc construct (e.g., 714). [Figure 8] 8 is an illustration of an Fc construct (construct 9) containing five Fc domains formed from six polypeptides. The first and second polypeptides (802 and 810) each contain three Fc domain monomers linked in tandem (804, 806, 808, and 812, 814, 816, respectively). In particular, in polypeptide 802 or 810, a first protuberance-containing Fc domain monomer (804 or 812) is bound to a second Fc domain monomer (806 or 814) that contains an amino acid with a different charge at the CH3-CH3 interface compared to the wt sequence, the second Fc domain monomer (806 or 814) being bound to a third protuberance-containing Fc domain monomer (808 or 816). The third through sixth polypeptides (818, 820, 822, and 824) each contain a cavity-containing Fc domain monomer to form an Fc domain together with each of the Fc domain monomers, 804, 808, 812, and 816, respectively. [Figure 9]9 is an illustration of an Fc construct (construct 10) containing five Fc domains formed from six polypeptides. The first and second polypeptides (902 and 910) each contain three Fc domain monomers linked in tandem (904, 906, 908, and 912, 914, 916, respectively). In particular, in polypeptide 902 or 910, a first protuberance-containing Fc domain monomer (904 or 912) is associated with a second protuberance-containing Fc domain monomer (906 or 914) which is associated with a third Fc domain monomer (908 or 916) that contains an amino acid with a different charge at the CH3-CH3 interface compared to the wt sequence. The third through sixth polypeptides (918, 920, 922, and 924) each contain a cavity-containing Fc domain monomer and together with each of the Fc domain monomers, 904, 906, 912, and 914, respectively, form an Fc domain. [Figure 10] Figure 10 is an illustration of an Fc construct (construct 11) containing three Fc domains formed from two polypeptides of identical sequence. The two polypeptides (1002 and 1010) each contain three Fc domain monomers linked in tandem (1004, 1006, 1008, and 1012, 1014, 1016, respectively). In particular, each polypeptide contains a first protuberance-containing Fc domain monomer (1004 or 1012) bound to a second cavity-containing Fc domain monomer (1006 or 1014) bound to a third Fc domain monomer (1008 or 1016) that has a different charged amino acid at the CH3-CH3 interface compared to the wt sequence. Fc domain monomers 1008 and 1016 associate to form a first Fc domain, Fc domain monomers 1004 and 1006 associate to form a second Fc domain, and Fc domain monomers 1012 and 1014 associate to form a third Fc domain. Construct 11 can be formed by expression of a single polypeptide sequence in a host cell. [Figure 11]11A-11B show reducing and non-reducing SDS-PAGE of Construct 4, respectively. [Figure 12] 12A-12B show reducing and non-reducing SDS-PAGE of Construct 6, respectively. [Figure 13] FIG. 13 is an SDS-PAGE of Construct 5 and the table shows the percentage of expressed protein with three Fc domains (trimer), two Fc domains (dimer), or one Fc domain (monomer) before and after purification of Construct 5. [Figure 14] 14A and 14B show activation assays (FIG. 14A) and inhibition assays (FIG. 14B) of THP-1 monocytes using constructs 1, 5, and 6. [Figure 15] FIG. 15 shows the effects of IVIG and Constructs 5 and 6 in the K / BxN model of rheumatoid arthritis. [Figure 16] FIG. 16 shows the effect of IVIG and Constructs 5 and 6 in a chronic ITP model. [Figure 17] FIG. 17 shows inhibition of phagocytosis by IVIG or Construct 5 in THP-1 monocytic cells. [Figure 18] FIG. 18 shows size distribution by non-reducing SDS-PAGE of clarified media obtained from expression of Construct 5 (SIF) and Construct 5-FcγRIIb+ mutant. [Figure 19] FIG. 19 shows the relative binding of IgG1 control, Construct 5 (SIF3), and Construct 5-FcγRIIb+ variant (FcfRIIB+) to Fc gamma receptors. [Figure 20] FIG. 20 shows CD86 surface expression on monocyte-derived dendritic cells (moDCs). [Figure 21] FIG. 21 shows CD86 surface expression on monocyte-derived dendritic cells (moDCs). DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0091] Detailed Description of the Invention Therapeutic proteins containing the Fc domain of IgG are used to treat inflammation and immune disorders and inflammation. The present disclosure relates to a method for treating infectious diseases, comprising administering to a patient a therapeutically effective amount of at least two (e.g., 2 to 10) F The present invention features compositions and methods for preparing various Fc constructs containing Fc domains. Let us assume that.

[0092] I. Fc domain monomer The Fc domain monomer consists of a hinge domain, C H 2 antibody constant domains, and C H 3 antibody determination Fc domain monomers are the most abundant Fc domains in the immunoglobulin antibody isotypes IgG, I The Fc domain monomer may be of IgE, IgM, IgA, or IgD. It also includes any immunoglobulin antibody isotype (e.g., IgG1, IgG2a, Ig The dimer of the Fc domain monomer may be of the same or different structure as the Fc domain monomer. It binds to an Fc receptor, e.g., FcγRIIIa, which is a receptor on the surface of a white blood cell. In the present disclosure, an Fc domain is an Fc domain (as further defined herein) capable of Domain monomer C H 3. Antibody constant domains H 3-C H 3. Antibody constant domain interface In some embodiments, the domains may contain amino acid substitutions in the ribozyme that promote association between the domains. So, the Fc domain monomer is, for example, C L and C H 1. Antibody constant domain, N-terminal In another embodiment, the Fc domain comprises two other constant domains linked together (Figure 7). The monomer may further comprise a further moiety attached to the C-terminus, e.g. an albumin-binding peptide. In the present disclosure, an Fc domain monomer can be, for example, a V H , V L , Complementarity Determining Region (CDR ), or hypervariable regions (HVRs). The domain monomers can be of various origins, e.g., human, mouse, or rat. It is possible.

[0093] II. Fc Domain As defined herein, an Fc domain is H 3. Interactions between Antibody Constant Domains Thus, it comprises two Fc domain monomers that dimerize. In the present disclosure, an Fc domain is, for example, For example, V H , V L The Fc domain does not include the variable regions of an antibody, which are the CDRs, HVRs, or CDRs. Fc receptors, e.g., Fc-gamma receptors (i.e., Fcγ receptors (FcγR)), Fc-alpha receptor (i.e., Fcα receptor (FcαR)), Fc-epsilon receptor (i.e., Fcε receptor (FcεR)) and / or fetal Fc receptor (FcRn) In some embodiments, the Fc domain of the present disclosure comprises an Fcγ Receptors (e.g., FcγRI (CD64), FcγRIIA (CD32), FcγRII B (CD32), FcγRIIIA (CD16a), and / or FcγRIIIB (CD 16b)) and / or fetal Fc receptor (FcRn).

[0094] III. Fc Domain Modifications The unmodified Fc domain monomer may be a naturally occurring human Fc domain monomer or a WT human Fc domain monomer. The Fc domain monomer can be an Fc domain monomer. The Fc domain monomer can be a hinge, a CH2 domain, A naturally occurring human Fc domain monomer containing the main and CH3 domains, or a directed Up to 16 (e.g., up to 2, 3, 4, 5, 6, 7, 8 , 9, 10, 11, 12, 13, 14, 15, or 16 amino acid modifications (e.g., In some cases, the Fc domain may be a variant thereof having a single amino acid modification. The base comprises at least one amino acid modification, the amino acid modification being (i) one or more (ii) the binding affinity of the Fc domain to the Fc receptor; (iii) the effector functions of the Fc domain; Level of sulfation, (iv) half-life, (v) protease resistance, (vi) Fc domain stability and / or (vii) susceptibility to degradation (e.g., For example, compared to an unmodified Fc domain. In some cases, the Fc domain is no more than two amino acid modifications (e.g., no more than two, no more than three, no more than four in the CH3 domain) , 5 or less, 6 or less, 7 or less, 8 or less, 9 or less, 10 or less, 11 or less, 12 1 or less, 13 or less, 14 or less, 15 or less, or 16 or less amino acid modifications .

[0095] The Fc domain of this disclosure is 1, 137-146, 169-175, 196, 199, 203, 206, 211, 21 4, 217, 219-341, 344-346, 349-370, 372-374, 37 6-380, 382-405, 407-422, 424, 426-442, and / or 4 At least one (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 30, 40, 50, 60, 70, 80, 90, or 1 In some embodiments, the amino acid modifications include at least one amino acid substitution. In some embodiments, the substituted amino acid is a natural or non-natural amino acid. In the present invention, the amino acid modification is a deletion of at least one amino acid (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 30, or 40) or more residues in the Fc domain In some embodiments, the Fc domain modification is an amino acid insertion At least one (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 20 , 30, or 40) or more residues are inserted into the Fc domain. The modifications may be a combination of modifications, such as one or more amino acid substitutions, deletions, and / or deletions. Or it may be a combination of insertions.

[0096] Fc receptor binding affinity The amino acid modifications of the present disclosure can include one or more (e.g., 1, 2, 3, 4, 5, or 6) Fc Receptors (e.g., Fc-gamma receptors (FcγR), Fc-alpha receptors (FcαR) , Fc-epsilon receptor (FcεR), and / or fetal Fc receptor (FcRn) The binding affinity of the Fc domain to The modified Fc domain exhibits an alteration (i.e., an increase) in the amount of glycerol in the glycerol-binding domain relative to the unmodified Fc domain. or decreased) affinity to FcγR (e.g., FcγRI (CD64), FcγRII A (CD32), FcγRIIB (CD32), FcγRIIIA (CD16a), and and / or FcγRIIIB (CD16b) and / or fetal Fc receptor (FcRn) The modified Fc domain may have one (e.g. For example, 1, 2, 3, 4, 5, or 6) or more Fc receptors are altered (i.e., increased or decreased). In addition, the modified Fc domain may have a modified dissociation constant (Kd). The altered (i.e., increased or decreased) levels of glycosylation compared to an unmodified Fc domain. Cosylation (e.g., glygan modifications (e.g., mannose, sialic acid, fucosylation) The Fc modification has one ( For example, affinity of the Fc domain for one, two, three, four, five, or six) or more Fc receptors. while varying at least one (e.g., 1, 2, 3, 4, 5, or 6) or more other It reverses the affinity for Fc receptors.

[0097] Table 1 lists exemplary Fc domains that can be modified to alter Fc receptor binding affinity. In some embodiments, the amino acid sequence is one of those listed in Table 1 (e.g., 1, 2, , 3, 4, 5, 6, 7, 8, 9, 10, 20, 30, or 40) or more residues The modified Fc domain has an Fc receptor that is more specific than an unmodified Fc domain. In some embodiments, the Fc domain modification has an altered binding affinity to the receptor. , one of the listed in Table 1 (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 2 In some embodiments, the amino acid substitutions occur at 0, 30, or 40 residues or more. In the present invention, the Fc domain modification is one of those listed in Table 1 (e.g., 1, 2, 3, 4, 5, Amino acid deletions occurring at 6, 7, 8, 9, 10, 20, 30, or 40 or more residues In some embodiments, the Fc domain modification is one listed in Table 1 (e.g., For example, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 30, or 40 or more residues The Fc domain modification is a combination of multiple modifications. For example, the modifications can include amino acid substitutions, deletions, and / or insertions. It is possible.

[0098] Table 2 lists exemplary amino acid modifications that alter the binding affinity of an Fc domain to an Fc receptor. The modified Fc domain may have one of the modifications listed in Table 2 (e.g., (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 30, or 40) The modifications in Table 2 can also be made by combining any one of the residues listed in Table 1 (e.g., 1 , 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 30, or 40 or more modifications and combinations You can also match them.

[0099] Fc domain modifications have at least 1-fold (e.g. For example, 2x, 3x, 4x, 5x, 6x, 7x, 8x, 9x, 10x, 20x, 30x, 4x 0x, 50x, 60x, 70x, 80x, 90x, 100x, 200x, 300x, 40 0, 500, or 100% specific to one or more Fc receptors (e.g., 2, 3, 4, 5, or 6) In some embodiments, the Fc domain modification can increase the binding affinity of the Fc domain. The decoration includes Fcγ receptors (e.g., FcγRI (CD64), FcγRIIA (CD32), FcγRIIB (CD32), FcγRIIIA (CD16a), and / or FcγRI IIB (CD16b) and / or fetal Fc receptor (FcRn) In some embodiments, the Fc domain modification increases FcγRIIIA (CD16a ) to increase binding affinity.

[0100] Fc domain modifications have at least 1-fold (e.g. For example, 2x, 3x, 4x, 5x, 6x, 7x, 8x, 9x, 10x, 20x, 30x, 4x 0x, 50x, 60x, 70x, 80x, 90x, 100x, 200x, 300x, 40 0, 500, or 100% specific to one or more Fc receptors (e.g., 2, 3, 4, 5, or 6) The binding affinity of the modified Fc domain may be decreased. In some embodiments, the Fc domain In-modifications include Fcγ receptors (e.g., FcγRI (CD64), FcγRIIA (CD3 2), FcγRIIB (CD32), FcγRIIIA (CD16a), and / or Fc γRIIIB (CD16b) and / or fetal Fc receptor (FcRn) binding affinity In some embodiments, the Fc domain modification reduces FcγRIIB (CD3 2) decreases the binding affinity to Fc. Decreases the binding affinity for Rn.

[0101] Exemplary Fc domains with altered binding affinity to Fc receptors include double mutants The Fc monomer contains the S267E / L328F variant. The difference has been shown to significantly and specifically enhance IgG1 binding to the FcγRIIb receptor. It has been proven that (Chu et al. Molecular Immunology .2008 Sep;45(15):3926-33).

[0102] Table 1. Fc domain residues that can be modified to alter Fc receptor binding affinity TIFF2025026880000004.tif155138

[0103] Table 2. Fc domain modifications that alter Fc receptor binding affinity TIFF2025026880000005.tif111170TIFF2025026880000006.tif228170TIFF202 5026880000007.tif227170TIFF2025026880000008.tif226170TIFF20250268800 00009.tif228170TIFF2025026880000010.tif228170TIFF2025026880000011.t if228170TIFF2025026880000012.tif226170TIFF2025026880000013.tif235170

[0104] Half-life Fc domain modifications that alter the half-life may reduce the binding of the modified Fc domain to FcRn, e.g. By changing the affinity of the interaction at pH 6.0 and / or pH 7.4, Amino acid modifications that alter half-life can affect the binding of the Fc domain to the FcRn receptor. Table 3 shows the half-life (e.g., pH dependence) of the Fc domain after modification. Exemplary Fc domain residues that can alter the binding of the Fc domain to the antibody (e.g., serum half-life) are listed below. In this embodiment, one of the peptides listed in Table 3 (e.g., 1, 2, 3, 4, 5, 6, 7, 8 In one embodiment, more than one residue (9, 10, 20, 30, or 40) can be modified, and the modified Fc domain can have The modified Fc domain has an altered half-life compared to the unmodified Fc domain. In embodiments, the Fc domain modification is one listed in Table 3 (e.g., 1, 2, 3, 4, , 5, 6, 7, 8, 9, 10, 20, 30, or 40) or more residues. In some embodiments, the Fc domain modification is one of the modifications listed in Table 3. For example, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 30, or 40) or more In some embodiments, the Fc domain modification is an amino acid deletion occurring in the group shown in Table 3. (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 3 Fc domain modifications are amino acid insertions occurring at residues 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, For example, the modifications may be amino acid substitutions, deletions, or combinations of modifications. , and / or insertions.

[0105] Table 4 lists exemplary modifications that alter the half-life of an Fc domain. is one of the modifications listed in Table 4 (e.g., 1, 2, 3, 4, 5, 6, 7, 8 , 9, 10, 20, 30, or 40 or more. This may be combined with modification of residue positions that have been modified.

[0106] In some embodiments, the Fc domain modification is compared to an unmodified Fc domain. , at least 0.5x (e.g., 1x, 2x, 3x, 4x, 5x, 6x, 7x, 8x, 9x fold, or 10 fold) may increase the half-life of the modified Fc domain.

[0107] In some embodiments, the Fc domain modification is compared to an unmodified Fc domain. , at least 0.5x (e.g., 1x, 2x, 3x, 4x, 5x, 6x, 7x, 8x, 9x fold, or 10-fold) may decrease the half-life of the modified Fc domain.

[0108] Table 3. Fc domain residues that can be modified to alter half-life TIFF2025026880000014.tif90128

[0109] Table 4. Fc domain modifications that alter half-life TIFF2025026880000015.tif151168TIFF2025026880000016.tif225168TIFF20250268800 00017.tif225168TIFF2025026880000018.tif225168TIFF2025026880000019.tif113168

[0110] Effector Function Table 5 lists modifications to enhance the effector function of the Fc domain (e.g., cell lysis (e.g., antibody dependent cell-mediated toxicity (ADCC) and / or complement-dependent cytotoxic activity (CDC), phagocytosis (e.g., antibody-dependent cellular phagocytosis (ADCP) and / or complement It can alter immune-dependent cytotoxicity (CDCC), immune activation and T-cell activation. In some embodiments, the Fc domain residues listed in Table 5 are exemplary Fc domain residues that may be present in the Fc domain. (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 30, or 40) The above residues can be modified, and the modified Fc domain can be compared to an unmodified Fc domain. Altered effector functions (e.g., ADCC, CDC, ADCP, CD In some embodiments, the Fc domain has a cytotoxicity (CC, immune activation, and / or T-cell activation). The main modification is one of those listed in Table 5 (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 30, or 40) or more amino acid substitutions. In this embodiment, the Fc domain modification is one listed in Table 5 (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 2 , 4, 5, 6, 7, 8, 9, 10, 20, 30, or 40) or more residues In some embodiments, the Fc modification is one listed in Table 5 (e.g., For example, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 30, or 40) or more residues. This is an amino acid insertion that occurs in

[0111] Table 6 lists the effector functions of the Fc domain (e.g., ADCC, CDC, ADCP, CD Exemplary modifications that alter CC, immune activation, and / or T-cell activation are listed. The modified Fc domain may have one of the modifications listed in Table 6 (e.g., 1, 2, 3, 4 , 5, 6, 7, 8, 9, 10, 20, 30, or 40) or more. The modifications may be combined with the residue modifications listed in Table 5.

[0112] The Fc domain modification increases the expression level by at least 0.5-fold compared to the unmodified Fc domain. (e.g. 1x, 2x, 3x, 4x, 5x, 6x, 7x, 8x, 9x, or 10x), Effector functions of decorated Fc domains (e.g., ADCC, CDC, ADCP, CDCC, immunoglobulins, etc.) The antibodies may increase immune activation and / or T-cell activation.

[0113] The Fc domain modification increases the expression level by at least 0.5-fold compared to the unmodified Fc domain. (e.g. 1x, 2x, 3x, 4x, 5x, 6x, 7x, 8x, 9x, or 10x), Effector functions of the decorated Fc domain (e.g., cell lysis (e.g., ADCC, CDC, AD CP, CDCC, immune activation, and / or T-cell activation).

[0114] Table 5. Fc domain residues that can be modified to alter effector function TIFF2025026880000020.tif112128

[0115] Table 6. Fc domain modifications that alter effector function TIFF2025026880000021.tif51138TIFF2025026880000022.tif227138TIFF2025026880000023.tif229138TIFF2025026880000024.tif79138

[0116] Stability Changes By altering the stability of the Fc domain, the thermal stability (e.g., melting temperature, Tm) and aggregation form can be improved. It is possible to affect the morphology of the aggregates (e.g., the aggregate morphology under acidic, i.e., low pH, conditions). be.

[0117] In some embodiments, the thermal stability of the modified Fc domain is compared to that of the unmodified Fc domain. At least about 0.1°C (e.g., about 0.25°C, about 0.5°C, about 0. 75°C, approx. 1°C, approx. 1.25°C, approx. 1.5°C, approx. 1.75°C, approx. 2°C, approx. 3 °C, approx. 4°C, approx. 5°C, approx. 6°C, approx. 7°C, approx. 8°C, approx. 9°C, approx. 10°C, approx. 20°C, about 30°C, about 40°C, or about 50°C), change (i.e., increase or decrease In some embodiments, the thermal stability of the modified Fc domain can be improved by modifying In some embodiments, the Fc domain is modified relative to an unmodified Fc domain. The thermal stability is decreased compared to the unmodified Fc domain. The modified Fc domain is at least as resistant to aggregation as the unmodified Fc domain. 1% (e.g., 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 20%, 3 0%, 40%, 50%, 60%, 70%, 80%, or 90%) or more (i.e., In some embodiments, the aggregation properties of the modified Fc domain are increased or decreased by the modified In some embodiments, the aggregation properties are increased compared to the unmodified Fc domain. The Fc domain is reduced in comparison to the unmodified Fc domain.

[0118] Table 7 lists exemplary Fc domains that can be modified to alter the stability of the Fc domain. In some embodiments, the amino acid sequence is one of those listed in Table 7 (e.g., 1, 2, , 3, 4, 5, 6, 7, 8, 9, 10, 20, 30, or 40) or more residues. and the modified Fc domain has an altered Fc domain structure compared to the unmodified Fc domain. In some embodiments, the Fc domain modification is one of those listed in Table 7. (e.g. 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 30, or 40) or more In some embodiments, the Fc domain modification is an amino acid substitution occurring at a residue of One of the numbers listed in 7 (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, In some embodiments, the amino acid deletion occurs at 30, 30, 40, or more residues. The Fc domain modification is one of those listed in Table 7 (e.g., 1, 2, 3, 4, 5, 6, 7 , 8, 9, 10, 20, 30, or 40) or more residues. .

[0119] Table 8 lists exemplary modifications that alter the stability of the Fc domain. The modification may be one of the modifications listed in Table 8 (e.g., 1, 2, 3, 4, 5, 6, 7, In addition, the modifications in Table 8 may be combined with those in Table 7. Modifications of the residues noted may be combined.

[0120] In some embodiments, the Fc domain modification is compared to an unmodified Fc domain. , at least 0.5x (e.g., 1x, 2x, 3x, 4x, 5x, 6x, 7x, 8x, 9x fold, or 10 fold) may increase the stability of the modified Fc domain.

[0121] The Fc domain modification increases the expression level by at least 0.5-fold compared to the unmodified Fc domain. (e.g. 1x, 2x, 3x, 4x, 5x, 6x, 7x, 8x, 9x, or 10x), This may decrease the stability of the decorated Fc domain.

[0122] Table 7. Fc domain residues that can be modified to alter stability TIFF2025026880000025.tif66128

[0123] Table 8. Fc domain modifications that alter stability TIFF2025026880000026.tif116170

[0124] Altered susceptibility to degradation Susceptibility to degradation influences how Fc domain-containing molecules are stored and transported The sensitivity of the Fc domain to environmental conditions such as temperature (e.g., temperature, humidity, pH) By reducing the binding affinity, Fc domain-containing molecules can be more easily transported and / or bind longer. It is possible to modify the Fc domain to protect it from degradation and make it shelf-stable for a long period of time. Exemplary Fc residues that can alter the susceptibility of Fc to IL-1 are 233, 234, 235, 236, 237, 238, 239, 240, 241, 242, 243, 244, 245, 246, 247, 248, 249, 250, 251, 252, 2 In some embodiments, residues 233, 241, and 249 are included. 34, 235, 236, 237, 239, 241, and 249 One (e.g., 1, 2, 3, 4, 5, 6, 7, or 8) residues can be modified. The modified Fc domain has altered resistance to degradation compared to the unmodified Fc domain. In some embodiments, the Fc modifications include 6, 237, 239, 241, and 249 (e.g., 1, 2, 3, 4, 5, 6, 7, or 8) of the amino acid substitutions. In some embodiments, the Fc modifications are 233, 234, 235, 236, 237, 239, 240, 241, 242, 243, 244, 245, 246, 247, 248, 249, 250, 251, 252, 253, 254, 255, 256, 25 41, and 249 (e.g., 1, 2, 3, 4, 5, 6, 7 In some embodiments, the amino acid deletion occurs at residue position 8 of Fc The modifications consisted of 233, 234, 235, 236, 237, 239, 241, and 249. One (e.g., 1, 2, 3, 4, 5, 6, 7, or 8) residue position selected from the group consisting of This is an amino acid insertion occurring at the position

[0125] Exemplary modifications that alter the susceptibility of an Fc domain to degradation include those in which X is any amino acid. , C233X, D234X, K235X, S236X, T236X, H237X, C2 39X, S241X, and G249X (e.g., 1, 2, The amino acid sequence may include modifications 3, 4, 5, 6, 7, or 8).

[0126] Fc domain modifications should be at least 1% (e.g. For example, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 20%, 30%, 4 0%, 50%, 60%, 70%, 80%, or 90%) or more of degradation of the modified Fc domain. In some embodiments, the Fc domain modification may reduce the At least 1% (e.g., 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, or 90% ), which may reduce degradation of the modified Fc domain upon heating. The domain must be updated for at least 1 hour (e.g., 2 hours, 3 hours, 4 hours, 5 hours, 6 hours, 7 hours, etc.) hours, 8 hours, 9 hours, 10 hours, 11 hours, 12 hours, 1 day, 2 days, 3 days, 4 days, 5 days In some embodiments, the Fc domain is heated for a period of time of at least 10 minutes. Heat the inn to a temperature of at least 45°C (50°C, 55°C, 60°C, 65°C, °C, 70 °C, 75 °C, 85 °C, or 95 °C) or higher. The level of degradation of the decorated Fc domain can be determined by, but not limited to, reduced capillary gel electrophoresis. electrophoresis (rCGE), sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS- and high performance size exclusion chromatography (HPSEC). Some methods can be used to measure the degree of aggregation, degradation, or fragmentation. It is possible.

[0127] Sulfation Exemplary Fc residues that can be modified to alter sulfation of the Fc domain include residue 2 41, 243, 246, 260, and 301. In some embodiments, 241, 24 3, 246, 260, and 301 (e.g., 1, 2, 3, 4) or 5) of the Fc domain residues can be modified, and the modified Fc domain can be In some embodiments, the Fc domain has altered sulfation compared to an unmodified Fc domain. The c domain modification is selected from the group consisting of 241, 243, 246, 260, and 301. The amino acid substitution occurs at one (e.g., 1, 2, 3, 4, or 5) residues in the In some embodiments, the Fc domain modifications are at residues 241, 243, 246, 260, and and / or one (e.g., 1, 2, 3, 4, or 5) residue selected from the group consisting of 301. In some embodiments, the Fc modification is an amino acid deletion occurring at Fc domain residue 2. 41, 243, 246, 260, and / or 301 (e.g., For example, amino acid insertions occurring at residue positions 1, 2, 3, 4, or 5.

[0128] Exemplary modifications that alter sulfation of the Fc domain include 241F, 243F, 246K, 26 0T, and / or 301R. The modified Fc domains include 241F, 243F, 246K, , 260T, and 301R (e.g., 1, 2, 3, 4, or Any one of these modifications may be added to residues 241, 243, 244, 245, 246, 247, 248, 249, 250, 251, 252, 253, 254, 255, 256, 257, 258, 259, 260, 261, 262, 263, 264, It may be combined with further modifications at 46, 260, and / or 301.

[0129] The Fc domain modification increases the expression level by at least 0.5-fold compared to the unmodified Fc domain. (e.g. 1x, 2x, 3x, 4x, 5x, 6x, 7x, 8x, 9x, or 10x), The decorated Fc domain may be sulfation-increased.

[0130] The Fc domain modification increases the expression level by at least 0.5-fold compared to the unmodified Fc domain. (e.g. 1x, 2x, 3x, 4x, 5x, 6x, 7x, 8x, 9x, or 10x), The sulfation of the decorated Fc domain may be reduced.

[0131] Protease resistance The Fc domain can be modified to inhibit, for example, endosomal proteases, extracellular proteases, (e.g., trypsin, chymotrypsin, plasmin), digestive proteases (e.g., pe pusin), serum proteases (e.g., clotting factors), proteases released from leukocytes (e.g., elastase and cathepsin G) and tissue-specific proteases (e.g., tumor Resistance to specific proteases (e.g., matrix metalloproteases) , which may increase protease resistance. Susceptibility to protease degradation is increased by the Fc domain. It is possible that the increased sensitivity is due to shorter half-lives. Altering protease resistance results in a longer half-life, whereas reduced sensitivity results in a longer half-life. To this end, the amino acid modification may include or affect a protease cleavage site. Alternatively, the glycosylation state of the Fc domain may be altered. Amino acid modifications that alter the protease resistance and / or sensitivity properties of the Fc domain obtain.

[0132] Exemplary Fc residues that can be modified to alter protease resistance are 4, 235, 236, 237, 239, 243, 267, 268, 292, 300, 32 4, 326, 332, and 333. In some embodiments, 233, 234, 235 , 236, 237, 239, 243, 267, 268, 292, 300, 324, 326 , 332, and 333 (e.g., 1, 2, 3, 4, 5, 6 , 7, 8, 9, 10, 11, 12, 13, 14, or 15) of the Fc domain residues. The modified Fc domain may have an altered Fc domain structure compared to the unmodified Fc domain. The Fc domain modifications are at residues 233, 234, 235, and 236. 36, 237, 239, 243, 267, 268, 292, 300, 324, 326, 3 32, and 333 (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, The amino acid substitution may occur at residue 10, 11, 12, 13, 14, or 15. Fc modifications have also been shown to be 7, 268, 292, 300, 324, 326, 332, and 333 (For example, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15 In some embodiments, the amino acid deletion may occur at residue 16 of Fc. Modifications: 233, 234, 235, 236, 237, 239, 243, 267, 268, 292, 300, 324, 326, 332, and 333 (e.g., 1 , 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15) In some embodiments, the Fc domain modification is an amino acid insertion occurring at position: Any combination of the above (e.g., amino acid substitutions, deletions, and / or insertions) (combination of).

[0133] Exemplary modifications that alter the protease resistance of the Fc domain include 233P, 234V, 23 5A, and 236del; 237A, 239D, and 332E; 237D, 239D, and and 332E;237P, 239D, and 332E;237Q, 239D, and 332E; 237S, 239D, and 332E;239D, 268F, 324T, and 332E;2 39D, 326A, and 333A; 239D and 332E; 243L, 292P, and 3 00L;267E, 268F, 324T, and 332E;267E and 332E;268 F, 324T, and 332E; 326A, 332E, and 333A; and 326A and 333A (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 1 0, 11, 12, 13, 14, or 15).

[0134] Fc domain modifications should be at least 1% (e.g. For example, 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 20%, 30 %, 40%, 50%, 60%, 70%, 80%, or 90%) or more of the modified Fc domain May increase protease resistance.

[0135] IV. Dimerization Selectivity Module In the present disclosure, the dimerization selectivity module is a selective selectivity module that selectively selects the preferred pair of two Fc domain monomers. It is the portion of the Fc domain monomer that facilitates the formation of the Fc domain. The dimerization selectivity module is a C domain that is formed by interacting with two Fc domain monomers. H 3 antibody determination C of Fc domain monomers containing amino acid substitutions located at the interface between the normal domains H 3 antibody determination In the dimerization selectivity module, the substitutions selected As a result of the compatibility of the selected amino acids, amino acid substitutions occur between the two C H 3. Antibody Constant Domains Dimerization is favored in Fc domain monomers that do not have a dimerization selectivity module. Fc domains with amino acid substitutions that do not fit within the selectivity modules that form or dimerize This preferred final form of the Fc domain is preferred over other Fc domains formed in monomelic monomers. This type of amino acid substitution is known as a QuikChange® mutation. This can be accomplished using conventional molecular cloning techniques well known in the art, such as mutagenesis. be.

[0136] In some embodiments, the dimer formation selectivity module is H 3 Within the antibody constant domain, In another embodiment, the dimerization selection The selectivity module is C H 3. Engineered protrusions (described further herein) within the antibody constant domain To selectively form an Fc domain, a suitable dimerization selectivity model is used. Two Fc domain monomers with different modules, e.g., one C domain containing an engineered cavity H 3 anti The other C domain contains the engineered protuberance. H 3. Binding to antibody constant domains This forms a pair in which the protrusion is inserted into the cavity of the Fc domain monomer.

[0137] In another embodiment, the dimerization selectivity module contains a positively charged amino acid substitution. The Fc domain monomers with negative charge and dimerization selectivity models containing negatively charged amino acid substitutions are Selectively binds to Fc domain monomers having a specific molecule, and selectively binds to the preferred amino acids having a charge. The Fc domain is formed via simple electrostatic steering (described further herein). Specific dimerization selectivity modules can be selected from the following Tables 1 and 2, which are further described below: and 2 further list, but are not limited to:

[0138] In another embodiment, the two Fc domain monomers are H3. Electric field at the interface between the domains containing identical opposite charge mutations at at least two positions within the ring of charged residues, Dimerization selectivity module. Complementary residues in two or more Fc domain monomers. The charge of both members of the complementary pair is reversed, resulting in a mutated Fc domain monomer with the same The Fc domain monomers are complementary to the mutant sequences, but not to the Fc domain monomers without these mutations. In one embodiment, the Fc domain is a doubly modified Variants K409D / D339K, K392D / D399K, E357K / K370E , D356K / K439D, K409E / D339K, K392E / D399K, E35 7K / K370D, or D356K / K439E. In the Fc domain, for example, K409D / D399K / E357K / K370E The Fc monomers include those containing quadruple mutants combining any pair of double mutants.

[0139] The formation of such Fc domains is H 3. Matching Amino Acid Substitutions in Antibody Constant Domains This is promoted by C H 3-C H At the three interfaces, two dimerization selectivity modules If they both contain incompatible amino acid substitutions, e.g., if both contain an engineered cavity, If both contain engineered protuberances, or if both contain amino acids with the same charge, In this case, formation of the Fc domain is not promoted.

[0140] In addition, a method for promoting the formation of an Fc domain from a defined Fc domain monomer is disclosed. Other methods that have been used include the synthesis of monomeric α-terminal leucine zippers, which allow for the formation of heterodimers. The LUZ-Y approach involves C-terminal fusions to each of the Fc domain monomers of LIX. (U.S. Patent Application Publication No. WO2011034605), and further, IgA and IgG C H Heterodimeric Fc domain monomers, each containing three alternating segments A strand-exchange engineered domain (SEED) approach to generate Fc domains (David s et al.,Protein Eng Des Sel.23:195-202, 2010), but are not limited to these.

[0141] V. ENGINEERED CAVITIES AND ENGINEERED PROJECTIONS The use of engineered cavities and engineered protrusions (i.e., "knob-in-hole") The "b-into-hole" strategy was developed by Carter and his colleagues (Ri dgway et al., Protein Eng.9:617-612,1996; Atwell et al., J Mol Biol. 270:26-35, 1997; Merchant et al., Nat Biotechnol. 16:677-68 The knob-hole interaction is described by While the knob-hole and hole-knob interactions favor the formation of the 3D structure, the 3D structure is not favored due to steric inconsistencies and favorable The lack of interactions prevents homodimer formation. This is also disclosed in US Pat. No. 5,731,168.

[0142] In the present disclosure, engineered cavities and engineered protrusions are incorporated into the Fc constructs described herein. The engineered cavity allows the natural amino acids in a protein to be substituted with lower amino acids. It is the void that is formed when a molecule is substituted with a different amino acid with a side chain of molecular weight. The protuberances are formed when a native amino acid in a protein is replaced by a different amino acid with a higher molecular weight side chain. Specifically, the protrusion is formed when the amino acid to be replaced is is the C of the Fc domain monomer. H 3. Within the antibody constant domain, two Fc domain monomers In some embodiments, one C H 3. Engineering Within Antibody Constant Domains The cavity is the other C H 3. To Accommodate Engineered Protuberances in Antibody Constant Domains Because both C H 3. Antibody constant domains form dimers of two Fc domain monomers It functions as a dimerization selectivity module (described above) that promotes or supports In one embodiment, H 3 The engineered cavity in the antibody constant domain is H 3 are designed to better accommodate the native amino acids in the antibody constant domain. In yet another embodiment, one C H 3 Engineered protuberances in antibody constant domains C H 3. Designed to form additional interactions with native amino acids in the antibody constant domain It has been made.

[0143] The engineered cavity is designed to accommodate amino acids containing longer side chains, such as tyrosine or tryptophan. with an amino acid containing a shorter side chain, such as alanine, valine, or threonine. In particular, some dimer-forming selectivity modules can be constructed by (described further above), C H 3. Antibody constant domains with engineered mutations such as Y407V Similarly, engineered protuberances allow amino acids containing shorter side chains to be more easily recruited. It is possible to construct a nucleotide sequence by substituting amino acids containing longer side chains. In some dimerization selectivity modules (described further above), H 3. Antibody Stationary In the present disclosure, the engineered space contains an engineered protuberance such as the T366W mutation in the domain. The sinuses and engineered projections also promote heterodimer formation. H 3-Domain Disruption Specifically, the hollow Fc contains a Y349C mutation and The protruding Fc contains the S354C mutation. The other engineered cavities and engineered protrusions were either combined with HA-TF (HA-TF mix) or with HA-TF (HA-TF mix). Examples of the above include, but are not limited to:

[0144] (Table 9) TIFF2025026880000027.tif67163

[0145] C H 3. Substituting a different amino acid residue for an original amino acid residue in the antibody constant domain This can be achieved by modifying the nucleic acid that codes for the native amino acid residue. The upper limit of the number of original amino acid residues that can be replaced is the number of amino acid residues that can be sufficiently substituted at the interface. Considering that we want to maintain the interactions, C H 3. Antibody constant domain interface is the total number of residues in the

[0146] VI. Electrostatic Steering Electrostatic steering is a method for controlling the formation of higher order protein molecules, peptides, proteins, and Protein domains and favorable electrostatic interactions between oppositely charged amino acids within a protein Electrostatic steering can be used to facilitate the selection of heterodimers for the generation of bispecific antibodies. The interactions of antibody domains are altered to favor dimer formation and reduce homodimer formation. A method for changing the use of the device is disclosed in U.S. Patent Application Publication No. 2014-0024111. do.

[0147] In the present disclosure, electrostatic steering is used to regulate the dimerization of Fc domain monomers and the Fc The formation of the construct is controlled by electrostatic steering. To control the dimerization of the cyclic amine monomer, H 3-C H 3. One or more interfaces that form The amino acid residues are replaced with positively or negatively charged amino acid residues, thereby Depending on the particular charged amino acid introduced, the interaction may be electrostatically favored or In some embodiments, lysine, arginine, or histidine are undesirable. The positively charged amino acids at the interface, such as aspartic acid or glutamic acid, are replaced by negatively charged amino acids, such as aspartic acid or glutamic acid. In another embodiment, the negatively charged amino acids at the interface are replaced with positively charged amino acids. The charged amino acid is replaced by one amino acid that interacts with the C H 3. Antibody Constant Domains The interacting C H 3. The antibody constant domain contains a charged amino acid By introducing an amino acid, a dimer formation selectivity module (described further above) can be obtained. The formation of the cyclic amines is due to electrostatic interactions between charged amino acids. Selective dimerization of Fc domain monomers as controlled by the binding effect It is possible.

[0148] In one particular embodiment, to form a dimer-forming selectivity module containing reversed charges, To, CH 3. Substitute amino acid Asp399 in the antibody constant domain with Lys, and The amino acid Lys409 is replaced by Asp. Heterodimerization of Fc domain monomers is achieved by Different but compatible mutations within the Fc domain monomer (including but not limited to those in Table 10) This can be enhanced by introducing charged residue pairs such as Homodimerization of the monomers was confirmed by the double mutants K409D / D339K or K392D / D39 By introducing the same mutation, such as 9K, into both Fc domain monomers in a symmetric manner, This can be promoted by:

[0149] (Table 10) TIFF2025026880000028.tif101146

[0150] VII. Linker In the present disclosure, polypeptide or protein domains and / or associated non-protein moieties A linker is used to describe a bond or connection between two molecules. In some embodiments, A linker is a bond or connection between at least two Fc domain monomers, and includes a first Fc Domain monomer C H 3. The C-terminus of the antibody constant domain is linked to the hin of a second Fc domain monomer. The two Fc domain monomers are linked in tandem to the N-terminus of the didomain. In another embodiment, the linker connects the Fc domain monomer to For example, a linker may be used to link an Fc domain to another protein domain. Main monomer C H 3. The C-terminus of the antibody constant domain is linked to the N-terminus of the albumin-binding peptide. In another embodiment, the linker can be H 1. Antibody constant domain The C-terminus can be joined to the N-terminus of the hinge domain of an Fc domain monomer. In yet another embodiment, the linker is a linker that separates two separate protein domains (Fc domain, It is possible to combine a cyclic amine such as C L C-terminus of antibody constant domain , via the linker H It is possible to attach it to the N-terminus of one antibody constant domain.

[0151] The linker may be a simple covalent bond, e.g., a peptide bond, or a polyethylene glycol bond. Synthetic polymers, e.g., PEG polymers, or polymers that are chemically linked, e.g., When the linker is a peptide bond, is a protein domain that is converted to a carboxyl group at the C-terminus of another protein domain through a condensation reaction. It reacts with the amino group at the N-terminus of a protein domain to form a peptide bond. Specifically, peptide bonds can be synthesized by synthetic means via conventional organic chemistry reactions well known in the art. or can be produced naturally from a host cell, e.g. For example, the DNA sequence encoding both tandem proteins, two Fc domain monomers. The polynucleotide sequence is encoded by a set of enzymes within the host cell, such as DNA polymerases and ribosomes. The necessary molecular machinery leads to adjacent polypeptides that code for both proteins. It can be directly transcribed or translated.

[0152] In cases where the linker is a synthetic polymer, such as a PEG polymer, the polymer may be Each end is functionalized with a reactive chemical functional group to form a terminal end at the connecting end of the two proteins. It is possible to react with amino acids.

[0153] When the linker (other than the peptide bond shown above) is formed by a chemical reaction, The substituents may be, for example, amines, carboxylic acids, esters, azides, or other substituents commonly used in the art. The other functional group, a chemical functional group, is located at the C-terminus of one protein and at the C-terminus of the other protein. These two functional groups can be synthetically linked to the N-terminus of each of the The two proteins are then reacted via synthetic chemistry to form a chemical bond. Such chemical ligation procedures are routine to those skilled in the art.

[0154] Spacer In the present disclosure, the linker between two Fc domain monomers may be 3 to 200 amino acids (e.g. , 3~150, 3~100, 3~60, 3~50, 3~40, 3~30, 3~20, 3~ 10, 3~8, 3~5, 4~30, 5~30, 6~30, 8~30, 10~20, 10~ 30, 12~30, 14~30, 20~30, 15~25, 15~30, 18~22, and The peptide may be an amino acid spacer containing 20 to 30 amino acids. Tide spacers are known in the art and include, for example, flexibilizers such as glycine and serine. In some embodiments, the spacer comprises a peptide linker that contains a sizable amino acid residue. For example, TIFF2025026880000029.tif4128, which may contain multiple motifs or repeating motifs. In some embodiments, the spacer can be, e.g., TIFF2025026880000030.tif10128, which may contain 2 to 12 amino acids including the GS motif. In embodiments, the spacer may be, for example, It is possible for the sequence to contain 3 to 12 amino acids including the GGS motif, which is TIFF2025026880000031.tif4150. In other embodiments, the spacer is, e.g. TIFF2025026880000032.tif4136, containing 4 to 12 amino acids including the motif of GGSG (SEQ ID NO: 12). In other embodiments, the spacer can be, for example, TIFF2025026880000033.tif4128, and may contain the motif GGGGS (SEQ ID NO: 16). In embodiments, the spacer may also contain amino acids other than glycine and serine. For example, In some embodiments of the present disclosure, a peptide spacer of 12 amino acids or 20 Two Fc domain monomers, each with the sequence TIFF2025026880000035.tif4134, a 12 amino acid peptide spacer and a 20 amino acid peptide spacer are in series In another embodiment, the array TIFF2025026880000036.tif4128 was used to construct the 18 amino acid peptide spacer C L and C H 1 antibody constant domain In one embodiment, the spacer is, for example, TIFF2025026880000037.tif10143, and may contain the motif GGGG (SEQ ID NO: 51). In the form, the spacer is The file is TIFF2025026880000038.tif4128.

[0155] VIII. Serum Protein-Binding Peptides Improving the pharmacokinetics of protein drugs by binding serum proteins to peptides In particular, the Fc constructs described herein can be used to bind serum protein binding peptides. It can be fused with a petide.

[0156] As an example, albumin binding peptides that can be used in the methods and compositions described herein are Peptides are generally known in the art. In one embodiment, albumin binding The peptide has the sequence In one embodiment, the albumin-binding domain comprises, consists of, or consists essentially of the sequence TIFF2025026880000039.tif4128. The combined peptides may be up to 10 (e.g., up to 9, 8, 7, 6, 5, 4, 3, 2, or 1). a single amino acid modification (e.g., a substitution, e.g., a conservative substitution) of the sequence It comprises, consists of, or consists essentially of the sequence TIFF2025026880000040.tif4128.

[0157] In the present disclosure, the albumin binding peptide is a specific polypeptide within the Fc construct. In one embodiment, the albumin binding peptide may be linked to the N-terminus or C-terminus of One or more of the following in tracts 1, 2, 3, or 7A (Figures 1, 2, 3, and 7A, respectively): In another embodiment, the albumin binding peptide may be linked to the C-terminus of the polypeptide. Two Fs connected in series in constructs 4, 5, and 6 (Figs. 4, 5, and 6, respectively) It is possible to fuse the c-domain monomer to the C-terminus of the polypeptide encoding it. In another embodiment, the albumin binding peptide is Construct 4 and Construct 6 (Figure 1, respectively). 4 and 6) A polypeptide encoding two tandemly connected Fc domain monomers. The Fc domain monomer can be linked to the C-terminus of a second Fc domain monomer in the Albumin-binding peptides can be genetically fused to Fc constructs, Alternatively, it can be linked to the Fc construct via chemical means, e.g., chemical linkage. If desired, a spacer may be inserted between the Fc construct and the albumin-binding peptide. Without being bound by theory, it is possible to insert a sequencer into the disclosed Fc construct. Inclusion of an albumin-binding peptide within the cytoplasmic vesicle enhances its binding to serum albumin. It is anticipated that this could result in extended retention of the therapeutic protein via

[0158] IX. Fc Construct Generally, this disclosure provides antibodies that contain 2-10 Fc domains (e.g., 2, 3, 4, 5, 6, 7, 8, 9, or 10 Fc domains; 2-8 Fc domains, 2-6 Fc domains , 2-4 Fc domains, 2-3 Fc domains, 5-10 Fc domains, 5- The Fc constructs have eight Fc domains or five to six Fc domains. These are, for example, single wild-type Fc receptors for FcγRIIIa. The present disclosure provides a method for the preparation of a polypeptide having a binding affinity and / or avidity greater than that ... Two Fc domain monomers of the c domain selectively form dimers with each other, thereby The two interacting Cs are then mixed together to avoid the formation of unwanted multimers or aggregates. H 3. Antibody Stationary Methods for engineering amino acids at the domain interface are disclosed. The Fc domain comprises a number of Fc domain monomers, each pair of which forms an Fc domain. The Fc construct is a functional construct formed from a dimer of two Fc domain monomers. At a minimum, it contains a suitable Fc domain.

[0159] In some embodiments, the Fc construct comprises a dimer of two Fc domain monomers. It contains a single Fc domain (Figures 1-3 and 7A). H 3. Antibody Constant The domains can be unmodified (Figure 1) or contain amino acids at their interface. In particular, amino acid substitutions can be made in engineered cavities (FIG. 2), engineered protrusions (FIG. 3), and / or engineered ribs (FIG. 4). The amino acids can be neutral (Figure 2) or charged (Figure 3).

[0160] In another embodiment, the Fc construct comprises two Fc polypeptides formed from three polypeptides. The first polypeptide contains the c domain (FIGS. 4 and 6). The second and third polypeptides each contain two Fc domain monomers linked in tandem. The second and third polypeptides contain two Fc domain monomers. The Fc polypeptide may be a polypeptide of interest or may be a different polypeptide. The figure shows an example of a construct. The first polypeptide is linked in series via a linker. The second and third polypeptides each contain two wild-type Fc domain monomers linked together, Each of the first and second polypeptides contains a wild-type Fc domain monomer. The first polypeptide and the second polypeptide form a first Fc domain, while the second polypeptide and the second polypeptide form a first Fc domain. The other Fc domain monomer in the polypeptide is combined with a third polypeptide to form a second Fc domain monomer. The second polypeptide and the third polypeptide form a domain. is not linked. Figure 6 illustrates an Fc construct similar to that in Figure 4. In 6, both Fc domain monomers in the first polypeptide are C H 3. Antibody Stationary Within the domain, the second and third polypeptides contain engineered protuberances, while the third and fourth polypeptides contain C H 3 The antibody constant domain contains an engineered cavity into which a protrusion has been inserted. C H 3-C H 3 The interface supports the formation of heterodimers of Fc domain monomers and prevents unwanted multimerization. This avoids the uncontrolled formation of dimers, as further described in Example 4 herein. Like the manipulated C H 3. A dimerization selectivity module comprising an antibody constant domain is formed in the dimer We explain the formation of Fc constructs from Fc domain monomers without a selectivity module. This prevents the formation of unwanted multimers as shown in Example 3 below.

[0161] Additionally, in other embodiments, the Fc construct is formed of four polypeptides. It is possible for the first and second polypeptides to contain three Fc domains (FIG. 5). can be the same or different, and for the third and fourth polypeptides In this embodiment, the first and second polypeptides can both be The Fc domain monomers were linked in series via a linker. One Fc domain monomer is C H 3. Charged amino acid substitutions in antibody constant domains The other Fc domain monomer contains C H 3. Antibody constant domain containing protuberances The third and fourth polypeptides both encode an Fc domain monomer having a cavity. The first and second polypeptides are H 3 Opposite Charges in Antibody Constant Domains Through interaction with each other, they form the first Fc domain. The second and third Fc domains In is a protrusion in the first and second polypeptides and a cavity in the third and fourth polypeptides. This is formed by the interaction between the Fc construct and the protrusions that are inserted into the cavity. Each Fc domain monomer contains a dimerization selectivity module that promotes the formation of a specific Fc domain. Contains a molecule.

[0162] In yet another embodiment, C H 3. Not through interactions between antibody constant domains, C L Constant domain and C H 1. A single polypeptide through interactions between the constant domains However, the construct may be a dimer (e.g., Construct 7A, FIG. 7A) or a multimer (e.g., Construct FIG. 7B shows the C of one Fc domain. L D The main C of the adjacent Fc domain H Multiple Fc domains interacting with one Fc domain 1 illustrates an Fc construct containing ribonuclease.

[0163] In yet another embodiment, the Fc construct comprises a pentameric Fc construct formed of six polypeptides. It is possible for the antibody to contain two Fc domains. Two examples are illustrated in Figures 8 and 9. These illustrated Fc constructs are composed of six polypeptides. Four of the peptides can be encoded by the same nucleic acid, and the remaining two The polypeptides can also be encoded by the same nucleic acid. These Fc constructs can be produced by expression of the two nucleic acids in a suitable host cell. do.

[0164] In another embodiment, the Fc constructs containing two or more Fc domains are Such a construct can be formed of two polypeptides having the same primary sequence. It can be formed by expression of a single polypeptide sequence in the host cell. An example is shown in Figure 1. In this example, an Fc construct containing three Fc domains is A single nucleic acid is sufficient to encode two Fc domains that are part of the same polypeptide. The main monomer contains a flexible linker of sufficient length and flexibility to form F The linker is a cleavable linker that allows the formation of the c domain. This same polypeptide may also be linked via any flexible linker. The third Fc domain monomer is linked to the third Fc domain monomer. , and linked to other Fc domain monomers to produce the Y-shaped Fc construct depicted in FIG. 10. The formation of the Fc domain can be achieved by dimerization, as shown in FIG. It is possible to control this by using selectivity modules. In some cases, three The Fc construct containing the Fc domain is, for example, a structure of two polypeptides as shown in FIG. Such constructs can be formed from two polypeptides in a host cell. The vector can be formed from expression of a plasmid p53.

[0165] In some embodiments, one or more Fc polypeptides in the Fc construct comprise a terminal In some embodiments, one or more of the Fc constructs contain terminal lysine residues. In some embodiments, the Fc polypeptide does not contain a terminal lysine residue. All of the Fc polypeptides in the construct contain a terminal lysine residue. In the Fc construct, all of the Fc polypeptides contain a terminal lysine residue. In one example, the terminal lysine residue in the Fc polypeptide is selected from SEQ ID NOs: 30, 32, 34, , 36, 38, 40, 42, 44, 45, and 46 (see Example 1). A nucleic acid sequence that comprises, consists of, or consists essentially of a sequence and is removed. to generate a corresponding Fc polypeptide that does not contain terminal lysine residues. In other examples, the terminal lysine residue comprises a sequence of SEQ ID NO: 48 or 50 (see Example 1). and binding to an Fc polypeptide consisting of, or consisting essentially of, the sequence, In another embodiment, a corresponding Fc polypeptide containing a sRNA residue can be generated. or the terminal lysine residue comprises the sequence of SEQ ID NO: 48 or 50 (see Example 1). and binding to an Fc polypeptide consisting of, or consisting essentially of, a sequence of up to 10 (e.g. up to 9, 8, 7, 6, 5, 4, 3, 2, or 1 single amino acid modification (e.g., substitution , e.g., a conservative substitution) It is possible.

[0166] X. Host Cells and Protein Production In the present disclosure, a host cell is a cell that expresses a polypeptide or construct as described herein. necessary for expression from the corresponding nucleic acid of the The term refers to a medium containing cellular components. Nucleic acids can be derived from any of the conventional techniques known in the art (e.g., transformation, transfection, etc.). by transfection, electroporation, calcium phosphate precipitation, direct microinjection, etc. The host cell may be a mammalian cell. Mammalian host cells can be of either mammalian or bacterial origin. CHO (or CHO derived cell lines, e.g., CHO-K1, CHO-DXB11, CHO-D G44), mouse host cells (e.g., NS0, Sp2 / 0), VERY, HEK (e.g. , HEK293), BHK, HeLa, COS, MDCK, 293, 3T3, W138, BT483, Hs578T, HTB2, BT20, and T47D, CRL7O3O, The proteins include, but are not limited to, HsS78Bst and HsS78Bst cells. Regulating expression of the construct or modifying and promoting the protein product in a desired specific manner It is possible to select a host cell for cloning. Various host cells are suitable for protein production. The cell line has specific properties and mechanisms for post-translational processing and modification of the product. Alternatively, a host system may be chosen to ensure correct modification and processing of the expressed protein. It is possible.

[0167] Expression of protein products from their corresponding DNA plasmid constructs and For secretion, promoters, enhancers, sequences, transcription terminators, polyadenylation The vector can be expressed by suitable expression control elements known in the art, including transcription sites and selectable markers. A host cell can be transfected or transformed with DNA controlled by the therapeutic tag. Methods for expressing proteins are known in the art. as,Argelia Lorence(eds.)Recombinant Gene Expression:Reviews and Protocols(Method s in Molecular Biology), Humana Press; 2nd ed.2004 edition (July 20,2004);Vladimir Voynov and Justin A. Caravella (eds.) Ther. apeutic Proteins:Methods and Protocols(M methods in Molecular Biology)Humana Press ;2nd ed.2012 edition(June 28,2012) stomach.

[0168] XI. Purification The Fc construct can be purified by any method known in the art of protein purification, such as by chromatography. Chromatography (e.g., ion exchange), affinity (e.g., protein A affinity) ), and size exclusion column chromatography), centrifugation, differential solubility, or protein The Fc polypeptide may be purified by other standard techniques for purification of Fc polypeptides. For constructing the protein, it is important to select an affinity column such as a protein A column appropriately. by combining chromatography columns, filtration, ultrafiltration, salting out and dialysis procedures. The product can be isolated and purified by, for example, Process Scaling. e Purification of Antibodies, Uwe Gottsch alk(ed.)John Wiley & Sons, Inc.,2009;and Subramanian(ed.) Antibodies-Volume I-Pro duction and Purification,Kluwer Academic / Plenum Publishers, New York (2004). In this example, the Fc construct is coupled to a tag sequence, such as a peptide, to facilitate purification. An example of a tagged amino acid sequence is a hexahistidine peptide, which is It binds to a nickel-functionalized agarose affinity column with micromolar affinity. Other peptide tags useful for purification include, but are not limited to, the hemagglutinin "HA" tag. The antibody includes a polypeptide that corresponds to an epitope derived from the influenza hemagglutinin protein. (Wilson et al., 1984, Cell 37:767).

[0169] For the Fc construct, the purification method was Protein A column chromatography. Protein A ligand can be linked to Fc constructs via the Fc region. Protein A chromatography is a method for detecting the presence of β-lactams in the majority of host cells. This is a highly selective capture method that can remove proteins. The Fc construct was purified using Protein A column chromatography as described in Example 2. The lactate can be purified.

[0170] XII. Pharmaceutical Compositions / Formulations The disclosure also provides pharmaceutical compositions comprising one or more of the Fc constructs described herein. In one embodiment, the pharmaceutical composition comprises a structurally identical or substantially identical Fc In various embodiments, the pharmaceutical composition comprises a substantially homogenous population of constructs. Tracts 1-10 and 5 * The present invention includes a substantially homogenous population of any one of the above.

[0171] The therapeutic protein constructs of the disclosure, for example Fc constructs, can be administered to pharmaceutical compositions. Pharmaceutical compositions containing therapeutic proteins can be incorporated into pharmaceutical compositions. The pharmaceutical compositions can be prepared by known methods. Parenteral administration is possible in injectable preparations including sterile solutions or suspensions in acceptable liquids. For example, the pharmaceutical composition may be water for injection (WFI), saline, an emulsifier, a suspending agent, a surfactant ... Pharmaceutically acceptable vehicles or solvents such as repellents, stabilizers, diluents, binders, excipients, etc. and Fc The constructs are then suitably combined and packaged in units required by commonly accepted pharmaceutical practice. The active ingredient contained in the pharmaceutical preparation can be prepared by mixing the active ingredient in the pharmaceutical preparation in a suitable amount. The amount of the portion is such that a suitable dose is given within the range specified.

[0172] Sterile compositions for injection are prepared according to conventional pharmaceutical practice using distilled water for injection as a vehicle. For example, glucose and D-sorbitol, D-mannose, D- Physiological saline or isotonic solutions, optionally containing other additives such as mannitol, sodium chloride, etc. In addition, a suitable solubilizing agent, for example an alcohol such as ethanol, which is commonly known in the art, may be used. and polyalcohols such as propylene glycol or polyethylene glycol, and polysols. Bet 80 TM (trademark), in combination with a non-ionic surfactant such as HCO-50, The preparation of therapeutic protein products can be carried out in aqueous solutions. and is known in, for example, Banga (ed.) Therapeutic Peptides s and Proteins:Banga(ed.)[4]Therapeutic Peptides and Proteins:Formulation, Proce ssing and delivery systems (2d ed.)Taylor &Francis Group, CRC Press (2006).

[0173] XIII.Dose Pharmaceutical compositions are administered in a manner compatible with the dosage formulation, and in such amount as will be therapeutically effective. The pharmaceutical composition may be administered, for example, in an intravenous or subcutaneous dosage form, resulting in an improvement or treatment of the symptoms. The drug may be administered in a variety of dosage forms, including oral dosage forms such as ingestible solutions, drug-releasing capsules, etc. The dosage appropriate for an individual subject will depend on the therapeutic objectives, the route of administration, and the condition of the patient. The recombinant protein may be administered at a concentration of 1 to 200 mg / kg, for example 1 to 100 mg / kg, for example For optimal therapeutic effect, the dose is usually 20-100 mg / kg. Healthcare providers can customize doses, titrate, and adjust route of administration as needed. It is necessary.

[0174] XIV. Indications The pharmaceutical compositions and methods of the disclosure reduce inflammation in a subject and inhibit autoantibodies in a subject. promotes clearance of, inhibits antigen presentation in a subject, and reduces immune responses (e.g., Preventing activation of immune complex-based immune responses in a subject, and Useful for treating immune conditions or disorders and inflammatory conditions or disorders. and diseases include rheumatoid arthritis (RA); systemic lupus erythematosus (SLE); ANCA-related Continuous vasculitis; antiphospholipid antibody syndrome; autoimmune hemolytic anemia; chronic inflammatory demyelinating neuropathy C; anti-allo in transplantation, anti-self in GVHD -self), anti-substitution, IgG treatment, clearance of IgG paraprotein; dermatomyositis; Goodpasture's syndrome; organ system targeting mediated by antibody-dependent cell-mediated cytotoxicity Type II hypersensitivity syndromes, such as Guillain-Barre syndrome, CIDP, dermatomyositis, Tee's syndrome, antibody-mediated rejection, autoimmune thyroid disease, ulcerative colitis, autoimmune liver Diseases; idiopathic thrombocytopenic purpura; myasthenia gravis, neuromyelitis optica; pemphigus and other autoimmune disorders Epidemic bullous disease; Sjogren's syndrome; mediated by antibody-dependent phagocytosis Autoimmune cytopenias and other disorders; e.g. synovitis, dermatomyositis, systemic vasculitis, glomerulitis , and other FcR-dependent inflammatory syndromes that are vasculitis. EXAMPLES

[0175] Example 1 - Design and cloning of DNA plasmid constructs A total of eight DNA plasmid constructs were used to construct eight Fc constructs. The DNA plasmid constructs were then used for protein production. Human embryonic kidney (HEK) 293 cells were transfected with eight coding fragments. The secreted polypeptide had the basic structure as shown below.

[0176] A.wt Fc: wild-type Fc domain monomers (Figure 1: 102 and 104, Figure 4: 408 and 410).

[0177] B.Protrusion Fc:C H 3. Fc domain monomers with engineered protuberances within the antibody constant domain Body (Figure 2:202).

[0178] C. Cavity Fc:C H 3. Fc domain monomer with engineered cavity in antibody constant domain Body (Fig. 2: 204, Fig. 5: 514 and 516).

[0179] C * .Cavity Fc * :C H 3. Fc domain with engineered cavity in antibody constant domain Monomer (Figure 2: 204, Figure 5: 514 and 516). Cavity Fc * is also compared to the cavity Fc. It contains additional amino acid substitutions.

[0180] D.Charge Fc:C H 3. Fc domain monomer with inverted charge in antibody constant domain ( Figure 3: 302 and 304).

[0181] E. wt-12-wt Fc2: Two Fc domain monomers, 12-amino acid GGG Linked in series via an S peptide linker (Figure 4:402).

[0182] F. protrusion-20-charge Fc2:C H 3. Fc domain with inverted charge in antibody constant domain The main monomer and C H 3. Fc Domain Monomers with Engineered Protuberances in the Antibody Constant Domain and linked in series via a 20-amino acid SGGG peptide linker (Figure 5:5 02 and 508).

[0183] F * .Protrusion-20-Charge Fc2 * :C H 3. F with inverted charge in antibody constant domain c domain monomer and C H 3. Single Fc domain with engineered protuberances in the antibody constant domain The 20-amino acid SGGG peptide linker was used to link the 20-amino acid SGGG peptide linker in series (Figure 5 :502 and 508). Projection-20-charge Fc2 * Also, compared to the protrusion Fc, there is an additional mesh. Contains an acid substitution.

[0184] G. Protrusion-20-Protrusion Fc2:C H 3. Both have engineered protuberances in the antibody constant domain. Two Fc domain monomers were linked in tandem via a 20-amino acid GGGS peptide linker. Connected to (Figure 6:602).

[0185] HC H C L Fc+:C H 1 and C L The Fc domain monomer with the constant domain is 7A: 702 and 704, and 7B: 706, 708, and 709. 10, 712, 714, and 716). L The constant domain is an 18 amino acid GGGS peptide. C via the chid linker H1 It is linked to the constant domain.

[0186] The Fc DNA sequence was derived from human IgG1 Fc. The mutations in the payload were substituted into the parent Fc sequence. The leader sequence was derived from the kappa light chain of human immunoglobulin. The DNA encoding the peptide was linked to the 5' region. H C L All except for IgG1A (IgG1A+), contain this encoded peptide on the amino terminus, Protein translocation to the endoplasmic reticulum for assembly and secretion was performed. It is understood that any one of the above techniques may be used in connection with the present disclosure. The leader peptide is usually cleaved off within the ER lumen. An 11-nucleotide sequence containing a 5'-terminal EcoR1 site was inserted above the ATG start codon. A 30 nucleotide sequence containing a 3'-terminal Xho1 site was added to the 3'-terminal TG The DNA sequence was optimized for expression in mammalian cells. and cloned into the pcDNA3.4 mammalian expression vector.

[0187] Mutations were numbered according to the EU Kabat numbering system (Kabat et al., Seq uences of Proteins of Immunological Inte rest,National Institutes of Health,Bethe wild-type amino acid residues according to their positions using the sda ​​(Md., ed. 5, 1991) and their followed by the replacement residue in single letter code.

[0188] The nucleotide and amino acid sequences of the secreted polypeptides A to H described above are shown below. (Cavity Fc * and protrusion-20-charge Fc2 * For those, only amino acid sequences are available. (Indicated below).

[0189] TIFF2025026880000041.tif160128TIFF2025026880000042.tif228109TIFF20250268800 00043.tif224127TIFF2025026880000044.tif204126TIFF2025026880000045.tif222111 TIFF2025026880000046.tif224108TIFF2025026880000047.tif207110TIFF20250268800 00048.tif223110TIFF2025026880000049.tif226130TIFF2025026880000050.tif255128

[0190] In some embodiments, the Fc polypeptide in the Fc construct described herein comprises: up to 10 (e.g. up to 9, 8, 7, 6, 5, 4, 3, 2, or 1) single amino acids Any of the sequences described herein, including those having modifications (e.g., substitutions, e.g., conservative substitutions). , consisting of, or consisting essentially of, any of said sequences.

[0191] In some embodiments, one or more Fc polypeptides in the Fc construct comprise a terminal In some embodiments, one or more of the Fc constructs contain terminal lysine residues. In some embodiments, the Fc polypeptide does not contain a terminal lysine residue. All of the Fc polypeptides in the construct contain a terminal lysine residue. In the Fc construct, all of the Fc polypeptides contain a terminal lysine residue. In one embodiment, the sequences of SEQ ID NOs: 30, 32, 34, 36, 38, 40, 42, 44, 45 and 46. The terminal lysine residues in an Fc polypeptide consisting of a nucleotide sequence are removed to comprise a terminal lysine residue. In another embodiment, a corresponding Fc polypeptide can be produced that does not contain terminal lysine residues. The group comprises, consists of or consists essentially of the sequence of SEQ ID NO: 48 or 50. a corresponding Fc polypeptide containing a terminal lysine residue, can be generated.

[0192] Example 2 - Expression of Fc construct proteins Regarding the expression of the Fc construct protein, select from A to H described in Example 1. Two of the DNA plasmid constructs were transfected into EXPI293 cells (Life Technologies, Inc.). The cells were transfected into liposome-transfected cells (Technologies). Plasmid DNA was introduced into EXPI293 cells using a transfection kit. The total amount of plasmid constructs used is fixed, while the amount of different plasmid constructs used is The ratio of constructs was varied to maximize the yield of the desired construct (Table 11 below). For each Fc construct, two DNA plasmid vectors were transfected. The ratios (mass ratios) of the constructs are shown in Table 11. Schematics of the constructs are shown in Figures 1 to 7B. It shows.

[0193] After protein expression, the expressed construct was purified by Protein A-based affinity chromatography. The culture supernatant was purified from the cell culture supernatant by column chromatography. vant Preparative Chromatography System (GE Healthcare Life Sciences Using Poros MabCapture A (LifeTech The captured Fc construct was then loaded onto a column (Humanities). phosphate-buffered saline (low salt wash), followed by phosphate-buffered saline supplemented with 500 mM NaCl (high salt wash). The Fc construct was washed with 100 mM glycine, 150 mM The protein solution removed from the column was eluted with 1M NaCl, pH 3 buffer. Neutralize to a final concentration of 100 mM by adding 100 mM TRIS pH 7.4. The construct was prepared using Poros® XS resin (Applied Biosciences, Inc.). For ion exchange chromatography, use a 100% glycerol ester (Nexases, Cat. # 4404336). The column was pre-equilibrated with 10 mM MES, pH 6 (Buffer A). and gradient with 10 mM MES, 500 mM sodium chloride, pH 6 (buffer B). The sample was eluted by chromatography.

[0194] A total of seven Fc constructs were obtained (see Table 11 below and Figures 1 to 7B). The Fc constructs were analyzed by SDS-PAGE under both reducing and non-reducing conditions. sodium silane sulfate polyacrylamide gel electrophoresis), followed by Coomassie blue staining Analysis by color confirmed the presence of a protein band of the expected size.

[0195] (Table 11) TIFF2025026880000051.tif70166

[0196] Example 3 - Preparation of Construct 4 and SDS-PAGE Analysis Two DNA plasmid constructs, wt-12-wt Fc2 (DNA A plasmid construct E) and wt Fc (DNA plasmid construct of Example 1) Construct 4 was expressed using the two plasmid constructs (Figure 4). The tract was transformed into HEK 293 cells for protein expression and purification as described in Example 2. 11A-B show the reduced SDS-PAGE and In reduced SDS-PAGE (Figure 11A), the wt A band at approximately 25 kDa (lanes 2 and 3, Fig. 11A) and a 50 kDa complex corresponding to a tandem dimer of wt-12-wt Fc2. Bands (lanes 1 to 3, FIG. 11A) and non-reducing SDS-PAGE (FIG. 11B) were observed. Lanes 2 and 3 are high protein (1 / 2) and low protein (1 / 2), respectively. 3) each containing the final protein product of Construct 4. wt-12-wt Fc2 tandem dimer and two wt Fc domain monomers form group 4 One major band at approximately 100 kDa corresponds to an association with wt The Fc domain monomers and the free wt-12-wt Fc2 tandem dimers were Another major band of roughly equivalent signal intensity at approximately 50 kDa corresponds to the was observed.

[0197] Furthermore, a multimer of wt-12-wt Fc2 and a wt Fc domain monomer was identified. , high molecular weight bands at approximately 150 kDa, 200 kDa, and 250 kDa (referred to as "high molecular weight bands"). Patterns 2 and 3, FIG. 11B) were also observed.

[0198] Example 4 - Preparation of Construct 6 and SDS-PAGE Analysis Two plasmid constructs, prong-20-prong Fc2 (DNA plus the DNA of Example 1) Cavity Fc (DNA plasmid construct G of Example 1) Construct 6 was expressed using the following plasmids (Figure 6). The resulting product was transfected into HEK 293 cells for protein expression and purification as described in Example 2. 12A-12B show reduced SDS-PAGE and non-reduced SDS-PAGE of Construct 6. The reduced SDS-PAGE (Figure 12A) shows that the cavity Fc A band at approximately 25 kDa corresponding to the domain monomer (lanes 2 and 3, FIG. 12 A) and a band at 50 kDa corresponding to a tandem dimer of prong-20-prong Fc2 ( In non-reducing SDS-PAGE (Fig. 12B), Sections 2 and 3 are high (1 / 2) and low (1 / 3) protein content, respectively, and The final protein product of lactate 6 contains 20-probe Fc2 in series. The molecular weight of the Fc domain at approximately 100 kDa corresponds to an association of a mer with two hollow Fc domain monomers. One major band was observed, as well as free bands that were not associated with any of the hollow Fc domain monomers. A weak signal at approximately 50 kDa corresponds to a distant 20-probe Fc2 tandem dimer. A band of low null intensity was observed.

[0199] A similar experiment was performed with construct 5 (Figure 13). , protrusion-20-charge Fc2 (DNA plasmid construct F in Example 1) and cavity Fc (DNA plasmid construct C of Example 1) to produce construct 5. The two plasmid constructs were then transferred to the cytoplasm using cationic lipid transferase. EXPI293 cells were transfected with 100% ribosomal RNA at empirically determined ratios by centrifugation. The transfected cultures are incubated in cell culture medium for 6-8 days. After the period, the cells were removed by centrifugation. The supernatant (medium, lane 1 in Figure 13) contained trans The infected cells secrete Construct 5 into the medium. Construct 5 is a protein A affinity The medium was purified by immunochromatography. At this point, the medium contained the three Fc domains. The desired construct 5 has two Fc domains (dimer) as well as a trimer. (approximately 10-15%) and one Fc domain (monomeric, 5-10%). The remaining part contained some of the proteins that were not present. Small amounts of contaminating host cell proteins were also present. The eluate from the Protein A column was buffer exchanged, concentrated, and purified using Strong The mixture was separated by Cation Exchange (SCX) chromatography. Construct 5 binds to an SCX column and is then eluted with a salt and pH gradient. This step resulted in the production of unassembled proteins with two or one Fc domains. Construct 5, which has undesired post-translational modifications, and contaminating host cell types. This allows the isolation of the desired construct 5, which has three Fc domains, from the protein. After another concentration and buffer exchange, pure final protein production of construct 5 was achieved. The product was obtained (pure column, lane 2 in FIG. 13).

[0200] FIG. 13 shows the results of the antibody produced from cultured host cells engineered to express Construct 5. SDS-PAGE of the culture medium (lane 1) and pure Construct 5 (lane 2). The percentage of the major band in SDS-PAGE for each sample is shown. In the medium sample (lane 1), the three Fc domains were identified. The major band of approximately 150 kDa corresponds to the final protein product of construct 5. The medium samples also contained a protein with two Fc domains. A small band with a weak signal intensity at 100 kDa and a single Fc domain A second minor band with the weakest signal intensity at 50 kDa corresponds to a protein After purification (lane 2), construct 5, which contains three Fc domains, The major band of approximately 150 kDa, which corresponds to the final protein product, was enhanced. Quantifying the signal intensity of protein bands on SDS-PAGE of construct 5 As a result, before protein purification, approximately 79% of the total protein in the medium was conjugated to the construct. After protein purification, the desired protein product was approximately 95% A substantially homogenous population of Construct 5 was observed, with a purity of .about.1.

[0201] These findings are H 3. Engineered protrusions or engineered cavities in antibody constant domains Selective dimerization modules containing either one of these reduce self-association and are controlled. It has been demonstrated that the antibody prevents the formation of aggregates or multimers mediated by Fc, which is not present in the present specification. The use of dimerization selectivity modules in the constructs described herein is in accordance with the Fc construct. This suggests that the method can be used to obtain a substantially homogeneous preparation of the drug. This observation supports the idea of ​​producing constructs to control, for example, biological activity and potency. This implies significant advantages in purification and purification.

[0202] Example 5 - Binding affinity and avidity Binding of the constructs to multiple Fcγ receptors was assessed using cell-based FRET competitive assays. The constructs 5 and 6 were evaluated using a ELISA kit (Cisbio Bioassays). 6 has FcγRIIa, FcγRIIb, and FcγRIIc compared to the wild-type Fc domain (construct 1). The IC50 values ​​for FcγRIIIa and FcγRIIIa were at least 10-fold lower (i.e. , increased binding).

[0203] Example 6 - Monocyte activation and inhibition assays Constructs 1, 5, and 6 contain one, three, and two Fc domains, respectively. The ability of three Fc constructs to activate THP-1 monocytes by themselves IL-8 release was used as an indicator of monocyte activation. Constructs 1, 5, and 6 were expressed and purified as described above. Each of the constructs was added to THP-1 monocytes. No substantial IL-8 release was observed, and the data are presented in Figure 14A.

[0204] The same three Fc constructs then inhibited Fc receptor-mediated monocyte activation. The ability of IgG1 (100 μg / mL) to bind to IgG1 was tested. and used to induce IL-8 release by THP-1 monocytes. 1, 5, and 6, or control material (intravenous immunoglobulin (IVIg), human serum Serial dilutions of serum albumin (HSA), and glycine buffer were performed in tissue culture plates. THP-1 monocytes (1.5x10 5 Cells) were added immediately with thorough mixing. Cultures were incubated for 18 hours and supernatants were analyzed for IL-8. Constructs 5 and 6 suppressed IL-8 release more effectively than construct 1 at low doses. Okay, the data is provided in Figure 14B.

[0205] Example 7 - K / BxN Arthritis Model Fc constructs 1, 5, and 6, and IVIg, K / BxN serum transfer The ability of the authors to protect mice from arthritis in a model was examined by Anthony, Proc. Nat. Acad.Sci.USA105:19571-19578(2008) Twelve-week-old K / BxN mice were cultured at 100 ng / mL in a 10-mL tube at 4 °C for 1 h. A total of 30 C57BL mice were purchased from Biosciences Laboratories. The mice were divided into five groups of six each. 200 μl of K / BxN serum (arthritis-inducing serum) was injected. One hour before injection, each group received 200 μl of Construct 6 at 0.1 g / kg. 200 μl of 0.1 g / kg of IVIg, 200 μl of 0.1 g / kg of IVI g at ​​1 g / kg, 230 μl, or 200 μl of phosphate-buffered saline (PBS), intravenously The patient was given an IV injection (day 0). Clinical examination of leg swelling and ankle thickness revealed Inflammation was scored. Paw swelling was scored from 0 to 3 for each paw (0 being no swelling, 3 being no swelling). For each mouse, the scores for all four paws were added together to determine the total clinical score. For ankle thickness, caliper measurements were used. Each mouse was The daily mean clinical scores for each group of six mice are plotted in FIG. I did.

[0206] As shown in FIG. 15, IVIg at 1 g / kg, Construct 5 at 0.1 g / kg Construct 6 at 0.1 g / kg and 0.1 g / kg provided similar levels of protection from inflammation. Constructs 5 and 6 were administered at a ten-fold lower dose compared to the IVIg dose. Considering these results, constructs 5 and 6 were found to be more potent than IVIg. It is clear.

[0207] Example 8 - Chronic ITP model Constructs 1 and 5 as well as IVIg were associated with immune thrombocytopenia (ITP) The researchers tested the ability of the drug to treat mice with ITP, an anti-inflammatory drug that causes thrombocytopenia. Platelet Abs were administered to 45 C57BL / 6 mice (18–22 g, Char and rat anti-CD41 antibody (Lauren Rivers Labs, Massachusetts, USA). (Ab) (clone MWReg30, BioLegend, catalog #133910) 1.5 μg / mouse, once a day for 4 days (days 1, 2, 3, and 4) Five mice were injected intraperitoneally (i.p.) with a rat IgG1,k isotype control. Injection of 1.5 μg / mouse of IgG antibody (BioLegend, Cat#400414) was performed. Antibody (Ab) was administered in 100 μl of PBS. All mice were given saline co-administration 2 hours after the third anti-CD41 Ab injection on the third day. control, IVIg at 1 g / kg, 0.02, 0.03, 0.1, and 0.3 g / kg Construct 1 at 0.004, 0.02, and 0.1 g / kg The mice were intravenously administered either lactate or lactate 5 in a volume of 200 μl once. -24 hours after CD41 Ab injection, blood was exsanguinated and the VetScan Instrument was used. Total platelet levels were quantified by nt. All procedures were performed in accordance with the Animal Welfare Act and the Care of Laboratory Animals. and the guidelines for use were followed.

[0208] As shown in FIG. 16, platelet levels were increased by 0.02 and 0.05 compared to saline controls. Significantly increased after treatment with Construct 5 at 0.1 g / kg ( **** p< 0.0001 for one-way ANOVA in multiple comparison tests Platelet levels in these groups were approximately 100% higher than those in the normal isotype-treated group. The results were similar to those of IVIg at 1 g / kg and cons Treatment with Tract 1 also significantly increased platelet levels compared to saline controls. However, the number of * p<0.05, ** p<0.01, single-comparison test The platelet levels in these groups were analyzed by one-way ANOVA. The levels were lower than those of the Construct 5 treatment groups at 0.02 and 0.1 g / kg. In this model, construct 5 was found to be approximately 50-fold more potent than IVIg. Either way.

[0209] Example 9 - Construct 5 * showed enhanced binding to FcγR compared to IVIg. The binding and binding activity are shown. Following the same protocol as described in Example 8, protrusion-20-charged Fc2 * (implementation Construct F in Example 1 * ) and hollow Fc * (Construct C of Example 1 * ) to the code Construct 5 was created using two plasmid constructs: * Express and purify The binding profile of this construct to various Fc receptors was analyzed using fluorescence resonance energy spectroscopy. The results were compared with those of IVIG in a FRET competitive binding assay.

[0210] Construct 5 * showed similar overall effects to IVIg against various Fcγ-receptors. The combined binding profile of FcγRIIb was shown to be the lowest, with FcγRIIb showing the lowest binding affinity. ), and binding to all low affinity FcγRs was greatly increased compared to IVIg. Increased binding to FcγR corresponds to higher avidity, which is It means the cumulative effect of the affinity of the individual binding interactions. Construct 5 * I The C50 values ​​were consistently lower than those of IVIg, compared with individual IgG molecules. This suggests that binding to low affinity FcγRs is significantly increased. Struct 5 * was approximately 1.0 times higher with FcγRIIa (H131 mutant) compared to IVIg. It showed a 70-fold increase in affinity and a 55-fold increase in affinity for FcγRIIb.

[0211] Example 10 - Inhibition of phagocytosis in THP-1 monocytic cells Construct 5 * and IVIg were tested in a model of phagocytosis.

[0212] Phagocytosis is when a cell (a phagocyte) engulfs a solid particle, such as a bacterium, and forms a phagosome. In the immune system, phagocytosis is the process of forming internal vesicles known as It is the primary mechanism used to eliminate pathogens and cellular debris. Monocytes and macrophages The mechanism of engagement is largely dependent on FcγR-mediated adhesion. Specialized in clearing opsonized (antibody-coated) particles from the immune system via gocytosis However, in autoimmune diseases, phagocytes are activated and This can lead to the harmful release of pro-inflammatory cytokines or other critical cell factors in the body. A pooled, multivitamin-containing IgG antibody was extracted from plasma of over 1000 donors. Autoimmune diseases are treated with IVIg, which contains high titer IgG antibodies.

[0213] The assay system uses latex beads coated with fluorescently labeled antibodies, opsonized Bacterial or viral mimics were administered to THP-1 cells, and construct 5 * and the existence of IVIg At the end of the incubation period, the cells were allowed to phagocytose in the presence and absence of IgG. Any external fluorescence was then quenched with trypan blue, and the amount of intracellular fluorescence was measured by flow cytometry. All groups were compared with their untreated controls (THP-1 cells and LaCross-Lysin cells). Results are representative of two separate experiments. be.

[0214] As shown in Figure 17, THP-1 monocytic cells phagocytose the opsonized beads. The system was administered IVIg and construct 5. * Although suppressed by treatment with both, Tract 5 * The IC50 value of was approximately 100-fold lower than that of IVIg. , Fc constructs of this disclosure, such as Construct 5 * Using "It is possible to treat not only pulmonary edema but also other indications treatable with IVIg," This suggests that...

[0215] Example 11 - Enhancement of Fc construct binding to FcγRIIb Previously, it was reported that the S267E / L328F mutation inhibits IgG1 binding to the FcγRIIb receptor. It has been shown that the antibody significantly and specifically enhances the binding of β-lactamase (Chu et al. Molecular ar Immunology 45 2008). The construct 5-FcγRIIb+ variant was engineered into the framework of SIF. The variants are well expressed and constructed (see FIG. 18) (SIF: Construct 5, Fc γRIIb+: Construct 5-FcγRIIb+ mutant). The results were obtained from transient expression of construct 5 (SIF) and construct 5-FcγRIIb+ mutant. Non-reducing sodium dodecyl sulfate polyacrylamide gel electrophoresis results for the clarified medium The results are shown in Fig. 1. The constructs 5-FcγRIIb and 5-FcγRIIb are encoded by the same plasmid. The mides were transfected into HEK293 cells at a ratio of 1 / 1 (w / w) or 2 / 1.

[0216] Binding of construct 5-FcγRIIb+ mutant to inhibitory FcγRIIb receptors However, binding was greatly enhanced compared to the construct 5 (SIF3) control ( >300-fold increase). Conversely, binding to activated FcγRIIa was relatively unaffected. However, binding to FcγRIIIa is reduced (see FIG. 19).

[0217] FIG. 19 shows the results of the IgG1 control, Construct 5, and Construct 5-Fcγ A graph summarizing the experimental results comparing the binding of RIIb+ mutants to Fc gamma receptors. A cell-based, competitive time-resolved fluorescence resonance energy transfer assay (CisBio Relative binding was measured using a ELISA kit (Bioassays, Bedford, MA). The results were expressed as EC50 values, which represent the percentage of antibodies that bind to specific cell surface Fc gamma receptors. The concentration of proband required to displace the fluorescently labeled antibody is shown. The higher the number, the less binding or affinity.

[0218] Example 12 - Inhibition of monocyte-derived dendritic cell (moDC) activation Construct 5-FcγRIIb+ variant suppresses the activity of monocyte-derived dendritic cells (moDCs) Dendritic cells, the most important population of professional antigen-presenting cells, DCs process antigenic material and present it on the cell surface for the purpose of initiating T cell responses. We present that FcγRs may play a major role in regulating the function of moDCs. Adhesion of immune complexes to activating FcγRs is mediated by immature human moDCs. Conversely, adhesion of inhibitory FcγRIIb may trigger maturation and activation of IL-1. (Boruchov AM et al. J Clin Invest 2005 115(10)). We demonstrated that Fc constructs inhibit the maturation and activity of moDCs. It has already been shown that the oxidative stress can suppress the oxidative stress (Ortiz et al. ci Transl Med 2016 and Figure 3). On the other hand, FcγRIIb+ mutations The Fc construct (e.g., construct 5 / SIF3) is immobilized on a plate. Exposure to immune complex surrogates such as IgG1 significantly enhanced moDC activation. Construct 5-FcγRIIb+ alone can be used to inactivate the 5-FcγRIIb construct (Figure 20). Incubation does not induce moDC activation (FIG. 21).

[0219] Immature human moDCs were cultured in 100 ng / mL GM-CSF and 50 ng / mL IL- DCs were generated from negatively selected CD14+ monocytes in the presence of 4. The cells were incubated with PBS, anti-CD32a antibody IV.3, Construct 5 (SIF3), or Construct 6 (SIF6). The mice were incubated in medium with either FcγRIIb+ or FcγRIIb+ for 20 min at 37°C. After blocking, the cell suspension was transferred to an IgG1-coated plate and additional GM- Culture medium supplemented with CSF and IL-4 was added. After 48 hours of incubation, the cells were gently The attached cells were harvested by washing the plates twice with ice-cold PBS. The cells were immunoblotted with anti-HLA-DR FITC Ab and anti-CD86-PE Cy7 Ab. The cells were stained using a FACSCanto (BD) and FlowJo Software ( The data were analyzed using TreeStar.

[0220] Figure 20: Construct 5 inhibits moDC activation by plate-immobilized IgG. On the other hand, construct 5-FcγRIIb+ enhances activation. gram, but moDCs cultured on untreated (UT) plates as a negative control. or an antibody that inhibits activating FcγRIIa (IV.3), Construct 5; or moDCs pretreated for 20 min with increasing concentrations of construct 5-FcγRIIB+. Above, CD86 surface expression is shown. Treated cells were then incubated with immobilized IgG1 (PB Tumor necrosis factor alpha (TNFa) treatment was performed as a positive control. Surface expression of CD86 was assessed by flow cytometry. Histograms of CD86 expression were gated using unstimulated cells as a control. The percentage of cells that are CD86 positive is shown on the y-axis versus treatment condition.

[0221] Figure 21: Construct 5-FcγRIIb+ by itself induces moDC activation However, plate-immobilized IgG enhances activation. Representative histograms are shown below. on moDCs cultured on untreated (UT) plates as a negative control, or Pre-treated with construct 5-FcγRIIB+ for 20 minutes and then transferred to an untreated plate. Figure 1 shows CD86 surface expression on moDCs that were FcγRIIb+ alone. PBS (PB IgG), antibody that blocks activated FcγRIIa (IV.3), and moDCs pretreated with construct 5-FcγRIIB+ (FcgRIIb+) The surface expression of CD86 was determined by flow cytometry. Histograms of CD86 expression were obtained using unstimulated cells as a control. The percentage of cells that were CD86 positive for each treatment condition is shown on the y-axis.

Claims

1. below: a) a first polypeptide having the formula A-L-B, wherein i) A comprises a first Fc domain monomer; ii) L is a linker; iii) B comprises a second Fc domain monomer; the first polypeptide; b) a second polypeptide comprising a third Fc domain monomer; c) a third polypeptide comprising a fourth Fc domain monomer; and An Fc construct comprising: The first Fc domain monomer and the third Fc domain monomer combine to form a first Fc domain monomer. forming a Fc domain, said second Fc domain monomer and said fourth Fc domain monomer; combine to form a second Fc domain, At least one of the Fc domains (i) binds to one or more Fc receptors (ii) binding affinity to the antibody; (iii) effector function; (iv) level of sulfation of the Fc domain; (iv) half-life; (v) protease resistance; (vi) Fc domain stability; and / or vii) at least one amino acid that alters one or more of the following susceptibility to degradation: Including modifications, The Fc construct.

2. The first Fc domain monomer and the third Fc domain monomer are and a complementary dimerization promoter between the Fc domain monomer and the third Fc domain monomer. Dimerization selectivity module The Fc construct of claim 1, comprising a nucleotide sequence (a) and a nucleotide sequence (b) of the Fc construct.

3. The second Fc domain monomer and the fourth Fc domain monomer are and a complementary dimerization promoter between said first Fc domain monomer and said second Fc domain monomer.

3. The Fc construct of claim 1 or 2, comprising a dimerization selectivity module.

4. The Fc construct according to any one of claims 1 to 3, wherein A consists of an Fc domain monomer. Struct.

5. The Fc construct according to any one of claims 1 to 4, wherein B consists of an Fc domain monomer. Struct.

6. 6. Any of claims 1 to 5, wherein the second polypeptide consists of an Fc domain monomer. An Fc construct according to one aspect.

7. 7. Any of claims 1 to 6, wherein the third polypeptide consists of an Fc domain monomer. An Fc construct according to one aspect.

8. at the N-terminus or C-terminus of one or more of the polypeptides via a linker The antibody according to any one of claims 1 to 7, further comprising a linked albumin binding peptide. Fc construct.

9. IgG C Ｌ Antibody constant domains and IgG C Ｈ 1 antibody constant domain, IgG C Ｈ one antibody constant domain is linked to A or the second polypeptide via a linker The Fc construct of any one of claims 1 to 8, wherein the Fc construct is linked to the N-terminus of

10. The second polypeptide and the third polypeptide have the same amino acid sequence.

10. An Fc construct according to any one of claims 1 to 9.

11. 2. The method of claim 1, wherein there are 14 or fewer amino acid modifications in at least one of the Fc domains.

11. An Fc construct according to any one of 1 to 10.

12. below: a) a first polypeptide having the formula A-L-B, wherein i) A comprises a first Fc domain monomer; ii) L is a linker; iii) B comprises a second Fc domain monomer; the first polypeptide; b) a second polypeptide having the formula A'-L'-B', wherein i) A' comprises a third Fc domain monomer; ii) L' is a linker; iii) B' comprises a fourth Fc domain monomer; the second polypeptide; c) a third polypeptide comprising a fifth Fc domain monomer; d) a fourth polypeptide comprising a sixth Fc domain monomer; and An Fc construct comprising: A of the first polypeptide and A' of the second polypeptide bind to form a first Fc A domain is formed, and B of the first polypeptide and the fifth Fc domain monomer are linked. and combining the second polypeptide with the sixth Fc domain to form a second Fc domain. and the main monomer combine to form a third Fc domain, At least one of the Fc domains (i) binds to one or more Fc receptors (ii) binding affinity to the antibody; (iii) effector function; (iv) level of sulfation of the Fc domain; (iv) half-life; (v) protease resistance; (vi) Fc domain stability; and / or vii) at least one amino acid that alters one or more of the following susceptibility to degradation: Including modifications, The Fc construct.

13. The first Fc domain monomer and the third Fc domain monomer are and a complementary dimerization promoter between the Fc domain monomer and the third Fc domain monomer. The Fc construct of claim 12, comprising a dimerization selectivity module.

14. The second Fc domain monomer and the fifth Fc domain monomer are and a complementary dimerization promoter between the fifth Fc domain monomer and the fifth Fc domain monomer.

14. The Fc construct of claim 12 or 13, comprising a dimerization selectivity module.

15. The fourth Fc domain monomer and the sixth Fc domain monomer are and a complementary dimerization promoter between the sixth Fc domain monomer and the sixth Fc domain monomer. The Fc polypeptide of any one of claims 12 to 14, comprising a dimerization selectivity module. Instruct.

16. A comprises an Fc domain monomer. construct.

17. 17. The Fc domain of any one of claims 12 to 16, wherein B consists of an Fc domain monomer. construct.

18. A' comprises an Fc domain monomer according to any one of claims 12 to 17. c construct.

19. B' consists of an Fc domain monomer. c construct.

20. 20. The method of claim 12, wherein the third polypeptide consists of an Fc domain monomer. An Fc construct described in any one of claims 1 to 4.

21. 21. The method of claim 12, wherein the fourth polypeptide consists of an Fc domain monomer. An Fc construct described in any one of claims 1 to 4.

22. IgG C Ｌ Antibody constant domains and IgG C Ｈ 1 antibody constant domain, IgG C Ｌ The antibody constant domain is linked to the IgG C Ｈ 1. Antibody constant domain The IgG C Ｈ 1 antibody constant domain is connected to A via a linker 22. The Fc construct of any one of claims 12 to 21, which is N-terminally linked. 。

23. Second IgG C Ｌ Antibody constant domain and second IgG C Ｈ 1. Antibody constant domain and the second IgG C Ｌ The antibody constant domain is connected to the second I via a linker. gG C Ｈ 1 antibody constant domain, and the second IgG C Ｈ 1. Antibody constant The Fc construct of claim 22, wherein the domain is linked to the N-terminus of A' via a linker. Tract.

24. 23. The composition according to any one of claims 12 to 22, further comprising an albumin binding peptide. c construct.

25. the first polypeptide and the second polypeptide have an identical amino acid sequence; The third polypeptide and the fourth polypeptide have the same amino acid sequence.

24. An Fc construct according to any one of claims 12 to 23.

26. The first polypeptide and the second polypeptide comprise the sequence of SEQ ID NO:

50. wherein the third polypeptide and the fourth polypeptide comprise the sequence of SEQ ID NO:

48.

26. The Fc construct of claim 25.

27. 2. The method of claim 1, wherein there are 14 or fewer amino acid modifications in at least one of the Fc domains.

26. An Fc construct according to any one of claims 12 to 25.

28. below: a) a first polypeptide having the formula A-L-B, wherein i) A comprises a first Fc domain monomer; ii) L is a linker; iii) B comprises an albumin binding peptide; the first polypeptide; b) a second polypeptide comprising a second Fc domain monomer; and An Fc construct comprising: The first Fc domain monomer and the second Fc domain monomer are combined to form an Fc domain. Forming an in At least one of the Fc domains (i) binds to one or more Fc receptors (ii) binding affinity to the antibody; (iii) effector function; (iv) level of sulfation of the Fc domain; (iv) half-life; (v) protease resistance; (vi) Fc domain stability; and / or vii) at least one amino acid that alters one or more of the following susceptibility to degradation: Including modifications, The Fc construct.

29. The first Fc domain monomer and the second Fc domain monomer are and a complementary dimerization promoter between the first Fc domain monomer and the second Fc domain monomer.

29. The Fc construct of claim 28, comprising a dimerization selectivity module.

30. 30. The Fc construct of claim 28 or 29, wherein A consists of an Fc domain monomer. 。

31. 31. Any of claims 28 to 30, wherein the second polypeptide consists of an Fc domain monomer. An Fc construct according to one aspect.

32. 2. The method of claim 1, wherein there are 14 or fewer amino acid modifications in at least one of the Fc domains.

32. An Fc construct according to any one of claims 28 to 31.

33. below: a) a first polypeptide having the formula A-L1-B-L2-C, wherein i) A is IgG C Ｌ comprising an antibody constant domain, ii) L1 and L2 are each a linker; iii) B is IgG C Ｈ 1 antibody constant domain, iv) C comprises a first Fc domain monomer; the first polypeptide; b) a second polypeptide having the formula A'-L1'-B'-L2'-C', 、 i) A' is IgG C Ｌ comprising an antibody constant domain, ii) L1′ and L2′ are each a linker; iii) B' is IgG C Ｈ 1 antibody constant domain, iv) C' comprises a second Fc domain monomer; The second polypeptide; An Fc construct comprising: The first Fc domain monomer and the second Fc domain monomer are combined to form an Fc domain. Forming an in At least one of the Fc domains (i) binds to one or more Fc receptors (ii) binding affinity to the antibody; (iii) effector function; (iv) level of sulfation of the Fc domain; (iv) half-life; (v) protease resistance; (vi) Fc domain stability; and / or vii) at least one amino acid that alters one or more of the following susceptibility to degradation: Including modifications, The Fc construct.

34. The first Fc domain monomer and the second Fc domain monomer are and a complementary dimerization promoter between the first Fc domain monomer and the second Fc domain monomer. The Fc construct of claim 33, comprising a dimerization selectivity module.

35. 35. The Fc construct of claim 33 or 34, wherein C consists of an Fc domain monomer. 。

36. Fc' of any one of claims 33 to 35 consists of an Fc domain monomer. c construct.

37. Further comprising an albumin binding peptide linked to the C-terminus of the C or C' via a linker.

37. An Fc construct according to any one of claims 33 to 36.

38. 2. The method of claim 1, wherein there are 14 or fewer amino acid modifications in at least one of the Fc domains.

38. An Fc construct according to any one of claims 33 to 37.

39. below: a) a first polypeptide having the formula A-L1-B-L2-C, wherein i) A is IgG C Ｌ comprising an antibody constant domain, ii) L1 and L2 are each a linker; iii) B is IgG C Ｈ 1 antibody constant domain, iv) C comprises a first Fc domain monomer; the first polypeptide; b) a second polypeptide having the formula A'-L1'-B'-L2'-C', 、 i) A' is IgG C Ｌ comprising an antibody constant domain, ii) L1′ and L2′ are each a linker; iii) B' is IgG C Ｈ 1 antibody constant domain, iv) C' comprises a second Fc domain monomer; the second polypeptide; c) a third polypeptide comprising a third Fc domain monomer; d) a fourth polypeptide comprising a fourth Fc domain monomer; and An Fc construct comprising: The first Fc domain monomer and the third Fc domain monomer combine to form a first Fc domain monomer. forming the c domain, The second Fc domain monomer and the fourth Fc domain monomer combine to form a second Forming an Fc domain, The IgG C of the first polypeptide Ｈ 1 antibody constant domain is The IgG C peptide Ｌ Binds to antibody constant domains, The IgG C of the second polypeptide Ｈ an antibody constant domain is provided in said first polypeptide; The IgG C peptide Ｌ Binds to antibody constant domains, At least one of the Fc domains (i) binds to one or more Fc receptors (ii) binding affinity to the antibody; (iii) effector function; (iv) level of sulfation of the Fc domain; (iv) half-life; (v) protease resistance; (vi) Fc domain stability; and / or vii) at least one amino acid that alters one or more of the following susceptibility to degradation: Including modifications, The Fc construct.

40. 2. The method of claim 1, wherein there are 14 or fewer amino acid modifications in at least one of the Fc domains.

39. The Fc construct according to claim 39.

41. below: a) a first polypeptide comprising a first Fc domain monomer; b) a second polypeptide comprising a second Fc domain monomer; and An Fc construct comprising: The first Fc domain monomer and the second Fc domain monomer are combined to form an Fc domain. and forming a first Fc domain monomer and a second Fc domain monomer, Promoting dimer formation between the first Fc domain monomer and the second Fc domain monomer. and a complementary dimerization selectivity module that selects between the nucleotides of At least one of the Fc domains (i) binds to one or more Fc receptors (ii) binding affinity to the antibody; (iii) effector function; (iv) level of sulfation of the Fc domain; (iv) half-life; (v) protease resistance; (vi) Fc domain stability; and / or vii) at least one amino acid that alters one or more of the following susceptibility to degradation: Including modifications, The Fc construct.

42. The Fc construct of claim 41, wherein the first polypeptide consists of an Fc domain monomer. Struct.

43. 43. The method of claim 41 or 42, wherein the second polypeptide consists of an Fc domain monomer. Fc construct.

44. 44. The composition of claim 41, further comprising an albumin binding peptide. c construct.

45. IgG C Ｌ Antibody constant domains and IgG C Ｈ 1 antibody constant domain, IgG C Ｈ an antibody constant domain is attached to the first polypeptide or 45. The method of claim 41, wherein the second polypeptide is linked to the N-terminus of the second polypeptide. Fc constructs as described above.

46. 2. The method of claim 1, wherein there are 14 or fewer amino acid modifications in at least one of the Fc domains.

46. ​​An Fc construct according to any one of claims 41 to 45.

47. below: a) a first polypeptide having the formula A-L-B, wherein i) A comprises a first Fc domain monomer; ii) L is a linker; iii) B comprises a second Fc domain monomer; the first polypeptide; b) a second polypeptide having the formula A'-L'-B', wherein: i) A' comprises a third Fc domain monomer; ii) L' is a linker; iii) B' comprises a fourth Fc domain monomer; The second polypeptide; An Fc construct comprising: the first Fc domain monomer and the second Fc domain monomer, C Ｈ and a third Fc domain monomer comprising an engineered cavity within said third Fc domain monomer and The fourth Fc domain monomers are Ｈ Within the three domains, engineered protrusions and the manipulated cavity and the manipulated protrusion form a pair in which the protrusion is inserted into the cavity. and wherein said first Fc domain monomer and said third Fc domain monomer are positioned so as to and the second Fc domain monomer binds to the fourth Fc domain monomer to form a first Fc domain. and an Fc domain monomer of the first Fc domain to form a second Fc domain; At least one of the Fc domains (i) binds to one or more Fc receptors (ii) binding affinity to the antibody; (iii) effector function; (iv) level of sulfation of the Fc domain; (iv) half-life; (v) protease resistance; (vi) Fc domain stability; and / or vii) at least one amino acid that alters one or more of the following susceptibility to degradation: Including modifications, The Fc construct.

48. Each of A, B, A', or B' consists of an Fc domain monomer.

48. The Fc construct of claim 47.

49. Further comprising an albumin-binding peptide linked to the C-terminus of B or B' via a linker.

49. An Fc construct according to claim 47 or 48.

50. IgG C Ｌ Antibody constant domains and IgG C Ｈ 1 antibody constant domain, IgG C Ｈ An antibody constant domain is linked to the N-terminus of A or A' via a linker.

50. The Fc construct of any one of claims 47 to 49,

51. 2. The method of claim 1, wherein there are 14 or fewer amino acid modifications in at least one of the Fc domains.

51. An Fc construct according to any one of claims 47 to 50.

52. The dimerization selectivity module is one of the C of the Fc domain monomer. Ｈ Within the three domains The engineered cavity and the other C of the Fc domain monomer Ｈ 3. Engineered protrusions within the domain wherein the engineered cavity and the engineered protrusion protrude into a cavity of an Fc domain monomer. are positioned to form an interdigitated pair. 9, 34, or 41. An Fc construct according to any one of claims 9, 34, or 41.

53. one of the Fc domain monomers comprises Y407V and Y349C, and the Fc domain The Fc construct of claim 44, wherein the other monomer comprises T366W and S354C. 。

54. The dimerization selectivity module is a C Ｈ Within the three domains A negatively charged amino acid and the other C of the Fc domain monomer. Ｈ Positive charge in three domains and an amino acid having a negative charge and a positive charge, 2, 3, 13, 14, 15, or 16, which is positioned to promote the formation of the c domain.

42. An Fc construct according to any one of claims 29, 34 or 41.

55. one of the Fc domain monomers comprises D399K and the other of the Fc domain monomers comprises 55. The Fc construct of claim 54, comprising K409D.

56. 6. The method of claim 1, wherein one or more linkers in the Fc construct are bonds.

5. An Fc construct according to any one of claims 1 to 4.

57. 1 , wherein one or more linkers in the Fc construct are spacers.

56. An Fc construct according to any one of claims 1 to 55.

58. The spacer comprises any one of SEQ ID NOs: 1 to 27.

7. The Fc construct according to claim 7.

59. 58. The method of claim 57, wherein the sequence is any one of SEQ ID NOs: 1, 2, and 3. Fc constructs as described above.

60. The albumin-binding peptide comprises the sequence of SEQ ID NO:

28.

50. An Fc construct described in any one of claims 37, 44 and 49.

61. 61. The albumin-binding peptide of claim 60, wherein the albumin-binding peptide consists of the sequence of SEQ ID NO:

28. c construct.

62. below: a) a first polypeptide having the formula A-L1-B-L2-C, wherein i) A comprises a first Fc domain monomer; ii) L1 is a linker; iii) B comprises a second Fc domain monomer; iv) L2 is a linker; v) C comprises a third Fc domain monomer; the first polypeptide; b) a second polypeptide having the formula A'-L1'-B'-L2'-C', 、 i) A' comprises a fourth Fc domain monomer; ii) L1′ is a linker; iii) B' comprises a fifth Fc domain monomer; iv) L2' is a linker; v) C' comprises a sixth Fc domain monomer; The second polypeptide; An Fc construct comprising: The first Fc domain monomer and the second Fc domain monomer are combined to form a first Fc domain monomer. forming a fourth Fc domain monomer and a fifth Fc domain monomer; combine to form a second Fc domain, and the third Fc domain monomer and the sixth F and a c domain monomer combine to form a third Fc domain, At least one of the Fc domains (i) binds to one or more Fc receptors (ii) binding affinity to the antibody; (iii) effector function; (iv) level of sulfation of the Fc domain; (iv) half-life; (v) protease resistance; (vi) Fc domain stability; and / or vii) at least one amino acid that alters one or more of the following susceptibility to degradation: Including modifications, The Fc construct.

63. The Fc construct of claim 62, wherein the linkers L1 and L1' are cleavable linkers. Tract.

64. 2. The method of claim 1, wherein there are 14 or fewer amino acid modifications in at least one of the Fc domains.

64. The Fc construct according to claim 62 or 63.

65. One or more of the Fc domain monomers may be an IgG hinge domain, an IgG C Ｈ 2 Antibody constant domains, and IgG C Ｈ 64. Any of claims 1 to 63 comprising three antibody constant domains. An Fc construct described in any one of claims 1 to 4.

66. Each of the Fc domain monomers comprises an IgG hinge domain, an IgG C Ｈ 2 Antibody constant Main, and IgG C Ｈ 66. The Fc construct of claim 65, comprising three antibody constant domains. Kut.

67. IgG is selected from the group consisting of IgG1, IgG2a, IgG2b, IgG3, and IgG4.

67. The Fc construct of claim 65 or 66, which is of a subtype selected from 。

68. 68. The Fc construct of any one of claims 1 to 67, having increased anti-inflammatory activity. Lacto.

69. 69. Any of claims 1 to 68, wherein each Fc domain monomer has 10 or fewer amino acid modifications. An Fc construct described in any one of claims 1 to 4.

70. One or more of the Fc domain monomers are human I having up to 10 amino acid modifications.

69. The Fc construct of any one of claims 1 to 68, which is a gG Fc domain monomer. Tract.

71. 71. A method for preparing an Fc construct according to any one of claims 1 to 70, comprising: 、 a) providing a host cell comprising a polynucleotide encoding said polypeptide; and, b) incubating said first antibody in said host cell under conditions that allow the formation of said Fc construct. expressing said polypeptide, said second polypeptide and said third polypeptide. and, c) recovering said Fc construct; The method comprising:

72. A host cell expressing an Fc construct according to any one of claims 1 to 70. wherein the host cell contains a polynucleotide encoding the polypeptide, The host cell, wherein a nucleotide is expressed within the host cell.

73. comprising a substantially homogenous population of Fc constructs having 2-10 Fc domains; Pharmaceutical compositions.

74. A plurality of the 2 to 10 Fc domains are capable of forming a dimer between two Fc domain monomers. -Fc-Domain Monomers Containing Complementary Dimerization Selectivity Modules Promoting Dimerization - Patent application 74. The pharmaceutical composition of claim 73, formed by the association of:

75. The medicament of claim 73, wherein the Fc construct has 2 to 3 Fc domains. composition.

76. A plurality of the two to three Fc domains are dimerized between two Fc domain monomers. The present invention relates to a method for the preparation of a dimerization selectivity module that is capable of forming a dimer of two Fc domain monomers.

76. The pharmaceutical composition of claim 75 formed by association.

77. The medicament of claim 73, wherein the Fc construct has 2 to 4 Fc domains. composition.

78. A plurality of the two to four Fc domains are dimerized between two Fc domain monomers. The present invention relates to a method for the preparation of a dimerization selectivity module that is capable of forming a dimer of two Fc domain monomers.

78. The pharmaceutical composition of claim 77 formed by association.

79. The pharmaceutical composition of claim 73, wherein the Fc construct has 5 to 10 Fc domains. Pharmaceutical composition.

80. A plurality of the 5 to 10 Fc domains are capable of forming a dimer between two Fc domain monomers. -Fc-Domain Monomers Containing Complementary Dimerization Selectivity Modules Promoting Dimerization - Patent application 80. The pharmaceutical composition of claim 79, formed by the association of:

81. 75. The method of claim 74, wherein two distinct polypeptides of the plurality have an identical amino acid sequence. The pharmaceutical composition described.

82. 75. The method of claim 74, wherein two distinct polypeptides of the plurality have different amino acid sequences. The pharmaceutical composition described.

83. 8. The complementary Fc monomer sequences have complementary dimerization selectivity modules.

5. The pharmaceutical composition described in 4.

84. The Fc construct has the following characteristics: a) at least one Fc domain monomer that is present in a different polypeptide; Partially formed, b) does not contain an additional domain that facilitates the association of the two polypeptides; c) Covalent bonds are formed only between the two Fc domain monomers that are linked to form the Fc domain. include, d) does not contain a covalent bond between the Fc domains; e) all or substantially all of the Fc domains in said Fc construct are expressed on the cell surface 1. Provide sufficient flexibility so that it can simultaneously interact with the above Fc receptors; f) comprising at least two Fc domains linked via a linker; 7. The method of claim 6, further comprising:

4. The pharmaceutical composition described in 3.

85. A substantially homogenous population of Fc constructs according to any one of claims 1 to 70. A pharmaceutical composition comprising:

86. 8. The substantially homogenous population of Fc constructs is at least 85% homogenous.

6. The pharmaceutical composition described in 5.

87. The Fc construct comprises 2 to 5 associated polypeptides, each polypeptide comprises at least one Fc domain monomer, and each Fc domain monomer of the construct 87. Any of claims 73 to 86, wherein the mers are identical or differ by no more than 10 amino acids. The pharmaceutical composition described in claim 1.

88. The Fc construct comprises two Fc domains comprising three associated polypeptides. one of the three polypeptides comprises two Fc domain monomers, each of the polypeptides of claim 73 comprises one Fc domain monomer.

87. A pharmaceutical composition according to any one of claims 1 to 86.

89. The Fc construct comprises three Fc domains comprising four associated polypeptides. wherein two of the four polypeptides each contain two Fc domain monomers. wherein two of the four polypeptides each contain one Fc domain monomer; 87. A pharmaceutical composition according to any one of claims 73 to 86.

90. The Fc construct comprises n Fc domains comprising 2n polypeptides. , each polypeptide is an Fc domain monomer, IgG C Ｌ Antibody constant domains, and IgG C Ｈ 87. The pharmaceutical composition of any one of claims 73 to 86, comprising one antibody constant domain. thing.

91. 71. A method of treating inflammation in a subject comprising administering to a subject a composition comprising the compound of claim 1 . The therapeutic use of the Fc construct or the pharmaceutical composition of any one of claims 73 to 90. The method comprises administering to the subject a pharmaceutical composition comprising an effective amount.

92. A method for reducing activation of an immune complex-based immune response in a subject, comprising: 1 to 58 or any one of claims 73 to 90. The method comprises administering to the subject a pharmaceutical composition described in any one of claims 1 to 4.

93. 93. The method of claim 92, wherein the subject has an autoimmune disease.

94. below: a) a first polypeptide having the formula A-L-B, wherein i) A comprises a first Fc domain monomer; ii) L is a linker; iii) B comprises a second Fc domain monomer; the first polypeptide; b) a second polypeptide having the formula A'-L'-B', wherein: i) A' comprises a third Fc domain monomer; ii) L' is a linker; iii) B' comprises a fourth Fc domain monomer; the second polypeptide; c) a third polypeptide comprising a fifth Fc domain monomer; d) a fourth polypeptide comprising a sixth Fc domain monomer; and An Fc construct consisting of: A and A' combine to form a first Fc domain, and B and the fifth Fc domain monomer and B' combine to form a second Fc domain monomer, and B' combines with the sixth Fc domain monomer to form a second Fc domain monomer. combine to form a third Fc domain; each of the first Fc domain monomer and the third Fc domain monomer Promoting dimer formation between the first Fc domain monomer and the third Fc domain monomer. and a complementary dimerization selectivity module that each of the second Fc domain monomer and the fifth Fc domain monomer Promoting dimer formation between the second Fc domain monomer and the fifth Fc domain monomer. and a complementary dimerization selectivity module that each of the fourth Fc domain monomer and the sixth Fc domain monomer Promoting dimer formation between the fourth Fc domain monomer and the sixth Fc domain monomer. and a complementary dimerization selectivity module that the Fc construct contains no more than three Fc domains; At least one of the Fc domains (i) binds to one or more Fc receptors (ii) binding affinity to the antibody; (iii) effector function; (iv) level of sulfation of the Fc domain; (iv) half-life; (v) protease resistance; (vi) Fc domain stability; and / or vii) at least one amino acid that alters one or more of the following susceptibility to degradation: Including modifications, The Fc construct.

95. below: a) a first polypeptide having the formula A-L-B, wherein i) A consists of a first Fc domain monomer; ii) L is a linker; iii) B consists of a second Fc domain monomer; the first polypeptide; b) a second polypeptide having the formula A'-L'-B', wherein: i) A' consists of a third Fc domain monomer; ii) L' is a linker; iii) B' consists of a fourth Fc domain monomer; the second polypeptide; c) a third polypeptide consisting of a fifth Fc domain monomer; d) a fourth polypeptide consisting of a sixth Fc domain monomer; and An Fc construct consisting of: A and A' combine to form a first Fc domain, and B and the fifth Fc domain monomer and B' combine to form a second Fc domain monomer, and B' combines with the sixth Fc domain monomer to form a second Fc domain monomer. combine to form a third Fc domain; each of the first Fc domain monomer and the third Fc domain monomer Promoting dimer formation between the first Fc domain monomer and the third Fc domain monomer. and a complementary dimerization selectivity module that each of the second Fc domain monomer and the fifth Fc domain monomer Promoting dimer formation between the second Fc domain monomer and the fifth Fc domain monomer. and a complementary dimerization selectivity module that each of the fourth Fc domain monomer and the sixth Fc domain monomer Promoting dimer formation between the fourth Fc domain monomer and the sixth Fc domain monomer. and a complementary dimerization selectivity module that the Fc construct contains no more than three Fc domains; At least one of the Fc domains (i) binds to one or more Fc receptors (ii) binding affinity to the antibody; (iii) effector function; (iv) level of sulfation of the Fc domain; (iv) half-life; (v) protease resistance; (vi) Fc domain stability; and / or vii) at least one amino acid that alters one or more of the following susceptibility to degradation: Including modifications, The Fc construct.

96. The complementary dimerization selectivity module of the first Fc domain monomer has a negative charge. and a complementary dimerization selectivity module of said third Fc domain monomer. The second Fc domain monomer comprises a positively charged amino acid substitution and a complementary The dimerization selectivity module comprises an engineered protrusion, and the fifth Fc domain monomer The complementary dimerization selectivity module of the fourth Fc domain comprises an engineered cavity. The complementary dimerization selectivity module of the in monomer comprises an engineered protuberance, and the sixth The complementary dimerization selectivity module of the Fc domain monomer of the present invention contains an engineered cavity.

96. An Fc construct according to claim 94 or 95.

97. 97. The method according to claim 94, wherein the linker L and / or L' is 3 to 200 amino acids in length. An Fc construct described in any one of claims 1 to 4.

98. The linker L and / or L' is any one of SEQ ID NOs: 1, 2, and 3.

97. The Fc construct of claim 96, comprising:

99. below: a) a first polypeptide having the formula A-L1-B-L2-C, wherein i) A comprises a first Fc domain monomer; ii) L1 is a linker; iii) B comprises a second Fc domain monomer; iv) L2 is a linker; v) C comprises a third Fc domain monomer; the first polypeptide; b) a second polypeptide having the formula A'-L1'-B'-L2'-C', Inside, i) A' comprises a fourth Fc domain monomer; ii) L1′ is a linker; iii) B' comprises a fifth Fc domain monomer; iv) L2' is a linker; v) C' comprises a sixth Fc domain monomer; the second polypeptide; c) a third polypeptide comprising a seventh Fc domain monomer; d) a fourth polypeptide comprising an eighth Fc domain monomer; e) a fifth polypeptide comprising a ninth Fc domain monomer; d) a sixth polypeptide comprising a tenth Fc domain monomer; and An Fc construct consisting of: A and the seventh Fc domain monomer combine to form a first Fc domain, and B and B ' binds to form a second Fc domain, and C binds to the eighth Fc domain monomer. A' combines with the ninth Fc domain monomer to form a third Fc domain, and A' combines with the ninth Fc domain monomer to form a third Fc domain. forming a fourth Fc domain, and C′ and the tenth Fc domain monomer combine to form a fifth Fc domain. Forming an Fc domain, each of the first Fc domain monomer and the seventh Fc domain monomer Promoting dimer formation between the first Fc domain monomer and the seventh Fc domain monomer. and a complementary dimerization selectivity module that each of the second Fc domain monomer and the fifth Fc domain monomer Promoting dimer formation between the second Fc domain monomer and the fifth Fc domain monomer. and a complementary dimerization selectivity module that each of the third Fc domain monomer and the eighth Fc domain monomer Promoting dimer formation between the third Fc domain monomer and the eighth Fc domain monomer. and a complementary dimerization selectivity module that each of the fourth Fc domain monomer and the ninth Fc domain monomer Promoting dimer formation between the fourth Fc domain monomer and the ninth Fc domain monomer. and a complementary dimerization selectivity module that each of the sixth Fc domain monomer and the tenth Fc domain monomer Promoting dimer formation between the sixth Fc domain monomer and the tenth Fc domain monomer. and a complementary dimerization selectivity module that the Fc construct contains five or fewer Fc domains; At least one of the Fc domains (i) binds to one or more Fc receptors (ii) binding affinity to the antibody; (iii) effector function; (iv) level of sulfation of the Fc domain; (iv) half-life; (v) protease resistance; (vi) Fc domain stability; and / or vii) at least one amino acid that alters one or more of the following susceptibility to degradation: Including modifications, The Fc construct.

100. below: a) a first polypeptide having the formula A-L1-B-L2-C, wherein i) A consists of a first Fc domain monomer; ii) L1 is a linker; iii) B consists of a second Fc domain monomer; iv) L2 is a linker; v) C consists of a third Fc domain monomer; the first polypeptide; b) a second polypeptide having the formula A'-L1'-B'-L2'-C', Inside, i) A' consists of a fourth Fc domain monomer; ii) L1′ is a linker; iii) B' consists of a fifth Fc domain monomer; iv) L2' is a linker; v) C' consists of a sixth Fc domain monomer; the second polypeptide; c) a third polypeptide consisting of a seventh Fc domain monomer; d) a fourth polypeptide consisting of an eighth Fc domain monomer; e) a fifth polypeptide consisting of a ninth Fc domain monomer; d) a sixth polypeptide consisting of a tenth Fc domain monomer; and An Fc construct consisting of: A and the seventh Fc domain monomer combine to form a first Fc domain, and B and B ' binds to form a second Fc domain, and C binds to the eighth Fc domain monomer. A' combines with the ninth Fc domain monomer to form a third Fc domain, and A' combines with the ninth Fc domain monomer to form a third Fc domain. forming a fourth Fc domain, and C′ and the tenth Fc domain monomer combine to form a fifth Fc domain. Forming an Fc domain, each of the first Fc domain monomer and the seventh Fc domain monomer Promoting dimer formation between the first Fc domain monomer and the seventh Fc domain monomer. and a complementary dimerization selectivity module that each of the second Fc domain monomer and the fifth Fc domain monomer Promoting dimer formation between the second Fc domain monomer and the fifth Fc domain monomer. and a complementary dimerization selectivity module that each of the third Fc domain monomer and the eighth Fc domain monomer Promoting dimer formation between the third Fc domain monomer and the eighth Fc domain monomer. and a complementary dimerization selectivity module that each of the fourth Fc domain monomer and the ninth Fc domain monomer Promoting dimer formation between the fourth Fc domain monomer and the ninth Fc domain monomer. and a complementary dimerization selectivity module that each of the sixth Fc domain monomer and the tenth Fc domain monomer Promoting dimer formation between the sixth Fc domain monomer and the tenth Fc domain monomer. and a complementary dimerization selectivity module that the Fc construct contains five or fewer Fc domains; At least one of the Fc domains (i) binds to one or more Fc receptors (ii) binding affinity to the antibody; (iii) effector function; (iv) level of sulfation of the Fc domain; (iv) half-life; (v) protease resistance; (vi) Fc domain stability; and / or vii) at least one amino acid that alters one or more of the following susceptibility to degradation: Including modifications, The Fc construct.

101. The complementary dimerization selectivity module of the second Fc domain monomer has a negative charge. and a complementary dimerization selectivity module of the fifth Fc domain monomer. The first Fc domain monomer, the third Fc domain monomer, and the third Fc domain monomer each contain a positively charged amino acid substitution. the first Fc domain monomer, the fourth Fc domain monomer, and the sixth Fc domain monomer. each complementary dimerization selectivity module of said dimers comprises an engineered protuberance; a seventh Fc domain monomer, an eighth Fc domain monomer, a ninth Fc domain monomer, and a complementary dimerization selectivity module of each of the tenth Fc domain monomers.

101. The Fc construct of claim 99 or 100, wherein the molecule comprises an engineered cavity.

102. below: a) a first polypeptide having the formula A-L1-B-L2-C, wherein i) A comprises a first Fc domain monomer; ii) L1 is a linker; iii) B comprises a second Fc domain monomer; iv) L2 is a linker; v) C comprises a third Fc domain monomer; the first polypeptide; b) a second polypeptide having the formula A'-L1'-B'-L2'-C', Inside, i) A' comprises a fourth Fc domain monomer; ii) L1′ is a linker; iii) B' comprises a fifth Fc domain monomer; iv) L2' is a linker; v) C' comprises a sixth Fc domain monomer; the second polypeptide; c) a third polypeptide comprising a seventh Fc domain monomer; d) a fourth polypeptide comprising an eighth Fc domain monomer; e) a fifth polypeptide comprising a ninth Fc domain monomer; d) a sixth polypeptide comprising a tenth Fc domain monomer; and An Fc construct consisting of: A and A' combine to form a first Fc domain, and B and the seventh Fc domain monomer and C combine to form the second Fc domain, and C combines with the eighth Fc domain monomer. B' combines with the ninth Fc domain monomer to form a third Fc domain, and B' combines with the ninth Fc domain monomer to form a third Fc domain. forming a fourth Fc domain, and C′ and the tenth Fc domain monomer combine to form a fifth Fc domain. Forming an Fc domain, each of the first Fc domain monomer and the fourth Fc domain monomer Promoting dimer formation between the first Fc domain monomer and the fourth Fc domain monomer. and a complementary dimerization selectivity module that each of the second Fc domain monomer and the seventh Fc domain monomer Promoting dimer formation between the second Fc domain monomer and the seventh Fc domain monomer. and a complementary dimerization selectivity module that each of the third Fc domain monomer and the eighth Fc domain monomer Promoting dimer formation between the third Fc domain monomer and the eighth Fc domain monomer. and a complementary dimerization selectivity module that each of the fifth Fc domain monomer and the ninth Fc domain monomer Promoting dimer formation between the fifth Fc domain monomer and the ninth Fc domain monomer. and a complementary dimerization selectivity module that each of the sixth Fc domain monomer and the tenth Fc domain monomer Promoting dimer formation between the sixth Fc domain monomer and the tenth Fc domain monomer. and a complementary dimerization selectivity module that the Fc construct contains five or fewer Fc domains; At least one of the Fc domains (i) binds to one or more Fc receptors (ii) binding affinity to the antibody; (iii) effector function; (iv) level of sulfation of the Fc domain; (iv) half-life; (v) protease resistance; (vi) Fc domain stability; and / or vii) at least one amino acid that alters one or more of the following susceptibility to degradation: Including modifications, The Fc construct.

103. below: a) a first polypeptide having the formula A-L1-B-L2-C, wherein i) A consists of a first Fc domain monomer; ii) L1 is a linker; iii) B consists of a second Fc domain monomer; iv) L2 is a linker; v) C consists of a third Fc domain monomer; the first polypeptide; b) a second polypeptide having the formula A'-L1'-B'-L2'-C', Inside, i) A' consists of a fourth Fc domain monomer; ii) L1′ is a linker; iii) B' consists of a fifth Fc domain monomer; iv) L2' is a linker; v) C' consists of a sixth Fc domain monomer; the second polypeptide; c) a third polypeptide consisting of a seventh Fc domain monomer; d) a fourth polypeptide consisting of an eighth Fc domain monomer; e) a fifth polypeptide consisting of a ninth Fc domain monomer; d) a sixth polypeptide consisting of a tenth Fc domain monomer; and An Fc construct consisting of: A and A' combine to form a first Fc domain, and B and the seventh Fc domain monomer and C combine to form the second Fc domain, and C combines with the eighth Fc domain monomer. B' combines with the ninth Fc domain monomer to form a third Fc domain, and B' combines with the ninth Fc domain monomer to form a third Fc domain. forming a fourth Fc domain, and C′ and the tenth Fc domain monomer combine to form a fifth Fc domain. Forming an Fc domain, each of the first Fc domain monomer and the fourth Fc domain monomer Promoting dimer formation between the first Fc domain monomer and the fourth Fc domain monomer. and a complementary dimerization selectivity module that each of the second Fc domain monomer and the seventh Fc domain monomer Promoting dimer formation between the second Fc domain monomer and the seventh Fc domain monomer. and a complementary dimerization selectivity module that each of the third Fc domain monomer and the eighth Fc domain monomer Promoting dimer formation between the third Fc domain monomer and the eighth Fc domain monomer. and a complementary dimerization selectivity module that each of the fifth Fc domain monomer and the ninth Fc domain monomer Promoting dimer formation between the fifth Fc domain monomer and the ninth Fc domain monomer. and a complementary dimerization selectivity module that each of the sixth Fc domain monomer and the tenth Fc domain monomer Promoting dimer formation between the sixth Fc domain monomer and the tenth Fc domain monomer. and a complementary dimerization selectivity module that the Fc construct contains five or fewer Fc domains; At least one of the Fc domains (i) binds to one or more Fc receptors (ii) binding affinity to the antibody; (iii) effector function; (iv) level of sulfation of the Fc domain; (iv) half-life; (v) protease resistance; (vi) Fc domain stability; and / or vii) at least one amino acid that alters one or more of the following susceptibility to degradation: Including modifications, The Fc construct.

104. The complementary dimerization selectivity module of the first Fc domain monomer has a negative charge. and a complementary dimerization selectivity module of said fourth Fc domain monomer. the second Fc domain monomer, the third Fc domain monomer, the first Fc domain monomer, the fifth Fc domain monomer, and the sixth Fc domain monomer. each complementary dimerization selectivity module of said dimers comprises an engineered protuberance; a seventh Fc domain monomer, an eighth Fc domain monomer, a ninth Fc domain monomer, and a complementary dimerization selectivity module of each of the tenth Fc domain monomers.

104. The Fc construct of claim 102 or 103, wherein the molecule comprises an engineered cavity.

105. the linkers L1, L2, L1', and / or L2' are 3 to 200 amino acids in length; 105. An Fc construct according to any one of claims 99 to 104.

106. The linkers L1, L2, L1', and / or L2' are selected from the group consisting of SEQ ID NOs: 1, 2, and 3. The Fc construct of claim 105, comprising any one of the sequences:

107. Promoting clearance of autoantibodies and enhancing antigen presentation in a subject in need thereof. suppressing, attenuating an immune response, or inhibiting the activation of immune complex-based immune responses Any of claims 1 to 70 or 94 to 106 for use in reducing An Fc construct according to any one of the preceding claims.

108. Inflammatory or autoimmune diseases or immune disorders (e.g., rheumatoid arthritis (RA); systemic endothelial cell carcinoma (ESC)); Systemic lupus erythematosus (SLE); ANCA-associated vasculitis; antiphospholipid syndrome; autoimmune lytic disorder Hemolytic anemia; chronic inflammatory demyelinating neuropathy; anti-allo ), anti-self in GVHD, anti-replacement, IgG therapy, IgG para Protein clearance; Dermatomyositis; Goodpasture's syndrome; Antibody-dependent cell-mediated Type II hypersensitivity syndromes targeting organ systems mediated by cytotoxicity, e.g., Guillain-Barre syndrome – syndrome, CIDP, dermatomyositis, Felty syndrome, antibody-mediated rejection, autoimmune ulcerative colitis, autoimmune liver disease; idiopathic thrombocytopenic purpura; myasthenia gravis , neuromyelitis optica; pemphigus and other autoimmune blistering diseases; Sjogren's syndrome; antibody-dependent Autoimmune cytopenias and other disorders mediated by endothelial phagocytosis; e.g., synovial Other FcR-dependent inflammatory syndromes that are vasculitis, dermatomyositis, systemic vasculitis, glomerulitis, or vasculitis.

20. The method of claim 1 for use in treating An Fc construct described in any one of claims 1 to 4.

109. The amino acid modifications that alter the binding affinity to one or more Fc receptors are those in Table 2. any one of claims 1, 12, 28, 33, 39, 41, 47, 62, 94, 95, 99, 100, 102, and 103. Struct.

110. The amino acid modification that alters binding affinity to one or more Fc receptors is S267E / L328F, and said modification increases binding affinity to FcγRIIb.

107. The Fc construct according to item 106.

111. The amino acid modification that changes the effector function is any one of the amino acid modifications in Table 6. Claims 1, 12, 28, 33, 39, 41, 47, 62, 94, 95, 99, 10 104. The Fc construct of any one of claims 0, 102 and 103.

112. Amino acid modifications that alter the level of sulfation in the Fc domain include 241F, 243F, 246 Claims 1, 12, 28, 33, 39, 41, 47, 62, 94, 95, 99, 100, 102, and 103. Struct.

113. The amino acid modification that alters the half-life is any one of the amino acid modifications in Table 4. Request 1, 12, 28, 33, 39, 41, 47, 62, 94, 95, 99, 100, 10 2 and 103. An Fc construct described in any one of claims 2 and 103.

114. The amino acid modifications that alter protease resistance were the following set: 233P, 234V, 23 5A, and 236del; 237A, 239D, and 332E; 237D, 239D, and and 332E; 237P, 239D, and 332E; 237Q, 239D, and 332E; 237S, 239D, and 332E; 239D, 268F, 324T, and 332E; 39D, 326A, and 333A; 239D and 332E; 243L, 292P, and 3 00L; 267E, 268F, 324T, and 332E; 267E and 332E; 268 F, 324T, and 332E; 326A, 332E, and 333A; and 326A and 333A. 94, 95, 99, 100, 102, and 103. Lacto.

115. The amino acid modification that alters the Fc domain stability is any one of the amino acid modifications in Table 8. Claims 1, 12, 28, 33, 39, 41, 47, 62, 94, 95, 99, 104. An Fc construct according to any one of claims 100, 102 and 103.

116. Amino acid modifications that alter the susceptibility of the Fc domain to degradation include C233X, D234X, , K235X, S236X, T236X, H237X, C239X, S241X, and G 249X, where X is any amino acid. , 47, 62, 94, 95, 99, 100, 102, and 103. Fc construct.

117. below: a) a first polypeptide having the formula A-L-B, wherein i) A comprises a first Fc domain monomer; ii) L is a linker; iii) B comprises a second Fc domain monomer; the first polypeptide; b) a second polypeptide having the formula A'-L'-B', wherein: i) A' comprises a third Fc domain monomer; ii) L' is a linker; iii) B' comprises a fourth Fc domain monomer; the second polypeptide; c) a third polypeptide comprising a fifth Fc domain monomer; d) a fourth polypeptide comprising a sixth Fc domain monomer; and An Fc construct comprising: A of the first polypeptide and A' of the second polypeptide bind to form a first Fc A domain is formed, and B of the first polypeptide and the fifth Fc domain monomer are linked. and combining the second polypeptide with the sixth Fc domain to form a second Fc domain. and the main monomer combine to form a third Fc domain, The first polypeptide and the second polypeptide comprise the sequence of SEQ ID NO:

50. wherein the third polypeptide and the fourth polypeptide comprise the sequence of SEQ ID NO:

48. 、 The Fc construct.

118. The first polypeptide and the second polypeptide have up to 10 single amino acid modifications. and wherein the third polypeptide and the fourth polypeptide comprise a sequence of SEQ ID NO: 50 having a sequence 118. The method of claim 117, comprising the sequence of SEQ ID NO: 48 with up to 10 single amino acid modifications. Fc construct.

119. One or more of the Fc domain monomers is an Fc an IgG1 Fc domain monomer.

118. The method of claim 1, wherein the compound is a carboxylate. Fc constructs as described.

Images