Compositions and methods relating to engineered Fc constructs

Abstract

Engineered Fc constructs, methods for their preparation and pharmaceutical compositions comprising the Fc constructs are provided. [Solution] The Fc constructs comprise one or more Fc domains. Such constructs can have a desired serum half-life and / or binding affinity and / or avidity for Fc receptors. These constructs are useful for treating, for example, reducing inflammation in a subject, promoting clearance of autoantibodies in a subject, suppressing antigen presentation in a subject, and inhibiting an immune response. The Fc constructs can be used to treat patients with immune and inflammatory diseases without significantly stimulating immune cells.

Description

[Background technology]

[0001] background Therapeutic proteins, such as therapeutic antibodies and Fc fusion proteins, are rapidly becoming a treatment for immune diseases and These drugs have become a clinically important class for patients with chronic and inflammatory diseases. Summary of the Invention

[0002] The present invention features biologically active Fc domain-containing therapeutic constructs. The construct may have a desired serum half-life and / or binding affinity and / or binding to Fc receptors. These constructs can have a binding activity, for example, to reduce inflammation in a subject, to block autoantibodies in a subject, Promoting clearance, suppressing antigen presentation in a subject, inhibiting an immune response in a subject, e.g. and inhibiting immune complex activation of the immune system and treating immune and inflammatory diseases in a subject (e.g., autoimmune diseases, The Fc constructs described herein are useful for treating autoimmune diseases. The present invention can be used to treat patients with immune and inflammatory diseases without the need for additional treatment.

[0003] Generally, the present invention provides Fc constructs having 2-10 Fc domains, e.g., 2, 3, 4, 5 The present invention features Fc constructs having one, six, seven, eight, nine, or ten Fc domains. In embodiments, the Fc construct comprises 2-10 Fc domains, 2-5 Fc domains, 3-5 Fc domains, 2- The constructs may contain 2 to 6 (e.g., 2, 3, 4 Each polypeptide may comprise at least one associated polypeptide. and each Fc domain monomer of the construct is identical to another monomer of the construct. or 20 or fewer amino acids (e.g., 15 or fewer, 10 or fewer amino acids), e.g., 20 or fewer, 15 Less than or equal to 10, Less than or equal to 8, Less than or equal to 7, Less than or equal to 6, Less than or equal to 5, Less than or equal to 4, Less than or equal to 3, or Less than or equal to 2 The following amino acids differ: In some embodiments, the Fc construct (or the Fc domains within the Fc construct) do not include different Formed in whole or in part by association of Fc domain monomers present in a given polypeptide In certain embodiments, the Fc construct contains an additional domain ( In other embodiments, the covalent bond does not include an Fc domain (e.g., an IgM tail or an IgA tail). In another embodiment, the Fc domain is present only between two Fc domain monomers of the Fc construct that form the main chain. In yet another embodiment, the Fc construct comprises: Provide sufficient structural flexibility so that all or substantially all of the Fc domains of the Fc construct can be In some embodiments, the Fc constructs can simultaneously interact with Fc receptors on the cell surface. The entity may comprise at least two amino acid residues joined by a linker (e.g., a flexible amino acid spacer). In one embodiment, the domain monomer comprises a dimerization selectivity module. The primary sequences differ from the wild type and from each other in that

[0004] The Fc constructs of the invention comprise an Fc domain having 2 to 10 Fc domains as described herein. c constructs, e.g., 2, 3, 4, 5, 6, 7, 8, 9, or 10 Fc domains Constructs having, e.g., a substantially homogenous population of constructs (e.g., at least 85%, 90%, The resulting pharmaceutical composition may be comprised of a pharmaceutical composition having a homogeneity of 95%, 98%, or 99%. to produce a pharmaceutical composition that does not have substantial aggregation or undesirable multimerization of the Fc construct. It is possible.

[0005] In one aspect, the Fc construct comprises three polypeptides forming two Fc domains. The polypeptide has the formula ALB, where A comprises a first Fc domain monomer; L comprises a linker. and B comprises a second Fc domain monomer. The second polypeptide comprises a third Fc domain monomer. In this aspect, the third polypeptide comprises a fourth Fc domain monomer. The first Fc domain monomer and the third Fc domain monomer combine to form the first Fc domain. Similarly, the second Fc domain monomer and the fourth Fc domain monomer combine to form the second Fc domain. Exemplary Fc constructs according to this aspect of the invention are illustrated in FIG. .

[0006] In certain embodiments, the first Fc domain monomer and the third Fc domain monomer are The domains each contain a complementary dimerization selectivity module that promotes dimerization between monomers. In the same manner, the second Fc domain monomer and the fourth Fc domain monomer are The dimers each contain a complementary dimerization selectivity module that promotes dimerization between the dimers.

[0007] In certain embodiments, one or more of A, B, the second polypeptide, and the third polypeptide. In one embodiment, the number is comprised of an Fc domain monomer. Each of the polypeptides consists of an Fc domain monomer.

[0008] In certain embodiments, the Fc construct comprises a heterologous moiety, e.g., a peptide, e.g., a serum protein. For example, the moiety may further comprise a peptide that binds to an albumin binding peptide. linked, e.g., by a linker, to the N-terminus or carboxy-terminus of B or a third polypeptide It is possible.

[0009] In a particular embodiment, the Fc construct comprises an IgG L Antibody constant domains and IgG C H 1. Antibody constant domain IgG C H One antibody constant domain can be linked to A or a second polypeptide, for example by a linker. The polypeptide may be attached to the N-terminus of the polypeptide.

[0010] In other embodiments, the second and third polypeptides of the Fc construct have the same amino acid sequence. .

[0011] In another aspect, the present invention provides an Fc construct comprising four polypeptides forming three Fc domains. The first polypeptide has the formula ALB, where A is a first Fc domain monomer. L is a linker; and B comprises a second Fc domain monomer. The peptide has the formula A'-L'-B', where A' comprises the third Fc domain monomer; L' is a linker. and B' comprises a fourth Fc domain monomer. The third polypeptide comprises a fifth Fc domain monomer. The fourth polypeptide comprises a sixth Fc domain monomer, and the fourth polypeptide comprises a sixth Fc domain monomer. In the Fc domain, A and A' combine to form the first Fc domain, and B and the fifth Fc domain monomer combine to form the third Fc domain. 2 Fc domain, and B' and the sixth Fc domain monomer combine to form the third Fc domain. An exemplary Fc construct according to this aspect of the invention is illustrated in FIG.

[0012] In certain embodiments, A and A' each promote dimerization between these Fc domain monomers. In another embodiment, the Fc domain monomer comprises a dimerization selectivity module that selectively induces a dimerization selectivity of the Fc domain. Each of these Fc domains contains a dimerization selectivity module that promotes dimerization between these Fc domain monomers. In yet other embodiments, the B' and sixth Fc domain monomers each comprise one of these Fc domain monomers. It contains a dimerization selectivity module that promotes dimerization between c domain monomers.

[0013] In certain embodiments, one of A, B, A', B', a third polypeptide, and a fourth polypeptide. or more than one comprises an Fc domain monomer. In one embodiment, the A, B, A', B', third polypeptide Each of the first polypeptide, the second polypeptide, and the fourth polypeptide consists of an Fc domain monomer.

[0014] In a particular embodiment, the Fc construct comprises an IgG L Antibody constant domains and IgG C H 1. Antibody constant domain IgG C L The antibody constant domains are linked to the IgG H 1. Antibody constant domain IgG C H 1. The antibody constant domain is connected to the N-terminus of A by, for example, a linker. In one embodiment, the Fc construct comprises a second IgG C L Antibody constant domain and second IgG C H 1 antibody constant domain and a second IgG C L Antibody constant domains include, for example, Second IgG C by Carr H 1 antibody constant domain and a second IgG C H 1 antibody The constant domain is attached to the N-terminus of A', for example by a linker.

[0015] In certain embodiments, the Fc construct is linked to a heterologous moiety, e.g., a peptide, e.g., a linker. It further comprises an albumin binding peptide conjugated to the N-terminus or C-terminus of B or B'.

[0016] 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.

[0017] In another aspect, the invention features an Fc construct that includes two polypeptides. The peptide has the formula ALB, where A comprises a first Fc domain monomer; L is a linker. and B comprises a serum protein binding moiety, e.g., an albumin binding peptide. The polypeptide comprises a second Fc domain monomer. In this aspect, the first Fc domain monomer and a second Fc domain monomer combine to form an Fc domain.

[0018] In certain embodiments, the first Fc domain monomer and the second Fc domain monomer are Complementary dimerization selectivity models that promote dimerization between the main monomer and the second Fc domain monomer. Includes Joules.

[0019] In certain embodiments, A and the second polypeptide each consist of an Fc domain monomer.

[0020] In yet another aspect, the invention features an Fc construct that includes two polypeptides. The polypeptide has the formula A-L1-B-L2-C, where A is IgG C L Contains the antibody constant domain; L1 and L2 are linkers; B is IgG C H 1 antibody constant domain; and C is and a first Fc domain monomer. The second polypeptide has the formula A'-L1'-B'-L2'-C' , A' is IgG C L each of L1' and L2' is a linker; B' IgG C H C' comprises a first antibody constant domain monomer; and C' comprises a second Fc domain monomer. In this case, a first Fc domain monomer and a second Fc domain monomer combine to form an Fc domain. An exemplary Fc construct according to this aspect of the invention is illustrated in Figure 7A.

[0021] In certain embodiments, the first Fc domain monomer and the second Fc domain monomer are A dimerization selectivity module that promotes dimerization between the main monomer and the second Fc domain monomer. Includes rules.

[0022] In certain embodiments, C and C' each consist of an Fc domain monomer.

[0023] In certain embodiments, the Fc construct is linked to a serum protein binding moiety, e.g., C, by a linker. or C'.

[0024] In yet another aspect, the invention features an Fc construct comprising four or more polypeptides. The first polypeptide has the formula A-L1-B-L2-C, where A is IgG C L Contains antibody constant domains L1 and L2 are linkers; B is IgG C H 1 antibody constant domain; and C comprises a first Fc domain monomer. The second polypeptide has the formula A'-L1'-B'-L2'-C' A' is IgG C L antibody constant domain; L1' and L2' are each a linker B' is IgG C H C' comprises one antibody constant domain; and C' comprises the second Fc domain monomer. In this aspect, the first Fc domain monomer is combined with a third Fc domain monomer to form the first Fc domain. and the second Fc domain monomer binds to a fourth Fc domain monomer to form a second Fc domain. In addition, the first polypeptide forms an IgG C domain. H 1 antibody constant domain is a second Polypeptide IgG C L The second polypeptide, IgG C, is linked to an antibody constant domain. H 1. Antibody Stationary The domain is an IgG C domain of the first polypeptide. L Combined with antibody constant domains, two or more Fc domains An exemplary Fc construct according to this aspect of the invention is illustrated in FIG. It is being done.

[0025] In another aspect, the invention features an Fc construct that includes two polypeptides. The peptide comprises a first Fc domain monomer and the second polypeptide comprises a second Fc domain monomer. In this aspect, a first and a second Fc domain monomer are combined to form an Fc domain. An exemplary Fc construct according to this aspect of the invention is illustrated in FIG. The first Fc domain monomer and the second Fc domain monomer each include a first Fc domain A dimerization selectivity module that promotes dimerization between the monomer and a second Fc domain monomer. Exemplary Fc constructs of this embodiment are illustrated in Figures 2 and 3.

[0026] In certain embodiments, the first and second polypeptides each consist of an Fc domain monomer. .

[0027] In certain embodiments, the Fc construct comprises a serum protein binding moiety, e.g., a The albumin-binding peptide is attached to the N-terminus or C-terminus of the first or second polypeptide. It further includes

[0028] In another aspect, the invention features an Fc construct that includes two polypeptides. The peptide has the formula ALB, where A comprises a first Fc domain monomer; L is a linker. and B comprises a second Fc domain monomer. The second polypeptide has the formula A'-L'-B'. wherein A' comprises a third Fc domain monomer; L' is a linker; and B' comprises a fourth Fc domain monomer. In this aspect, the first and second Fc domain monomers each comprise Each C H 3. comprising an engineered recess in the antibody constant domain and a second and Each of the four Fc domain monomers is H 3. Engineering Antibody Constant Domains engineered recesses and engineered protrusions The portions are arranged to form protrusion and recess pairs, with the protrusions fitting into the recesses. In one aspect, 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 and the fourth Fc domain monomer combine to form a second Fc domain. Form.

[0029] In certain embodiments, one or more of A, B, A', and B' consist of an Fc domain monomer. In one embodiment, each of A, B, A', and B' consists of an Fc domain monomer.

[0030] In certain embodiments, the Fc construct comprises a serum protein binding moiety, e.g., a and further comprising an albumin binding peptide joined to the N-terminus or C-terminus of B or B'.

[0031] In a particular embodiment, the Fc construct comprises an IgG L Antibody constant domains and IgG C H 1. Antibody constant domain IgG C L The antibody constant domains are linked to the IgG C H 1. Antibody Stationary Attached to the N-terminus of the domain and IgG C H 1. The antibody constant domain is connected to the N-terminus of A by a linker. In one embodiment, the Fc construct comprises a second IgG C L Antibody constant domain and second IgG C H 1 antibody constant domain and a second IgG C L The antibody constant domain is Therefore, the second IgG C H 1 antibody constant domain and a second IgG C H 1. Antibody Constant The main is attached to the N-terminus of A' by a linker.

[0032] In another aspect, the invention provides (a) a first polypeptide having the formula ALB; comprising or consisting of a first Fc domain monomer; L is a linker and B comprises or consists of a second Fc domain monomer. (b) a second polypeptide having the formula A'-L'-B', wherein A' is L' comprises or consists of a third Fc domain monomer; L' is a linker and B' comprises or is composed of a fourth Fc domain monomer. (c) a second polypeptide comprising a fifth Fc domain monomer or a fifth Fc domain. (d) a third polypeptide consisting of a sixth Fc domain monomer; or and an Fc construct comprising a fourth polypeptide comprising a sixth Fc domain monomer. A of the polypeptide and A' of the second polypeptide combine to form a first Fc domain; B of the polypeptide and the fifth Fc domain monomer combine to form a second Fc domain; and The B' of the polypeptide and the sixth Fc domain monomer combine to form the third Fc domain. Each of the first and third Fc domain monomers comprises a first Fc domain monomer and a third Fc domain monomer. and a complementary dimerization selectivity module that promotes dimerization between the second and Each of the fifth Fc domain monomers is a second Fc domain monomer and a fifth Fc domain monomer. and a fourth and sixth Fc Each of the domain monomers is between a fourth Fc domain monomer and a sixth Fc domain monomer. and a complementary dimerization selectivity module that promotes dimerization; the Fc construct comprises three or fewer Fc Includes the domain.

[0033] In some embodiments of this aspect, either 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 Fc domain monomer contains a positively charged The second and fourth Fc domain monomers or the fifth and sixth Fc domain monomers each contain an amino acid substitution selected from the group consisting of One of the monomers contains an engineered protrusion, and the other Fc domain The monomer comprises an engineered recess. In some embodiments, the linkers L1, L2, L1' and / or L2' are 3 to 200 amino acids in length. and / or L' consists of any one of SEQ ID NOs: 1, 2, and 3.

[0034] In another aspect, the invention provides: (a) a first polypeptide having the formula A-L1-B-L2-C. A comprises or consists of a first Fc domain monomer; L1 is , a linker; B comprises a second Fc domain monomer, or L2 is a linker; and C comprises a third Fc domain monomer, or a third and (b) a first polypeptide consisting of an Fc domain monomer of the formula A'-L1'-B'-L2'-C' and A' comprises a fourth Fc domain monomer, or a fourth L1' is a linker; B' is a fifth Fc domain monomer. L2' is a linker; and C' is a sixth Fc domain monomer. a second polypeptide comprising a sixth Fc domain monomer or consisting of a sixth Fc domain monomer; (c) a third polypeptide comprising or consisting of a seventh Fc domain monomer; (d) a peptide comprising or consisting of an eighth Fc domain monomer. a fourth polypeptide; (e) comprising or comprising a ninth Fc domain monomer; and (f) a fifth polypeptide comprising a tenth Fc domain monomer, or The present invention features an Fc construct comprising a sixth polypeptide comprising an Fc domain monomer. The A peptide and the seventh Fc domain monomer combine to form the first Fc domain; B of the first polypeptide and B' of the second polypeptide combine to form a second Fc domain; The C of the peptide and the eighth Fc domain monomer combine to form the third Fc domain, which then forms the second polypeptide. A' of the polypeptide and the ninth Fc domain monomer combine to form the fourth Fc domain, and the second polypeptide The C' peptide and the tenth Fc domain monomer combine to form the fifth Fc domain. Each of the seventh Fc domain monomers is a first Fc domain monomer and a seventh Fc domain monomer. and a complementary dimerization selectivity module that promotes dimerization between the second and fifth Fc Each of the domain monomers is between a second Fc domain monomer and a fifth Fc domain monomer. The third and eighth Fc domains contain complementary dimerization selectivity modules that promote dimerization. Each of the monomers undergoes dimerization between the third and eighth Fc domain monomers. and a complementary dimerization selectivity module that promotes dimerization of the fourth and ninth Fc domain monomers. Each of these promotes dimerization between the fourth and ninth Fc domain monomers. and a complementary dimerization selectivity module for the sixth and tenth Fc domain monomers; Each of these promotes dimerization between the sixth and tenth Fc domain monomers. and a complementary dimerization selectivity module; the Fc construct comprises five or fewer Fc domains.

[0035] In some embodiments of this aspect, each of the first, third, fourth, and sixth Fc domain monomers The second Fc domain monomer comprises an engineered protrusion and a negatively charged amino acid. the fifth Fc domain monomer comprises a positively charged amino acid substitution, and Each of the seventh, eighth, ninth, and tenth Fc domain monomers was engineered In some embodiments, the linkers L1, L2, L1', and / or L2' include 3 to 200 amino acids. In some embodiments, the linkers L1, L2, L1', and / or L2' are selected from the group consisting of SEQ ID NO: NO: Consists of any one of the sequences 1, 2, and 3.

[0036] In another aspect, the invention provides: (a) a first polypeptide having the formula A-L1-B-L2-C. A comprises or consists of a first Fc domain monomer; L1 is , a linker; B comprises a second Fc domain monomer, or L2 is a linker; and C comprises a third Fc domain monomer, or a third and (b) a first polypeptide consisting of an Fc domain monomer of the formula A'-L1'-B'-L2'-C' and A' comprises a fourth Fc domain monomer, or a fourth L1' is a linker; B' is a fifth Fc domain monomer. L2' is a linker; and C' is a sixth Fc domain monomer. a second polypeptide comprising a sixth Fc domain monomer or consisting of a sixth Fc domain monomer; (c) a third polypeptide comprising or consisting of a seventh Fc domain monomer; (d) a peptide comprising or consisting of an eighth Fc domain monomer. a fourth polypeptide; (e) comprising or comprising a ninth Fc domain monomer; (f) a fifth polypeptide comprising a tenth Fc domain monomer or a tenth Fc domain monomer. The present invention also features an Fc construct comprising a sixth polypeptide comprising a first polypeptide and a second polypeptide comprising a second monomer. A of the first polypeptide and A' of the second polypeptide combine to form a first Fc domain; The B and seventh Fc domain monomers of the first polypeptide combine to form a second Fc domain; C and the eighth Fc domain monomer combine to form a third Fc domain, and B' of the second polypeptide and a ninth Fc domain monomer combined to form a fourth Fc domain, and a second polypeptide The C' and the tenth Fc domain monomer combine to form the fifth Fc domain. Each of the c domain monomers is between a first Fc domain monomer and a fourth Fc domain monomer. The second and seventh Fc domains contain complementary dimerization selectivity modules that promote dimerization of the Each of the Fc domain monomers is a dimer between a second Fc domain monomer and a seventh Fc domain monomer. and a third and eighth Fc domain monomer, each of which contains a complementary dimerization selectivity module that promotes dimerization. Each of these promotes dimerization between the third Fc domain monomer and the eighth Fc domain monomer. The fifth and ninth Fc domain monomers each contain a complementary dimerization selectivity module; This is a complementarity that promotes dimerization between the fifth and ninth Fc domain monomers. and each of the sixth and tenth Fc domain monomers. is a complementary domain that promotes dimerization between the sixth and tenth Fc domain monomers. The Fc construct comprises a dimerization selectivity module; the Fc construct comprises five or fewer Fc domains.

[0037] In some embodiments of this aspect, the first Fc domain monomer comprises a negatively charged amino acid substitution. the fourth Fc domain monomer comprises a positively charged amino acid substitution and the second, third, fifth and each of the sixth Fc domain monomers comprises an engineered protrusion; and each of the seventh, eighth, ninth, and tenth Fc domain monomers is engineered. In some embodiments, the linkers L1, L2, L1', and / or L2' include a recess formed between 3 and 200 In some embodiments, the linkers L1, L2, L1', and / or L2' are of amino acid length. It consists of any one of the sequences of Q ID NO: 1, 2, and 3.

[0038] In another aspect, the invention features an Fc construct that includes one or more Fc domains, The Fc construct of is constructed from a single polypeptide sequence. This polypeptide has the formula ALB wherein A comprises a first Fc domain monomer; L comprises a linker (optionally, e.g., one or more is a cleavable linker having two or more cleavage sites; and B is a second Fc domain alone. The linker comprises a first Fc domain monomer and a second Fc domain monomer of the polypeptide. Sufficient length for the monomers to combine to form an Fc domain (e.g., at least 15 amino acids, Preferably, the length is at least about 20 amino acid residues, e.g., 15 to 200 amino acids) and In certain embodiments, the first Fc domain monomer and The second Fc domain monomer is a dimer between the first Fc domain monomer and the second Fc domain monomer. Such constructs contain complementary dimerization selectivity modules that promote merization in host cells. In one embodiment, the polypeptide can be formed from the expression of a single polypeptide sequence in a cell. The peptide has the formula A-L1-B-L2-C, where A comprises a first Fc domain monomer; L1 comprises a phosphoryl group; a cleavable linker (optionally, e.g., a cleavable linker having one or more cleavage sites) B comprises a second Fc domain monomer; L2 is a linker; and C comprises a third Fc domain monomer. The linker is a first Fc domain monomer and a second Fc domain monomer of the polypeptide. Sufficient length for the monomers to combine to form an Fc domain (e.g., at least 15 amino acids, Preferably, the nucleic acid has a length of at least about 20 amino acid residues, e.g., 15 to 200 amino acids, and is flexible. In certain embodiments, the first Fc domain monomer and the second Fc domain monomer may be a spacer of amino acid sequence. The Fc domain monomer is a dimer between a first Fc domain monomer and a second Fc domain monomer. This form contains three Fc domains and a complementary dimerization selectivity module that promotes dimerization. An example of such an Fc construct is shown in FIG.

[0039] In any of the Fc constructs described herein, the Fc domain monomers of the Fc domain of the construct For example, both Fc domain monomers of an Fc domain may have the same primary amino acid sequence. Either the Fc domain can be a native sequence or both Fc domain monomers have the same dimerization selection For example, both Fc domain monomers of an Fc domain may have a functional module. H 3 Domain At least two positions within the ring of charged residues at the interface between the cations are subjected to identical reverse charge mutations. e mutation).

[0040] In any of the Fc constructs described herein, the Fc domain monomers of the Fc domain of the construct , between two Fc monomers (i.e., between an Fc domain monomer and another monomer of an Fc construct), A sequence of 20 or fewer amino acids (e.g., 15 or fewer, 10 or fewer amino acids), e.g., 20 or less, 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 The Fc subunits of the constructs described herein may have sequences that differ by no more than two amino acids. The dimerization sequence is such that the complementary dimerization selectivity module of any Fc construct is selected from one domain monomer. C for body H 3. Engineered Recesses in Antibody Constant Domains and the Other Fc Domain Monomer C H 3. Different antibody constant domains may contain engineered protrusions. The engineered recess and engineered protrusion can be an Fc domain. The protrusions of the monomers are positioned to form protrusion-recess pairs that fit into the recesses. Suitable engineered recesses and protrusions are shown in Table 1. In another embodiment, complementary The selective dimerization module is a C dimer of one of the domain monomers. H 3. Antibody Constant Domain Engineered (substituted) negatively charged amino acids and other Fc domain monomers C H 3. Engineered (substituted) positively charged amino acids in antibody constant domains The negatively and positively charged amino acids are present between the complementary domain monomers. Exemplary complementary amino acid changes are shown in Table 2. It has been done.

[0041] In some embodiments, a dimerization selectivity module (e.g., an engineered recess and and overhangs, or engineered positively and negatively charged amino acids (e.g., In addition to the exemplary amino acid changes in Tables 1 and 2, the Fc structures described herein may also be modified to include those described herein. The construct may be, for example, to help stabilize the Fc construct or to prevent protein aggregation. Additionally, the Fc monomer sequence may also include additional amino acid substitutions from the wild-type sequence.

[0042] In some embodiments, the Fc constructs described herein comprise between 2 and 10 Fc domains (e.g., 2, 3 The Fc domain of this construct contains one, four, five, six, seven, eight, nine, ten domains. At least two of the constructs have different dimerization selectivity modules. For example, constructs 5, 8, 9, and 10, comprising at least one Fc domain containing engineered recesses and protrusions. The antibody has at least one Fc domain containing a complementary opposite charge mutation and a complementary opposite charge mutation.

[0043] In other embodiments, one or more of the linkers of the Fc constructs described herein is a bond.

[0044] In other embodiments, one or more linkers of the Fc constructs described herein are selected from the group consisting of a spacer For example, an amino acid spacer of 2 to 200 amino acids.

[0045] In certain embodiments, the amino acid spacer is a spacer rich in glycine and / or serine. For example, the spacer may be selected from the sequences GS, GGS, GGGGS (SEQ ID NO: 1), GGSG ( It contains two or more motifs of the following: SEQ ID NO: 2), or SGGG (SEQ ID NO: 3).

[0046] In certain embodiments, when the Fc construct comprises an albumin-binding peptide, the albumin The binding peptide has the sequence DICLPRWGCLW (SEQ ID NO: 28).

[0047] In other embodiments, one or more Fc domain monomers of the Fc constructs described herein are IgG Hinge domain, IgG C H 2 antibody constant domains, and IgG C H 3 antibody constant domains.

[0048] In a particular embodiment, each of the Fc domain monomers of the aforementioned Fc constructs comprises an IgG hinge domain. IgG C H 2 antibody constant domains, and IgG C H 3 antibody constant domains.

[0049] In certain embodiments, the IgG is selected from the group consisting of IgG1, IgG2a, IgG2b, IgG3, and IgG4. It is a subtype that is used.

[0050] In yet another aspect, the present invention provides a substantially homogenous ( For example, a pharmaceutical composition comprising a population of at least 85%, 90%, 95%, 97%, 98%, 99% homogeneous In one aspect, the present invention relates to a sterile syringe or vial suitable for pharmaceutical use. and a composition comprising an Fc construct as the sole or primary active ingredient, the Fc construct comprising any one of the Fc constructs described herein. A substantially homogeneous (e.g., at least 85%, 90%, 95%, 98%, or 99% homogeneous) collection of The pharmaceutical composition may contain, for example, salts, detergents, surfactants, fillers, polymers, preservatives, and other pharmaceutical excipients. In embodiments, a substantially homogenous pharmaceutical composition comprises less than 10%, less than 5%, less than 4%, less than 3% of the Fc construct. %, less than 2%, less than 1%, or less than 0.5% aggregates or undesired multimers.

[0051] In another aspect, the invention features a method of preparing any one of the aforementioned Fc constructs. The method includes the step of: 1) cloning one or more polypeptides encoding the polypeptides required for assembly of the Fc construct. providing a host cell containing nucleotides, hosting the host cell under conditions allowing for the formation of the Fc construct; Expressing the polypeptide in host cells and recovering the Fc construct (e.g., purifying (including the

[0052] In some embodiments, the Fc construct comprises an association of Fc domain monomers present on different polypeptides. In certain embodiments, the Fc construct is formed at least in part by different polypeptides. In these embodiments, the Fc construct is formed by the association of Fc domain monomers present in the tid. The construct may contain an additional domain (e.g., an IgM tail or an IgA tail) that facilitates association of the two polypeptides. In other embodiments, the covalent bond (e.g., a disulfide bridge) is not included in the bond. It is present only between the two Fc domain monomers that form the Fc domain. The composition does not contain a covalent bond (e.g., a disulfide bridge) between the Fc domains. In this manner, the Fc construct provides sufficient structural flexibility so that all or part of the Fc construct may be Qualitatively, all Fc domains can simultaneously interact with Fc receptors on the cell surface. In a particular example of any of these embodiments, the Fc construct may comprise a linker (e.g., a flexible amino acid It comprises at least two Fc domains joined via a spacer.

[0053] In one embodiment, the Fc domain monomers of the Fc domain are different monomers that assemble to form the Fc domain. For example, the constructs shown in Figures 4 and 6 have three associations. The Fc domain contains two polypeptides. One of the three polypeptides contains two The other two of the three polypeptides each contain one Fc domain monomer. The construct shown in FIG. 5 contains three Fc integrin monomers containing four associated polypeptides. two of the four polypeptides contain two Fc domain monomers and four The other two of the polypeptides each contain one Fc domain monomer. The Fc construct can have n Fc domains, which contain 2n polypeptides, where n is 2 to 10. , each polypeptide is an Fc domain monomer, an IgG C L , antibody constant domains, and IgG C H 1 anti The constructs shown in Figures 8 and 9 each contain six assembly polypeptides. It has five Fc domains that contain peptides. Two of the six polypeptides contain three Fc domains. The main monomer is the Fc domain, and the other four of the six polypeptides each contain one Fc domain. The construct shown in Figure 10 contains three Fc domains containing two associated polypeptides. Each of the two polypeptides has three Fc domain monomers joined in series. Including the body.

[0054] In another aspect, the present invention provides a composition for promoting selective dimerization of Fc domain monomers. The present invention features methods and methods for the preparation of antibodies that contain an Fc domain, the Fc domain comprising two Fc domains. The yne monomer is C HAt least two positions within the ring of charged residues at the interface between the three antibody constant domains The present invention also includes a method for producing such an Fc domain, The law is C H 3. Two sites at least in two positions within the ring of charged residues at the interface between antibody constant domains A complementary dimerization selectivity module with identical mutations was introduced into the Fc domain monomer sequence of C H The interface between the three antibody constant domains is surrounded by a ring of charged residues. Consists of a hydrophobic patch. H 3. Antibody constant domain is another C H 3 antibody constant domains When these charged residues are combined, they pair with residues of the opposite charge. By reversing the charges of both members of the pair, mutant Fc domain monomers can be synthesized with the same mutant The Fc domain monomers that maintain the sequence complementarity to the Fc domain monomer but do not have these mutations are In this embodiment, the same dimerization selectivity module has low complementarity to the dimer. , which promotes homodimerization. Exemplary Fc domains include the double mutant K409D / D339K, K392D / D39 9K, E357K / K370E, D356K / K439D, K409E / D339K, K392E / D399K, E357K / K370D, or D356K In another embodiment, the Fc domain comprises an Fc monomer comprising any pair of double mutants. The Fc monomers include quadruple mutants, e.g., Fc monomers containing K409D / D399K / E357K / K370E. In an embodiment, in addition to the same dimerization selectivity module, the Fc domain monomer of the Fc domain , non-identical, which promotes specific associations (e.g., associations of engineered recesses and protrusions) The result is a single Fc domain with complementary dimerization selectivity modules with mutations. The dimer comprises two dimerization selectivity modules and maintains complementarity to each other. However, it has low complementarity to other Fc domain monomers. In one example, both of the Fc domain monomers are The same mutations in the charge pair residues of one Fc domain monomer are located in the lobe and and combines with the recess of the other Fc domain monomer.

[0055] In another aspect, the invention features a method of reducing inflammation in a subject in need thereof. In one aspect, the present invention provides a method for promoting clearance of autoantibodies in a subject in need thereof. In another aspect, the present invention provides a method for inhibiting antigen presentation in a subject in need thereof. In another aspect, the present invention provides a method for reducing an immune response in a subject in need thereof, e.g. For example, the present invention features a method for reducing immune complex activation of an immune response in a subject in need thereof. These methods include administering to a subject an Fc construct or pharmaceutical composition described herein. Includes.

[0056] In another aspect, the invention provides an Fc construct or pharmaceutical composition described herein (e.g., Buildings 1-10 and 5 * The administration of any one of the above to a subject treats an inflammatory disease or The present invention features methods of treating an autoimmune or immune disease. Exemplary diseases include: Rheumatoid arthritis (RA); systemic lupus erythematosus (SLE); ANCA-associated vasculitis; antiphospholipid Somatic syndrome; Autoimmune hemolytic anemia; Chronic inflammatory demyelinating neuropathy; Anti-allo-transplantation, GV Clearance of anti-self, anti-substitution, IgG therapeutics, and IgG paraproteins in HD; dermatomyositis ;Goodpasture's syndrome; Organ system targeting mediated by antibody-dependent cell-mediated cytotoxicity Type II hypersensitivity syndromes, e.g. Guillain-Barre syndrome, CIDP, dermatomyositis, Felty's syndrome , antibody-mediated rejection, autoimmune thyroid disease, ulcerative colitis, autoimmune liver disease; idiopathic Thrombocytopenic purpura; myasthenia gravis, neuromyelitis optica; pemphigus and other autoimmune bullous Disease; Sjogren's syndrome; Autoimmune cytopenia mediated by antibody-dependent phagocytosis and other disorders; other FcR-dependent inflammatory syndromes, e.g., synovitis, dermatomyositis, systemic vascular These include inflammatory bowel disease, glomerulitis, and vasculitis.

[0057] In another aspect, the present invention relates to a method for the treatment of a subject in need thereof, comprising administering to said subject a therapeutically effective amount of a compound as described herein for use in reducing inflammation in a subject in need thereof. The Fc construct or pharmaceutical composition (e.g., constructs 1 to 10 and 5) * (one of the following) In another aspect, the present invention provides a method for the clearance of autoimmune antibodies in a subject in need thereof. The Fc constructs or pharmaceutical compositions described herein (e.g., Constructs 1-1) for use in promoting 0 and 5 * In another aspect, the present invention provides a method for treating a cancer, comprising: The Fc constructs or pharmaceutical compositions described herein (e.g., , constructs 1 to 10 and 5 * In another aspect, the present invention relates to a method for producing a medicament for a medicament comprising the steps of: Reducing an immune response in a subject in need thereof, e.g., an immune complex in the immune response in a subject in need thereof. The Fc constructs or pharmaceutical compositions described herein (e.g., , constructs 1 to 10 and 5 * The present invention is characterized by the following:

[0058] In another aspect, the present invention provides a method for treating an inflammatory disease or an autoimmune disease or an immune disease in a subject. The Fc constructs or pharmaceutical compositions described herein (e.g., constructs 1-10 and 5 * Exemplary diseases include: rheumatoid arthritis (RA); Sexual lupus erythematosus (SLE); ANCA-associated vasculitis; antiphospholipid syndrome; autoimmune hemolysis anemia; chronic inflammatory demyelinating neuropathy; anti-allo in transplantation, anti-autologous in GVHD, anti-replacement , IgG therapeutics, IgG paraprotein clearance; dermatomyositis; Goodpasture's syndrome Organ system-targeted type II hypersensitivity syndromes mediated by antibody-dependent cell-mediated cytotoxicity, e.g. For example, Guillain-Barre syndrome, CIDP, dermatomyositis, Felty syndrome, antibody-mediated rejection, autoimmune diseases, and Autoimmune thyroid disease, ulcerative colitis, autoimmune liver disease; idiopathic thrombocytopenic purpura; severe myasthenia gravis, neuromyelitis optica; pemphigus and other autoimmune blistering diseases; Sjogren's syndrome ;Autoimmune cytopenias and other disorders mediated by antibody-dependent phagocytosis;Other FcR Dependent inflammatory syndromes, such as synovitis, dermatomyositis, systemic vasculitis, glomerulitis, and vascular Flames are mentioned.

[0059] In any of the Fc constructs described herein, the order of the Fc domain monomers is interchangeable. For example, in a polypeptide having the formula ALB, the carboxy terminus of A can be attached to the amino terminus of L, and L can be attached to the amino terminus of B by its carboxy terminus. Alternatively, the carboxy terminus of B can be joined to the amino terminus of L, where L is Its carboxy terminus is joined to the amino terminus of C. Both of these structures are represented by the formula ALB. is encompassed by this.

[0060] In a related aspect, the invention features a host cell expressing any one of the aforementioned Fc constructs. The host cell contains polypeptides encoding the polypeptides necessary to construct the Fc construct. The polynucleotide comprises a nucleotide sequence that is expressed in a host cell.

[0061] definition As used herein, the term "Fc domain monomer" refers to an Fc domain monomer that contains at least a hinge domain. and the second and third antibody constant domains (C H 2 and C H 3) or a functional fragment thereof (e.g. (i) capable of dimerizing with another Fc domain monomer to form an Fc domain; and i) a fragment capable of binding to an Fc receptor. The mers may be any immunoglobulin antibody isotype, including IgG, IgE, IgM, IgA, or IgD. In addition, the Fc domain monomer may be an IgG subtype (e.g., IgG1, IgG2a, IgG2 The Fc domain monomer can be either IgG, IgG2, or IgG4. The Fc domain monomer functions as the antigen recognition region. Part of an immunoglobulin that can be used to identify a target gene, such as a variable domain or a complementarity determining region (CDR The Fc domain monomer does not contain any A wild-type Fc domain monomer sequence (e.g., amino acid substitutions 1-10, 1-8, 1-6, 1-4) that Examples of suitable alterations are known in the art. .

[0062] As used herein, the term "Fc domain" refers to a domain that is capable of binding to an Fc receptor. A dimer of two Fc domain monomers. In the wild-type Fc domain, two Fc domain monomers But two C's H Interactions between three antibody constant domains and two dimerizing Fc domain monomers Dimerization occurs via one or more disulfide bonds formed between the hinge domains of the ribosome. do.

[0063] In the present invention, the term "Fc construct" refers to a construct that contains 2 to 10 Fc domains as described herein. The Fc constructs described herein refer to associated polypeptide chains formed between the same or An Fc construct may contain Fc domain monomers with different sequences. For example, an Fc construct may contain two Fc domains. one Fc domain of which is an IgG1 or IgG1-derived Fc domain. and a second Fc domain monomer, the second Fc domain of which is an IgG2 or IgG2-derived Fc domain monomer. In another example, the Fc construct can have two Fc domains, one of which The Fc domain contains a "protrusion-recess pair in which the protrusion fits into the recess," of which the second F The Fc domain does not contain a "protrusion-recess pair in which the protrusion fits into the recess." The c domain is the variable region of an antibody, e.g., the V H , V L It does not contain any CDRs, HVRs, or Fc domains. The receptors include Fc receptors, e.g., FcγRI, FcγRIIa, FcγRIIb, FcγRIIIa, FcγRIIIb, FcγRI It forms a minimal structure that binds to V.

[0064] As used herein, the term "antibody constant domain" refers to the constant region domain of an antibody (e.g. For example, C LAntibody constant domain, C H 1. Antibody constant domain, C H 2 antibody constant domain, or C H 3 anti It refers to a polypeptide corresponding to an endothelial constant domain.

[0065] The term "promote" as used herein means to encourage and facilitate, e.g., to promote the development of Two Fc domain monomers that have a higher binding affinity for each other than the Fc domain monomers themselves. As described herein, the result is to promote the formation of an Fc domain from the host. Two Fc domain monomers that combine to form an Fc domain are H 3. Antibody Constant Domains Amino acid modifications compatible with the interface of the Compatible amino acid modifications can include those that incorporate such amino acid modifications. This is preferred over other Fc domain monomers that do not have this or that have incompatible amino acid modifications. This promotes or encourages the selective interaction of Fc domain monomers with each other, such as is two interacting C H 3. Amino acid modification at the interface of antibody constant domains allows for the formation of Fc domains alone Fc domain monomers have higher affinity to each other than to other Fc domain monomers not having the amino acid modification. This is because it has high affinity.

[0066] As used herein, the term "dimerization selectivity module" refers to a dimerization selectivity module that is a combination of two Fc Refers to the sequence of Fc domain monomers that facilitates pairing between the domain monomers. "Complementary" dimer The selectivity module promotes the selective interaction of two Fc domain monomers with each other. or a promoting dimerization selectivity module. The complementary dimerization selectivity module is Exemplary complementary dimerization selectivity modules are described herein. It is stated in the book.

[0067] As used herein, the term "engineered recess" means a recess that is H 3. Antibody constant domain At least one of the original amino acid residues of the inverted amino acid has a smaller side chain volume than the original amino acid residue. By substitution of different amino acid residues having C H 3. Forming a Three-Dimensional Recess in an Antibody Constant Domain The term "original amino acid residue" refers to the wild-type C H 3. Genetics of Antibody Constant Domains The amino acid residues refer to naturally occurring amino acid residues encoded by the amino acid sequence.

[0068] As used herein, the term "engineered protrusion" refers to a H 3. Antibody Constant At least one of the main original amino acid residues has a larger side chain volume than the original amino acid residue By substitution with a different amino acid residue having the H 3. Formation of three-dimensional protrusions in antibody constant domains The term "original amino acid residue" refers to the wild-type C H 3. Antibody Constant Domains Refers to naturally occurring amino acid residues encoded by the genetic code.

[0069] As used herein, the term "protrusion-recess pair in which the protrusion fits into the recess" refers to a first The Fc domain monomer of H 3. Engineered Recesses in Antibody Constant Domains and a second Fc domain monomer comprising: H 3 Engineered into antibody constant domains The Fc domain comprises two Fc domain monomers, each of which includes a protrusion disposed within a recess. In the pair of lobes and recesses that enter, the C of the first Fc domain monomer H 3. Enzyme-linked Antibody Constant Domain The ginniered protrusions are H Normal association of dimers at the interface between three antibody constant domains. of the second Fc domain monomer without significantly impeding the synthesis of H 3. Engineering of Antibody Constant Domains The recess is disposed so as to interact with the recess.

[0070] As used herein, the term "conjugated" includes chemical conjugation, recombinant and by chemical bonds, including disulfide bonds and amide bonds. The binding of two or more elements, components, or protein domains, e.g., polypeptides or Used to describe attachment: chemical conjugation, chemical bond, peptide linker or other covalent means to join, for example, two single polypeptides to form a In some embodiments, the first Fc domain can form one continuous protein structure. The Fc domain monomer is joined to a second Fc domain monomer by a peptide linker, forming a peptide linker. The N-terminus of the anchor is linked to the C of the first Fc domain monomer by a chemical bond, e.g., a peptide bond. and the C-terminus of the peptide linker is joined to a chemical bond, e.g., a peptide bond. In another embodiment, the albumin binding peptide is attached to the N-terminus of the second Fc domain monomer. The N-terminus of the peptide is linked to the C of the Fc domain monomer by a linker in the same manner as described above. H 3 antibody determination It is joined to the C-terminus of the common domain.

[0071] As used herein, the term "associated" refers to a polypeptide (or a single polypeptide) that is The peptide is arranged to form an Fc construct having at least one Fc domain. interactions between polypeptides (or sequences within one single polypeptide), such as For example, it can be used to represent hydrogen bonds, hydrophobic interactions, or ionic interactions. For example, two polypeptides each containing one Fc domain monomer can assemble to form an Fc construct. In some embodiments, for example, one polyimide may be formed (e.g., as shown in Figures 1-3). The peptide contains two Fc domain monomers, and the two polypeptides each contain one Fc domain. Three polypeptides containing the ribonucleic acid monomers assemble to form an Fc construct having two Fc domains. In some embodiments, e.g., two polysaccharides are used (e.g., as shown in FIG. 4 and FIG. 6). Each peptide contains two Fc domain monomers, and each of the two polypeptides contains one Fc domain monomer. Four polypeptides containing c domain monomers assemble to form an Fc structure having three Fc domains. In other embodiments, for example, each of the pols may be formed into a construct (e.g., as shown in FIG. 5). The peptide is an Fc domain monomer, an IgG C L Antibody constant domains, and IgG C H 1. Antibody Constant 2n polypeptides each containing a polypeptide are assembled to form an Fc construct having n Fc domains. The two polypeptides are composed of respective Fc domain monomers (shown in FIG. 7B). The association can be via the ribosome or via other moieties of the polypeptide. For example, in FIG. wherein polypeptide 708 associates with polypeptide 706 via its Fc domain monomer; and Polypeptide 710 C H 1 domain and its associated C L Polypeptide synthesis via domain association The association between the polypeptides does not include covalent interactions. For example, In FIG. 10, a single polypeptide is shown such that Fc monomer sequences 1004 and 1006 associate to form an Fc domain. The Fc monomer sequences 1014 and 1012 in the peptide associate to form an Fc domain.

[0072] As used herein, the term "linker" refers to a linker that is a structural unit that separates two elements, e.g., a protein domain. A linker may be a covalent bond or a spacer. The term refers to a chemical bond, e.g., an amide bond or a disulfide bond, or a chemical reaction, e.g. For example, this refers to any type of bond formed by chemical conjugation. The term "polypeptide" refers to a polypeptide that is present between two polypeptides or polypeptide domains and that providing space and / or flexibility between two polypeptides or polypeptide domains A moiety (e.g., a polyethylene glycol (PEG) polymer) or an amino acid sequence (e.g., , a sequence of 3 to 200 amino acids, a sequence of 3 to 150 amino acids, or a sequence of 3 to 100 amino acids) Amino acid spacers can be used to (e.g., polypeptide or or a portion of the primary sequence of a polypeptide (or a polypeptide domain). , the two hinge regions that form the Fc domain or the disulfide between the two Fc domain monomers The formation of the bond is not considered a linker.

[0073] As used herein, the term "cleavable linker" refers to a cleavable linker that is capable of binding to, for example, Refers to a linker that contains one or more elements that can be selectively cleaved later, e.g., cleavage Possible linkers include polypeptide sequences that can be selectively cleaved by a prosthesis.

[0074] As used herein, the term "albumin-binding peptide" refers to a peptide that binds to serum albumin. The amino acid sequence of 12 to 16 amino acids that has the affinity and function of binding to serum albumin is Albumin-binding peptides are those of different origins, e.g., human, mouse, or rat origin. In some embodiments of the present invention, the albumin binding peptide may be an Fc domain monomer. The albumin binding peptide is fused to the C-terminus of Fc to increase the serum half-life of the Fc construct. The c-domain can be fused directly to the N- or C-terminus of the monomer or fused via a linker. obtain.

[0075] As used herein, the term "multimer" refers to an association of at least two of the molecules described herein. The term "Fc construct" refers to a molecule comprising an Fc construct comprising an Fc construct that is

[0076] As used herein, the term "polynucleotide" refers to an oligonucleotide or a nucleic acid. nucleotides, and fragments or portions thereof, and may be single-stranded or double-stranded, or refers to DNA or RNA of genomic or synthetic origin that represents one sense or antisense strand A single polynucleotide is translated into a single polypeptide.

[0077] As used herein, the term "polypeptide" refers to a group of monomers linked together by amide bonds. A polypeptide refers to a single polymer of amino acid residues joined together by a single amino acid. It is intended to encompass any amino acid sequence that is recombinantly or synthetically produced.

[0078] As used herein, the term "amino acid position" refers to a sequence of an amino acid in a protein or protein domain. The amino acid positions of the antibody or Fc construct refer to the amino acid position number in the Kabat Numbering system (Kabat numbering system) (Kabat et al., Sequences of Proteins of Immun ological interest, National Institutes of Health, Bethesda, Md., ed 5, 1991). The numbers are assigned using this method.

[0079] As used herein, the term "host cell" refers to a cell that is capable of expressing a protein from a corresponding nucleic acid. Nucleus refers to a vehicle that contains essential cellular components, e.g., organelles, that are necessary for the cell to function. The acid is typically produced by conventional techniques known in the art (e.g., transformation, transfection, electroporation, etc.). into host cells via troporation, calcium phosphate precipitation, direct microinjection, etc. The host cell can be a prokaryotic cell, such as a bacterial cell, or a nucleic acid vector that can be used to express the vector. The cell may be a eukaryotic cell, for example a mammalian cell (e.g., a CHO cell). Thus, the host cell is able to generate the desired domains that can be combined to form the desired Fc construct. The vector is used to express one or more polypeptides encoding the

[0080] As used herein, the term "pharmaceutical composition" refers to an active ingredient as well as a pharmaceutical composition for administering the active ingredient. The pharmaceutical formulation or The pharmaceutical composition of the present invention refers to a pharma- ceutical formulation comprising a pharma- ceutical acceptable carrier compatible with the Fc construct. The pharmaceutical composition typically comprises a water-soluble component for intravenous or subcutaneous administration. It is the form.

[0081] As used herein, the term "substantially homogenous population" of polypeptides or Fc constructs comprises at least 85% of the polypeptide or Fc construct in a composition (e.g., a pharmaceutical composition). of polypeptides or Fc constructs having the same number of Fc domains and the same Fc domain structure. In various embodiments, at least 90% of the polypeptides or Fc constructs in the composition , 92%, 95%, 97%, 98%, 99%, or 99.5% are the same. A pharmaceutical composition comprising a substantially homogenous population is one in which at least 85% of the Fc constructs in the composition are of the same number of Fc A substantially homogenous population of Fc constructs is a pharmaceutical composition having the same structure as the Fc domain. , does not contain more than 10% multimers or aggregates of the Fc construct (e.g., 8% or less, 5% or less, 2% or less, or 1% or less).

[0082] As used herein, the term "pharmaceutical acceptable carrier" refers to an additive in a pharmaceutical composition. A pharma- ceutically acceptable carrier refers to an agent or diluent that is compatible with the other ingredients of a formulation. In the present invention, pharma- ceutically acceptable The carrier used must provide sufficient pharmaceutical stability for the Fc construct. The dosage will vary depending on the mode of administration. For example, for oral administration, solid carriers are preferred; for intravenous administration, For administration, aqueous carriers (e.g., WFI and / or buffer) are generally used. .

[0083] As used herein, the term "therapeutically effective amount" refers to a therapeutically effective amount that is sufficient to deliver a desired biological effect to a subject or patient. An amount effective to induce an effect or to treat a patient having a condition or disorder described herein, e.g. For example, a pharmaceutical dose. Also, as used herein, a "therapeutically effective amount" refers to a single or any Whether administered in a dose or alone or in combination with other therapeutic agents, the desired It is understood that this can be interpreted as an amount that provides a therapeutic effect. [The present invention 1001] (a) a first polypeptide having the formula ALB; (i) A comprises a first Fc domain monomer; (ii) L is a linker; and (iii) a first polypeptide, wherein B comprises a second Fc domain monomer; (b) a second polypeptide comprising a third Fc domain monomer; and (c) a third polypeptide comprising a fourth Fc domain monomer. Including, The first Fc domain monomer and the third Fc domain monomer are combined to form a first Fc domain. and the second Fc domain monomer and the fourth Fc domain monomer are bound to each other to form a The Fc construct forms the second Fc domain. [The present invention 1002] The first Fc domain monomer and the third Fc domain monomer are Complementary dimerization selection to promote dimerization between the first Fc domain monomer and the third Fc domain monomer. The Fc construct of the present invention comprises a functional module. [The present invention 1003] The second Fc domain monomer and the fourth Fc domain monomer are Complementary dimerization selection to promote dimerization between the first Fc domain monomer and the fourth Fc domain monomer. The Fc construct of the present invention 1001 or 1002, comprising a sex module. [The present invention 1004] The Fc construct of any one of 1001 to 1003, wherein A consists of an Fc domain monomer. [The present invention 1005] The Fc construct of any one of 1001 to 1004, wherein B consists of an Fc domain monomer. [The present invention 1006] Any of claims 1001 to 1005, wherein the second polypeptide comprises an Fc domain monomer. Fc constructs. [The present invention 1007] Any of claims 1001 to 1006, wherein the third polypeptide comprises an Fc domain monomer. Fc constructs. [The present invention 1008] linked to the N-terminus or C-terminus of one or more of said polypeptides by a linker The Fc construct of any of claims 1001 to 1007, further comprising an albumin binding peptide. [The present invention 1009] IgG C L Antibody constant domains and IgG C H IgG C1 antibody constant domain, H 1 anti a constant domain is attached to the N-terminus of A or the second polypeptide by a linker; The Fc construct of any one of claims 1001 to 1008, [The present invention 1010] the second polypeptide and the third polypeptide have the same amino acid sequence; Any of the Fc constructs of 1001 to 1009 of the present invention. [The present invention 1011] (a) a first polypeptide having the formula ALB; (i) A comprises a first Fc domain monomer; (ii) L is a linker; and (iii) a first polypeptide, wherein B comprises a second Fc domain monomer; (b) a second polypeptide having the formula A'-L'-B'; (i) A' comprises a third Fc domain monomer; (ii) L' is a linker; and (iii) a second polypeptide, wherein B' comprises a fourth Fc domain monomer; (c) a third polypeptide comprising a fifth Fc domain monomer; and (d) a fourth polypeptide comprising a sixth Fc domain monomer. Including, A of the first polypeptide and A' of the second polypeptide bind to form a first Fc domain and B of the first polypeptide and the fifth Fc domain monomer bind to form a second Fc domain. and B' of the second polypeptide and the sixth Fc domain monomer are linked to form a third Fc domain monomer. 3 Fc domains, forming an Fc construct. [The present invention 1012] The first Fc domain monomer and the third Fc domain monomer are Complementary dimerization selection to promote dimerization between the first Fc domain monomer and the third Fc domain monomer. The Fc construct of the present invention comprises a functional module. [The present invention 1013] The second Fc domain monomer and the fifth Fc domain monomer are Complementary dimerization selection to promote dimerization between the 5th Fc domain monomer and the 6th Fc domain monomer. The Fc construct of the present invention 1011 or 1012, comprising a sex module. [The present invention 1014] The fourth Fc domain monomer and the sixth Fc domain monomer are Complementary dimerization selection to promote dimerization between the α-amino acid monomer and the sixth Fc domain monomer. An Fc construct according to any one of claims 1011 to 1013, comprising a functional module. [The present invention 1015] Any of the Fc constructs of the present invention 1011 to 1014, wherein A consists of an Fc domain monomer. [The present invention 1016] Any of the Fc constructs of 1011 to 1015, wherein B consists of an Fc domain monomer. [The present invention 1017] The Fc construct of any of claims 1011 to 1016, wherein A' consists of an Fc domain monomer. [The present invention 1018] The Fc construct of any of claims 1011 to 1017, wherein B' consists of an Fc domain monomer. [The present invention 1019] Any of the methods according to claims 1011 to 1018, wherein the third polypeptide comprises an Fc domain monomer. Fc constructs. [The present invention 1020] Any of the methods according to claims 1011 to 1019, wherein the fourth polypeptide comprises an Fc domain monomer. Fc constructs. [The present invention 1021] IgG C L Antibody constant domains and IgG C H IgG C1 antibody constant domain, L anti The IgG C constant domain is linked to the H 1 attached to the N-terminus of an antibody constant domain, And the IgG C H 1. The antibody constant domain is attached to the N-terminus of A by a linker. An Fc construct according to any one of claims 1011 to 1020. [The present invention 1022] Second IgG C L Antibody constant domains and second IgG C H 1 further comprises an antibody constant domain, The second IgG C L The antibody constant domain is linked to the second IgG C H 1. Antibody constant domain and the second IgG C H 1. An antibody constant domain is linked to A' by a linker The Fc construct of the present invention, wherein the Fc construct is attached to the N-terminus of The IgG C H 1. An antibody constant domain is attached to the N-terminus of A or A' by a linker . [The present invention 1023] The Fc construct of any of claims 1011 to 1021, further comprising an albumin binding peptide. [The present invention 1024] The first polypeptide and the second polypeptide have the same amino acid sequence, wherein the third polypeptide and the fourth polypeptide have the same amino acid sequence; Any of the Fc constructs of the present invention 1011 to 1022. [The present invention 1025] (a) a first polypeptide having the formula ALB; (i) A comprises a first Fc domain monomer; (ii) L is a linker; and (iii) a first polypeptide, wherein B comprises an albumin binding peptide; and (b) a second polypeptide comprising a second Fc domain monomer. Including, The first Fc domain monomer and the second Fc domain monomer combine to form an Fc domain. The Fc construct comprises: [The present invention 1026] The first Fc domain monomer and the second Fc domain monomer are Complementary dimerization selection that promotes dimerization between the first Fc domain monomer and the second Fc domain monomer. The Fc construct of the present invention comprises a functional module. [The present invention 1027] The Fc construct of the present invention 1025 or 1026, wherein A consists of an Fc domain monomer. [The present invention 1028] The Fc domain of any one of 1025 to 1027 of the present invention, wherein the second polypeptide is composed of an Fc domain monomer. Construction. [The present invention 1029] (a) a first polypeptide having the formula A-L1-B-L2-C; (i) A is IgG C LContains an antibody constant domain; (ii) L1 and L2 are each a linker; (iii) B is IgG C H 1 antibody constant domain; and (iv) a first polypeptide, wherein C comprises a first Fc domain monomer; and (b) a second polypeptide having the formula A'-L1'-B'-L2'-C'; (i) A' is IgG C L Contains an antibody constant domain; (ii) L1' and L2' are each a linker; (iii) B' is IgG C H 1 antibody constant domain; and (iv) a second polypeptide, wherein C' comprises a second Fc domain monomer. Including, The first Fc domain monomer and the second Fc domain monomer combine to form an Fc domain. The Fc construct comprises: [The present invention 1030] The first Fc domain monomer and the second Fc domain monomer are Complementary dimerization selection that promotes dimerization between the first Fc domain monomer and the second Fc domain monomer. 1029 Fc constructs of the present invention comprising a functional module. [The present invention 1031] The Fc construct of the present invention 1029 or 1030, wherein C consists of an Fc domain monomer. [The present invention 1032] The Fc construct of any of claims 1029 to 1031, wherein C' consists of an Fc domain monomer. [The present invention 1033] Further comprising an albumin binding peptide joined to the C-terminus of the C or C' by a linker. , any one of Fc constructs 1029 to 1032 of the present invention. [The present invention 1034] (a) a first polypeptide having the formula A-L1-B-L2-C; (i) A is IgG C L Contains an antibody constant domain; (ii) L1 and L2 are each a linker; (iii) B is IgG C H 1 antibody constant domain; and (iv) a first polypeptide, wherein C comprises a first Fc domain monomer; and (b) a second polypeptide having the formula A'-L1'-B'-L2'-C'; (i) A' is IgG C L Contains an antibody constant domain; (ii) L1' and L2' are each a linker; (iii) B' is IgG C H 1 antibody constant domain; and (iv) a second polypeptide, wherein C' comprises a second Fc domain monomer; (c) a third polypeptide comprising a third Fc domain monomer; and (d) a fourth polypeptide comprising a fourth Fc domain monomer. Including, The first Fc domain monomer and the third Fc domain monomer are combined to form a first Fc domain. the second Fc domain monomer and the fourth and third Fc domain monomers combine to form a second Forms the Fc domain; The IgG C of the first polypeptide H 1 antibody constant domain is an IgG C L binds to an antibody constant domain; and The second polypeptide is IgG H 1 antibody constant domain is an IgG C domain of the first polypeptide L An Fc construct that combines antibody constant domains. [The present invention 1035] (a) a first polypeptide comprising a first Fc domain monomer; and (b) a second polypeptide comprising a second Fc domain monomer. Including, The first Fc domain monomer and the second Fc domain monomer combine to form an Fc domain. and the first Fc domain monomer and the second Fc domain monomer are A complementary dimer that promotes dimerization between a c domain monomer and said second Fc domain monomer. An Fc construct comprising a cytosine selectivity module. [The present invention 1036] The Fc construct of the present invention 1035, wherein said first polypeptide consists of an Fc domain monomer. [The present invention 1037] The Fc construct of the invention 1035 or 1036, wherein said second polypeptide consists of an Fc domain monomer. thing. [The present invention 1038] The Fc construct of any of claims 1035 to 1037, further comprising an albumin binding peptide. [The present invention 1039] IgG C L Antibody constant domains and IgG C H IgG C1 antibody constant domain, H 1 anti a constant domain is attached to the N-terminus of the first or second polypeptide by a linker; The Fc construct of any one of 1035 to 1038, wherein the Fc construct is attached to a polypeptide of the present invention. [The present invention 1040] (a) a first polypeptide having the formula ALB; (i) A comprises a first Fc domain monomer; (ii) L is a linker; and (iii) a first polypeptide, wherein B comprises a second Fc domain monomer; (b) a second polypeptide having the formula A'-L'-B'; (i) A' comprises a third Fc domain monomer; (ii) L' is a linker; and (iii) a second polypeptide, wherein B' comprises a fourth Fc domain monomer. Including, The first and second Fc domain monomers each have a respective C H There are three domains: wherein the third and fourth Fc domain monomers each comprise an aligned recess; C H 3 domain, comprising an engineered protrusion in said engineered The recess and the engineered protrusion are arranged in a protrusion-recess arrangement, where the protrusion enters the recess. and wherein the first Fc domain monomer and the third Fc domain monomer are positioned to form a pair. The first Fc domain monomer is linked to the second Fc domain monomer to form a fourth Fc domain monomer. and a cytochrome P450 monomer that combines to form a second Fc domain. [The present invention 1041] Each of the A, B, A', or B' is composed of an Fc domain monomer. Ming1040 Fc construct. [The present invention 1042] Further comprising an albumin binding peptide joined to the C-terminus of B or B' by a linker. , the Fc construct of the present invention 1040 or 1041. [The present invention 1043] IgG C L Antibody constant domains and IgG C H IgG C1 antibody constant domain, H 1 anti The present invention relates to an antibody constant domain attached to the N-terminus of A or A' by a linker. 1042 Fc constructs. [The present invention 1044] The dimerization selectivity module comprises one C of the Fc domain monomer. H In the three domains The engineered recess and the other C of the Fc domain monomer H 3 Domains and a recess formed in said recess and said engineered protrusion. The ringed protrusion forms a protrusion-recess pair in which the protrusion of the Fc domain monomer fits into the recess. The present invention 1002, 1003, 1012, 1013, 1014, 1026, 1030, and 1035 Fc constructs. [The present invention 1045] one of the Fc domain monomers comprises Y407V and Y349C, and the other of the Fc domain monomers comprises The Fc construct of the present invention comprises T336W and S354C. [The present invention 1046] The dimerization selectivity module is a C H 3 Domains with negative and the other C of the Fc domain monomer. H There are three positively charged domains and the negatively charged amino acid and the positively charged amino acid are included in the Fc domain. The present invention is directed to the formation of a phospholipid complex. 1030, or 1035 Fc constructs. [The present invention 1047] one of the Fc domain monomers comprises D399K and the other of the Fc domain monomers comprises K409D The Fc construct of the present invention comprising: [The present invention 1048] Any of claims 1001 to 1047, wherein one or more linkers of said Fc construct are bonds. Fc constructs. [The present invention 1049] Any of the present inventions 1001 to 1047, wherein one or more linkers of said Fc construct are spacers. Either Fc construct. [The present invention 1050] The Fc structure of the present invention 1049, wherein the spacer comprises any one of the sequences of SEQ ID NO: 1 to 27. building. [The present invention 1051] The Fc construct of the present invention 1050, wherein the sequence is any one of SEQ ID NOs: 1, 2, and 3. thing. [The present invention 1052] The present invention relates to a method for the preparation of a medicament for the preparation of an albumin-binding peptide comprising the sequence of SEQ ID NO: 28. 5, 1033, 1038, and 1042 Fc constructs. [The present invention 1053] The Fc construct of the present invention 1052, wherein the albumin binding peptide has the sequence of SEQ ID NO: 28. thing. [The present invention 1054] (a) a first polypeptide having the formula A-L1-B-L2-C; (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) a first polypeptide, wherein C comprises a third Fc domain monomer; and (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) a second polypeptide, wherein C' comprises a sixth Fc domain monomer. Including, The first Fc domain monomer and the second Fc domain monomer are bound to each other to form a first Fc domain. and the fourth Fc domain monomer and the fifth Fc domain monomer combine to form a second Fc domain monomer. and the third Fc domain monomer and the sixth Fc domain monomer form an Fc domain of combine to form a third Fc domain. [The present invention 1055] The Fc construct of the present invention 1054, wherein the linkers L1 and L1' are cleavable linkers. [The present invention 1056] One or more of the Fc domain monomers may comprise an IgG hinge domain, an IgG C H 2. Antibody constant domain In, and IgG C H 3. An Fc construct according to any one of claims 1001 to 1055, comprising an antibody constant domain . [The present invention 1057] Each of the Fc domain monomers comprises an IgG hinge domain, an IgG C H 2 antibody constant domains, and IgG C H The Fc construct of the present invention comprises three antibody constant domains. [The present invention 1058] The IgG is a subtype selected from the group consisting of IgG1, IgG2a, IgG2b, IgG3, and IgG4. The Fc construct of the present invention is of the 1056 or 1057 type. [The present invention 1059] A method for preparing any one of Fc constructs 1001 to 1058 of the present invention, comprising: (a) providing a host cell containing a polynucleotide encoding said polypeptide; ; (b) infecting the host cell with the first, second, and third nucleotides under conditions that allow formation of the Fc construct. and expressing said third polypeptide; (c) recovering the Fc construct. A method comprising: [The present invention 1060] A host cell expressing any one of the Fc constructs of the present invention, a polynucleotide encoding a polypeptide, said polynucleotide being expressed in said host cell; A host cell. [The present invention 1061] A pharmaceutical composition comprising a substantially homogenous population of Fc constructs having 2-10 Fc domains. [The present invention 1062] The plurality of 2 to 10 Fc domains are complementary to promote dimerization between two Fc domain monomers. formed by the association of two Fc domain monomers containing a selective dimerization selectivity module, Pharmaceutical compositions of the present invention. [The present invention 1063] The two separate polypeptides of the multiple Fc domains have the same amino acid sequence. The pharmaceutical composition of invention 1062. [The present invention 1064] wherein two separate polypeptides of the Fc domains have different amino acid sequences. Pharmaceutical compositions of the present invention. [The present invention 1065] The complementary Fc monomer sequence has a complementary dimerization selectivity module. chemical composition. [The present invention 1066] The Fc construct comprises one or more (e.g., two, three, four, five, or six) of the following: The pharmaceutical composition of the present invention has the following characteristics: (a) at least in part by association of Fc domain monomers present on different polypeptides formed in a systematic way; (b) does not contain an additional domain that promotes association of the two polypeptides; (c) contains a covalent bond only between two Fc domain monomers that join to form the Fc domain; (d) does not contain a covalent bond between the Fc domains; (e) all or substantially all of the Fc domains in the Fc construct are linked to a cell surface F c) provide sufficient flexibility so that the polypeptides can simultaneously interact with the receptors; and / or or (f) comprises at least two Fc domains joined by a linker. [The present invention 1067] A pharmaceutical composition comprising a substantially homogenous population of any of the Fc constructs of the present invention 1001 to 1058. [The present invention 1068] The Fc construct comprises 2-5 associated polypeptides, each polypeptide comprising at least and each Fc domain monomer of the construct is the same or 10 or less amino acids differ from each other. [The present invention 1069] The Fc construct comprises two Fc domains comprising three associated polypeptides, one of the polypeptides contains two Fc domain monomers, and the three polypeptides Any of the methods of the present invention 1061 to 1067, wherein two of the peptides each contain one Fc domain monomer. A pharmaceutical composition. [The present invention 1070] The Fc construct comprises three Fc domains comprising four associated polypeptides, Two of the polypeptides each contain two Fc domain monomers, and the four polypeptides Any of the peptides according to the present invention, wherein two of the peptides each contain one Fc domain monomer. Any pharmaceutical composition. [The present invention 1071] The Fc construct comprises n Fc domains comprising 2n polypeptides, each polypeptide The Fc domain monomer, IgG C L Antibody constant domains, and IgG C H1. Antibody constant domain The pharmaceutical composition of any one of claims 1061 to 1067, comprising [The present invention 1072] A method for treating inflammation in a subject, comprising administering to a subject an Fc construct of any one of claims 1001 to 1058 or an A pharmaceutical composition comprising a therapeutically effective amount of any one of the pharmaceutical compositions of inventions 1061 to 1071 is administered to the subject. The method includes providing. [The present invention 1073] A method for reducing immune complex activation of an immune response in a subject, comprising the steps of: Any of the Fc constructs of the present invention or any of the pharmaceutical compositions of the present inventions 1061 to 1071 is administered to the subject. The method includes providing. [The present invention 1074] The method of claim 1073, wherein the subject has an autoimmune disease. [The present invention 1075] (a) a first polypeptide having the formula ALB; (i) A comprises a first Fc domain monomer; (ii) L is a linker; and (iii) a first polypeptide, wherein B comprises a second Fc domain monomer; (b) a second polypeptide having the formula A'-L'-B'; (i) A' comprises a third Fc domain monomer; (ii) L' is a linker; and (iii) a second polypeptide, wherein B' comprises a fourth Fc domain monomer; (c) a third polypeptide comprising a fifth Fc domain monomer; and (d) a fourth polypeptide comprising a sixth Fc domain monomer. It consists of: A and A' bind to form the first Fc domain, and B binds to the fifth Fc domain monomer. and B' combines with the sixth Fc domain monomer to form a third Fc domain. forming the c domain; each of the first and third Fc domain monomers is a first Fc domain monomer; and the third Fc domain monomer. Includes each of the second and fifth Fc domain monomers is a second Fc domain monomer; and the fifth Fc domain monomer. contains the rule, and each of the fourth and sixth Fc domain monomers is a fourth Fc domain monomer; and the sixth Fc domain monomer. contains the rule; and An Fc construct comprising three or fewer Fc domains. [The present invention 1076] (a) a first polypeptide having the formula ALB; (i) A consists of a first Fc domain monomer; (ii) L is a linker; and (iii) a first polypeptide, wherein B consists of a second Fc domain monomer; (b) a second polypeptide having the formula A'-L'-B'; (i) A' consists of a third Fc domain monomer; (ii) L' is a linker; and (iii) a second polypeptide, wherein B' consists of a fourth Fc domain monomer; (c) a third polypeptide consisting of a fifth Fc domain monomer; and (d) a fourth polypeptide consisting of a sixth Fc domain monomer. It consists of: A and A' bind to form the first Fc domain, and B binds to the fifth Fc domain monomer. and B' combines with the sixth Fc domain monomer to form a third Fc domain. forming the c domain; each of the first and third Fc domain monomers is a first Fc domain monomer; and the third Fc domain monomer. Includes each of the second and fifth Fc domain monomers is a second Fc domain monomer; and the fifth Fc domain monomer. contains the rule, and each of the fourth and sixth Fc domain monomers is a fourth Fc domain monomer; and the sixth Fc domain monomer. contains the rule; and An Fc construct comprising three or fewer Fc domains. [The present invention 1077] The complementary dimerization selectivity module of the first Fc domain monomer is negatively charged. The complementary dimerization selectivity module of the third Fc domain monomer comprises an amino acid substitution. wherein the complementary dimer of the second Fc domain monomer comprises a positively charged amino acid substitution. The selective selectivity module comprises an engineered protrusion, and the fifth Fc domain the complementary dimerization selectivity module of the monomer comprises an engineered recess; The complementary dimerization selectivity module of the fourth Fc domain monomer is and the complementary dimerization selectivity domain of the sixth Fc domain monomer. The Fc construct of the present invention 1075 or 1076, wherein the module comprises an engineered recess. [The present invention 1078] The linker L and / or L' has a length of 3 to 200 amino acids. Any Fc construct. [The present invention 1079] The linker L and / or L' is selected from the group consisting of any one of SEQ ID NOs: 1, 2, and 3. The Fc construct of the present invention 1077. [The present invention 1080] (a) a first polypeptide having the formula A-L1-B-L2-C; (i) A comprises a first Fc domain monomer; (ii) L1 is a linker; (iii) B comprises a second Fc domain monomer; and (iv) L2 is a linker; (v) a first polypeptide, wherein C comprises a third Fc domain monomer; and (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; and (iv) L2' is a linker; (v) a second polypeptide, wherein C' comprises a sixth Fc domain monomer; (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. It consists of: A and the seventh Fc domain monomer bind to form the first Fc domain, and B and B' bind to and C and the eighth Fc domain monomer combine to form a third Fc domain. A' and the ninth Fc domain monomer combine to form a fourth Fc domain; and C' and the tenth Fc domain monomer combine to form a fifth Fc domain; each of the first and the seventh Fc domain monomer is a first Fc domain monomer; and the seventh Fc domain monomer. Includes each of the second and fifth Fc domain monomers is a second Fc domain monomer; and the fifth Fc domain monomer. Includes each of the third and eighth Fc domain monomers is a third Fc domain monomer; and the eighth Fc domain monomer. Includes; each of the fourth and ninth Fc domain monomers is a fourth Fc domain monomer; and the ninth Fc domain monomer. contains the rule; and each of the sixth and tenth Fc domain monomers is linked to the sixth domain monomer; A complementary dimerization selectivity module that promotes dimerization between the tenth Fc domain monomer. and An Fc construct comprising five or fewer Fc domains. [The present invention 1081] (a) a first polypeptide having the formula A-L1-B-L2-C; (i) A consists of a first Fc domain monomer; (ii) L1 is a linker; (iii) B consists of a second Fc domain monomer; and (iv) L2 is a linker; (v) a first polypeptide, wherein C consists of a third Fc domain monomer; and (b) a second polypeptide having the formula A'-L1'-B'-L2'-C'; (i) A' consists of a fourth Fc domain monomer; (ii) L1' is a linker; (iii) B' consists of a fifth Fc domain monomer; and (iv) L2' is a linker; (v) a second polypeptide, wherein C' consists of a sixth Fc domain monomer; (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. It consists of: A and the seventh Fc domain monomer bind to form the first Fc domain, and B and B' bind to and C and the eighth Fc domain monomer combine to form a third Fc domain. A' and the ninth Fc domain monomer combine to form a fourth Fc domain; and C' and the tenth Fc domain monomer combine to form a fifth Fc domain; each of the first and the seventh Fc domain monomer is a first Fc domain monomer; and the seventh Fc domain monomer. Includes each of the second and fifth Fc domain monomers is a second Fc domain monomer; and the fifth Fc domain monomer. Includes each of the third and eighth Fc domain monomers is a third Fc domain monomer; and the eighth Fc domain monomer. Includes; each of the fourth and ninth Fc domain monomers is a fourth Fc domain monomer; and the ninth Fc domain monomer. contains the rule; and each of the sixth and tenth Fc domain monomers is linked to the sixth domain monomer; A complementary dimerization selectivity module that promotes dimerization between the tenth Fc domain monomer. and An Fc construct comprising five or fewer Fc domains. [The present invention 1082] The complementary dimerization selectivity module of the second Fc domain monomer is negatively charged. The complementary dimerization selectivity module of the fifth Fc domain monomer comprises an amino acid substitution. the first, the third, the fourth, and the sixth amino acid substitutions each contain a positively charged amino acid substitution; The complementary dimerization selectivity modules of each of the Fc domain monomers are engineered the seventh, eighth, ninth, and tenth Fc domains comprising a segmented overhang; Each of the monomers has a complementary dimerization selectivity module that occupies an engineered recess. The Fc construct of the present invention comprising 1080 or 1081. [The present invention 1083] (a) a first polypeptide having the formula A-L1-B-L2-C; (i) A comprises a first Fc domain monomer; (ii) L1 is a linker; (iii) B comprises a second Fc domain monomer; and (iv) L2 is a linker; (v) a first polypeptide, wherein C comprises a third Fc domain monomer; and (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; and (iv) L2' is a linker; (v) a second polypeptide, wherein C' comprises a sixth Fc domain monomer; (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. It consists of: A and A' bind to form the first Fc domain, and B binds to the seventh Fc domain monomer. and C and the eighth Fc domain monomer combine to form a third Fc domain. A' and the ninth Fc domain monomer combine to form a fourth Fc domain; and C' and the tenth Fc domain monomer combine to form a fifth Fc domain; each of the first and fourth Fc domain monomers is a first Fc domain monomer; and the fourth Fc domain monomer. Includes each of the second and the seventh Fc domain monomers is a second Fc domain monomer; and the seventh Fc domain monomer. Includes each of the third and eighth Fc domain monomers is a third Fc domain monomer; and the eighth Fc domain monomer. Includes; each of the fifth and ninth Fc domain monomers is a fifth Fc domain monomer; and the ninth Fc domain monomer. contains the rule; and each of the sixth and tenth Fc domain monomers is linked to the sixth domain monomer; A complementary dimerization selectivity module that promotes dimerization between the tenth Fc domain monomer. and An Fc construct comprising five or fewer Fc domains. [The present invention 1084] (a) a first polypeptide having the formula A-L1-B-L2-C; (i) A consists of a first Fc domain monomer; (ii) L1 is a linker; (iii) B consists of a second Fc domain monomer; and (iv) L2 is a linker; (v) a first polypeptide, wherein C consists of a third Fc domain monomer; and (b) a second polypeptide having the formula A'-L1'-B'-L2'-C'; (i) A' consists of a fourth Fc domain monomer; (ii) L1' is a linker; (iii) B' consists of a fifth Fc domain monomer; and (iv) L2' is a linker; (v) a second polypeptide, wherein C' consists of a sixth Fc domain monomer; (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. It consists of: A and A' bind to form the first Fc domain, and B binds to the seventh Fc domain monomer. and C and the eighth Fc domain monomer combine to form a third Fc domain. A' and the ninth Fc domain monomer combine to form a fourth Fc domain; and C' and the tenth Fc domain monomer combine to form a fifth Fc domain; each of the first and fourth Fc domain monomers is a first Fc domain monomer; and the fourth Fc domain monomer. Includes each of the second and the seventh Fc domain monomers is a second Fc domain monomer; and the seventh Fc domain monomer. Includes each of the third and eighth Fc domain monomers is a third Fc domain monomer; and the eighth Fc domain monomer. Includes; each of the fifth and ninth Fc domain monomers is a fifth Fc domain monomer; and the ninth Fc domain monomer. contains the rule; and each of the sixth and tenth Fc domain monomers is linked to the sixth domain monomer; A complementary dimerization selectivity module that promotes dimerization between the tenth Fc domain monomer. and An Fc construct comprising five or fewer Fc domains. [The present invention 1085] The complementary dimerization selectivity module of the first Fc domain monomer is negatively charged. The complementary dimerization selectivity module of the fourth Fc domain monomer comprises an amino acid substitution. the second, third, fifth, and sixth amino acid substitutions each contain a positively charged amino acid substitution, The complementary dimerization selectivity modules of each of the Fc domain monomers are engineered the seventh, eighth, ninth, and tenth Fc domains comprising a segmented overhang; Each of the monomers has a complementary dimerization selectivity module that occupies an engineered recess. The Fc construct of the present invention, comprising 1083 or 1084. [The present invention 1086] The linkers L1, L2, L1', and / or L2' are 3 to 200 amino acids in length. Any of the Fc constructs, 1080 to 1085. [The present invention 1087] The linkers L1, L2, L1', and / or L2' are any of SEQ ID NOs: 1, 2, and 3. The Fc construct of the present invention, which consists of one or more sequences. [The present invention 1088] Accelerating clearance of autoantibodies, suppressing antigen presentation, and reducing immune responses in subjects who need them The present invention relates to a method for reducing the activation of immune responses by immune complexes, and or any of the Fc constructs 1075-1087. [The present invention 1089] Inflammatory or autoimmune diseases or immune disorders (e.g., rheumatoid arthritis (RA); systemic lupus erythematosus (SLE); ANCA-associated vasculitis; antiphospholipid syndrome; autoimmune hemolytic Anemia; chronic inflammatory demyelinating neuropathy; anti-allo in transplantation, anti-autologous in GVHD, anti-substitution, IgG therapeutics, IgG paraprotein clearance; dermatomyositis; Goodpasture's syndrome; Organ system-targeted type II hypersensitivity syndromes mediated by antibody-dependent cell-mediated cytotoxicity, e.g. , Guillain-Barre syndrome, CIDP, dermatomyositis, Felty syndrome, antibody-mediated rejection, autoimmune Immune thyroid disease, ulcerative colitis, autoimmune liver disease; idiopathic thrombocytopenic purpura; severe myasthenia gravis, neuromyelitis optica; pemphigus and other autoimmune blistering diseases; Sjogren's syndrome; Autoimmune cytopenias and other disorders mediated by antibody-dependent phagocytosis; other FcR-dependent Persistent inflammatory syndromes, e.g., synovitis, dermatomyositis, systemic vasculitis, glomerulitis, and vasculitis ) Any of the Fc constructs of 1001 to 1058 or 1075 to 1087 of the present invention for use in the treatment of [Brief description of the drawings]

[0084] [Figure 1] FIG. 1 is an illustration of an Fc construct (construct 1) comprising a dimer of two wild-type (wt) Fc domain monomers (102 and 104). [Diagram 2] 2 is an illustration of an Fc construct (construct 2) comprising a dimer of two Fc domain monomers: the first Fc domain monomer (202) comprises a protrusion in its CH3 antibody constant domain, and the second Fc domain monomer (204) comprises a recess in juxtaposed position in its CH3 antibody constant domain. [Diagram 3] Figure 3 is an illustration of another Fc construct (construct 3), which comprises a dimer of two Fc domain monomers (302 and 304), both of which contain different charged amino acids in their CH3-CH3 interface than the wt sequence, which promotes favorable electrostatic interactions between the two Fc domain monomers. [Figure 4] Figure 4 is an illustration of an Fc construct (construct 4) comprising two Fc domains. The construct is formed from three polypeptides. The first polypeptide (402) comprises two wt Fc domain monomers (404 and 406) joined in tandem. The second and third polypeptides (408 and 410, respectively) each comprise a wt Fc domain monomer. [Diagram 5]FIG. 5 is an illustration of an Fc construct (construct 5 or construct 5*) comprising three Fc domains formed from four polypeptides. The first polypeptide (502) comprises one Fc domain monomer comprising a different charged amino acid at the CH3-CH3 interface than the wt sequence (506) joined in tandem with a lobe-containing Fc domain monomer (504). The second polypeptide (508) comprises an Fc domain monomer comprising a different charged amino acid at the CH3-CH3 interface than the wt sequence (5126) joined in tandem with another lobe-containing Fc domain monomer (510). The third and fourth polypeptides (514 and 516, respectively) each comprise a recess-containing Fc domain monomer. [Figure 6] 6 is an illustration of an Fc construct (construct 6) comprising two Fc domains formed from three polypeptides: a first polypeptide (602) comprises two lobe-containing Fc domain monomers (604 and 606) joined in tandem, and a second and third polypeptide (608 and 610, respectively) each comprise an Fc domain monomer engineered with a corresponding recess. [Figure 7] FIG. 7A is an illustration of another Fc construct (construct 7). This Fc construct comprises a dimer of two CL-CH1-Fc domain monomers (702 and 704). In this embodiment, a CL antibody constant domain is joined to an adjacent CH1 antibody constant domain. FIG. 7B is an illustration of an Fc construct (construct 8) comprising a multimer of CL-CH1-Fc domain monomers (e.g., 706, 708, and 710) comprising 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 CH1 antibody constant domain of a second adjacent Fc construct (e.g., 714). [Figure 8]8 is an illustration of an Fc construct (construct 9) comprising five Fc domains formed from six polypeptides. The first and second polypeptides (802 and 810) each comprise three Fc domain monomers joined in series (804, 806, 808, and 812, 814, 816, respectively). Specifically, in polypeptides 802 or 810, a first lobe-containing Fc domain monomer (804 or 812) is connected to a second Fc domain monomer that comprises a different charged amino acid at the CH3-CH3 interface than the wt sequence (806 or 814), which is connected to a third lobe-containing Fc domain monomer (808 or 816). The third to sixth polypeptides (818, 820, 822, and 824) each contain a recess-containing Fc domain monomer and form an Fc domain with Fc domain monomers 804, 808, 812, and 816, respectively. [Figure 9] Figure 9 is an illustration of an Fc construct (construct 10) comprising five Fc domains formed from six polypeptides. The first and second polypeptides (902 and 910) each comprise three Fc domain monomers joined in series (904, 906, 908, and 912, 914, 916, respectively). Specifically, in polypeptides 902 or 910, a first lobe-containing Fc domain monomer (904 or 912) is connected to a second lobe-containing Fc domain monomer (906 or 914), which is connected to a third Fc domain monomer that comprises a different charged amino acid at the CH3-CH3 interface than the wt sequence (908 or 916). The third to sixth polypeptides (918, 920, 922, and 924) each contain a recess-containing Fc domain monomer and form an Fc domain with Fc domain monomers 904, 906, 912, and 916, respectively. [Figure 10]Figure 10 is an illustration of an Fc construct (construct 11) comprising three Fc domains formed from two polypeptides of identical sequence. The two polypeptides (1002 and 1010) each comprise three Fc domain monomers joined in series (1004, 1006, 1008, and 1012, 1014, 1016, respectively). Specifically, each polypeptide comprises a first lobe-containing Fc domain monomer (1004 or 1012) connected to a second recess-containing Fc domain monomer (1006 or 1014), which is connected to a third Fc domain monomer that has a different charged amino acid at the CH3-CH3 interface than the wt sequence (1008 or 1016). 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 from 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-B 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 blocking assays (FIG. 14B) of THP-1 monocytes with constructs 1, 5, and 6. [Figure 15] FIG. 15 shows the effect 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. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0085] Detailed Description of the Invention Therapeutic proteins containing the Fc domain of IgG are being used to treat inflammation, immune disorders, and inflammatory diseases. The present invention relates to a method for producing an Fc domain comprising the steps of: Compositions and methods for preparing various Fc constructs are featured.

[0086] I. Fc domain monomer The Fc domain monomer consists of a hinge domain, C H 2 antibody constant domains, and C H 3. Antibody constant domain Fc domain monomers are the three main classes of immunoglobulin antibody isotypes: IgG, IgE, IgM, and IgA. The Fc domain monomer may also be any immunoglobulin antibody isotype. The Fc domain monomer may be of any type (e.g., IgG1, IgG2a, IgG2b, IgG3, or IgG4). The dimer comprises an Fc domain (as described herein) capable of binding to an Fc receptor, e.g., FcγRIIIa. Fc receptors are receptors located on the surface of white blood cells (as further defined in this document). In the present invention, the C of the Fc domain monomer H The three antibody constant domains promote their association with each other. The amino acid substitutions that promote H 3-C H In some embodiments, the Fc domain may be present at the interface of the Fc domain and the Fc domain of the antibody. A domain monomer consists of two other constant domains attached to the N-terminus, e.g., C L and CH 1 antibody In another embodiment, the Fc domain monomer comprises an additional constant domain attached to the C-terminus (Figure 7). In the present invention, the Fc domain monomer includes any portion thereof, such as an albumin binding peptide. Any type of antibody variable region, e.g., V H , V L , complementarity determining regions (CDRs), or hypervariable It does not include the region (HVR).

[0087] II. Fc Domain As defined herein, an Fc domain is H 3 Antibody constant domain interactions In the present invention, the Fc domain comprises two Fc domain monomers that are dimerized by the Fc domain of an antibody. Variable regions, e.g., V H , V L The Fc domain does not include any CDRs, CDRs, or HVRs. For example, the smallest structure that binds to FcγRI, FcγRIIa, FcγRIIb, FcγRIIIa, FcγRIIIb, and FcγRIV Form.

[0088] III. Dimerization Selectivity Module In the present invention, the dimerization selectivity module is a dimerization selectivity module that is a combination of two Fc domains forming an Fc domain. It is the portion of the Fc domain monomer that facilitates the preferred pairing of the dimer. The antibody selectivity module is a C domain that interacts with two Fc domains. H 3. At the interface between antibody constant domains C of Fc domain monomers containing amino acid substitutions located at H 3. Part of the antibody constant domain. In the merization selectivity module, the amino acid substitution is determined by the amino acid selected for that amino acid substitution. As a result of the compatibility of the amino acids, two C H 3. Favorable Fc domain dimerization. The main ultimate formation does not have a dimerization selectivity module. The dimerization selectivity module is provided with non-compatible amino acid substitutions from the Fc domain monomer or in the dimerization selectivity module. This type of Fc domain is selective for other Fc domains formed from Fc domain monomers that Amino acid substitutions can be made using conventional molecular cloning techniques well known in the art, for example, QuikChange ( This can be accomplished using RT-PCR mutagenesis.

[0089] In some aspects, the dimerization selectivity module is H 3. Antibody constant domain contains an engineered In another embodiment, the dimerization selection The selectivity module is C H 3. Engineered protrusions in antibody constant domains (as described herein) (further defined in the document). Two Fc domain monomers with selective cleavage modules, e.g., engineered One C including the recess H 3. The other C contains the antibody constant and an engineered protrusion H 3. Antibody Stationary bind to form a protrusion-recess pair in which the protrusion of the Fc domain monomer fits into the recess. .

[0090] In another embodiment, an Fc having a dimerization selectivity module comprising a positively charged amino acid substitution. It has a dimerization selectivity module that contains domain monomers and negatively charged amino acid substitutions. The Fc domain monomers selectively bind to each other, providing favorable electrostatic attraction of charged amino acids (referred to herein as (further defined) to form the Fc domain. Specific dimerization selectivity modules include: These are further listed, without limitation, in Tables 1 and 2, which are further described below.

[0091] In other embodiments, the two Fc domain monomers are H A small number of charged residues in the ring at the interface between the three domains The dimerization selectivity module contains identical opposite charge mutations at at least two positions. Reverse the charge of both members of two or more pairs of complementary residues in a domain monomer Thus, the mutant Fc domain monomers exhibit complementary binding to Fc domain monomers of the same mutant sequence. These mutants maintain their complementarity to the Fc domain monomers that do not have these mutations, but have low complementarity to the Fc domain monomers that do not have these mutations. In embodiments, the Fc domain is a double mutant K409D / D339K, K392D / D399K, E357K / K370E, D356K / K Contains Fc monomers containing 439D, K409E / D339K, K392E / D399K, E357K / K370D, or D356K / K439E In another embodiment, the Fc domain is a quadruple mutant combining any pair of double mutants, e.g. For example, an Fc monomer comprising K409D / D399K / E357K / K370E.

[0092] The formation of such an Fc domain is H 3. Compatible Amino Acid Substitutions in Antibody Constant Domains For example, co-engineered recesses, C with or without protrusions H 3-C H 3 Incompatible amino acids with the same charge at the interface The two dimerization selectivity modules containing amino acid substitutions do not promote the formation of an Fc domain.

[0093] Additionally, it can be used to promote the formation of Fc domains using defined Fc domain monomers. Other methods that may be used include, but are not limited to, the use of Fc domain monoclonal antibodies that allow for the formation of heterodimers. LUZ contains a C-terminal fusion of a monomeric α-helix of a leucine zipper to each of the mers. -Y approach (U.S. Patent Application Publication No. WO 2011 / 034605), and IgA sequences and IgG C H Heterodimeric Fc domain monomers each containing three alternating segments were used to synthesize F The strand-exchange recombination domain (SEED)-based approach to generate c domains (Davis et al., Proc. tein Eng Des Sel. 23:195-202, 2010).

[0094] IV. Engineered Recesses and Engineered Protrusions The use of engineered recesses and engineered protrusions (or “no The "into-hole" strategy is published by Carter and colleagues (Ridgway et al., Protein Eng. 9:617-612, 1996; Atwell et al., J Mol Biol. 270:26-35, 1997 (Merchant et al., Nat Biotechnol. 16:677-681, 1998). The knob-hole interaction The knob-knob and hole-hole interactions promote the formation of heterodimers, resulting in steric clashes and favorable The lack of essential interactions prevents homodimer formation. The technique is also disclosed in U.S. Pat. No. 5,731,168.

[0095] In the present invention, the engineered recesses and engineered protrusions are The engineered recess is used to prepare the Fc constructs described herein. It is formed when the original amino acid of a substance is replaced by a different amino acid that has a smaller side chain volume. The engineered overhang is a gap created by replacing the original amino acids of the protein with It is the protuberance that forms when a different amino acid with a larger side chain volume is substituted. Specifically, the amino acid to be replaced is C of the Fc domain monomer. H 3 in the antibody constant domain, and is involved in the dimerization of two Fc domain monomers. In some embodiments, one C H 3. Antibody Stationary The engineered recesses in the domains are separate C H 3. Engineering of Antibody Constant Domains The casing is shaped to accommodate the grooved protrusions, allowing both C H 3. Antibody constant domains , a dimerization selectivity module ( In another embodiment, one C H 3. Engineered Antibody Constant Domains The recess is another C H 3. The antibody constant domain is designed to better accommodate the original amino acids. In yet another embodiment, one C H 3. Engineered Protrusions of Antibody Constant Domains is another C H 3. Formed to allow additional interactions with the original amino acids of the antibody constant domain will be done.

[0096] The engineered recess may be modified to accommodate amino acids containing large side chains, such as tyrosine or Tryptophan can be substituted with amino acids containing small side chains, such as alanine, valine, or threonine. Specifically, some dimerization selection can be performed by substituting onine. The sex module (described in detail above) is H 3 Engineered into antibody constant domains Similarly, engineered protrusions include small recesses, e.g., Y407V mutations. It can be constructed by replacing amino acids containing large side chains with amino acids containing large side chains. In particular, some dimerization selectivity modules (described in detail above) can be H 3 antibodies It includes engineered protrusions in the constant domain, such as the T366W mutation. In the case of engineered recesses and engineered protrusions, H 3D Combined with interdomain disulfide bond engineering to form heterodimers Specifically, the recessed Fc contains a Y349C mutation and the protruding Fc contains a S354C mutation. Other engineered recesses and engineered protrusions include disulfides. Combined with hybrid engineering or structural calculation (mixed HA-TF), are included in Table 1 without being

[0097] [Table 1]

[0098] C H 3 Replacement of an original amino acid residue in an antibody constant domain with a different amino acid residue results in This can be achieved by modifying the nucleic acid encoding the amino acid residue. The upper limit of the number of amino acid residues to be obtained is C H 3. Sufficient interactions at the antibody constant domain interface If retained, this is the total number of residues at this interface.

[0099] V. Electrostatic Induction Electrostatic induction is a method for controlling the formation of peptide and protein domains. and taking advantage of favorable electrostatic interactions between oppositely charged amino acids in proteins. The electrostatic induction effect is used to modify the interactions of antibody domains, thereby forming bispecific A method for promoting heterodimer formation and reducing homodimer formation in the production of specific antibodies has been proposed. , as disclosed in U.S. Patent Application Publication No. 2014 / 0024111.

[0100] In the present invention, electrostatic induction controls the dimerization of Fc domain monomers and the formation of Fc constructs. In particular, it is used to control the dimerization of Fc domain monomers using electrostatic induction. Therefore, C H 3-C H 3. One or more amino acid residues that make up the interface are positively or negatively charged. The amino acid residues are replaced, so that the interaction depends on the specific charged amino acid that is introduced. , electrostatically favorable or unfavorable. In some embodiments, positively charged amino acids at the interface , e.g., lysine, arginine, or histidine, and negatively charged amino acids, e.g., In another embodiment, the negatively charged amino acid at the interface is replaced with aspartic acid or glutamic acid. The amino acid that is replaced by a positively charged amino acid is the C H 3 Charged amino acids can be introduced into one or both of the antibody constant domains. H 3 By introducing it into the antibody constant domain, electrostatic induction resulting from interactions between charged amino acids Dimers capable of selectively forming dimers of Fc domain monomers controlled by the effect of A solvated selectivity module (described in detail above) is formed.

[0101] In one particular example, to form a dimerization selectivity module containing opposite charges, C H 3 anti The amino acid Asp399 in the human constant domain is replaced by Lys, and the amino acid Lys409 is replaced by Asp. Heterodimerization of Fc domain monomers is a process that involves the formation of different but suitable ligands in the two Fc domain monomers. By introducing a complementary mutation, for example, but not limited to, the charged residue pairs shown in Table 2, Homodimerization of Fc domain monomers can be promoted by the symmetric introduction of the same mutation into the host, e.g., the double mutant K409D / D339K or K392D / D399K This can be facilitated by:

[0102] [Table 2]

[0103] VI. Linker In the present invention, linkers are used to link polypeptide or protein domains and / or In some embodiments, the linkage or connection between the phosphoryl group and the associated non-protein moiety is described. A linker is a link or connection between at least two Fc domain monomers, and this linker is , the C of the first Fc domain monomer such that the two Fc domain monomers are joined in tandem with each other. H 3. Connect the C-terminus of an antibody constant domain to the N-terminus of the hinge domain of a second Fc domain monomer In other embodiments, the linker is a linker that is attached to an Fc domain monomer and For example, a linker may be used to link an Fc domain monomer to another protein domain. C H 3 The C-terminus of the antibody constant domain can be attached to the N-terminus of the albumin-binding peptide. In another example, the linker can be H 1. The C-terminus of the antibody constant domain is linked to the hinb of the Fc domain monomer. In yet another embodiment, the linker can be connected to the N-terminus of the didomain. It is possible to connect individual protein domains (not including the Fc domain), e.g. L anti The C-terminus of the constant domain is linked to H 1. Attach to the N-terminus of an antibody constant domain It is possible.

[0104] The linker may be a simple covalent bond, e.g., a peptide bond, a synthetic polymer, e.g., a polyethylene ethylene glycol (PEG) polymers, or chemical reactions, e.g., chemical conjugation. If the linker is a peptide bond, In this case, the carboxyl group at the C-terminus of one protein domain is condensed with that of another protein domain. It can react with the amino group at the N-terminus of an amino acid to form a peptide bond. The peptide bonds can be synthesized by synthetic means using conventional organic chemistry reactions well known in the art, or by inducing synthesis by the host cell. They can be formed by natural synthesis from vesicles, where both proteins in tandem A polynucleotide sequence encoding a protein, e.g., DNA sequences of two Fc domain monomers, Directly by the necessary molecular machinery of the host cell, e.g., DNA polymerases and ribosomes. It can be transcribed and translated into a contiguous polypeptide encoding both proteins.

[0105] When the linker is a synthetic polymer, such as a PEG polymer, the polymer may be a polymer having two tethers. Functionalized with reactive chemical groups at each end to react with the terminal amino acids at the connecting ends of the protein. It is possible.

[0106] If the linker (other than the peptide bond discussed above) is formed from a chemical reaction, a chemical functional group , for example, amines, carboxylic acids, esters, azides, or other groups commonly used in the art. The functional groups were synthetically attached to the C-terminus of one protein and the N-terminus of another protein. The two functional groups can then be reacted by synthetic chemical means to form A chemical bond can be formed between the two proteins, which connects the two proteins together. Such chemical conjugation procedures are routine to those of skill in the art.

[0107] Spacer In the present invention, the linker between two Fc domain monomers is an amino acid sequence containing 3 to 200 amino acids. Suitable peptide spacers are known in the art, for example: The peptide linkers include flexible amino acid residues, such as glycine and serine. In certain embodiments, the spacer comprises a motif, e.g., GS, GGS, GGGGS (SEQ ID NO: 1), GG Multiple or repeated motifs of SG (SEQ ID NO: 2) or SGGG (SEQ ID NO: 3) In certain embodiments, the spacer may comprise 2 to 12 amino acids including a GS motif, e.g. For example, GS, GSGS(SEQ ID NO: 4), GSGSGS(SEQ ID NO: 5), GSGSGSGS(SEQ ID NO: 6) , GSGSGSGSGS (SEQ ID NO: 7), or GSGSGSGSGSGS (SEQ ID NO: 8). In another embodiment, the spacer is a 3-12 amino acid sequence including a GGS motif, e.g., GGS, G GSGGS(SEQ ID NO: 9), GGSGGSGGS(SEQ ID NO: 10), and GGSGGSGGSGGS(SEQ ID NO: In yet another embodiment, the spacer may comprise the motif GGSG (SEQ ID NO: 12). GGSG (SEQ ID NO: 13), GGSGGGSG (SEQ ID NO: 14), 4), or GGSGGGSGGGSG (SEQ ID NO: 15). In another embodiment, the spacer may comprise GGGGS (SEQ ID NO: 16) motif, e.g., GGGSGGGGSGGGGS (SEQ ID NO: 17). In other embodiments, the spacer is an amino acid other than glycine and serine, e.g., GE NLYFQSGG(SEQ ID NO: 18), SACYCELS(SEQ ID NO: 19), RSIAT(SEQ ID NO: 20), RP ACKIPNDLKQKVMNH(SEQ ID NO: 21), GGSAGGSGSGSSGGSSGASGTGTAGGTGSGSGTGSG(SEQ ID N O: 22), AAANSSIDLISVPVDSR(SEQ ID NO: 23), or GGSGGGSEGGGSEGGGGSEGGGSEGGGSEG GGSGGGS (SEQ ID NO: 24). In particular embodiments of the invention, the 12 or 20 amino acid sequence A peptide spacer is used to connect two Fc domain monomers in series (Figures 4-6), The 12 or 20 amino acid peptide spacer has the sequence GGGSGGGSGGGS (SEQ ID NO: In another embodiment, the sequence GG An 18 amino acid peptide spacer consisting of SGGGSGGGSGGGSGGS (SEQ ID NO: 27) was used. C L and C H 1. Connect the antibody constant domains (Figure 7A to Figure 7B).

[0108] VII. Serum protein-binding peptides Conjugation to serum protein peptides can improve the pharmacokinetics of protein therapeutics In particular, the Fc constructs described herein can be fused to serum protein binding peptides. can.

[0109] As an example, albumin binding agents that can be used in the methods and compositions described herein. Such peptides are generally known in the art. In one embodiment, the albumin binding peptide comprises: It contains the sequence DICLPRWGCLW (SEQ ID NO: 28).

[0110] In the present invention, the albumin binding peptide is attached to the N-terminus or can be attached to the C-terminus. In one embodiment, the albumin binding peptide is Construct 1, 2, 3, or 7A (FIG. 1, FIG. 2, FIG. 3, or FIG. 7A, respectively). In another embodiment, the albumin binding peptide can be attached to the C-terminus of construct 4. , 5, and 6 (Fig. 4, Fig. 5, and Fig. 6, respectively) The polypeptide may be fused to the C-terminus of the polypeptide encoding the amino acid sequence of the amino acid sequence of the cyclic amine monomer. In the present study, the albumin binding peptides are shown in constructs 4 and 6 (Figures 4 and 6, respectively). The second polypeptide encoding two Fc domain monomers linked in tandem as shown in It can be attached to the C-terminus of an Fc domain monomer that is joined to an Fc domain monomer. Albumin-binding peptides can be genetically fused to the Fc construct or by chemical means, e.g. , can be attached to the Fc construct by chemical conjugation. Alternatively, a spacer can be inserted between the Fc construct and the albumin binding peptide. Without being bound by theory, the inclusion of an albumin-binding peptide in the Fc construct of the present invention This allows the retention of a therapeutic protein by binding to serum albumin. It is anticipated that continued retention may result.

[0111] VIII. Fc constructs In general, the present invention features Fc constructs having 2-10 Fc domains. Higher binding affinity and affinity to receptors, e.g., FcγRIIIa, than a single wild-type Fc domain. The present invention relates to a method for producing an Fc domain comprising the steps of: (1) synthesizing an Fc domain having a binding activity and / or a binding activity to an Fc domain; To selectively form dimers and thereby prevent the formation of undesirable multimers or aggregates. So, two interacting C H 3. Engineering Amino Acids at the Interface of Antibody Constant Domains The method disclosed herein is directed to an Fc construct that includes an even number of Fc domain monomers, each of which is A pair of Fc domains form the Fc domain. The Fc construct consists, at a minimum, of a dimer of two Fc domain monomers. It contains one functional Fc domain formed from

[0112] In some embodiments, the Fc construct comprises one Fc domain that comprises a dimer of two Fc domain monomers. (Figures 1 to 3 and 7A). H 3 Antibody constant domains can be used without modification. 1) or may contain amino acid substitutions at the interface. The replacements are engineered recesses (Figure 2), engineered protrusions (Figure 2), or a charged amino acid (Figure 3).

[0113] In other embodiments, the Fc construct comprises two Fc domains formed from three polypeptides (Figure 4). and FIG. 6). The first polypeptide is joined by a linker and joined in tandem and the second and third polypeptides each comprise two Fc domain monomers, and The second and third polypeptides may be the same polypeptide or or a different polypeptide. Figure 4 shows an example of such an Fc construct. The first polypeptide comprises two wild-type Fc domain monomers joined in tandem by a linker. and the second and third polypeptides each comprise one wild-type Fc domain monomer. One Fc domain monomer of the first polypeptide comprises a second polypeptide and a first Fc domain, and the other Fc domain monomer of the first polypeptide is a third polypeptide and a second Fc domain. The second and third polypeptides are attached or linked to each other. FIG. 6 shows an Fc construct similar to that of FIG. 4. In FIG. 6, the first poly Both Fc domain monomers of the peptide are C H 3. Engineered into the antibody constant domain the second and third polypeptides contain a C H 3. Antibody constant domains Includes engineered recesses. Engineered protrusions enter recesses. H 3-C H 3 The interface promotes the formation of heterodimers of Fc domain monomers and prevents the formation of undesired multimers. As further described herein, in Example 4, the engineered C H The dimerization selectivity module containing three antibody constant domains is shown in Example 3. Prevents the formation of desired multimers. Example 3 shows an Fc that does not have a dimerization selectivity module. 1 illustrates the formation of an Fc construct from domain monomers.

[0114] In yet another embodiment, the Fc construct comprises three Fc domains formed from four polypeptides. The first and second polypeptides may comprise a third and fourth polypeptide (FIG. 5). In this example, the first and second polypeptides are Both peptides encode two Fc domain monomers connected in tandem by a linker. The Fc domain monomer is C H 3. Containing charged amino acid substitutions in the antibody constant domain, whereas the other F The c domain monomer is C H 3. The antibody constant domain includes a protrusion. The first and second polypeptides together encode an Fc domain monomer having a recess. Those C H The opposite charge interactions of the three antibody constant domains form the first Fc domain. The second and third Fc domains are composed of the first and second polypeptide lobes and the third and and the recess of the fourth polypeptide, the protrusion is formed from an interaction between the protrusion and the recess of the fourth polypeptide, the protrusion being inserted into the recess. Each Fc domain monomer in this Fc construct is regulated by a dimerization selector that promotes the formation of a specific Fc domain. Includes a selectivity module.

[0115] In yet another embodiment, the single polypeptide is H 3 Antibody constant domain interactions Not te, but C L Constant domain and C H 1. Dimerization (e.g., Construct 7A; FIG. 7A) or multimers (e.g., construct 7B; FIG. 7B). Figure 7B shows the C of one Fc domain. L C of the Fc domain adjacent to the domain H 1. Domains and interactions 1 shows an Fc construct comprising multiple Fc domains that

[0116] In yet another embodiment, the Fc construct comprises five Fc domains formed from six polypeptides. Two examples are shown in Figures 8 and 9. These illustrated Fc constructs It is composed of six polypeptides, four of which are linked by the same nucleic acid. and the remaining two polypeptides can be encoded by the same nucleic acid. As a result, these Fc constructs can be synthesized by expression of the two nucleic acids in a suitable host cell. It can be produced by the above method.

[0117] In another embodiment, an Fc construct comprising two or more Fc domains comprises two polypeptides having the same primary sequence. Such constructs can be formed from a single polypeptide in a host cell. An example is shown in FIG. 10. In this example, A single nucleic acid is sufficient to encode an Fc construct containing two Fc domains. The two Fc domain monomers that are part of the peptide are linked together by a flexible linker of sufficient length and flexibility. The inclusion of a linker allows the formation of an Fc domain; this linker can be cleaved This same polypeptide can be linked by an optional flexible linker. The third Fc domain monomer is joined to another Fc domain. It is possible to join the .alpha.-amino acids to the .alpha.-amino acids to form the Y-shaped Fc construct shown in FIG. 10. The formation of the Fc domain is similarly shown in FIG. 10, in that the dimerization selectivity module It can be controlled by use.

[0118] IX. Host Cells and Protein Production In the present invention, host cells can be used to produce the polypeptides and constructs described herein from the corresponding nucleic acids. A vehicle that contains essential cellular components, e.g., organelles, that are necessary for expression of the product. The nucleic acid may be synthesized by conventional techniques known in the art (e.g., transformation, transfection, electrophoresis, etc.). The drug is introduced into the host cell by various methods, such as poration, calcium phosphate precipitation, or direct microinjection. The host cell may be of mammalian or bacterial origin. Mammalian host cells include, but are not limited to, CHO (or CHO-derived cell lines). , e.g., CHO-K1, CHO-DXB11, CHO-DG44), mouse host cells (e.g., NS0, Sp2 / 0) , VERY, HEK (e.g., HEK293), BHK, HeLa, COS, MDCK, 293, 3T3, W138, BT483, Hs57 8T, HTB2, BT20 and T47D, CRL7O3O, and HsS78Bst cells. Regulating expression of a construct or modifying and processing a protein product in a desired specific manner It is also possible to select a host cell that is suitable for post-translational processing of the protein product. Different host cells may differ in Suitable cell lines or host systems include those that have characteristic specific mechanisms for transcription and modification. Selection should be made to ensure correct modification and processing of the expressed protein. can.

[0119] For expression and secretion of protein products from their corresponding DNA plasmid constructs In addition, the host cell may contain promoters, enhancers, sequences, transcription terminators, polyadenylation sequences, and The expression vector is controlled by suitable expression control elements known in the art, including transcriptional site, and selection marker. The cells can be transfected or transformed with DNA that is capable of expressing therapeutic proteins. The methods are known in the art. For example, see Paulina Balbas, Argelia Lorence (eds.) Rec. ombinant Gene Expression: Reviews and Protocols (Methods in Molecular Biology), Humana Press; 2nd ed. 2004 edition (July 20, 2004); Vladimir Voynov and Justin A Caravella (eds.) Therapeutic Proteins: Methods and Protocols (Methods in Molec (Clinical Biology) Humana Press; 2nd ed. 2012 edition (June 28, 2012).

[0120] X. Purification The Fc construct can be purified by any method of protein purification known in the art, for example, by chromatography. Chromatography (e.g., ion exchange), affinity (e.g., protein A affinity) (and size exclusion column chromatography), centrifugation, differential solubility, or tandem The protein can be purified by other standard techniques for protein purification. For example, Fc The constructs can be purified by affinity column chromatography, e.g., by protein chromatography. By appropriately selecting and combining the A column, filtration, ultrafiltration, salting out, and dialysis methods, The antibody can be isolated and purified by the process scale purification method (e.g., Process Scale Purification of Antibodies). dies, Uwe Gottschalk (ed.) John Wiley & Sons, Inc., 2009; and Subramanian (ed.) Antibodies-Volume I-Production and Purification, Kluwer Academic / Plenum Publisher rs, New York (2004). In some cases, the Fc construct comprises a marker sequence, For example, they can be conjugated to a peptide to facilitate purification. An example of a sequence of amino acids is a hexa-histidine peptide. The peptide was bound to a nickel-functionalized agarose gel affinity column with micromolar affinity. Other peptide tags useful for purification include, but are not limited to, hemagglutinin. Examples of such tags include the "HA" tag, which is derived from the influenza hemagglutinin protein. It corresponds to a peptide (Wilson et al., 1984, Cell 37:767).

[0121] For Fc constructs, Protein A column chromatography is used as the purification process. Protein A ligand can interact with Fc constructs via the Fc region and bind to proteins. The TEIN A chromatography is a high-performance chromatography method that can remove most of the host cell proteins. In the present invention, the Fc construct is a protein A as described in Example 2. Purification can be achieved using column chromatography.

[0122] XI. Pharmaceutical Compositions / Formulations The invention features pharmaceutical compositions that include one or more Fc constructs described herein. In one embodiment, the pharmaceutical composition comprises a substantially identical or substantially identical Fc construct. In various examples, the pharmaceutical composition comprises a substantially homogenous population of constructs 1-10 and 5. * Either or one substantially homogeneous population.

[0123] The therapeutic protein constructs of the present invention, e.g., Fc constructs, can be included in pharmaceutical compositions. Pharmaceutical compositions containing therapeutic proteins can be formulated by methods known to those skilled in the art. The pharmaceutical composition may be a sterile solution or suspension in water or another pharma- ceutically acceptable solvent. It can be administered parenterally in the form of an injection preparation, including a suspension. For example, the pharmaceutical composition The vehicle or medium may be a pharma- ceutically acceptable vehicle or medium, such as sterile water for injection (WFI), saline, or the like. Saline, emulsifier, suspending agent, surfactant, stabilizer, diluent, binder, additive and Fc construct are suitable and then blended into a unit dosage form required for generally accepted pharmaceutical practice. The amount of active ingredient contained in the pharmaceutical preparation may be any suitable dosage within the specified range. is the amount provided.

[0124] Sterile compositions for injection are prepared according to conventional pharmaceutical practice using distilled water for injection as the vehicle. For example, saline, or glucose and other supplements, e.g. , D-sorbitol, D-mannose, D-mannitol, and sodium chloride, etc. The tonicity solution may optionally contain a suitable solubilizing agent, such as an alcohol, e.g., ethanol, and polyisoprene. Alcohols, such as propylene glycol or polyethylene glycol, and non-isopropyl alcohols. Ionic surfactants such as polysorbate 80™, HCO-50, and those commonly used in the art It can be used as an injectable aqueous solution in combination with those known in the art. Methods for formulating protein products for use are known in the art. Peptides and Proteins: Formulation, Processing and Delivery Systems (2d ed.) Tay See Lor & Francis Group, CRC Press (2006).

[0125] XII. Administration The pharmaceutical composition should be administered in a manner compatible with the dosage formulation to provide therapeutic benefit, resulting in amelioration or treatment of the symptoms. The pharmaceutical compositions are administered in various dosage forms, for example, intravenous dosage forms, subcutaneous dosage forms, The drug is administered in oral dosage forms, such as ingestible solutions, and drug-release capsules. The dosage appropriate for an individual subject will depend on the therapeutic objectives, the route of administration, and the condition of the patient. Generally, the recombinant protein is administered at a concentration of 1 to 200 mg / kg, for example, 1 to 100 mg / kg, for example, 20 to 10 0 mg / kg; therefore, your healthcare provider will need to adjust the dose to achieve the specified concentration. and alter the route of administration as necessary to obtain the maximal therapeutic effect.

[0126] XIII. Indications The pharmaceutical compositions and methods of the present invention provide for the reduction of inflammation in a subject, clearance of autoantibodies in a subject, suppression of antigen presentation in a subject; reduction of an immune response, e.g., an immune complex of an immune response in a subject. These compounds are useful for inhibiting activation by the body and treating immune and inflammatory conditions or diseases in a subject. Exemplary conditions and diseases include, but are not limited to, rheumatoid arthritis (RA); systemic lupus erythematosus SLE; ANCA-associated vasculitis; antiphospholipid syndrome; autoimmune hemolytic anemia; chronic inflammation Anti-allo in transplantation, anti-autolog in GVHD, anti-substitution, IgG therapeutics, IgG Paraprotein clearance; Dermatomyositis; Goodpasture's syndrome; Antibody-dependent cellular Organ system-targeted type II hypersensitivity syndromes mediated by endothelial cell cytotoxicity, e.g., Guillain-Barre syndrome syndrome, CIDP, dermatomyositis, Felty syndrome, antibody-mediated rejection, autoimmune thyroid disease ulcerative colitis, autoimmune liver disease; idiopathic thrombocytopenic purpura; myasthenia gravis, optic nerve disorder Transmyelitis; Pemphigus and other autoimmune blistering diseases; Sjogren's syndrome; Antibody-dependent phagocytosis Autoimmune cytopenias and other disorders mediated by FcR; other FcR-dependent inflammatory syndromes Groups include, for example, synovitis, dermatomyositis, systemic vasculitis, glomerulitis, and vasculitis. EXAMPLES

[0127] Example 1 - Design and cloning of DNA plasmid constructs A total of eight DNA plasmid constructs were used to construct the eight Fc constructs (Figures 1 to 7B). The A plasmid construct was transformed into human embryonic kidney (HEK) 293 cells for protein production. The eight encoded secreted polypeptides have the general structures shown below. Ta: A. wt Fc: wild-type Fc domain monomers (Figure 1: 102 and 104; Figure 4: 408 and 410). B. Projection Fc:C H 3. Fc domain with engineered protrusions in the antibody constant domain In monomer (Figure 2:202). C. Recess Fc:C H 3. Fc domain with engineered protrusions in the antibody constant domain monomers (Figure 2: 204, Figure 5: 514 and 516). C * . Recess Fc * :C H 3. Fc domain with an engineered recess in the antibody constant domain monomer (Figure 2: 204; Figure 5: 514 and 516). * represents an additional amino acid sequence for the recessed Fc. Acid substitutions are also included. D. Charge Fc:C H 3. Fc domain monomers with opposite charges in the antibody constant domain (Figure 3: 302 and 304). E. wt-12-wt Fc2: Two 12-amino acid GGGS peptide linkers joined in tandem. Fc domain monomer (Figure 4:402). F. Overhang-20-charged Fc2: 20 amino acids joined in tandem by a SGGG peptide linker , C H 3. Fc domain monomers with opposite charges in antibody constant domains and C H 3. Antibody Constant Domains Fc domain monomers with engineered protrusions in the . F * .Protrusion-20-Charge Fc2 * : joined in tandem by a 20 amino acid SGGG peptide linker T, C H 3. Fc domain monomers with opposite charges in antibody constant domains and C H 3. Antibody Constant Domain Fc domain monomers with engineered protrusions in the domain (Figure 5: 502 and 508 ). Protrusion-20-Charge Fc2 * also contains additional amino acid substitutions to the Fc protrusions. G. Lobe-20-lobe Fc2: 20 amino acids joined in tandem by a GGGS peptide linker T, C H3 Two Fc domains, each with engineered protrusions in the antibody constant domain Domain monomer (Figure 6:602). H.C. H C L Fc+: C attached to the hinge domain H 1 and C L Fc domain with constant domains Monomers (Figure 7A: 702 and 704; Figure 7B: 706, 708, 710, 712, 714, and 716). C L Stationary The main chain is bounded by an 18 amino acid GGGS peptide linker. H 1 attached to a constant domain do.

[0128] The Fc DNA sequence was derived from human IgG1 Fc. The lobes, recesses, and charge mutations were c sequence, which encodes a leader peptide derived from the human immunoglobulin kappa light chain. The DNA was attached to the 5' region. All polypeptides except one (C H C L Fc+) is a structure To induce translocation of proteins into the endoplasmic reticulum for assembly and secretion All of the various leader peptides contained this encoded peptide at their amino termini. It should be understood that either one of these may be used in conjunction with the present invention. The 11-nucleotide sequence containing the 5'-terminal EcoR1 site is usually cleaved in the lumen of the endoplasmic reticulum. A 30-nucleotide sequence containing the 3'-terminal Xho1 site was added upstream of the initiation codon. The DNA sequence was optimized for expression in mammalian cells. and cloned into the pcDNA3.4 mammalian expression vector.

[0129] Mutations are indicated by the wild-type amino acid residue followed by the position of the mutation according to the EU Kabat numbering system. (Kabat et al., Sequences of Proteins of Immunological Interest, National Institute The amino acid sequence is shown using the nucleotide sequence of the nucleotides in the Table of Contents of Health, Bethesda, Md., ed. 5, 1991), and the substituted residues are shown in one sentence. The nucleotide and amino acid sequences of the above secreted polypeptides A to H are shown in the alphabetic code. The columns are shown below (only the amino acid sequence is shown in the recess Fc * and protrusion-20-charge Fc2 * (Excluding.)

[0130] wtFc TIFF0007600172000003.tif82128TIFF0007600172000004.tif43128 Projection Fc TIFF0007600172000005.tif140128 Recess Fc TIFF0007600172000006.tif132128 Recess Fc * SEQ ID NO:45: TIFF0007600172000007.tif41128 Charge Fc TIFF0007600172000008.tif127128wt-12-wt Fc2 TIFF0007600172000009.tif210112 Protrusion-20-Charge Fc2 TIFF0007600172000010.tif146128TIFF0007600172000011.tif66128Protrusion-20-Charge Fc2 * TIFF0007600172000012.tif66128Protrusion-20-ProtrusionFc2 TIFF0007600172000013.tif147128TIFF0007600172000014.tif66128C H CL Fc+ TIFF0007600172000015.tif135128TIFF0007600172000016.tif65128

[0131] Example 2 - Expression of Fc construct proteins For the expression of the Fc construct protein, a DNA fragment selected from A to H described in Example 1 is used. Two of the mide constructs were transfected into EXPI293 cells (Life Technologies). Plasmid DNA was introduced into EXPI293 cells using genome transfection. The total amount of plasmid construct infected was kept constant, but the amount of plasmid was increased to maximize the yield of the desired construct. The ratios of the different plasmid constructs were varied to achieve a For each Fc construct, the ratio (by mass) of the two transfected DNA plasmid constructs is These are shown in Table 3. Exemplary constructs are shown in Figures 1-7B.

[0132] After protein expression, the expressed construct was incubated with affinity chromatography using Protein A. The medium supernatant was purified from the cell culture supernatant by column chromatography using AKTA Avant Poros MabCapt using a preparative chromatography system (GE Healthcare Life Sciences) The captured Fc construct was then loaded onto a ureA (LifeTechnologies) column. Wash with low-salt wash, followed by phosphate buffered saline supplemented with 500 mM NaCl. The Fc construct was incubated in 100 mM Glycine, 150 mM NaCl, and washed with high-salt wash. The protein solution eluted from the column was diluted with 1M TRIS pH 7.4 and eluted with 1M TRIS pH 7.4. Neutralize by adding 0.5% PBS to a final concentration of 100 mM. The Fc constructs were incubated with Poros® XS resin (Ap and by ion exchange chromatography using a 100% pure 1-chlorophenyl 2-(2-methylphenyl)pyridine dinucleotide (Plied Biosciences Cat. # 4404336). The column was pre-equilibrated with 10 mM MES, pH 6 (buffer A), and the sample was diluted with 10 mM Elution was performed with a gradient of MES, 500 mM sodium chloride, pH 6 (buffer B).

[0133] Applicants obtained a total of seven Fc constructs (see Table 3 below and Figures 1-7B). The purified Fc constructs were subjected to SDS-PAGE (SDS-PAGE) under both reducing and non-reducing conditions. Sodium polyacrylamide gel electrophoresis (SPC) followed by Coomassieve The presence of a protein band of the expected size was confirmed by Louw staining.

[0134] [Table 3]

[0135] Example 3 - Preparation of Construct 4 and SDS-PAGE Analysis Two DNA plasmid constructs, wt-12-wt Fc2 (DNA plasmid construct E in Example 1) and and wt Fc (DNA plasmid construct A in Example 1) were used to express construct 4 (Figure 4). The two plasmid constructs were used for protein expression and purification as described in Example 2. 11A-11B show the reduced and non-reduced expression of construct 4. In the reduced SDS-PAGE (FIG. 11A), the applicants observed that the wt Fc domain The bands of approximately 25 kDa (lanes 2 and 3, Fig. 11A) correspond to the Fc2 monomer and the wt-12-wt Fc2 A band of approximately 50 kDa (lanes 1 to 3, Figure 11A) corresponding to the tandem dimer was observed. In DS-PAGE (Figure 11B), lanes 2 and 3 contain high (1 / 2) and low (1 / 2) amounts of protein, respectively. The final protein product of construct 4 contains less (1 / 3) and less protein. The association of the wt-12-wt Fc2 tandem dimer to form product 4 into two wt Fc domain monomers was One major band of approximately 100 kDa corresponding to the free wt Fc domain not conjugated to the wt Fc domain monomer was observed. One major band of approximately equal signal intensity at approximately 50 kDa corresponds to the t-12-wt Fc2 tandem dimer. The results were observed.

[0136] In addition, applicants have identified a multimer of wt-12-wt Fc2 and wt Fc domain monomers. High molecular weight bands were observed at approximately 150 kDa, 200 kDa, and 250 kDa (lanes 2 and 3, Figure 11B). I understand.

[0137] Example 4 - Preparation of Construct 6 and SDS-PAGE Analysis Two plasmid constructs, Lobe-20-LobeFc2 (DNA plasmid construct G in Example 1) and recess-Fc (DNA plasmid construct C of Example 1) was used to express construct 6 (Figure 6 The two plasmid constructs were used for protein expression and The construct was transfected into HEK 293 cells for purification. In reduced SDS-PAGE (FIG. 12A), the applicants observed a 100% reduction in the recess Fc The band of approximately 25 kDa (lanes 2 and 3, FIG. 12A) corresponds to the domain monomer and the protrusion-2 A 50 kDa band (lanes 1 to 3, FIG. 12A) corresponding to the 0-protruding Fc2 tandem dimer was observed. In non-reducing SDS-PAGE (Figure 12B), lanes 2 and 3 each contained a large amount of protein. The final protein product of construct 6 includes the 1 / 2 and 1 / 3 protein-rich constructs. They found that the association of a lobe-20-lobe Fc2 tandem dimer into two recessed Fc domain monomers There was one major band of approximately 100 kDa corresponding to the concave Fc domain bound to any monomers. Weak signal intensity of approximately 50 kDa corresponding to the free lobe-20-lobe Fc2 tandem dimer One minor band was observed.

[0138] A similar experiment was carried out with construct 5 (Figure 13). Two plasmid constructs, overhang -20- Charge Fc2 (DNA plasmid construct F of Example 1) and recess Fc (DNA plasmid construct F of Example 1) Construct 5 was expressed using construct C) (Figure 5). The two plasmid constructs were then transfected with cationic lipids. The transfection was carried out in EXPI293 cells at empirically determined ratios by plasmid transfection. The transfected cultures were incubated in cell culture medium for 6–8 days. The cells were then removed by centrifugation. The supernatant (medium, lane 1 in Figure 13) contained trastuzumab. The construct 5 is secreted into the medium by the transfected cells. Construct 5 was purified by Protein A affinity chromatography. At this point, the medium contained three Fc domains (trimers). The desired construct 5, as well as two Fc domains (dimer, approximately 10-15%) and one Fc domain It contains a certain percentage of misassembled proteins with a monomeric form (5-10%). Small amounts of contaminating host cell proteins were still present. The Protein A column eluate was purified using Strontium nitrate. The mixture was buffer exchanged, concentrated, and separated by Cation Exchange (SCX) chromatography. Briefly, construct 5 was bound to an SCX column and then eluted with a salt and pH gradient. This step resulted in the production of two or one of the desired constructs 5 having three Fc domains. Separation of most misassembled proteins with Fc domains, unwanted post-translational modifications This allows for the separation of the construct 5 carrying the After another round of concentration and buffer exchange, the pure final protein product of construct 5 was obtained. (pure, lane 2 in FIG. 13).

[0139] FIG. 13 shows the results of the antibody produced from cultured host cells engineered to express construct 5. The SDS-PAGE of the culture medium (lane 1) and purified construct 5 (lane 2) is shown. A table showing the percentage of the main band in SDS-PAGE for the samples is also shown. In (lane 1), a major band of approximately 150 kDa was observed, corresponding to construct 5 having three Fc domains. This medium sample corresponded to the final protein product. A minor band of weak signal intensity at 100 kDa corresponding to the protein and one Fc domain were observed. The purified product also contained a second minor band of weak intensity at 50 kDa corresponding to a protein containing After purification (lane 2), approximately 1 μM of the construct was purified, corresponding to the final protein product of construct 5 with three Fc domains. The main band at 50 kDa was concentrated. Signal intensity of protein bands on SDS-PAGE of construct 5 Quantification of the total protein in the culture medium revealed that, prior to protein purification, approximately 79% of the total protein was The desired protein product was shown to be 5. After purification of the protein, it was found to be approximately 95% pure. A substantially homogenous population of construct 5 carrying the construct was obtained.

[0140] These findings suggest that C H 3. Engineered Protuberances in Antibody Constant Domains Selected dimerization modules containing either the α- or α-amino acid sequence or an engineered recess are It has been demonstrated that the antibody reduces the binding of IgG1 to IgG1, preventing unrestricted Fc-mediated aggregation or multimer formation. The use of dimerization selectivity modules in the constructs described herein can be used to substantially enhance the selectivity of Fc constructs. This observation suggests that it is possible to produce substantially homogeneous formulations. For advantages in construct production, yield, and purity, to control for efficacy and efficacy. Has a significant impact.

[0141] Example 5 - Binding affinity and avidity Binding of the constructs to multiple Fcγ receptors was assessed using a cell-based FRET competition assay (Cisbio Bio Constructs 5 and 6 were evaluated using the wild-type Fc domain (construct 1). , at least a 10-fold decrease in IC50 for FcγRIIa, FcγRIIb, and FcγRIIIa ( i.e., increased binding).

[0142] Example 6 - Monocyte activation and blocking assays Three Fc constructs, constructs 1, 5, and 6, containing one, three, and two Fc domains, respectively The compounds were tested for their ability to promote the activation of their own THP-1 monocytes. 8 release was used as an indicator of monocyte activation. Constructs 1, 5, and 6 were used as described in Examples 1 and 2. Each of the purified Fc constructs was added to THP-1 monocytes. No substantial IL-8 release was observed with any of the three constructs. This data is shown in Figure 1. As shown in FIG. 14A.

[0143] The same three Fc constructs were then assayed for their ability to inhibit Fc receptor-mediated monocyte activation. IgG1 (100 μg / mL) was immobilized on a 96-well plate and used to measure the activity of TH IL-8 release was induced by P-1 monocytes. Constructs 1, 5, and 6 or control substances (immunoglobulins) were then administered. Injectable immunoglobulin (IVIg), human serum albumin (HSA), and glycine buffer Serial dilutions were performed in tissue culture plates. THP-1 monocytes (1.5 × 10 5 of cells) with thorough mixing. Cultures were incubated for 18 hours and supernatants were analyzed for IL-8. Constructs 5 and 6 were found to inhibit IL-8 release more effectively than construct 1 at low doses. The data are shown in Figure 14B.

[0144] Example 7 - K / BxN Arthritis Model Fc constructs 1, 5, and 6 and IVIg were synthesized as described by Anthony, Proc. Natl. Acad. Sci. USA 10 5:19571-19578 (2008) using the method described in The 12-week K / BxN mice of age 18 years were purchased from Jackson Laboratories. A total of 30 C57BL mice were The mice were divided into five groups of six mice each. Each group received 200 μl of Construct 6 at 0.1 g / kg, 200 0.1 g / kg of construct 5, 200 μl of IVIg at 0.1 g / kg, 230 μl of IVIg at 1 g / kg, or 200 μl of phosphate-buffered saline (PBS) and 200 μl of K / BxN serum (arthritis-inducing serum) The injection was intravenously (iv) 1 hour before the start of treatment (day 0). Inflammation was assessed clinically as swelling of the legs and ankle circumference. The examination was scored. For paw swelling, each paw was scored from 0 to 3 (0 being no swelling). (none; 3 being maximal swelling). The scores for the four paws were added to the total clinical score for each individual mouse. For neck thickness, we used caliper measurements. Each mouse was The mean daily clinical scores for each group of six mice are plotted in FIG. As shown in FIG. 15, IVIg at 1 g / kg, construct 5 at 0.1 g / kg, and Construct 6 provided similar levels of inflammation protection. Given that constructs 5 and 6 were administered at a 10-fold lower dose compared to IVIg, appears to be more effective.

[0145] Example 8 - Chronic ITP model Constructs 1 and 5 and IVIg were used to treat mice with immune thrombocytopenia (ITP). ITP is a condition induced by antiplatelet Abs that causes thrombocytopenia. Forty-five C57BL / 6 mice (18–22 g, Charles Rivers Labs, MA) were administered 1.5 μg / mouse of 100 mg of 10 ... Rat anti-CD41 antibody (Ab) (clone MWReg30 BioLegend cat#133910) was injected intraperitoneally every day for 4 days. Intravenous injections of 1.5 μg / mouse were administered to five mice (days 1, 2, 3, and 4). Injection of anti-IgG1, k isotype control Ab (BioLegend cat# 400414) resulted in normal platelet levels Ab was injected in 100 μl of PBS. 2 hours after the third injection of anti-CD41 Ab on day 3 All mice received 200 μl of saline control, 1 g / kg IVIg, 0.02 g / kg, 0.03 g / kg, or 0 Construct 1 at 0.1 g / kg, and 0.3 g / kg, and construct 2 at 0.004 g / kg, 0.02 g / kg, and 0.1 g / kg Mice were intravenously injected once with construct 5. On day 5 (24 hours after the fourth injection of anti-CD41 Ab), Bleeding was performed and total platelet levels were quantified by VetScan Instrument. Comply with the Animal Welfare Act and follow the Guide for the Care and Use of Laboratory Animals. And so he went.

[0146] As shown in FIG. 16, platelet levels increased by 0.02 g / kg compared to saline controls. The results were significantly increased after treatment with 0.1 g / kg and 0.2 g / kg of construct 5 (On By e-way ANOVA, **** p< 0.0001). Platelet levels in these groups were higher than normal isotypes. The levels were similar in the 1 g / kg IVIg and 0.1 and 0.3 g / kg configuration treatment groups. Treatment with construct 1 also significantly increased platelet levels compared to saline controls. (By one-way ANOVA using multiple comparison tests, * p < 0.05; ** p<0.01), Platelet levels in these groups were lower than the 0.02 g / kg and 0.1 g / kg Construct 5 treatment groups. In this model, construct 5 appears to be approximately 50-fold more potent than IVIg.

[0147] Example 9 - Construct 5 * Enhanced binding and avidity to FcγR compared with IVIg Show Following the same protocol as described in Example 8, overhang-20-charged Fc2 * (implementation Construct F of Example 1 * ) and recess Fc * (Construct C of Example 1 * ) two plasmids encoding Using construct 5 * The construct was expressed and purified. Binding of this construct to various Fc receptors The profile was determined by binding the Fc of IVIg in a fluorescence resonance energy transfer (FRET) competitive binding assay. The binding profiles for the receptors were compared.

[0148] Construct 5 * showed a similar overall binding profile to IVIg for different Fcγ receptors. However, compared with IVIg, all low binding affinities were significantly higher (lowest binding affinity was observed for FcγRIIb). The binding to high affinity FcγRs was significantly enhanced. This corresponds to a high binding activity, implying a cumulative effect of the cumulative affinity of each binding interaction. Structure 5 * The IC50 values ​​of IVIg were always lower than those of IVIg and were lower compared to individual IgG molecules. These results suggest a marked increase in binding to FcγRs with low affinity compared to IVIg. , Construct 5 * showed an approximately 170-fold increase in affinity for FcγRIIa (H131 mutant) and Fcγ It showed a 55-fold increase in affinity for RIIb.

[0149] Example 10 - Inhibition of phagocytosis in THP-1 monocytic cells Construct 5 * and IVIg were tested in a model of phagocytosis.

[0150] Phagocytosis is the process by which a cell (a phagocyte) engulfs a solid particle, e.g., a bacterium, and creates a cell called a phagosome. In the immune system, phagocytosis is the process of forming internal vesicles that are absorbed by the cells and destroy pathogens and cell debris. Monocytes and macrophages are among the major mechanisms used to remove inflammatory cytokines. Among these, opsonization by phagocytosis ( However, in autoimmune diseases, Phagocytosis is activated to prevent the harmful release of inflammatory cytokines or other important cells in the body. A pooled plasma sample extracted from over 1000 blood donors was IVIg containing multivalent IgG antibodies is used to treat autoimmune diseases.

[0151] In this assay system, a fluorescently labeled antibody that mimics opsonized bacteria or viruses is used. Body-coated latex beads are used in construct 5. * and THP-1 monocytes in the presence and absence of IVIg. At the end of the incubation period, external fluorescence was detected with trypan blue. The fluorescence is quenched and the amount of intracellular fluorescence is quantified by flow cytometry. All groups were normalized to their untreated controls (THP-1 cells and latex beads only). Results are representative of two separate experiments.

[0152] As shown in Figure 17, phagocytosis of opsonized beads by THP-1 monocytic cells is inhibited by treatment with both IVIg and construct 5*, but the IC50 value of construct 5* is approximately 100-fold lower than that of IVIg, suggesting that Fc constructs of the invention, such as construct 5*, can be used to treat autoimmune conditions and other conditions treatable with IVIg. The inventions described in the original claims of this application are listed below. [Invention 1] (a) a first polypeptide having the formula ALB; (i) A comprises a first Fc domain monomer; (ii) L is a linker; and (iii) a first polypeptide, wherein B comprises a second Fc domain monomer; (b) a second polypeptide having the formula A'-L'-B'; (i) A' comprises a third Fc domain monomer; (ii) L' is a linker; and (iii) a second polypeptide, wherein B' comprises a fourth Fc domain monomer; (c) a third polypeptide comprising a fifth Fc domain monomer; and (d) a fourth polypeptide comprising a sixth Fc domain monomer. Including, A of the first polypeptide and A' of the second polypeptide combine to form a first Fc domain, B of the first polypeptide and a fifth Fc domain monomer combine to form a second Fc domain, and B' of the second polypeptide and a sixth Fc domain monomer combine to form a third Fc domain, (I) the first Fc domain monomer and the third Fc domain monomer comprise complementary dimerization selectivity modules that promote dimerization between the first Fc domain monomer and the third Fc domain monomer; (II) the second Fc domain monomer and the fifth Fc domain monomer comprise complementary dimerization selectivity modules that promote dimerization between the second Fc domain monomer and the fifth Fc domain monomer; and / or (III) the fourth Fc domain monomer and the sixth Fc domain monomer comprise complementary dimerization selectivity modules that promote dimerization between the fourth Fc domain monomer and the sixth Fc domain monomer; Fc construct. [Invention 2] The Fc construct of invention 1, wherein A, B, A', B', the third polypeptide, and / or the fourth polypeptide consist of an Fc domain monomer. [Invention 3] (a) IgG C L Antibody constant domains and IgG C H 1 antibody constant domain, L The antibody constant domain is linked to the IgG C H 1 antibody constant domain and attached to the N-terminus of said IgG C H IgG C, in which an antibody constant domain is attached to the N-terminus of A by a linker L Antibody constant domains and IgG C H an antibody constant domain; or (b) IgG C L Antibody constant domain, second IgG CL Antibody constant domain, IgG C H 1 antibody constant domain, and the second IgG C H 1 antibody constant domain, L The antibody constant domain is linked to the IgG C H 1 antibody constant domain and attached to the N-terminus of said IgG C H A second IgG C L The antibody constant domain is linked to the second IgG C H attached to the N-terminus of one antibody constant domain and said second IgG H IgG C1 antibody constant domain is attached to the N-terminus of A' by a linker L Antibody constant domain, second IgG C L Antibody constant domain, IgG C H 1 antibody constant domain, and the second IgG C H 1. Antibody Constant Domain 3. The Fc construct according to claim 1 or 2, further comprising: [Invention 4] 4. The Fc construct according to any one of claims 1 to 3, further comprising an albumin binding peptide. [Invention 5] The Fc construct according to any one of Inventions 1 to 4, wherein the first polypeptide and the second polypeptide have the same amino acid sequence, and the third polypeptide and the fourth polypeptide have the same amino acid sequence. [Invention 6] The dimerization selectivity module comprises one C of the Fc domain monomer. H A recess engineered into the Fc domain and another C of the Fc domain monomer. H 6. An Fc construct according to any one of claims 1 to 5, comprising an engineered protrusion in the three domains, said engineered recess and said engineered protrusion being positioned such that the protrusion of the Fc domain monomer fits into the recess to form a protrusion-recess pair. [Invention 7] 7. The Fc construct of claim 6, wherein one of the Fc domain monomers comprises Y407V and Y349C, and the other of the Fc domain monomers comprises T366W and S354C. [Invention 8] The dimerization selectivity module is a C H The negatively charged amino acid in the Fc domain and the other C H 6. The Fc construct according to any one of claims 1 to 5, comprising positively charged amino acids in the three domains, the negatively charged amino acids and the positively charged amino acids being positioned so as to promote the formation of an Fc domain. [Invention 9] 9. The Fc construct of invention 8, wherein one of said Fc domain monomers comprises D399K and another of said Fc domain monomers comprises K409D. [Invention 10] 10. The Fc construct according to any one of claims 1 to 9, wherein one or more linkers of the Fc construct are bonds or spacers. [Invention 11] 11. The Fc construct of claim 10, wherein the spacer comprises any one of the sequences of SEQ ID NOs: 1 to 27, or consists of any one of SEQ ID NOs: 1, 2, and 3. [Invention 12] 12. The Fc construct according to any one of claims 4 to 11, wherein the albumin binding peptide comprises or consists of the sequence of SEQ ID NO:28. [Invention 13] one or more of said Fc domain monomers, and / or each of said Fc domain monomers, (a) IgG hinge domain, IgG C H 2 antibody constant domains, and IgG C H 3 antibody constant domains; or (b) IgG hinge domain, IgG C H 2 antibody constant domains, and IgG C H 3 antibody constant domains, wherein the IgG is a subtype selected from the group consisting of IgG1, IgG2a, IgG2b, IgG3, and IgG4; H 2 antibody constant domains, and IgG C H 3 anti Constant domain 13. The Fc construct according to any one of claims 1 to 12, comprising: [Invention 14] (a) a first polypeptide having the formula ALB; (i) A comprises a first Fc domain monomer; (ii) L is a linker; and (iii) a first polypeptide, wherein B comprises a second Fc domain monomer; (b) a second polypeptide having the formula A'-L'-B'; (i) A' comprises a third Fc domain monomer; (ii) L' is a linker; and (iii) a second polypeptide, wherein B' comprises a fourth Fc domain monomer; (c) a third polypeptide comprising a fifth Fc domain monomer; and (d) a fourth polypeptide comprising a sixth Fc domain monomer. It consists of: A and A' combine to form a first Fc domain, B and said fifth Fc domain monomer combine to form a second Fc domain, and B' and said sixth Fc domain monomer combine to form a third Fc domain; each of the first and third Fc domain monomers comprises a complementary dimerization selectivity module that promotes dimerization between the first Fc domain monomer and the third Fc domain monomer; each of the second and fifth Fc domain monomers comprises a complementary dimerization selectivity module that promotes dimerization between the second Fc domain monomer and the fifth Fc domain monomer; and each of the fourth and sixth Fc domain monomers comprises a complementary dimerization selectivity module that promotes dimerization between the fourth Fc domain monomer and the sixth Fc domain monomer; and An Fc construct comprising three or fewer Fc domains. [Invention 15] (a) a first polypeptide having the formula ALB; (i) A consists of a first Fc domain monomer; (ii) L is a linker; and (iii) a first polypeptide, wherein B consists of a second Fc domain monomer; (b) a second polypeptide having the formula A'-L'-B'; (i) A' consists of a third Fc domain monomer; (ii) L' is a linker; and (iii) a second polypeptide, wherein B' consists of a fourth Fc domain monomer; (c) a third polypeptide consisting of a fifth Fc domain monomer; and (d) a fourth polypeptide consisting of a sixth Fc domain monomer. It consists of: A and A' combine to form a first Fc domain, B and said fifth Fc domain monomer combine to form a second Fc domain, and B' and said sixth Fc domain monomer combine to form a third Fc domain; each of the first and third Fc domain monomers comprises a complementary dimerization selectivity module that promotes dimerization between the first Fc domain monomer and the third Fc domain monomer; each of the second and fifth Fc domain monomers comprises a complementary dimerization selectivity module that promotes dimerization between the second Fc domain monomer and the fifth Fc domain monomer; and each of the fourth and sixth Fc domain monomers comprises a complementary dimerization selectivity module that promotes dimerization between the fourth Fc domain monomer and the sixth Fc domain monomer; and An Fc construct comprising three or fewer Fc domains. [Invention 16] 16. The Fc construct of claim 14 or 15, wherein the complementary dimerization selectivity module of the first Fc domain monomer comprises a negatively charged amino acid substitution, the complementary dimerization selectivity module of the third Fc domain monomer comprises a positively charged amino acid substitution, the complementary dimerization selectivity module of the second Fc domain monomer comprises an engineered protrusion, the complementary dimerization selectivity module of the fifth Fc domain monomer comprises an engineered recess, the complementary dimerization selectivity module of the fourth Fc domain monomer comprises an engineered protrusion, and the complementary dimerization selectivity module of the sixth Fc domain monomer comprises an engineered recess. [Invention 17] 17. The Fc construct according to any one of claims 14 to 16, wherein the linker L and / or L' is 3 to 200 amino acids in length and / or has a sequence selected from the group consisting of SEQ ID NOs: 1, 2, and 3. [Invention 18] A method for preparing an Fc construct according to any one of claims 1 to 17, comprising: (a) providing a host cell containing a polynucleotide encoding the polypeptide; (b) expressing the first, second, and third polypeptides in the host cell under conditions that allow formation of the Fc construct; (c) recovering the Fc construct. A method comprising: [Invention 19] A host cell expressing an Fc construct according to any one of inventions 1 to 17, comprising a polynucleotide encoding said polypeptide, said polynucleotide being expressed in said host cell. [Invention 20] A pharmaceutical composition comprising a substantially homogenous population of Fc constructs according to any one of claims 1 to 17. [Invention 21] 21. The pharmaceutical composition of invention 20, wherein said Fc construct comprises three Fc domains comprising four associated polypeptides, two of said four polypeptides each comprising two Fc domain monomers, and two of said four polypeptides each comprising one Fc domain monomer. [Invention 22] A pharmaceutical composition comprising an Fc construct according to any one of Inventions 1 to 17 or a pharmaceutical composition according to Invention 20 or 21 for treating an inflammatory disease or an autoimmune disease or an immune disease, promoting clearance of autoantibodies, inhibiting antigen presentation, or reducing an immune response in a subject.

Claims

1. a) a first polypeptide comprising a first Fc domain monomer and a second Fc domain monomer linked by a linker; b) a second polypeptide comprising a third Fc domain monomer and a fourth Fc domain monomer linked by a linker; c) a third polypeptide comprising a fifth Fc domain monomer; and d) a fourth polypeptide comprising a sixth Fc domain monomer.

2. An Fc construct comprising: said first and third domain monomers combine to form a first Fc domain, said second and fifth domain monomers combine to form a second Fc domain, and said fourth and sixth domain monomers combine to form a third Fc domain; the first polypeptide comprises the same amino acid sequence as the second polypeptide, the third polypeptide comprises the same amino acid sequence as the fourth polypeptide, the second Fc domain monomer comprises a different amino acid sequence from the fifth Fc domain monomer, and the fourth Fc domain monomer comprises a different amino acid sequence from the sixth Fc domain monomer; And, Each of the first, second, third, fourth, fifth and sixth Fc domain monomers is a human IgG1 C H 2-domain monomer and human IgG1 C H 3 domain monomer, wherein said C H each of the three domain monomers has ten or fewer single amino acid substitutions to promote complementary dimerization of the Fc domain monomers; The C of the first and third Fc domain monomers H each of the three domain monomers comprises a complementary dimerization selectivity module that promotes dimerization between said first Fc domain monomer and said third Fc domain monomer; The C of the second and fifth Fc domain monomers H each of the three domain monomers comprises a complementary dimerization selectivity module that promotes dimerization between said second Fc domain monomer and said fifth Fc domain monomer; And, The C of the fourth and sixth Fc domain monomers H each of the three domain monomers comprises a complementary dimerization selectivity module that promotes dimerization between the fourth Fc domain monomer and the sixth Fc domain monomer; The Fc construct.

2. Each of the complementary dimerization selectivity modules of the first and third Fc domain monomers is a complementary dimerization selectivity module of the first and third Fc domain monomers. H The Fc construct of claim 1 , comprising opposite charge mutations at at least two positions within the ring of charged residues at the interface between the three domain monomers.

3. 3. The Fc construct of claim 2, wherein the reverse charge mutations at the at least two positions are K409D / D339K, K392D / D399K, E357K / K370E, D356K / K439D, K409E / D339K, K392E / D399K, E357K / K370D, or D356K / K439E.

4. The complementary dimerization selectivity modules of the first and third Fc domain monomers are selected from the C of the first and third Fc domain monomers. H The Fc construct of claim 1 , comprising quadruple opposite charge mutations at four positions in the interface between the three domain monomers.

5. 5. The Fc construct of claim 4, wherein the quadruple reverse charge mutation is any combination of double mutants selected from K409D / D339K, K392D / D399K, E357K / K370E, D356K / K439D, K409E / D339K, K392E / D399K, E357K / K370D, and D356K / K439E.

6. one of the complementary dimerization selectivity modules of the second and fifth Fc domain monomers comprises an engineered overhang; another of the complementary dimerization selectivity modules of the second and fifth Fc domain monomers comprises an engineered recess; The Fc construct of claim 1.

7. one of the complementary dimerization selectivity modules of the fourth and sixth Fc domain monomers comprises an engineered overhang; another of the complementary dimerization selectivity modules of the fourth and sixth Fc domain monomers comprises an engineered recess; The Fc construct of claim 1.

8. each of the complementary dimerization selectivity modules of the second and fourth Fc domain monomers comprises an engineered overhang; each of the complementary dimerization selectivity modules of the fifth and sixth Fc domain monomers comprises an engineered recess; The Fc construct of claim 1.

9. the engineered overhang comprises at least one mutation selected from the group consisting of S354C, T366W, T366Y, T394W, T394F, and F405W; the engineered recess comprises at least one mutation selected from the group consisting of Y349C, T366S, L368A, Y407V, Y407T, Y407A, F405A, and T394S; The Fc construct of claim 8.

10. each of the complementary dimerization selectivity modules of the second and fourth Fc domain monomers comprises an engineered recess; each of the complementary dimerization selectivity modules of the fifth and sixth Fc domain monomers comprises an engineered protrusion; The Fc construct of claim 1.

11. the engineered recess comprises at least one mutation selected from the group consisting of Y349C, T366S, L368A, Y407V, Y407T, Y407A, F405A, and T394S; the engineered overhang comprises at least one mutation selected from the group consisting of S354C, T366W, T366Y, T394W, T394F, and F405W; The Fc construct of claim 10.

12. The Fc construct of claim 1 , wherein one or more linkers of the Fc construct are spacers.

13. The C of the first and third Fc domain monomers H and the three domain monomers dimerize, and the complementary dimerization selectivity modules of the first and third Fc domain monomers are coupled to the C of the first and third Fc domain monomers, respectively. H comprising opposite charge mutations at at least two positions within the ring of charged residues at the interface between the three-domain monomers; The C of the second and fifth Fc domain monomers H the three domain monomers dimerize, the complementary dimerization selectivity module of the second Fc domain monomer comprises an engineered protrusion, the complementary dimerization selectivity module of the fifth Fc domain monomer comprises an engineered recess, and The C of the fourth and sixth Fc domain monomers H the three domain monomers dimerize, the complementary dimerization selectivity module of the fourth Fc domain monomer comprises an engineered protrusion, and the complementary dimerization selectivity module of the sixth Fc domain monomer comprises an engineered recess. The Fc construct of claim 1.

14. The C of the first and third Fc domain monomers H the three domain monomers dimerize, the complementary dimerization selectivity module of the first Fc domain monomer comprises an engineered protrusion and the complementary dimerization selectivity module of the third Fc domain monomer comprises an engineered recess; The C of the second and fifth Fc domain monomers H and the complementary dimerization selectivity modules of the second and fifth Fc domain monomers are coupled to the C of the second and fifth Fc domain monomers, respectively. H comprising opposite charge mutations at at least two positions within the ring of charged residues at the interface between the three-domain monomers; and The C of the fourth and sixth Fc domain monomers H and the C3 domain monomers dimerize, and the complementary dimerization selectivity modules of the fourth and sixth Fc domain monomers are coupled to the C4 of the fourth and sixth Fc domain monomers, respectively. H comprising opposite charge mutations at at least two positions within a ring of charged residues at the interface between the three-domain monomers; The Fc construct of claim 1.

15. The C of the first and third Fc domain monomers H the three domain monomers dimerize, the complementary dimerization selectivity module of the first Fc domain monomer comprises an engineered recess and the complementary dimerization selectivity module of the third Fc domain monomer comprises an engineered protrusion; The C of the second and fifth Fc domain monomers H and the complementary dimerization selectivity modules of the second and fifth Fc domain monomers are coupled to the C of the second and fifth Fc domain monomers, respectively. H comprising opposite charge mutations at at least two positions within the ring of charged residues at the interface between the three-domain monomers; and The C of the fourth and sixth Fc domain monomers H and the C3 domain monomers dimerize, and the complementary dimerization selectivity modules of the fourth and sixth Fc domain monomers are coupled to the C4 of the fourth and sixth Fc domain monomers, respectively. H comprising opposite charge mutations at at least two positions within a ring of charged residues at the interface between the three-domain monomers; The Fc construct of claim 1.

16. The Fc construct of claim 1, wherein the amino acid sequences of the second and fifth Fc domain monomers differ by two amino acids, and the amino acid sequences of the fourth and sixth Fc domain monomers differ by two amino acids.

17. The Fc construct of claim 1, wherein each of the first, second, third, fourth, fifth, and sixth Fc domain monomers further comprises a hinge domain or a functional fragment thereof.

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