Compositions and methods relating to engineered Fc constructs

Abstract

The present invention provides Fc constructs containing biologically active Fc domains with long serum half-lives and / or binding affinity and / or activity to Fc receptors. [Solution] An Fc construct is provided which comprises a first polypeptide comprising a first Fc domain monomer and a second Fc domain monomer, a second polypeptide comprising a third Fc domain monomer and a fourth Fc domain monomer, a third polypeptide comprising a fifth Fc domain monomer, and a fourth polypeptide comprising a sixth Fc domain monomer, wherein the first Fc domain monomer and the fifth Fc domain monomer are combined to form the first Fc domain, the second Fc domain monomer and the fourth Fc domain monomer are combined to form the second Fc domain, and the third Fc domain monomer and the sixth Fc domain monomer are combined to form the third Fc domain.

Description

[Technical field]

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS This application is a continuation of U.S. Provisional Patent Application No. 62 / 443,495, filed January 6, 2017. , U.S. Provisional Patent Application No. 62 / 510,228, filed May 23, 2017; This application claims the benefit of U.S. Provisional Patent Application No. 62 / 589,473, filed November 21, 2007. The contents of each of these applications are incorporated herein by reference in their entirety. It is assumed that. [Background technology]

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

[0003] overview The present disclosure features therapeutic constructs that contain a biologically active Fc domain. Such constructs may be used to provide the desired serum half-life and / or binding to Fc receptors. It may have affinity and / or avidity.

[0004] Generally, the present disclosure provides Fc constructs having 2-10 Fc domains, e.g., 2 Fc constructs having 3, 4, 5, 6, 7, 8, 9 or 10 Fc domains. In some embodiments, the Fc construct comprises 2-10 Fc domains, 2-5 Fc domains, 2-4 Fc domains, 2-3 Fc domains, 3-5 Some embodiments contain 1 Fc domain, 2 to 8 Fc domains, or 2 to 6 Fc domains. In some embodiments, the Fc construct comprises two to four Fc domains. In some embodiments, the Fc construct comprises 5-10 Fc domains (e.g., 5-6, 5-7, 5-8, 5-9, 5-10, 5-11, 5-12, 5-13, 5-14, 5-15, 5-16, 5-17, 5-18, 5-19, 5-20, 5-21, 5-22, 5-23 8, 5-9 or 5-10 Fc domains).

[0005] In some embodiments, a construct of the disclosure (e.g., 2-4 Fc domains, e.g. (e.g., Fc constructs having two, three or four Fc domains) and homogeneous pharmaceutical compositions. (e.g., an Fc domain having 2 to 4 Fc domains, e.g., an Fc domain having 2, 3, or 4 Fc domains) The present invention relates to a method for preventing an immune response and inhibiting an immune disorder in a subject. and treating inflammatory diseases (e.g., autoimmune diseases), e.g., reducing inflammation in a subject. promoting clearance of autoantibodies in a subject; and suppressing antigen presentation in a subject. In one embodiment, the present invention provides a method for preventing immune complex-based activation of an immune response in a subject, comprising: The Fc constructs described herein are useful in significantly stimulating immune cells. It can be used to treat patients with immune and inflammatory diseases without any side effects. In embodiments, a construct of the disclosure (e.g., 5-10 Fc domains, e.g. Fc constructs having 5, 6, 7, 8, 9 or 10 Fc domains) and homogenous A pharmaceutical composition (e.g., a pharmaceutical composition having 5 to 10 Fc domains, e.g., 5, 6, 7, 8, 9 or 10 The Fc construct having the Fc domain of Inducing immune cell activation of an immune response, target cells (i.e., cancer cells) in a subject or infected cells) and treating cancer and infection in a subject. These compounds are useful in treating diseases such as rheumatoid arthritis.

[0006] The nature of these constructs allows for the efficient production of substantially homogenous compositions. The degree of homogeneity of the composition affects the pharmacokinetics and in vivo performance of the composition. To ensure the safety, efficacy, uniformity, and reliability of the product, Homogeneity is desirable. The Fc constructs of the present disclosure are substantially homogeneous (e.g., at least 85%, 90%, 95%, 98% or 99% homogeneity) It is possible.

[0007] As described in more detail herein, the present disclosure provides a method for producing a fusion protein comprising the steps of: The present invention relates to a method for producing a Fc construct comprising administering to a subject a subject a disease comprising administering to a subject a disease comprising said ... The present invention features a method for preparing a qualitatively homogeneous composition.

[0008] In a first aspect, the disclosure provides a method for producing an Fc domain comprising: a) i) a first Fc domain monomer; ii) a second Fc domain monomer; iii) a first Fc domain monomer linked to a second Fc domain monomer; a) a first polypeptide comprising a linker connecting the first polypeptide and a second Fc domain monomer; i) a fourth Fc domain monomer, and iii) a third Fc domain monomer. c) a second polypeptide comprising a linker that links the main monomer to a fifth Fc domain; a third polypeptide comprising a sixth Fc domain monomer; and d) a fourth polypeptide comprising a sixth Fc domain monomer. and a peptide, wherein the first Fc domain monomer and The fifth Fc domain monomer is combined to form the first Fc domain, and the second Fc domain The main monomer and the fourth Fc domain monomer combine to form the second Fc domain. and the third Fc domain monomer and the sixth Fc domain monomer are combined. forming a third Fc domain, wherein the first and second polypeptides each have the sequence TIFF0007611880000001.tif46165, the third and fourth polypeptides each having the sequence Contains TIFF0007611880000002.tif22164.

[0009] In another aspect, the disclosure provides a method for producing an Fc domain comprising: a) i) a first Fc domain monomer; ii) a second Fc domain monomer; and iii) binding the first Fc domain monomer to the second Fc domain monomer. and b) a first polypeptide comprising a linker that binds i) a third Fc domain monomer, ii ) a fourth Fc domain monomer, and iii) a third Fc domain monomer. c) a second polypeptide comprising a linker that connects the Fc domain to the Fc monomer; and a third polypeptide comprising a first Fc domain monomer; and d) a fourth polypeptide comprising a sixth Fc domain monomer. and a peptide, wherein the peptide comprises a first Fc domain monomer and The five Fc domain monomers are combined to form a first Fc domain, and a second Fc domain is The intermolecular monomer and the fourth Fc domain monomer combine to form a second Fc domain. and the third Fc domain monomer and the sixth Fc domain monomer are combined to form the third Fc domain monomer and the sixth Fc domain monomer. and forming three Fc domains, wherein at least one of the Fc domains is an amino acid at position I253. In another aspect, the present disclosure provides a method for the preparation of a medicament for the treatment of a pulmonary arthritis, comprising: a) i) a first Fc domain monomer, ii) a second Fc domain monomer, and iii) a first Fc domain monomer comprising a linker that connects the first Fc domain monomer to the second Fc domain monomer; and b) i) a third Fc domain monomer, ii) a fourth Fc domain monomer. and iii) a linker that links the third Fc domain monomer to the fourth Fc domain monomer. and c) a third polypeptide comprising a fifth Fc domain monomer. and d) a fourth polypeptide comprising a sixth Fc domain monomer. tract, wherein the first Fc domain monomer and the fifth Fc domain monomer are are combined to form a first Fc domain, a second Fc domain monomer and a fourth Fc The domain monomers combine to form a second Fc domain, as well as a third Fc domain. The α-amino acid monomer and the sixth Fc domain monomer combine to form a third Fc domain. , wherein at least one Fc domain monomer comprises an amino acid substitution at position I253.

[0010] In some cases, the first and second polypeptides are identical to one another and the third and fourth polypeptides are identical to one another. In some embodiments, the first Fc domain has a polypeptide at position I2 In some instances, the first and fifth Fc domain monomers include an amino acid modification at position 53. One or both contain an amino acid substitution at position 1253. In some embodiments, the second and fourth domains comprise an amino acid modification at position I253. One or both of the Fc domain monomers contain an amino acid substitution at position I253. In embodiments, the third Fc domain comprises an amino acid modification at position I253. In one embodiment, one or both of the third and sixth Fc domain monomers have an agonist at position I253. In some embodiments, each amino acid modification at position I253 (e.g., substitution ) are I253A, I253C, I253D, I253E, I253F, I253G, I253H, I253I, I253K, I253L, I253M, I253N, I253 P, I253Q, I253R, I253S, I253T, I253V, I253W, and In some embodiments, the aryl group at position I253 is independently selected from the group consisting of I253Y. Each amino acid modification (e.g., substitution) is I253A. The tract (e.g., at least one Fc domain monomer) has at least one amino acid sequence at position R292. In some embodiments, at least one Fc domain monomer comprises one or more amino acid modifications. In some embodiments, the first Fc domain comprises an amino acid substitution at position R292. In some embodiments, the first Fc domain (e.g., For example, one or both of the first Fc domain monomer and the fifth Fc domain monomer are In some embodiments, the second Fc domain comprises an amino acid substitution at position R2 In some embodiments, the second Fc domain (e.g., a second one or both of the Fc domain monomer and the fourth Fc domain monomer) at position R292 In some embodiments, the third Fc domain comprises an amino acid substitution at position R292. In some embodiments, the third Fc domain (e.g., the third Fc domain) comprises a cyclic acid modification. The sixth Fc domain monomer or both of the sixth Fc domain monomers have an amino acid substitution at position R292. In some embodiments, each of the first, second, and third Fc domains comprises a substitution at position R In some instances, the first and fifth Fc domains include an amino acid modification (e.g., substitution) at 292. One or both of the main monomers contain an amino acid substitution at position R292, and the second and fourth F One or both of the c domain monomers contains an amino acid substitution at position R292, and and one or both of the sixth Fc domain monomers comprises an amino acid substitution at position R292. In some embodiments, each of the first, second, and third Fc domains comprises an amino acid modification (e.g., For example, the substitution R292P. (e.g., substitutions) include R292D, R292E, R292L, R292P, R292Q, R292R, R292T, and R292Y are independently selected from the group consisting of In embodiments, each amino acid modification (eg, substitution) at position R292 is R292P.

[0011] In some embodiments of all aspects, each of the Fc domains comprises a human IgG1 Fc sequence. Based on the above, and including the modifications described herein (i.e., variants of the human IgG Fc sequence). In some embodiments, the base IgG1 Fc sequence is SEQ ID NO:42. and contains up to 10 single amino acid modifications. Each of the Fc domains of the c constructs contains an IgA domain with up to 10 single amino acid modifications. In some embodiments, the IG1 Fc sequence is an IG1 Fc sequence (e.g., SEQ ID NO:42). The gG1 Fc sequence is SEQ ID NO:42, and contains an engineered cavity, engineered protrusion Modifications of the ribozyme, ribozyme, and / or electrostatic steering modifications to control the assembly of the polypeptide. and / or modifying the binding-related mutations to form the construct as described herein. Thus, up to 10 single amino acid modifications may be I253 (e.g., I253A) and / or R292 (e.g., R292P) modifications (e.g., substitutions) and engineered cavities to control the assembly of the polypeptide. , engineered protrusions, and / or electrostatic steering modifications (e.g., In some instances, each Fc domain monomer can include an I253 and an R 292 substitutions in either or both of the following: up to 10, 9, 8, 7, 6, 5, 4, 3, 2, or one single amino acid modification. Modifications that provide cavities, engineered protrusions, and / or electrostatic steering modifications are preferred. is located within the CH3 domain of the Fc domain.

[0012] In another aspect, the disclosure provides a method for producing an Fc domain comprising: a) i) a first Fc domain monomer; ii) a second Fc domain monomer; and iii) binding the first Fc domain monomer to the second Fc domain monomer. and b) a first polypeptide comprising a linker that binds i) a third Fc domain monomer, ii ) a fourth Fc domain monomer, and iii) a third Fc domain monomer. c) a second polypeptide comprising a linker that connects the Fc domain to the Fc monomer; and a third polypeptide comprising a first Fc domain monomer; and d) a fourth polypeptide comprising a sixth Fc domain monomer. and a peptide, wherein the peptide comprises a first Fc domain monomer and The five Fc domain monomers are combined to form a first Fc domain, and a second Fc domain is The intermolecular monomer and the fourth Fc domain monomer combine to form a second Fc domain. and the third Fc domain monomer and the sixth Fc domain monomer are combined to form the third Fc domain monomer and the sixth Fc domain monomer. and forming three Fc domains, wherein at least one of the Fc domains has an amino acid sequence at position R292. In another aspect, the present disclosure provides a method for the preparation of a medicament comprising the steps of: a) i) a first amino acid modification (e.g., a single amino acid modification); ii) a second Fc domain monomer, and iii) a first Fc domain monomer. a first polypeptide comprising a linker connecting the in monomer to a second Fc domain monomer; and b) i) a third Fc domain monomer, ii) a fourth Fc domain monomer, and iii) ) a third Fc domain monomer comprising a linker connecting the third Fc domain monomer to the fourth Fc domain monomer; c) a third polypeptide comprising a fifth Fc domain monomer; and d) and a fourth polypeptide comprising a sixth Fc domain monomer. wherein the first Fc domain monomer and the fifth Fc domain monomer are combined forming a first Fc domain, a second Fc domain monomer and a fourth Fc domain monomer; are combined to form a second Fc domain, and a third Fc domain monomer and The sixth Fc domain monomers combine to form a third Fc domain, where at least Both Fc domain monomers contain an amino acid substitution at position R292.

[0013] In some embodiments, the first Fc domain comprises an amino acid modification at position R292. In some embodiments, one or both of the first and fifth Fc domain monomers have an amino acid sequence at position R2 In some embodiments, the second Fc domain comprises an amino acid substitution at position R292. In some embodiments, the second and fourth Fc domain monomers include an amino acid modification. One or both contain an amino acid substitution at position R292. In some embodiments, the third and sixth domains comprise an amino acid modification at position R292. One or both of the Fc domain monomers contain an amino acid substitution at position R292. In embodiments, each of the first, second, and third Fc domains has the amino acid sequence at position R292. In some embodiments, the first, second, and third Fc domains include Each of the Fc monomers contains the amino acid modification (e.g., substitution) R292P (i.e., For example, SEQ ID NO:42 has an R292P modification. In embodiments, one or both of the first and fifth Fc domain monomers comprises an amino acid substitution R2 92P, and one or both of the second and fourth Fc domain monomers contain the amino acid substitution R2 92P, and one or both of the third and sixth Fc domain monomers comprises the amino acid Contains the substitution R292P.

[0014] In some embodiments, each amino acid modification (e.g., substitution) at position R292 is D, R292E, R292L, R292P, R292Q, R292R, R292T, or In some embodiments, each amino acid at position R292 is independently selected from R292Y. In some embodiments, the modification (e.g., substitution) is R292P. Each of the domains includes the amino acid modification (e.g., substitution) I253A, and the first, second, and Each of the three Fc domains contains the amino acid modification (e.g., substitution) R292P. In embodiments, one or both of the first and fifth Fc domain monomers comprise the amino acid substitution I25 3A, and one or both of the third and sixth Fc domain monomers contain the amino acid substitution I25 3A, and one or both of the first and fifth Fc domain monomers comprise the amino acid substitution R29 2P, and one or both of the second and fourth Fc domain monomers have the amino acid substitution R29 2P, and one or both of the third and sixth Fc domain monomers have the amino acid sequence In some embodiments, each of the first, second, and third Fc domains comprises the recombinant R292P. comprises the amino acid modifications (e.g., substitutions) I253A and R292P. In one embodiment, one or both of the first and fifth Fc domain monomers comprises the amino acid substitution I253A. and one or both of the second and fourth Fc domain monomers comprises the amino acid substitution I253A. and one or both of the third and sixth Fc domain monomers contain the amino acid substitution I253A. and one or both of the first and fifth Fc domain monomers comprises the amino acid substitution R292P. and one or both of the second and fourth Fc domain monomers comprises the amino acid substitution R292P. and one or both of the third and sixth Fc domain monomers have the amino acid substitution R29 Includes 2P.

[0015] In some embodiments, the sequence of each Fc domain has no more than 10 single amino acid modifications. In some embodiments, the sequence of each Fc domain is based on the human IgG1 Fc domain sequence. The array is based on SEQ ID NO:42 with no more than 10 single amino acid modifications.

[0016] In some embodiments, each of the first Fc domain and the third Fc domain comprises the amino acid the second Fc domain contains the amino acid substitution R292P; In some instances, one or both of the first and fifth Fc domain monomers comprise the amino acid substitution I253A, and one or both of the first and fifth Fc domain monomers have the amino acid substitution R292P, and one or both of the third and sixth Fc domain monomers contain the amino acid substitution I253A, and one or both of the third and sixth Fc domain monomers contain the amino acid substitution R292P, and one or both of the second and fourth Fc domain monomers Contains the amino acid substitution R292P.

[0017] In some embodiments, the second Fc domain comprises the amino acid substitution I253A. In embodiments, one or both of the second and fourth Fc domain monomers comprises an amino acid substitution In some embodiments, the first Fc domain and the third Fc domain comprise Each comprises the amino acid substitution I253A. In some embodiments, the first and fifth Fc domains One or both of the ribosome monomers contain the amino acid substitution I253A and are associated with the third and sixth Fc domains. In some embodiments, one or both of the amino acid monomers comprises the amino acid substitution I253A. Each of the first Fc domain, the second Fc domain, and the third Fc domain has an amino acid sequence In some embodiments, one or more of the first and fifth Fc domain monomers comprises a recombinant I253A. Both contain the amino acid substitution I253A and one or both of the second and fourth Fc domain monomers. Both contain the amino acid substitution I253A and one or both of the third and sixth Fc domain monomers. Both contain the amino acid substitution I253A.

[0018] In some embodiments, the second Fc domain comprises the amino acid substitution R292P. In embodiments, one or both of the second and fourth Fc domain monomers comprises an amino acid substitution In some embodiments, the second Fc domain comprises the amino acid substitution I253R292P. In some embodiments, one of the second and fourth Fc domain monomers comprises R292A and R292P. One or both of the second and fourth Fc domain monomers contain the amino acid substitution I253A. In some embodiments, one or both of the first Fc domains comprise the amino acid substitution R292P. Each of the first and third Fc domains contains the amino acid substitution I253A, and the second Fc domain In some embodiments, the first and fifth Fc domains comprise the amino acid substitution R292P. One or both of the monomers contain the amino acid substitution I253A and the third and sixth Fc domains One or both of the monomers contain the amino acid substitution I253A and the second and fourth Fc domains One or both of the monomers contains the amino acid substitution R292P.

[0019] In some embodiments, each of the first Fc domain and the third Fc domain comprises the amino acid the second Fc domain contains the amino acid substitutions I253A and R292P In some embodiments, one or both of the first and fifth Fc domain monomers comprise an amino acid. and one or both of the third and sixth Fc domain monomers comprise an amino acid substitution I253A. and one or both of the second and fourth Fc domain monomers comprise an amino acid substitution I253A. and one or both of the second and fourth Fc domain monomers comprise an amino acid substitution I253A. In some embodiments, the first Fc domain and the third Fc domain comprise a ribozyme substitution R292P. Each of the first and fifth domains comprises the amino acid substitution R292P. One or both of the Fc domain monomers contain the amino acid substitution R292P, and the third and sixth One or both of the Fc domain monomers contains the amino acid substitution R292P.

[0020] In some embodiments, the first Fc domain and the third Fc domain comprise the amino acid substitution R 292P, and the second Fc domain comprises the amino acid substitution I253A. In the present invention, one or both of the first and fifth Fc domain monomers have the amino acid substitution R292P. wherein one or both of the third and sixth Fc domain monomers comprise the amino acid substitution R292P. wherein one or both of the second and fourth Fc domain monomers comprise the amino acid substitution I253A. In some embodiments, each of the first Fc domain and the third Fc domain comprises I2 53A and R292P (e.g., containing the amino acid substitutions I253A and R292P) In some embodiments, one or both of the first and fifth Fc domain monomers are substitution I253A, and one or both of the first and fifth Fc domain monomers have the amino acid substitution R292P, and one or both of the third and sixth Fc domain monomers contain the amino acid and wherein one or both of the third and sixth Fc domain monomers comprise the substitution I253A. Contains the amino acid substitution R292P.

[0021] In some embodiments, each of the first Fc domain and the third Fc domain comprises the amino acid the second Fc domain contains the amino acid substitution I253A and R292P; In some embodiments, one or both of the first and fifth Fc domain monomers comprise an amino acid. and one or both of the first and fifth Fc domain monomers comprise an amino acid substitution I253A. and one or both of the third and sixth Fc domain monomers comprise an amino acid substitution R292P. and one or both of the third and sixth Fc domain monomers comprise an amino acid substitution I253A. and one or both of the second and fourth Fc domain monomers. In some embodiments, the first Fc domain, the second Fc domain, The Fc domain and the third Fc domain each contain the amino acid substitution R292P. In one embodiment, one or both of the first and fifth Fc domain monomers comprises the amino acid substitution R 292P, and one or both of the second and fourth Fc domain monomers contain the amino acid substitution R 292P, and one or both of the third and sixth Fc domain monomers contain the amino acid substitution R Contains 292 pages.

[0022] In some embodiments, each of the first Fc domain and the third Fc domain comprises the amino acid the second Fc domain contains the amino acid substitutions I253A and R292P In some embodiments, one or both of the first and fifth Fc domain monomers comprise an amino acid. and one or both of the third and sixth Fc domain monomers comprise an amino acid substitution R292P. and one or both of the second and fourth Fc domain monomers comprises an amino acid substitution R292P. and one or both of the second and fourth Fc domain monomers comprise an amino acid substitution I253A. In some embodiments, the first Fc domain, the second Fc domain, The first and third Fc domains each contain the amino acid substitutions I253A and R292P. In some embodiments, one or both of the first and fifth Fc domain monomers are substitution I253A, and one or both of the first and fifth Fc domain monomers have the amino acid substitution R292P, and one or both of the second and fourth Fc domain monomers have the amino acid substitution I253A, and one or both of the second and fourth Fc domain monomers contain the amino acid substitution R292P; and one or both of the third and sixth Fc domain monomers contain the amino acid and one or both of the third and sixth Fc domain monomers comprises the acid substitution I253A. , containing the amino acid substitution R292P.

[0023] In some embodiments, the first Fc domain monomer and the fifth Fc domain monomer are Complementation promoting dimer formation between one Fc domain monomer and a fifth Fc domain monomer In some embodiments, the second Fc domain monomer comprises a dimerization selection module. and the fourth Fc domain monomer is a second Fc domain monomer and a fourth Fc domain monomer. In some embodiments, the complementary dimerization selection module promotes dimerization between the dimers. In one embodiment, the third Fc domain monomer and the sixth Fc domain monomer are Complementary dimerization forms that promote dimerization between the first and sixth Fc domain monomers In some embodiments, the first polypeptide and the second polypeptide include a selection module. The peptides comprise, consist of, or consist essentially of the same amino acid sequence. The third polypeptide and the fourth polypeptide contain the same amino acid sequence, In some embodiments, the second Fc domain monomer comprises, or consists essentially of, the sequence. and the fourth Fc domain monomer each having D399K and either K409D or K409E. In some embodiments, the second Fc domain monomer and the fourth Fc domain monomer are Each of the monomers comprises K392D and D399K. The main monomer and the fourth Fc domain monomer each contain E357K and K370E. In some embodiments, each of the second Fc domain monomer and the fourth Fc domain monomer In some embodiments, the second Fc domain monomer comprises D356K and K439D. Each of the first and fourth Fc domain monomers contains K392E and D399K. In an embodiment, each of the second Fc domain monomer and the fourth Fc domain monomer is selected from the group consisting of E35 In some embodiments, the second Fc domain monomer and the fourth Fc domain monomer include Each of the Fc domain monomers comprises D356K and K439E. Each of the first Fc domain monomer and the third Fc domain monomer is S354C and T3 66W, and the fifth Fc domain monomer and the sixth Fc domain monomer each comprise Y 349C, T366S, L368A, and Y407V. and the fourth polypeptide each comprises S354C and T366W and a first Fc domain. The first Fc domain monomer and the third Fc domain monomer have the amino acid sequence Y349C, T366S, and L368, respectively. In some embodiments, the first Fc domain monomer and the third Fc domain monomer include Each of the Fc domain monomers contains E357K or E357R, and the fifth Fc domain monomer The sixth Fc domain monomer and the sixth Fc domain monomer each contain K370D or K370E. In one embodiment, the first Fc domain monomer and the third Fc domain monomer are D or K370E, and the fifth Fc domain monomer and the sixth Fc domain monomer are In some embodiments, the first Fc domain alone comprises E357K or E357R. Each of the third Fc domain monomer and the fifth Fc domain monomer contains K409D or K409E, The Fc domain monomer and the sixth Fc domain monomer are each D399K or D399 In some embodiments, the first Fc domain monomer and the third Fc domain monomer Each of the antibodies comprises D399K or D399R and a fifth Fc domain monomer and a sixth Fc domain monomer. Each c domain monomer contains K409D or K409E.

[0024] In some embodiments, the linker (e.g., spacer) is In another embodiment, the polypeptide comprises an Fc construct as described herein. One or more linkers in the above are spacers, and may be, for example, 2 to 200 amino acids (e.g. For example, 2~100, 3~200, 3~150, 3~100, 3~60, 3~50, 3~4 0, 3~30, 3~20, 3~10, 3~8, 3~5, 4~30, 5~30, 6~30, 8~30, 10~20, 10~30, 12~30, 14~30, 20~30, 15~25 , 15-30, 18-22 and 20-30 amino acids). In this case, the amino acid spacer may contain only glycines, only serine, or Contains only phosphorus and glycine. In some embodiments, the amino acid spacer contains only glycine. Includes.

[0025] In some embodiments, each of the first polypeptide and the second polypeptide comprises: In some embodiments, the nucleic acid comprises, consists of, or consists essentially of the sequence of TIFF0007611880000004.tif46165. Each of the first polypeptide and the second polypeptide may have a maximum of 10 (9, 8, 7, 6, 5 , 4, 3, 2, or 1 amino acid modification (e.g., substitution, e.g., conservative substitution). do, In some embodiments, the nucleic acid comprises, consists of, or consists essentially of the sequence of TIFF0007611880000005.tif46165. Each of the first polypeptide and the second polypeptide may include a region outside the linker (e.g., Block arrangement Outside TIFF0007611880000006.tif4128), a maximum of 10 (for example, 1 or less, 2 or less, 3 or less, 4 or less, 5 1 or less, 6 or less, 7 or less, 8 or less, 9 or less) single amino acid modifications, Q comprises, consists of, or consists essentially of the sequence of ID NO:78. The linker in EQ ID NO:78 can be replaced with an alternative linker. In some embodiments, each of the first polypeptide and the second polypeptide comprises up to 10 Consists of the sequence of SEQ ID NO: 78, comprising the sequence having a single amino acid modification; or consisting essentially of the sequence, provided that position 33 of SEQ ID NO:78 is an alanine; Position 72 of SEQ ID NO:78 is a proline, and position 72 of SEQ ID NO:78 is a proline 319 is a proline.

[0026] In some embodiments, each of the first polypeptide and the second polypeptide comprises: In some embodiments, the nucleic acid comprises, consists of, or consists essentially of the sequence of TIFF0007611880000007.tif46165. The first polypeptide and the second polypeptide may be up to 10 (e.g., up to 9, 8, 7, 6, 5, 4, 3, 2 or 1 single amino acid modification (e.g., substitution, e.g., conservative substitution) comprising, consisting of, or consisting essentially of the sequence of SEQ ID NO:49. In some embodiments, each of the first polypeptide and the second polypeptide comprises at least The sequence of SEQ ID NO:49 having up to 10 single amino acid modifications is or consisting essentially of said sequence, but containing up to 10 (e.g., 1 or less, 2 or less , 3 or less, 4 or less, 5 or less, 6 or less, 7 or less, 8 or less, 9 or less) Any of the amino acid modifications may be made to the outer regions of the linker (e.g., the subsequence TIFF0007611880000008.tif4128) Replace the linker in SEQ ID NO:49 with an alternative linker In some embodiments, the first polypeptide and the second polypeptide Each comprises the sequence of SEQ ID NO:49, with up to 10 single amino acid modifications. comprising, consisting of or consisting essentially of the sequence of SEQ ID NO:49 Position 134 is a cysteine, position 137 is a lysine in SEQ ID NO:49; Position 146 of SEQ ID NO:49 is tryptophan; position 426 of SEQ ID NO:49 is tryptophan is a lysine, and position 436 of SEQ ID NO:49 is an aspartic acid.

[0027] In some embodiments, each of the first polypeptide and the second polypeptide comprises: In some embodiments, the nucleic acid comprises, consists of, or consists essentially of the sequence of TIFF0007611880000009.tif46165. The first polypeptide and the second polypeptide may be up to 10 (e.g., up to 9, 8, 7, 6, 5, 4, 3, 2 or 1 single amino acid modification (e.g., substitution, e.g., conservative substitution) comprising, consisting of, or consisting essentially of the sequence of SEQ ID NO:62. In some embodiments, each of the first polypeptide and the second polypeptide comprises The outer region of the anchor (e.g., the subsequence Outside TIFF0007611880000010.tif4128), a maximum of 10 (for example, 1 or less, 2 or less, 3 or less, 4 or less, 5 1 or less, 6 or less, 7 or less, 8 or less, 9 or less) single amino acid modifications, Q comprises, consists of, or consists essentially of the sequence of ID NO:62. The linker in EQ ID NO:62 can be replaced with an alternative linker. In some embodiments, each of the first polypeptide and the second polypeptide comprises up to 10 Consists of the sequence of SEQ ID NO: 62, with a single amino acid modification; or consisting essentially of the sequence, provided that position 134 of SEQ ID NO:62 is a cysteine. position 137 of SEQ ID NO:62 is a lysine; position 137 of SEQ ID NO:62 is a lysine 146 is a tryptophan, position 280 of SEQ ID NO:62 is an alanine, Position 426 of SEQ ID NO:62 is a lysine, and position 436 of SEQ ID NO:62 is a Aspartic acid.

[0028] In some embodiments, each of the first polypeptide and the second polypeptide comprises: In some embodiments, the nucleic acid comprises, consists of, or consists essentially of the sequence of TIFF0007611880000011.tif46165. Each of the first polypeptide and the second polypeptide may have up to 10 (e.g., 1 or less, 2 or less, 3 or less, 4 or less, 5 or less, 6 or less, 7 or less, 8 or less, 9 or less) having a single amino acid modification (e.g., a substitution, e.g., a conservative substitution) of SEQ ID NO. In some embodiments, the sequence comprises, consists of, or consists essentially of the sequence: Each of the first polypeptide and the second polypeptide has an outer region of the linker (e.g., , subarray TIFF0007611880000012.tif4128), the sequence of SEQ ID NO:64 with up to 10 single amino acid modifications. The sequence of SEQ ID NO:64 may be any sequence comprising, consisting of, or consisting essentially of the sequence. The linker may be replaced with an alternative linker. The peptide and the second polypeptide each have up to 10 single amino acid modifications. comprising, consisting of or consisting essentially of the sequence of EQ ID NO:64; However, position 33 of SEQ ID NO:64 is an alanine.

[0029] In some embodiments, each of the first polypeptide and the second polypeptide comprises: In some embodiments, the nucleic acid comprises, consists of, or consists essentially of the sequence of TIFF0007611880000013.tif46165. The first polypeptide and the second polypeptide may be up to 10 (e.g., up to 9, 8, 7, 6, 5, 4, 3, 2 or 1 single amino acid modification (e.g., substitution, e.g., conservative substitution) comprising, consisting of, or consisting essentially of the sequence of SEQ ID NO:65. In some embodiments, each of the first polypeptide and the second polypeptide comprises anchors (e.g., subsequences TIFF0007611880000014.tif4128) in the outer area, up to 10 (e.g., 1 or less, 2 or less, 3 or less, 4 or less , 5 or less, 6 or less, 7 or less, 8 or less, 9 or less) single amino acid modifications; Comprising, consisting of, or consisting essentially of the sequence of SEQ ID NO:65 The linker in SEQ ID NO:65 can be replaced with an alternative linker. In some embodiments, each of the first polypeptide and the second polypeptide is at most 10 The sequence of SEQ ID NO: 65, comprising the sequence of SEQ ID NO: 65, having one single amino acid modification. wherein position 33 of SEQ ID NO:65 is an alanine amino acid sequence. position 134 of SEQ ID NO:65 is a cysteine; Position 137 is a lysine, position 146 is a tryptophan, Position 280 of SEQ ID NO:65 is an alanine, and position 426 of SEQ ID NO:65 is an alanine. is a lysine, and position 436 of SEQ ID NO:65 is an aspartic acid.

[0030] In some embodiments, each of the first polypeptide and the second polypeptide comprises: In some embodiments, the nucleic acid comprises, consists of, or consists essentially of the sequence of TIFF0007611880000015.tif46165. Each of the first polypeptide and the second polypeptide may have up to 10 (e.g., 1 or less, 2 or less, 3 or less, 4 or less, 5 or less, 6 or less, 7 or less, 8 or less, 9 or less) having a single amino acid modification (e.g., a substitution, e.g., a conservative substitution) of SEQ ID NO. In some embodiments, the nucleic acid comprises, consists of, or consists essentially of the sequence: Each of the first polypeptide and the second polypeptide has an outer region of the linker (e.g., , subarray TIFF0007611880000016.tif4128), the sequence of SEQ ID NO:66 with up to 10 single amino acid modifications. The sequence of SEQ ID NO:66 may be any sequence comprising, consisting of, or consisting essentially of the sequence. The linker may be replaced with an alternative linker. The peptide and the second polypeptide each have up to 10 single amino acid modifications. comprising, consisting of or consisting essentially of the sequence of EQ ID NO:66; However, position 134 of SEQ ID NO:66 is a cysteine. position 137 of SEQ ID NO:66 is a lysine; position 146 of SEQ ID NO:66 is a tryptophan; Position 319 of SEQ ID NO:66 is a proline; position 42 of SEQ ID NO:66 is a 6 is a lysine, and position 436 of SEQ ID NO:66 is an aspartic acid.

[0031] In some embodiments, each of the first polypeptide and the second polypeptide comprises: In some embodiments, the nucleic acid comprises, consists of, or consists essentially of the sequence of TIFF0007611880000017.tif46165. The first polypeptide and the second polypeptide may be up to 10 (e.g., up to 9, 8, 7, 6, 5, 4, 3, 2 or 1 single amino acid modification (e.g., substitution, e.g., conservative substitution) comprising, consisting of, or consisting essentially of the sequence of SEQ ID NO:67. In some embodiments, each of the first polypeptide and the second polypeptide comprises anchors (e.g., subsequences TIFF0007611880000018.tif4128) in the outer area, up to 10 (e.g., 1 or less, 2 or less, 3 or less, 4 or less , 5 or less, 6 or less, 7 or less, 8 or less, 9 or less) single amino acid modifications; Comprising, consisting of, or consisting essentially of the sequence of SEQ ID NO:67 The linker in SEQ ID NO:67 can be replaced with an alternative linker. In some embodiments, each of the first polypeptide and the second polypeptide is at most 10 The sequence of SEQ ID NO:67, comprising the sequence of SEQ ID NO:67, having one single amino acid modification. wherein the nucleic acid sequence is a sequence similar to that of SEQ ID NO:67, except that position 134 of SEQ ID NO:67 is a cis-nucleotide sequence. lysine, position 137 of SEQ ID NO:67 is a lysine, Position 146 is a tryptophan, position 280 is an alanine in SEQ ID NO:67 Position 318 of SEQ ID NO:67 is a proline, and position 426 of SEQ ID NO:67 is a proline. is a lysine, and position 436 of SEQ ID NO:67 is an aspartic acid.

[0032] In some embodiments, each of the first polypeptide and the second polypeptide comprises: In some embodiments, the nucleic acid comprises, consists of, or consists essentially of the sequence of TIFF0007611880000019.tif46165. The first polypeptide and the second polypeptide may be up to 10 (e.g., up to 9, 8, 7, 6, 5, 4, 3, 2 or 1 single amino acid modification (e.g., substitution, e.g., conservative substitution) comprising, consisting of, or consisting essentially of the sequence of SEQ ID NO:68. In some embodiments, each of the first polypeptide and the second polypeptide comprises anchors (e.g., subsequences A maximum of 10 (e.g., 1 or less, 2 or less, 3 or less, 4 or less) in the outer region of TIFF0007611880000020.tif4128 , 5 or less, 6 or less, 7 or less, 8 or less, 9 or less) single amino acid modifications; Comprising, consisting of, or consisting essentially of the sequence of SEQ ID NO:68 The linker in SEQ ID NO:68 can be replaced with an alternative linker. In some embodiments, each of the first polypeptide and the second polypeptide is at most 10 The sequence of SEQ ID NO:68, comprising the sequence of SEQ ID NO:68, having one single amino acid modification. wherein position 33 of SEQ ID NO:68 is an alanine amino acid sequence. position 134 of SEQ ID NO:68 is a cysteine; Position 137 is a lysine, position 146 is a tryptophan in SEQ ID NO:68 Position 319 of SEQ ID NO:68 is a proline, position 426 of SEQ ID NO:68 is a A lysine, and position 436 of SEQ ID NO:68 is an aspartic acid.

[0033] In some embodiments, each of the first polypeptide and the second polypeptide comprises: In some embodiments, the nucleic acid comprises, consists of, or consists essentially of the sequence of TIFF0007611880000021.tif46165. The first polypeptide and the second polypeptide may be up to 10 (e.g., up to 9, 8, 7, 6, 5, 4, 3, 2 or 1 single amino acid modification (e.g., substitution, e.g., conservative substitution) comprising, consisting of, or consisting essentially of the sequence of SEQ ID NO:69. In some embodiments, each of the first polypeptide and the second polypeptide comprises anchors (e.g., subsequences TIFF0007611880000022.tif4128) in the outer area, up to 10 (e.g., 1 or less, 2 or less, 3 or less, 4 or less , 5 or less, 6 or less, 7 or less, 8 or less, 9 or less) single amino acid modifications; Comprising, consisting of, or consisting essentially of the sequence of SEQ ID NO:69 The linker in SEQ ID NO:69 can be replaced with an alternative linker. In some embodiments, each of the first polypeptide and the second polypeptide is at most 10 The sequence of SEQ ID NO:69, comprising the sequence of SEQ ID NO:69, having one single amino acid modification. wherein position 33 of SEQ ID NO:69 is an alanine amino acid sequence. position 134 of SEQ ID NO:69 is a cysteine; Position 137 is a lysine, position 146 is a tryptophan in SEQ ID NO:69 Position 280 of SEQ ID NO:69 is an alanine, position 319 of SEQ ID NO:69 is an alanine proline, position 426 of SEQ ID NO:69 is a lysine, and Position 436 of 69 is an aspartic acid.

[0034] In some embodiments, each of the first polypeptide and the second polypeptide comprises: In some embodiments, the nucleic acid comprises, consists of, or consists essentially of the sequence of TIFF0007611880000023.tif46165. The first polypeptide and the second polypeptide may be up to 10 (e.g., up to 9, 8, 7, 6, 5, 4, 3, 2 or 1 single amino acid modification (e.g., substitution, e.g., conservative substitution) comprising, consisting of, or consisting essentially of the sequence of SEQ ID NO:71. In some embodiments, each of the first polypeptide and the second polypeptide comprises anchors (e.g., subsequences TIFF0007611880000024.tif4128) in the outer area, up to 10 (e.g., 1 or less, 2 or less, 3 or less, 4 or less , 5 or less, 6 or less, 7 or less, 8 or less, 9 or less) single amino acid modifications; Comprising, consisting of, or consisting essentially of the sequence of SEQ ID NO:71 The linker in SEQ ID NO:71 can be replaced with an alternative linker. In some embodiments, each of the first polypeptide and the second polypeptide is at most 10 The sequence of SEQ ID NO: 71, comprising the sequence of SEQ ID NO: 72, having one single amino acid modification. wherein position 71 of SEQ ID NO:71 is a nucleic acid sequence consisting essentially of said sequence, position 134 of SEQ ID NO:71 is a cysteine; Position 137 is a lysine, position 146 is a tryptophan, SEQ ID NO:71 Position 426 of SEQ ID NO:71 is a lysine, and position 436 of SEQ ID NO:71 is an alanine. It is paraginic acid.

[0035] In some embodiments, each of the first polypeptide and the second polypeptide comprises: In some embodiments, the nucleic acid comprises, consists of, or consists essentially of the sequence of TIFF0007611880000025.tif46164. The first polypeptide and the second polypeptide may be up to 10 (e.g., up to 9, 8, 7, 6, 5, 4, 3, 2 or 1 single amino acid modification (e.g., substitution, e.g., conservative substitution) comprising, consisting of, or consisting essentially of the sequence of SEQ ID NO:72. In some embodiments, each of the first polypeptide and the second polypeptide comprises anchors (e.g., subsequences A maximum of 10 (e.g., 1 or less, 2 or less, 3 or less, 4 or less) in the outer region of TIFF0007611880000026.tif4128 , 5 or less, 6 or less, 7 or less, 8 or less, 9 or less) single amino acid modifications; Comprising, consisting of, or consisting essentially of the sequence of SEQ ID NO:72 The linker in SEQ ID NO:72 can be replaced with an alternative linker. In some embodiments, each of the first polypeptide and the second polypeptide is at most 10 The sequence of SEQ ID NO: 72, comprising the sequence of SEQ ID NO: 72, having one single amino acid modification. wherein position 72 of SEQ ID NO:72 is a proline position 134 of SEQ ID NO:72 is cystine; position 134 of SEQ ID NO:72 is cystine Position 137 is a lysine, position 146 is a tryptophan, and position 150 is a lysine. Position 426 of SEQ ID NO:72 is a lysine, and position 436 of SEQ ID NO:72 is a Aspartic acid.

[0036] In some embodiments, each of the first polypeptide and the second polypeptide comprises: In some embodiments, the nucleic acid comprises, consists of, or consists essentially of the sequence of TIFF0007611880000027.tif46165. The first polypeptide and the second polypeptide may be up to 10 (e.g., up to 9, 8, 7, 6, 5, 4, 3, 2 or 1 single amino acid modification (e.g., substitution, e.g., conservative substitution) comprising, consisting of, or consisting essentially of the sequence of SEQ ID NO:74. In some embodiments, each of the first polypeptide and the second polypeptide comprises anchors (e.g., subsequences TIFF0007611880000028.tif4128) in the outer area, up to 10 (e.g., 1 or less, 2 or less, 3 or less, 4 or less , 5 or less, 6 or less, 7 or less, 8 or less, 9 or less) single amino acid modifications; Comprising, consisting of, or consisting essentially of the sequence of SEQ ID NO:74 The linker in SEQ ID NO:74 can be replaced with an alternative linker. In some embodiments, each of the first polypeptide and the second polypeptide is at most 10 The sequence of SEQ ID NO: 74, comprising the sequence of SEQ ID NO: 74, having one single amino acid modification. wherein position 33 of SEQ ID NO:74 is an alanine amino acid sequence. position 72 of SEQ ID NO:74 is a proline; position 74 of SEQ ID NO:74 is a nucleotide 134 is a cysteine; position 137 of SEQ ID NO:74 is a lysine; Position 146 of SEQ ID NO:74 is tryptophan; position 426 of SEQ ID NO:74 is riboflavin. syn, and position 436 of SEQ ID NO:74 is an aspartic acid.

[0037] In some embodiments, each of the first polypeptide and the second polypeptide comprises: In some embodiments, the nucleic acid comprises, consists of, or consists essentially of the sequence of TIFF0007611880000029.tif46165. The first polypeptide and the second polypeptide may be up to 10 (e.g., up to 9, 8, 7, 6, 5, 4, 3, 2 or 1 single amino acid modification (e.g., substitution, e.g., conservative substitution) comprising, consisting of, or consisting essentially of the sequence of SEQ ID NO:75. In some embodiments, each of the first polypeptide and the second polypeptide comprises anchors (e.g., subsequences TIFF0007611880000030.tif4128) in the outer area, up to 10 (e.g., 1 or less, 2 or less, 3 or less, 4 or less , 5 or less, 6 or less, 7 or less, 8 or less, 9 or less) single amino acid modifications; Comprising, consisting of, or consisting essentially of the sequence of SEQ ID NO:75 The linker in SEQ ID NO:75 can be replaced with an alternative linker. In some embodiments, each of the first polypeptide and the second polypeptide is at most 10 A sequence comprising the sequence of SEQ ID NO:75, said sequence having one single amino acid modification. wherein position 33 of SEQ ID NO:75 is an alanine amino acid sequence. position 72 of SEQ ID NO:75 is a proline; position 73 of SEQ ID NO:75 is a proline 134 is a cysteine, position 137 of SEQ ID NO:75 is a lysine, Position 146 of SEQ ID NO:75 is tryptophan, and position 280 of SEQ ID NO:75 is a lanine, position 426 of SEQ ID NO:75 is a lysine, and Position 436 of 5 is an aspartic acid.

[0038] In some embodiments, each of the first polypeptide and the second polypeptide comprises: In some embodiments, the nucleic acid comprises, consists of, or consists essentially of the sequence of TIFF0007611880000031.tif46165. The first polypeptide and the second polypeptide may be up to 10 (e.g., up to 9, 8, 7, 6, 5, 4, 3, 2 or 1 single amino acid modification (e.g., substitution, e.g., conservative substitution) comprising, consisting of, or consisting essentially of the sequence of SEQ ID NO:76. In some embodiments, each of the first polypeptide and the second polypeptide comprises anchors (e.g., subsequences TIFF0007611880000032.tif4128) in the outer area, up to 10 (e.g., 1 or less, 2 or less, 3 or less, 4 or less , 5 or less, 6 or less, 7 or less, 8 or less, 9 or less) single amino acid modifications; Comprising, consisting of, or consisting essentially of the sequence of SEQ ID NO:76 The linker in SEQ ID NO:76 can be replaced with an alternative linker. In some embodiments, each of the first polypeptide and the second polypeptide is at most 10 The sequence of SEQ ID NO: 76, comprising the sequence having one single amino acid modification. wherein the nucleic acid sequence of SEQ ID NO:76 is a nucleic acid sequence consisting essentially of the nucleic acid sequence of SEQ ID NO:76, and position 319 of SEQ ID NO:76 is a proline. each of the first polypeptide and the second polypeptide comprises In some embodiments, the nucleic acid comprises, consists of, or consists essentially of the sequence of TIFF0007611880000033.tif46164. The first polypeptide and the second polypeptide may be up to 10 (e.g., up to 9, 8, 7, 6, 5, 4, 3, 2 or 1 single amino acid modification (e.g., substitution, e.g., conservative substitution) comprising, consisting of, or consisting essentially of the sequence of SEQ ID NO:77. In some embodiments, each of the first polypeptide and the second polypeptide comprises anchors (e.g., subsequences TIFF0007611880000034.tif4128) in the outer area, up to 10 (e.g., 1 or less, 2 or less, 3 or less, 4 or less , 5 or less, 6 or less, 7 or less, 8 or less, 9 or less) single amino acid modifications; Comprising, consisting of, or consisting essentially of the sequence of SEQ ID NO:77 The linker in SEQ ID NO:77 can be replaced with an alternative linker. In some embodiments, each of the first polypeptide and the second polypeptide is at most 10 The sequence of SEQ ID NO: 77, comprising the sequence having one single amino acid modification. wherein position 72 of SEQ ID NO:77 is a proline. position 134 of SEQ ID NO:77 is a cysteine; Position 137 is a lysine, position 146 is a tryptophan in SEQ ID NO:77 Position 280 of SEQ ID NO:77 is an alanine, position 319 of SEQ ID NO:77 is an alanine proline, position 426 of SEQ ID NO:77 is a lysine, and Position 436 of 77 is an aspartic acid.

[0039] In some embodiments, each of the first polypeptide and the second polypeptide comprises: In some embodiments, the nucleic acid comprises, consists of, or consists essentially of the sequence of TIFF0007611880000035.tif46165. The first polypeptide and the second polypeptide may be up to 10 (e.g., up to 9, 8, 7, 6, 5, 4, 3, 2 or 1 single amino acid modification (e.g., substitution, e.g., conservative substitution) comprising, consisting of, or consisting essentially of the sequence of SEQ ID NO:79. In some embodiments, each of the first polypeptide and the second polypeptide comprises anchors (e.g., subsequences TIFF0007611880000036.tif4128) in the outer area, up to 10 (e.g., 1 or less, 2 or less, 3 or less, 4 or less , 5 or less, 6 or less, 7 or less, 8 or less, 9 or less) single amino acid modifications; Comprising, consisting of, or consisting essentially of the sequence of SEQ ID NO:79 The linker in SEQ ID NO:79 can be replaced with an alternative linker. In some embodiments, each of the first polypeptide and the second polypeptide is at most 10 The sequence of SEQ ID NO: 79, comprising the sequence of SEQ ID NO: 79, having one single amino acid modification. wherein position 33 of SEQ ID NO:79 is an alanine amino acid sequence. position 72 of SEQ ID NO:79 is a proline; position 73 of SEQ ID NO:79 is a proline 280 is an alanine, and position 319 of SEQ ID NO:79 is a proline.

[0040] In some embodiments, each of the third polypeptide and the fourth polypeptide comprises: In some embodiments, the nucleic acid comprises, consists of, or consists essentially of the sequence of TIFF0007611880000037.tif22165. The third polypeptide and the fourth polypeptide may each be up to 10 (e.g., up to 9, 8, 7, 6, 5, 4, 3, 2 or 1 single amino acid modification (e.g., substitution, e.g., conservative substitution) comprising, consisting of, or consisting essentially of the sequence of SEQ ID NO:73. In some embodiments, each of the third polypeptide and the fourth polypeptide comprises at least The sequence of SEQ ID NO: 73, which has 10 or more single amino acid modifications. wherein position 33 of SEQ ID NO:73 is alanine, and position 72 of SEQ ID NO:73 is a proline.

[0041] In some embodiments, each of the third polypeptide and the fourth polypeptide comprises: In some embodiments, the nucleic acid comprises, consists of, or consists essentially of the sequence of TIFF0007611880000038.tif22165. The third polypeptide and the fourth polypeptide may each be up to 10 (e.g., up to 9, 8, 7, 6, 5, 4, 3, 2 or 1 single amino acid modification (e.g., substitution, e.g., conservative substitution) comprising, consisting of, or consisting essentially of the sequence of SEQ ID NO:61. In some embodiments, each of the third polypeptide and the fourth polypeptide comprises at least The sequence of SEQ ID NO:61, which has up to 10 single amino acid modifications. wherein the sequence is a sequence identical to or essentially identical to position 129 of SEQ ID NO:61. is a cysteine, position 146 of SEQ ID NO:61 is a serine, position 148 of SEQ ID NO:61 is an alanine, and position 150 of SEQ ID NO:61 is an aspartic acid and position 187 of SEQ ID NO:61 is a valine.

[0042] In some embodiments, each of the third polypeptide and the fourth polypeptide comprises: In some embodiments, the nucleic acid comprises, consists of, or consists essentially of the sequence of TIFF0007611880000039.tif22165. The third polypeptide and the fourth polypeptide may each be up to 10 (e.g., up to 9, 8, 7, 6, 5, 4, 3, 2 or 1 single amino acid modification (e.g., substitution, e.g., conservative substitution) comprising, consisting of, or consisting essentially of the sequence of SEQ ID NO:63. In some embodiments, each of the third polypeptide and the fourth polypeptide comprises at least The sequence of SEQ ID NO:63, which has 10 or more single amino acid modifications. wherein position 33 of SEQ ID NO:63 is It is alanine.

[0043] In some embodiments, each of the third polypeptide and the fourth polypeptide comprises: In some embodiments, the nucleic acid comprises, consists of, or consists essentially of the sequence of TIFF0007611880000040.tif22164. The third polypeptide and the fourth polypeptide may each be up to 10 (e.g., up to 9, 8, 7, 6, 5, 4, 3, 2 or 1 single amino acid modification (e.g., substitution, e.g., conservative substitution) comprising, consisting of, or consisting essentially of the sequence of SEQ ID NO:70. In some embodiments, each of the third polypeptide and the fourth polypeptide comprises at least The sequence of SEQ ID NO:70, which has up to 10 single amino acid modifications. 70, comprising, or consisting essentially of, the sequence at position 72 of SEQ ID NO:70; is proline.

[0044] In some embodiments, each of the first and second polypeptides comprises: a third and fourth polypeptide comprising, consisting of, or consisting essentially of the sequence of TIFF0007611880000041.tif46165; Each of the petites is In some embodiments, the nucleic acid comprises, consists of, or consists essentially of the sequence of TIFF0007611880000042.tif22165. Each of the first and second polypeptides may have up to 10 (e.g., up to 9, 8, 7, 6, 5, 4, 3, 2 or 1) single amino acid modification (e.g., substitution, e.g., conservative substitution), comprising, consisting of or consisting essentially of the sequence of EQ ID NO:78; Each of the third and fourth polypeptides may have a maximum of 10 (9, 8, 7, 6, 5, 4, 3, 2 or 1) single amino acid modification (e.g., substitution, e.g., conservative substitution), DNO:73 In an embodiment, each of the first and second polypeptides has up to 10 single amino acid modifications. The present invention relates to a method for the preparation of a nucleic acid sequence comprising, consisting of, or consisting essentially of the sequence of SEQ ID NO:78. However, the maximum number of items is 10 (for example, 1 or less, 2 or less, 3 or less, 4 or less, 5 or less). , 6 or less, 7 or less, 8 or less, or 9 or less) of any single amino acid modification in the linker (For example, a subarray TIFF0007611880000043.tif4128) outside the region of SEQ ID NO: 78. The linker in SEQ ID NO: 78 is replaced with an alternative linker. In some embodiments, each of the first and second polypeptides can be The sequence of SEQ ID NO: 78, which has up to 10 single amino acid modifications. wherein position 33 of SEQ ID NO:78 is alanine, position 72 of SEQ ID NO:78 is proline, and position 319 of 78 is a proline, and each of the third and fourth polypeptides contains up to 10 The sequence of SEQ ID NO: 73, comprising the sequence of SEQ ID NO: 73, having a single amino acid modification of or consisting essentially of the sequence, provided that position 33 of SEQ ID NO:73 is an alanine and position 72 of SEQ ID NO:73 is a proline.

[0045] In some embodiments, each of the first and second polypeptides comprises: a third and fourth polypeptide comprising, consisting of, or consisting essentially of the sequence of TIFF0007611880000044.tif46165; Each of the petites is In some embodiments, the nucleic acid comprises, consists of, or consists essentially of the sequence of TIFF0007611880000045.tif22165. Each of the first and second polypeptides may have up to 10 (9, 8, 7, 6, 5, 4, 3, 2 or or one amino acid sequence) having a single amino acid modification (e.g., a substitution, e.g., a conservative substitution), comprising, consisting of, or consisting essentially of the sequence of SEQ ID NO:49, and Each of the fourth polypeptides may have up to 10 (9, 8, 7, 6, 5, 4, 3, 2, or 1) SEQ ID NO:6, having a single amino acid modification (e.g., a substitution, e.g., a conservative substitution) of In some embodiments, the nucleic acid comprises, consists of, or consists essentially of the sequence of Each of the first and second polypeptides may have a maximum (e.g., 1 or less, 2 or less, 3 or less, 4 or less, 5 or less, 6 or less, 7 or less, 8 or less, 9 or less) single amino acid modifications Linkers (e.g., subsequences The linker in TIFF0007611880000046.tif4128, SEQ ID NO:76, can be replaced with an alternative linker. The present invention relates to a method for producing a nucleic acid sequence comprising the sequence of SEQ ID NO: 49, the sequence of which is located in the outer region of the nucleic acid sequence of SEQ ID NO: 49. and each of the third and fourth polypeptides consists essentially of said sequence and includes up to 10 single amino acids. comprising, consisting of, or essentially of the sequence of SEQ ID NO:61 with an acid modification. In some embodiments, each of the first and second polypeptides comprises up to 10 The sequence of SEQ ID NO: 49, comprising the sequence of SEQ ID NO: 49, having one single amino acid modification. wherein the nucleic acid sequence is a sequence similar to that of SEQ ID NO:49, except that position 134 of SEQ ID NO:49 is a cis-nucleotide sequence. lysine at position 137 of SEQ ID NO:49; Position 146 is a tryptophan, position 426 is a lysine in SEQ ID NO:49, and Position 436 of EQ ID NO:49 is an aspartic acid, and the third and fourth polypeptides Each of the peptides has a sequence of SEQ ID NO:61 with up to 10 single amino acid modifications. comprising, consisting of, or consisting essentially of the sequence, provided that SEQ ID NO: position 129 of SEQ ID NO:61 is a cysteine; position 146 of SEQ ID NO:61 is a serine; Position 148 of SEQ ID NO:61 is an alanine, and position 150 of SEQ ID NO:61 is an alanine. is an aspartic acid, and position 187 of SEQ ID NO:61 is a valine.

[0046] In some embodiments, each of the first and second polypeptides comprises: a third and fourth polypeptide comprising, consisting of, or consisting essentially of the sequence of Each of the petites is In some embodiments, the nucleic acid comprises, consists of, or consists essentially of the sequence of TIFF0007611880000048.tif22165. Each of the first and second polypeptides may have up to 10 (9, 8, 7, 6, 5, 4, 3, 2 or or one amino acid sequence) having a single amino acid modification (e.g., a substitution, e.g., a conservative substitution), comprising, consisting of, or consisting essentially of the sequence of SEQ ID NO:62, and Each of the fourth polypeptides may have up to 10 (9, 8, 7, 6, 5, 4, 3, 2, or 1) SEQ ID NO:6, having a single amino acid modification (e.g., a substitution, e.g., a conservative substitution) of In some embodiments, the nucleic acid comprises, consists of, or consists essentially of the sequence of Each of the first and second polypeptides may have up to 10 (e.g., 1 or less, 2 or less, 3 or less) 4 or less, 5 or less, 6 or less, 7 or less, 8 or less, 9 or less) single amino acids The modification can be added to a linker (e.g., a subsequence The linker in TIFF0007611880000049.tif4128, SEQ ID NO:62, can be replaced with an alternative linker. The present invention relates to a method for producing a nucleic acid sequence comprising the sequence of SEQ ID NO: 62, the sequence being located in the outer region of the nucleic acid sequence of SEQ ID NO: 62. and each of the third and fourth polypeptides consists essentially of said sequence and includes up to 10 single amino acids. comprising, consisting of, or essentially of the sequence of SEQ ID NO:61 with an acid modification. In some embodiments, each of the first and second polypeptides comprises up to 10 The sequence of SEQ ID NO: 62, comprising the sequence of SEQ ID NO: 62, having one single amino acid modification. wherein the nucleic acid sequence is a sequence similar to that of SEQ ID NO:62, except that position 134 of SEQ ID NO:62 is a cis-nucleotide sequence. lysine at position 137 of SEQ ID NO:62; Position 146 is a tryptophan, position 280 is an alanine, Position 426 of SEQ ID NO:62 is a lysine, and position 43 of SEQ ID NO:62 is a 6 is aspartic acid, and each of the third and fourth polypeptides has up to 10 single amines. The present invention relates to a method for the preparation of a nucleic acid sequence comprising the sequence of SEQ ID NO:61, the sequence having amino acid modifications, wherein position 129 of SEQ ID NO:61 is a cysteine, S Position 146 of SEQ ID NO:61 is serine, position 148 of SEQ ID NO:61 is alanine, position 150 of SEQ ID NO:61 is aspartic acid, and Position 187 of DNO:61 is a valine.

[0047] In some embodiments, each of the first and second polypeptides comprises: a third and fourth polypeptide comprising, consisting of, or consisting essentially of the sequence of TIFF0007611880000050.tif46165; Each of the petites is In some embodiments, the nucleic acid comprises, consists of, or consists essentially of the sequence of TIFF0007611880000051.tif22165. Each of the first and second polypeptides may have up to 10 (9, 8, 7, 6, 5, 4, 3, 2 or or one amino acid sequence) having a single amino acid modification (e.g., a substitution, e.g., a conservative substitution), comprising, consisting of, or consisting essentially of the sequence of SEQ ID NO:64, and Each of the fourth polypeptides may have up to 10 (9, 8, 7, 6, 5, 4, 3, 2, or 1) SEQ ID NO:6, having a single amino acid modification (e.g., a substitution, e.g., a conservative substitution) of In some embodiments, the nucleic acid comprises, consists of, or consists essentially of the sequence of: Each of the first and second polypeptides may have up to 10 (e.g., 1 or less, 2 or less, 3 or less) 4 or less, 5 or less, 6 or less, 7 or less, 8 or less, 9 or less) single amino acids The modification can be added to a linker (e.g., a subsequence The linker in TIFF0007611880000052.tif4128, SEQ ID NO:64, can be replaced with an alternative linker. The present invention relates to a method for producing a nucleic acid sequence comprising the sequence of SEQ ID NO: 64, the sequence being located in the outer region of the nucleic acid sequence of SEQ ID NO: 64. and each of the third and fourth polypeptides consists essentially of said sequence and includes up to 10 single amino acids. comprising, consisting of, or essentially of the sequence of SEQ ID NO:63 with acid modifications. In some embodiments, each of the first and second polypeptides comprises up to 10 The sequence of SEQ ID NO:64, comprising the sequence of SEQ ID NO:64, wherein position 33 of SEQ ID NO:64 is an alanine amino acid sequence. and each of the third and fourth polypeptides has up to 10 single amino acid modifications. comprising, consisting of, or consisting essentially of the sequence of SEQ ID NO:63. where position 33 of SEQ ID NO:63 is an alanine.

[0048] In some embodiments, each of the first and second polypeptides comprises: TIFF0007611880000053.tif46165, Each of the four polypeptides is In some embodiments, the nucleic acid comprises, consists of, or consists essentially of the sequence of TIFF0007611880000054.tif22165. Each of the first and second polypeptides may have up to 10 (9, 8, 7, 6, 5, 4, 3, 2 or or one amino acid sequence) having a single amino acid modification (e.g., a substitution, e.g., a conservative substitution), comprising, consisting of, or consisting essentially of the sequence of SEQ ID NO:65, and Each of the fourth polypeptides may have up to 10 (9, 8, 7, 6, 5, 4, 3, 2, or 1) SEQ ID NO:6, having a single amino acid modification (e.g., a substitution, e.g., a conservative substitution) of In some embodiments, the nucleic acid comprises, consists of, or consists essentially of the sequence of: Each of the first and second polypeptides may have up to 10 (e.g., 1 or less, 2 or less, 3 or less) 4 or less, 5 or less, 6 or less, 7 or less, 8 or less, 9 or less) single amino acids The modification can be added to a linker (e.g., a subsequence The linker in TIFF0007611880000055.tif4128, SEQ ID NO:65, can be replaced with an alternative linker. The present invention relates to a method for producing a nucleic acid sequence comprising the sequence of SEQ ID NO: 65, the sequence of which is contained in the outer region of the nucleic acid sequence of SEQ ID NO: 65. and each of the third and fourth polypeptides consists essentially of said sequence and includes up to 10 single amino acids. comprising, consisting of, or essentially of the sequence of SEQ ID NO:63 with acid modifications. In some embodiments, each of the first and second polypeptides comprises up to 10 The sequence of SEQ ID NO: 65, comprising the sequence of SEQ ID NO: 65, having one single amino acid modification. wherein position 33 of SEQ ID NO:65 is an alanine amino acid sequence. position 134 of SEQ ID NO:65 is a cysteine; Position 137 is a lysine, position 146 is a tryptophan, SEQ ID NO:65 Position 280 of SEQ ID NO:65 is an alanine, position 426 of SEQ ID NO:65 is an alanine lysine, and position 436 of SEQ ID NO:65 is aspartic acid; Each of the fourth polypeptides has up to 10 single amino acid modifications, comprising, consisting of, or consisting essentially of the sequence of SEQ ID NO:63, provided that Position 33 of ID NO:63 is an alanine.

[0049] In some embodiments, each of the first and second polypeptides comprises: a third and fourth polypeptide comprising, consisting of, or consisting essentially of the sequence of TIFF0007611880000056.tif46165; Each of the petites is In some embodiments, the nucleic acid comprises, consists of, or consists essentially of the sequence of TIFF0007611880000057.tif22165. Each of the first and second polypeptides may have up to 10 (9, 8, 7, 6, 5, 4, 3, 2 or or one amino acid sequence) having a single amino acid modification (e.g., a substitution, e.g., a conservative substitution), comprising, consisting of, or consisting essentially of the sequence of SEQ ID NO:66, and Each of the fourth polypeptides may have up to 10 (9, 8, 7, 6, 5, 4, 3, 2, or 1) SEQ ID NO:6, having a single amino acid modification (e.g., a substitution, e.g., a conservative substitution) of In some embodiments, the nucleic acid comprises, consists of, or consists essentially of the sequence of Each of the first and second polypeptides may have up to 10 (e.g., 1 or less, 2 or less, 3 or less) 4 or less, 5 or less, 6 or less, 7 or less, 8 or less, 9 or less) single amino acids The modification can be added to a linker (e.g., a subsequence The linker in TIFF0007611880000058.tif4128, SEQ ID NO:66, can be replaced with an alternative linker. The present invention relates to a method for producing a nucleic acid sequence comprising the sequence of SEQ ID NO: 66, the sequence being located in the outer region of the nucleic acid sequence of SEQ ID NO: 66. and each of the third and fourth polypeptides consists essentially of said sequence and includes up to 10 single amino acids. comprising, consisting of, or essentially of the sequence of SEQ ID NO:61 with an acid modification. In some embodiments, each of the first and second polypeptides comprises up to 10 The sequence of SEQ ID NO:66, comprising the sequence of SEQ ID NO:66, having one single amino acid modification. wherein the nucleotide sequence of SEQ ID NO:66 is a nucleotide sequence consisting essentially of the nucleotide sequence of SEQ ID NO:66 at position 134. lysine at position 137 of SEQ ID NO:66; Position 146 is a tryptophan; position 319 is a proline; Position 426 of SEQ ID NO:66 is a lysine, and position 43 of SEQ ID NO:66 is a 6 is aspartic acid, and each of the third and fourth polypeptides has up to 10 single amines. The present invention relates to a method for the preparation of a nucleic acid sequence comprising the sequence of SEQ ID NO:61, the sequence having amino acid modifications, wherein position 129 of SEQ ID NO:61 is a cysteine, S Position 146 of SEQ ID NO:61 is serine, position 148 of SEQ ID NO:61 is alanine, position 150 of SEQ ID NO:61 is aspartic acid, and Position 187 of DNO:61 is a valine.

[0050] In some embodiments, each of the first and second polypeptides comprises: a third and fourth polypeptide comprising, consisting of, or consisting essentially of the sequence of Each of the petites is In some embodiments, the nucleic acid comprises, consists of, or consists essentially of the sequence of TIFF0007611880000060.tif22165. Each of the first and second polypeptides may have up to 10 (9, 8, 7, 6, 5, 4, 3, 2 or or one amino acid sequence) having a single amino acid modification (e.g., a substitution, e.g., a conservative substitution), comprising, consisting of, or consisting essentially of the sequence of SEQ ID NO:67, and Each of the fourth polypeptides may have up to 10 (9, 8, 7, 6, 5, 4, 3, 2, or 1) SEQ ID NO:6, having a single amino acid modification (e.g., a substitution, e.g., a conservative substitution) of In some embodiments, the nucleic acid comprises, consists of, or consists essentially of the sequence of Each of the first and second polypeptides may have up to 10 (e.g., 1 or less, 2 or less, 3 or less) 4 or less, 5 or less, 6 or less, 7 or less, 8 or less, 9 or less) single amino acids The modification can be added to a linker (e.g., a subsequence The linker in TIFF0007611880000061.tif4128, SEQ ID NO:67, can be replaced with an alternative linker. The present invention relates to a method for producing a nucleic acid sequence comprising the sequence of SEQ ID NO: 67, the sequence of which is contained in the outer region of the nucleic acid sequence of SEQ ID NO: 67. and each of the third and fourth polypeptides consists essentially of said sequence and includes up to 10 single amino acids. comprising, consisting of, or essentially of the sequence of SEQ ID NO:61 with an acid modification. In some embodiments, each of the first and second polypeptides comprises up to 10 The sequence of SEQ ID NO:67, comprising the sequence of SEQ ID NO:67, having one single amino acid modification. wherein the nucleic acid sequence is a sequence similar to that of SEQ ID NO:67, except that position 134 of SEQ ID NO:67 is a cis-nucleotide sequence. lysine, position 137 of SEQ ID NO:67 is a lysine, Position 146 is a tryptophan, position 280 is an alanine in SEQ ID NO:67 Position 318 of SEQ ID NO:67 is a proline, and position 426 of SEQ ID NO:67 is a proline. is lysine, and position 436 of SEQ ID NO:67 is aspartic acid; and the fourth polypeptide each having up to 10 single amino acid modifications. comprising, consisting of, or consisting essentially of the sequence of SEQ ID NO:61, provided that Position 129 of SEQ ID NO:61 is a cysteine; position 14 of SEQ ID NO:61 is a cysteine 6 is a serine, position 148 of SEQ ID NO:61 is an alanine, position 150 of SEQ ID NO:61 is an aspartic acid, and position 187 of SEQ ID NO:61 is a valency It is.

[0051] In some embodiments, each of the first and second polypeptides comprises: a third and fourth polypeptide comprising, consisting of, or consisting essentially of the sequence of TIFF0007611880000062.tif46165; Each of the petites is In some embodiments, the nucleic acid comprises, consists of, or consists essentially of the sequence of TIFF0007611880000063.tif22165. Each of the first and second polypeptides may have up to 10 (9, 8, 7, 6, 5, 4, 3, 2 or or one amino acid sequence) having a single amino acid modification (e.g., a substitution, e.g., a conservative substitution), comprising, consisting of, or consisting essentially of the sequence of NO:68, and a third and Each of the fourth polypeptides may have up to 10 (9, 8, 7, 6, 5, 4, 3, 2, or 1) SEQ ID NO:6, having a single amino acid modification (e.g., a substitution, e.g., a conservative substitution) of In some embodiments, the nucleic acid comprises, consists of, or consists essentially of the sequence of: Each of the first and second polypeptides may have up to 10 (e.g., 1 or less, 2 or less, 3 or less) 4 or less, 5 or less, 6 or less, 7 or less, 8 or less, 9 or less) single amino acids The modification can be added to a linker (e.g., a subsequence The linker in TIFF0007611880000064.tif4128, SEQ ID NO:68, can be replaced with an alternative linker. The present invention relates to a method for producing a nucleic acid sequence comprising the sequence of SEQ ID NO: 68, the sequence being located in the outer region of the nucleic acid sequence of SEQ ID NO: 68. and each of the third and fourth polypeptides consists essentially of said sequence and includes up to 10 single amino acids. comprising, consisting of, or essentially of the sequence of SEQ ID NO:63 with acid modifications. In some embodiments, each of the first and second polypeptides comprises up to 10 The sequence of SEQ ID NO:68, comprising the sequence of SEQ ID NO:68, having one single amino acid modification. wherein position 33 of SEQ ID NO:68 is an alanine amino acid sequence. position 134 of SEQ ID NO:68 is a cysteine; Position 137 is a lysine, position 146 is a tryptophan in SEQ ID NO:68 Position 319 of SEQ ID NO:68 is a proline, position 426 of SEQ ID NO:68 is a lysine, and position 436 of SEQ ID NO:68 is aspartic acid; Each of the fourth polypeptides has up to 10 single amino acid modifications, comprising, consisting of, or consisting essentially of the sequence of SEQ ID NO:63, provided that Position 33 of ID NO:63 is an alanine.

[0052] In some embodiments, each of the first and second polypeptides comprises: a third and fourth polypeptide comprising, consisting of, or consisting essentially of the sequence of TIFF0007611880000065.tif46165; Each of the petites is In some embodiments, the nucleic acid comprises, consists of, or consists essentially of the sequence of TIFF0007611880000066.tif22165. Each of the first and second polypeptides may have up to 10 (9, 8, 7, 6, 5, 4, 3, 2 or or one amino acid sequence) having a single amino acid modification (e.g., a substitution, e.g., a conservative substitution), comprising, consisting of, or consisting essentially of the sequence of SEQ ID NO:69, and Each of the fourth polypeptides may have up to 10 (9, 8, 7, 6, 5, 4, 3, 2, or 1) SEQ ID NO:6, having a single amino acid modification (e.g., a substitution, e.g., a conservative substitution) of In some embodiments, the nucleic acid comprises, consists of, or consists essentially of the sequence of: Each of the first and second polypeptides may have up to 10 (e.g., 1 or less, 2 or less, 3 or less) 4 or less, 5 or less, 6 or less, 7 or less, 8 or less, 9 or less) single amino acids The modification can be added to a linker (e.g., a subsequence The linker in TIFF0007611880000067.tif4128, SEQ ID NO:69, can be replaced with an alternative linker. The present invention relates to a method for producing a nucleic acid sequence comprising the sequence of SEQ ID NO: 69, the sequence of which is located in the outer region of the nucleic acid sequence of SEQ ID NO: 69. and each of the third and fourth polypeptides consists essentially of said sequence and includes up to 10 single amino acids. comprising, consisting of, or essentially of the sequence of SEQ ID NO:63 with acid modifications. In some embodiments, each of the first and second polypeptides comprises up to 10 The sequence of SEQ ID NO:69, comprising the sequence of SEQ ID NO:69, having one single amino acid modification. wherein position 33 of SEQ ID NO:69 is an alanine amino acid sequence. position 134 of SEQ ID NO:69 is a cysteine; Position 137 is a lysine, position 146 is a tryptophan in SEQ ID NO:69 Position 280 of SEQ ID NO:69 is an alanine, position 319 of SEQ ID NO:69 is an alanine proline, position 426 of SEQ ID NO:69 is a lysine, and position 436 of 69 is aspartic acid, and each of the third and fourth polypeptides has a maximum The sequence of SEQ ID NO:63, which has 10 single amino acid modifications, wherein position 33 of SEQ ID NO:63 is a It's Ranin.

[0053] In some embodiments, each of the first and second polypeptides comprises: a third and fourth polypeptide comprising, consisting of, or consisting essentially of the sequence of TIFF0007611880000068.tif46165; Each of the petites is In some embodiments, the first and second polypeptides each comprise a sequence of up to 10 (9, 8, 7, 6, 5, 4, 3, 2 or 1) single amino acid modifications (e.g., substitutions, e.g. The present invention relates to a method for producing a nucleic acid sequence comprising the sequence of SEQ ID NO: 71, the sequence being selected from the group consisting of a nucleotide sequence having a nucleotide sequence similar to that of SEQ ID NO: 71, the ... and each of the third and fourth polypeptides consists essentially of said sequence, and , 7, 6, 5, 4, 3, 2 or 1 single amino acid modification (e.g., substitution, e.g., conservative substitution) The present invention relates to a method for producing a nucleic acid sequence comprising the sequence of SEQ ID NO: 70, the sequence of which is a nucleotide sequence of the present invention. In some embodiments, each of the first and second polypeptides comprises up to 10 (For example, 1 or less, 2 or less, 3 or less, 4 or less, 5 or less, 6 or less, 7 or less, 8 or less, 9 or less) of a single amino acid modification in the linker (e.g., subsequence The linker in TIFF0007611880000070.tif4128, SEQ ID NO:71, can be replaced with an alternative linker. The present invention relates to a method for producing a nucleic acid sequence comprising the sequence of SEQ ID NO: 71, the sequence being located in the outer region of the nucleic acid sequence of SEQ ID NO: 71. and each of the third and fourth polypeptides consists essentially of said sequence and includes up to 10 single amino acids. comprising, consisting of, or essentially of the sequence of SEQ ID NO:70 with acid modifications. In some embodiments, each of the first and second polypeptides comprises up to 10 The sequence of SEQ ID NO: 71, comprising the sequence of SEQ ID NO: 72, having one single amino acid modification. wherein position 71 of SEQ ID NO:71 is a nucleic acid sequence consisting essentially of said sequence, position 134 of SEQ ID NO:71 is a cysteine; Position 137 is a lysine, position 146 is a tryptophan, SEQ ID NO:71 Position 426 of SEQ ID NO:71 is a lysine, and position 436 of SEQ ID NO:71 is a lysine is aspartic acid, and each of the third and fourth polypeptides is up to 10 single amino acids. comprising, consisting of, or essentially of the sequence of SEQ ID NO:70, wherein position 72 of SEQ ID NO:70 is a proline.

[0054] In some embodiments, each of the first and second polypeptides comprises: a third and fourth polypeptide comprising, consisting of, or consisting essentially of the sequence of TIFF0007611880000071.tif46164; Each of the petites is In some embodiments, the sequence comprises, consists of, or consists essentially of the sequence of TIFF0007611880000072.tif22164. Each of the first and second polypeptides may have up to 10 (9, 8, 7, 6, 5, 4, 3, 2 or or one amino acid sequence) having a single amino acid modification (e.g., a substitution, e.g., a conservative substitution), comprising, consisting of, or consisting essentially of the sequence of SEQ ID NO:72, and Each of the fourth polypeptides may have up to 10 (9, 8, 7, 6, 5, 4, 3, 2, or 1) SEQ ID NO:7 having a single amino acid modification (e.g., a substitution, e.g., a conservative substitution) of In some embodiments, the nucleic acid comprises, consists of, or consists essentially of the sequence: Each of the first and second polypeptides may have up to 10 (e.g., 1 or less, 2 or less, 3 or less) 4 or less, 5 or less, 6 or less, 7 or less, 8 or less, 9 or less) single amino acids The modification can be added to a linker (e.g., a subsequence The linker in TIFF0007611880000073.tif4128, SEQ ID NO:72, can be replaced with an alternative linker. The present invention relates to a method for producing a nucleic acid sequence comprising the sequence of SEQ ID NO: 72, the sequence being located in the outer region of the nucleic acid sequence of SEQ ID NO: 72. and each of the third and fourth polypeptides consists essentially of said sequence and includes up to 10 single amino acids. comprising, consisting of, or essentially of the sequence of SEQ ID NO:70 with acid modifications. In some embodiments, each of the first and second polypeptides comprises up to 10 The sequence of SEQ ID NO: 72, comprising the sequence of SEQ ID NO: 72, having one single amino acid modification. wherein position 72 of SEQ ID NO:72 is a proline position 134 of SEQ ID NO:72 is cystine; position 134 of SEQ ID NO:72 is cystine Position 137 is a lysine, position 146 is a tryptophan, and position 150 is a lysine. Position 426 of SEQ ID NO:72 is a lysine, and position 436 of SEQ ID NO:72 is a aspartic acid, and each of the third and fourth polypeptides is up to 10 single amino acids. comprising, consisting of, or essentially consisting of the sequence of SEQ ID NO:70, with an acid modification. where position 72 of SEQ ID NO:70 is a proline.

[0055] In some embodiments, each of the first and second polypeptides comprises: a third and fourth polypeptide comprising, consisting of, or consisting essentially of the sequence of TIFF0007611880000074.tif46165; Each of the petites is In some embodiments, the nucleic acid comprises, consists of, or consists essentially of the sequence of TIFF0007611880000075.tif22165. Each of the first and second polypeptides may have up to 10 (9, 8, 7, 6, 5, 4, 3, 2 or or one amino acid sequence) having a single amino acid modification (e.g., a substitution, e.g., a conservative substitution), comprising, consisting of, or consisting essentially of the sequence of SEQ ID NO:74, and Each of the fourth polypeptides may have up to 10 (9, 8, 7, 6, 5, 4, 3, 2, or 1) SEQ ID NO:7 having a single amino acid modification (e.g., a substitution, e.g., a conservative substitution) of In some embodiments, the nucleic acid comprises, consists of, or consists essentially of the sequence of: Each of the first and second polypeptides may have up to 10 (e.g., 1 or less, 2 or less, 3 or less) 4 or less, 5 or less, 6 or less, 7 or less, 8 or less, 9 or less) single amino acids The modification can be added to a linker (e.g., a subsequence The linker in TIFF0007611880000076.tif4128, SEQ ID NO:74, can be replaced with an alternative linker. The present invention relates to a method for producing a nucleic acid sequence comprising the sequence of SEQ ID NO: 74, the sequence of which is located in the outer region of the nucleic acid sequence of SEQ ID NO: 74. and each of the third and fourth polypeptides consists essentially of said sequence and includes up to 10 single amino acids. comprising, consisting of, or essentially of the sequence of SEQ ID NO:73 with acid modifications. In some embodiments, each of the first and second polypeptides comprises up to 10 The sequence of SEQ ID NO: 74, comprising the sequence of SEQ ID NO: 74, having one single amino acid modification. wherein position 33 of SEQ ID NO:74 is an alanine amino acid sequence. position 72 of SEQ ID NO:74 is a proline; position 1 of SEQ ID NO:74 is a cyclic nucleotide 34 is a cysteine, position 137 of SEQ ID NO:74 is a lysine, Position 146 of SEQ ID NO:74 is tryptophan, position 426 of SEQ ID NO:74 is lysine and position 436 of SEQ ID NO:74 is an aspartic acid, and the third and fourth Each of the polypeptides of SEQ ID NO. 73, comprising, consisting of or consisting essentially of the sequence of SEQ ID NO: 73, Position 33 of SEQ ID NO:73 is an alanine, and position 72 of SEQ ID NO:73 is a proline. It's Rin.

[0056] In some embodiments, each of the first and second polypeptides comprises: a third and fourth polypeptide comprising, consisting of, or consisting essentially of the sequence of Each of the petites is In some embodiments, the nucleic acid comprises, consists of, or consists essentially of the sequence of TIFF0007611880000078.tif22165. Each of the first and second polypeptides may have up to 10 (9, 8, 7, 6, 5, 4, 3, 2 or or one amino acid sequence) having a single amino acid modification (e.g., a substitution, e.g., a conservative substitution), comprising, consisting of, or consisting essentially of the sequence of SEQ ID NO:75, and Each of the fourth polypeptides may have up to 10 (9, 8, 7, 6, 5, 4, 3, 2, or 1) SEQ ID NO:7 having a single amino acid modification (e.g., a substitution, e.g., a conservative substitution) of In some embodiments, the nucleic acid comprises, consists of, or consists essentially of the sequence of: Each of the first and second polypeptides may have up to 10 (e.g., 1 or less, 2 or less, 3 or less) 4 or less, 5 or less, 6 or less, 7 or less, 8 or less, 9 or less) single amino acids The modification can be added to a linker (e.g., a subsequence The linker in TIFF0007611880000079.tif4128, SEQ ID NO:75, can be replaced with an alternative linker. The present invention relates to a method for producing a nucleic acid sequence comprising the steps of: and each of the third and fourth polypeptides consists essentially of said sequence and includes up to 10 single amino acids. comprising, consisting of, or essentially of the sequence of SEQ ID NO:73 with acid modifications. In some embodiments, each of the first and second polypeptides comprises up to 10 A sequence comprising the sequence of SEQ ID NO:75, said sequence having one single amino acid modification. wherein position 33 of SEQ ID NO:75 is an alanine amino acid sequence. position 72 of SEQ ID NO:75 is a proline; position 1 of SEQ ID NO:75 is a 34 is a cysteine, position 137 of SEQ ID NO:75 is a lysine, Position 146 of SEQ ID NO:75 is tryptophan, position 280 of SEQ ID NO:75 is arabinopeptide nin, position 426 of SEQ ID NO: 75 is lysine, and position 436 of the third and fourth polypeptides is aspartic acid, The sequence of SEQ ID NO: 73, comprising the sequence of SEQ ID NO: 73, having one single amino acid modification. wherein position 33 of SEQ ID NO:73 is an alanine amino acid sequence. and position 72 of SEQ ID NO:73 is a proline.

[0057] In some embodiments, each of the first and second polypeptides comprises: a third and fourth polypeptide comprising, consisting of, or consisting essentially of the sequence of TIFF0007611880000080.tif46165; Each of the petites is In some embodiments, the first and second polypeptides each comprise a sequence of up to 10 (9, 8, 7, 6, 5, 4, 3, 2 or 1) single amino acid modifications (e.g., substitutions, e.g. The present invention relates to a method for producing a nucleic acid sequence comprising the sequence of SEQ ID NO: 76, the sequence of which is a conserved substitution. and each of the third and fourth polypeptides consists essentially of said sequence, and , 7, 6, 5, 4, 3, 2 or 1 single amino acid modification (e.g., substitution, e.g., conservative substitution) The present invention relates to a method for producing a nucleic acid sequence comprising the sequence of SEQ ID NO: 70, the sequence of which is a nucleotide sequence of the present invention. In some embodiments, each of the first and second polypeptides comprises up to 10 (For example, 1 or less, 2 or less, 3 or less, 4 or less, 5 or less, 6 or less, 7 or less, 8 or less, 9 or less) of a single amino acid modification in the linker (e.g., subsequence The linker in TIFF0007611880000082.tif4128, SEQ ID NO:76, can be replaced with an alternative linker. The present invention relates to a method for producing a nucleic acid sequence comprising the sequence of SEQ ID NO: 76, the sequence of which is contained in the outer region of the nucleic acid sequence of SEQ ID NO: 76. and each of the third and fourth polypeptides consists essentially of said sequence and includes up to 10 single amino acids. comprising, consisting of, or essentially of the sequence of SEQ ID NO:70 with acid modifications. In some embodiments, each of the first and second polypeptides comprises up to 10 The sequence of SEQ ID NO: 76, comprising the sequence having one single amino acid modification. wherein the nucleic acid sequence of SEQ ID NO:76 is a nucleic acid sequence consisting essentially of the nucleic acid sequence of SEQ ID NO:76, and position 319 of SEQ ID NO:76 is a proline, and the third and fourth polynucleotides Each of the peptides has up to 10 single amino acid modifications, SEQ ID NO:70 comprising, consisting of or consisting essentially of the sequence of SEQ ID N Position 72 of O:70 is a proline.

[0058] In some embodiments, each of the first and second polypeptides comprises: a third and fourth polypeptide comprising, consisting of, or consisting essentially of the sequence of TIFF0007611880000083.tif46165; Each of the petites is It comprises, consists of, or consists essentially of the sequence of TIFF0007611880000084.tif22164.

[0059] In some embodiments, each of the first and second polypeptides comprises: a third and fourth polypeptide comprising, consisting of, or consisting essentially of the sequence of TIFF0007611880000085.tif47163; Each of the petites is In some embodiments, the first and second polypeptides each comprise a sequence of up to 10 (9, 8, 7, 6, 5, 4, 3, 2 or 1) single amino acid modifications (e.g., substitutions, e.g. The present invention relates to a method for producing a nucleic acid sequence comprising the sequence of SEQ ID NO: 77, the sequence having a conserved substitution ( and each of the third and fourth polypeptides consists essentially of said sequence, and , 7, 6, 5, 4, 3, 2 or 1 single amino acid modification (e.g., substitution, e.g., conservative substitution) The present invention relates to a method for producing a nucleic acid sequence comprising the sequence of SEQ ID NO: 70, the sequence of which is a nucleotide sequence of the present invention. In some embodiments, each of the first and second polypeptides comprises up to 10 (For example, 1 or less, 2 or less, 3 or less, 4 or less, 5 or less, 6 or less, 7 or less, 8 or less, 9 or less) of a single amino acid modification in the linker (e.g., subsequence The linker in TIFF0007611880000087.tif4128, SEQ ID NO:77, can be replaced with an alternative linker. The present invention relates to a method for producing a nucleic acid sequence comprising the sequence of SEQ ID NO: 77, the sequence of which is located in the outer region of the nucleic acid sequence of SEQ ID NO: 77. and each of the third and fourth polypeptides consists essentially of said sequence and includes up to 10 single amino acids. comprising, consisting of, or essentially of the sequence of SEQ ID NO:70 with acid modifications. In some embodiments, each of the first and second polypeptides comprises up to 10 The sequence of SEQ ID NO: 77, comprising the sequence having one single amino acid modification. wherein position 72 of SEQ ID NO:77 is a proline. position 134 of SEQ ID NO:77 is a cysteine; Position 137 is a lysine, position 146 is a tryptophan in SEQ ID NO:77 Position 280 of SEQ ID NO:77 is an alanine, position 319 of SEQ ID NO:77 is an alanine proline, position 426 of SEQ ID NO:77 is a lysine, and position 436 of 77 is aspartic acid, and each of the third and fourth polypeptides has a maximum The sequence of SEQ ID NO:70, which has 10 single amino acid modifications, wherein position 72 of SEQ ID NO:70 is a sequence consisting of, or consisting essentially of, the sequence It's Lorin.

[0060] In some embodiments, each of the first and second polypeptides comprises: a third and fourth polypeptide comprising, consisting of, or consisting essentially of the sequence of TIFF0007611880000088.tif46165; Each of the petites is In some embodiments, the nucleic acid comprises, consists of, or consists essentially of the sequence of TIFF0007611880000089.tif22165. Each of the first and second polypeptides may have up to 10 (9, 8, 7, 6, 5, 4, 3, 2 or or one amino acid sequence) having a single amino acid modification (e.g., a substitution, e.g., a conservative substitution), comprising, consisting of, or consisting essentially of the sequence of SEQ ID NO:79, and Each of the fourth polypeptides may have up to 10 (9, 8, 7, 6, 5, 4, 3, 2, or 1) SEQ ID NO:7 having a single amino acid modification (e.g., a substitution, e.g., a conservative substitution) of In some embodiments, the nucleic acid comprises, consists of, or consists essentially of the sequence of: Each of the first and second polypeptides may have up to 10 (e.g., 1 or less, 2 or less, 3 or less) 4 or less, 5 or less, 6 or less, 7 or less, 8 or less, 9 or less) single amino acids The modification can be added to a linker (e.g., a subsequence The linker in TIFF0007611880000090.tif4128, SEQ ID NO:79, can be replaced with an alternative linker. The present invention relates to a method for producing a nucleic acid sequence comprising the sequence of SEQ ID NO: 79, the sequence of which is located in the outer region of the nucleic acid sequence of SEQ ID NO: 79. and each of the third and fourth polypeptides consists essentially of said sequence and includes up to 10 single amino acids. comprising, consisting of, or essentially of the sequence of SEQ ID NO:73 with acid modifications. In some embodiments, each of the first and second polypeptides comprises up to 10 The sequence of SEQ ID NO: 79, comprising the sequence of SEQ ID NO: 79, having one single amino acid modification. wherein position 33 of SEQ ID NO:79 is an alanine amino acid sequence. position 72 of SEQ ID NO:79 is a proline; position 73 of SEQ ID NO:79 is a proline 280 is an alanine, and position 319 of SEQ ID NO:79 is a proline; and the fourth polypeptide each having up to 10 single amino acid modifications, D NO:73, comprising, consisting of, or consisting essentially of the sequence, provided that Position 33 of SEQ ID NO:73 is an alanine, and position 7 of SEQ ID NO:73 is an alanine. 2 is proline. In some embodiments, the Fc construct comprises construct 4, Construct 5, Construct 6, Construct 7, Construct 8, Construct Construct 9, Construct 10, Construct 11, Construct 12, Construct Construct 13, Construct 14, Construct 15, Construct 16, Construct Construct 17, Construct 18, or Construct 19.

[0061] In some embodiments, the Fc construct is Construct 5, Construct 6, Construct 7, Construct 9, Construct 10, Construct 11, Construct 13, Construct 14, Construct 15, Construct 16, Construct 17, Construct 18, or Construct 19.

[0062] In some embodiments, the first polypeptide, the second polypeptide, the third polypeptide , and / or the fourth polypeptide each comprises an N-terminal D to Q amino acid substitution.

[0063] In a second aspect, the Fc construct of the present disclosure comprises: (i) a first Fc domain monomer and and (ii) a first Fc domain monomer, the first Fc domain monomer comprising a second Fc domain monomer, and and a second Fc domain comprising a first Fc domain monomer and a fourth Fc domain monomer, In another embodiment, the Fc domain of the present disclosure comprises an amino acid modification at position 1253. The c construct comprises: (i) a first Fc domain monomer and a second Fc domain monomer; (ii) a first Fc domain monomer and a fourth Fc domain monomer. and a second Fc domain comprising at least one Fc domain monomer. In some embodiments, each amino acid at position I253 comprises an amino acid substitution. Modifications (e.g., substitutions) include I253A, I253C, I253D, I253E, I253 F, I253G, I253H, I253I, I253K, I253L, I253M, I2 53N, I253P, I253Q, I253R, I253S, I253T, I253V, In some embodiments, the amino acid sequence is independently selected from the group consisting of I253W, I253Y, and I253Y. The amino acid modification (e.g., substitution) at position I253 is I253A. In one embodiment, the Fc construct comprises at least one amino acid modification at position R292 (e.g., substitution In some embodiments, the amino acid modification (e.g., substitution) at position R292 includes R292D, R292E, R292L, R292P, R292Q, R292R, R292 In some embodiments, the amino acid at position R292 is selected from the group consisting of R292Y, R292T, and R292Y. The amino acid modification (eg, substitution) is R292P.

[0064] In some embodiments, the first Fc domain monomer and the third Fc domain monomer are In some embodiments, the second F is linked by a linker (e.g., a spacer). The c domain monomer and the fourth Fc domain monomer are linked to a linker (e.g., a spacer). In some embodiments, each linker (e.g., spacer) is TIFF0007611880000091.tif77165.

[0065] In some embodiments, the first Fc domain monomer and the second Fc domain monomer are Complementation promotes dimer formation between one Fc domain monomer and a second Fc domain monomer. In some embodiments, the third Fc domain monomer comprises a dimerization selection module. and the fourth Fc domain monomer is a third Fc domain monomer and a fourth Fc domain monomer. The complementary dimerization selection modules promote dimerization between the dimers.

[0066] In another aspect, the disclosure provides a method for producing an Fc domain monomer comprising: (i) a first F domain monomer and a second Fc domain monomer; (ii) a first Fc domain monomer and a fourth Fc domain monomer. and a second Fc domain, wherein at least one Fc domain is located at a position In another aspect, the present invention provides an Fc construct comprising an amino acid modification at position R292. The illustration shows (i) a first Fc domain monomer comprising a first F domain monomer and a second Fc domain monomer. domain; and (ii) a third Fc domain monomer and a fourth Fc domain monomer. and a second Fc domain, wherein at least one Fc domain monomer is at position R292. In some embodiments, an Fc construct is provided that comprises an amino acid substitution at position R292. Each amino acid modification (e.g., substitution) in 2P, R292Q, R292R, R292T, and R292Y. In some embodiments, the amino acid modification (e.g., substitution) at position R292 is selected from R2 In some embodiments, the first Fc domain monomer and the third Fc domain monomer are The monomers are joined by a linker.

[0067] In some embodiments, the second Fc domain monomer and the fourth Fc domain monomer are In some embodiments, each linker is In some embodiments, the linker is independently selected from a polypeptide having the following structure: The file is TIFF0007611880000093.tif4128.

[0068] In some embodiments, the first Fc domain monomer and the second Fc domain monomer are Complementation promotes dimer formation between one Fc domain monomer and a second Fc domain monomer. In some embodiments, the third Fc domain monomer comprises a dimerization selection module. and the fourth Fc domain monomer is a third Fc domain monomer and a fourth Fc domain monomer. The complementary dimerization selection modules promote dimerization between the dimers.

[0069] In another aspect, the disclosure features a method of preparing an Fc construct described herein. The method includes: a) creating a host cell comprising a polynucleotide encoding a polypeptide of the present disclosure; b) providing a host cell under conditions that permit the formation of an Fc construct; and c) expressing the polypeptide in the Fc construct. do.

[0070] In another aspect, the disclosure provides a method for producing an Fc domain comprising: a) i) a first Fc domain monomer; ii) a second Fc domain monomer; and iii) binding the first Fc domain monomer to the second Fc domain monomer. and b) a first polypeptide comprising a linker that binds i) a third Fc domain monomer, ii ) a fourth Fc domain monomer, and iii) a third Fc domain monomer. c) a second polypeptide comprising a linker that connects the Fc domain to the Fc monomer; and a third polypeptide comprising a first Fc domain monomer; and d) a fourth polypeptide comprising a sixth Fc domain monomer. and a peptide, wherein the first Fc domain monomer and the fifth Fc domain monomer are The Fc domain monomers combine to form a first Fc domain and a second Fc domain. the first Fc domain monomer and the fourth Fc domain monomer combine to form a second Fc domain; and the third Fc domain monomer and the sixth Fc domain monomer are combined to form a third wherein at least one Fc domain has an amino acid sequence at position I253. and wherein each of the first and second polypeptides comprises an acid modification. TIFF0007611880000094.tif46165, wherein each of the third and fourth polypeptides comprises In another aspect, the present disclosure provides a method for the production of a fusion protein comprising: a) i) a first Fc domain monomer; ii) a second Fc domain monomer; and iii) a first Fc domain monomer and a second Fc domain monomer. a) a first polypeptide comprising a linker that connects the monomers; and b) i) a third Fc domain. monomer, ii) a fourth Fc domain monomer, and iii) a third Fc domain monomer. a second polypeptide comprising a linker linking the four Fc domain monomers; and c) a fifth a third polypeptide comprising an Fc domain monomer; and d) a sixth polypeptide comprising an Fc domain monomer. and a fourth polypeptide, wherein the first Fc domain alone is The fifth Fc domain monomer is combined to form a first Fc domain, and the fifth Fc domain monomer is combined to form a second Fc domain. The first Fc domain monomer and the fourth Fc domain monomer are combined to form a second Fc domain. and the third Fc domain monomer and the sixth Fc domain monomer are combined to form a combined to form a third Fc domain, wherein each of the first and second polypeptides comprises: TIFF0007611880000096.tif46165, wherein each of the third and fourth polypeptides comprises Contains sequence TIFF0007611880000097.tif22165.

[0071] In another aspect, the disclosure provides a method for producing an Fc domain comprising: a) i) a first Fc domain monomer; ii) a second Fc domain monomer; and iii) binding the first Fc domain monomer to the second Fc domain monomer. and b) a first polypeptide comprising a linker that binds i) a third Fc domain monomer, ii ) a fourth Fc domain monomer, and iii) a third Fc domain monomer. c) a second polypeptide comprising a linker that connects the Fc domain to the Fc monomer; and a third polypeptide comprising a first Fc domain monomer; and d) a fourth polypeptide comprising a sixth Fc domain monomer. and a peptide, wherein the first Fc domain monomer and the fifth Fc domain monomer are The Fc domain monomers combine to form a first Fc domain and a second Fc domain. the first Fc domain monomer and the fourth Fc domain monomer combine to form a second Fc domain; and the third Fc domain monomer and the sixth Fc domain monomer are combined to form a third wherein at least one Fc domain has an amino acid sequence at position I253. and wherein each of the first and second polypeptides comprises an acid modification. TIFF0007611880000098.tif46165, wherein each of the third and fourth polypeptides comprises In another aspect, the present disclosure provides a method for the production of a fusion protein comprising: a) i) a first Fc domain monomer; ii) a second Fc domain monomer; and iii) a first Fc domain monomer and a second Fc domain monomer. a) a first polypeptide comprising a linker that connects the monomers; and b) i) a third Fc domain. monomer, ii) a fourth Fc domain monomer, and iii) a third Fc domain monomer. a second polypeptide comprising a linker linking the four Fc domain monomers; and c) a fifth a third polypeptide comprising an Fc domain monomer; and d) a sixth polypeptide comprising an Fc domain monomer. and a fourth polypeptide, wherein the first Fc domain alone is The fifth Fc domain monomer is combined to form a first Fc domain, and the fifth Fc domain monomer is combined to form a second Fc domain. The first Fc domain monomer and the fourth Fc domain monomer are combined to form a second Fc domain. and the third Fc domain monomer and the sixth Fc domain monomer are combined to form a combined to form a third Fc domain, wherein each of the first and second polypeptides comprises: TIFF0007611880000100.tif46165, wherein each of the third and fourth polypeptides comprises Contains the sequence TIFF0007611880000101.tif22165.

[0072] In another aspect, the disclosure provides a method for producing an Fc domain comprising: a) i) a first Fc domain monomer; ii) a second Fc domain monomer; and iii) binding the first Fc domain monomer to the second Fc domain monomer. and b) a first polypeptide comprising a linker that binds i) a third Fc domain monomer, ii ) a fourth Fc domain monomer, and iii) a third Fc domain monomer. c) a second polypeptide comprising a linker that connects the Fc domain to the Fc monomer; and a third polypeptide comprising a first Fc domain monomer; and d) a fourth polypeptide comprising a sixth Fc domain monomer. and a peptide, wherein the first Fc domain monomer and the fifth Fc domain monomer are The Fc domain monomers combine to form a first Fc domain and a second Fc domain. the first Fc domain monomer and the fourth Fc domain monomer combine to form a second Fc domain; and the third Fc domain monomer and the sixth Fc domain monomer are combined to form a third wherein at least one Fc domain has an amino acid sequence at position R292. and wherein each of the first and second polypeptides comprises an acid modification. and each of the third and fourth polypeptides comprises the sequence of In another aspect, the present disclosure provides a method for the production of a fusion protein comprising: a) i) a first Fc domain monomer; ii) a second Fc domain monomer; and iii) a first Fc domain monomer and a second Fc domain monomer. a) a first polypeptide comprising a linker that connects the monomers; and b) i) a third Fc domain. monomer, ii) a fourth Fc domain monomer, and iii) a third Fc domain monomer. a second polypeptide comprising a linker linking the four Fc domain monomers; and c) a fifth a third polypeptide comprising an Fc domain monomer; and d) a sixth polypeptide comprising an Fc domain monomer. and a fourth polypeptide, wherein the first Fc domain alone is The fifth Fc domain monomer is combined to form a first Fc domain, and the fifth Fc domain monomer is combined to form a second Fc domain. The first Fc domain monomer and the fourth Fc domain monomer are combined to form a second Fc domain. and the third Fc domain monomer and the sixth Fc domain monomer are combined to form a combined to form a third Fc domain, wherein each of the first and second polypeptides comprises: and each of the third and fourth polypeptides comprises the sequence of Contains the sequence TIFF0007611880000105.tif22164.

[0073] In some embodiments of any of the Fc constructs described herein, each Fc domain The Fc domains are independently IgG1 Fc domain, IgG2 Fc domain, and IgG3 Fc domain. The antibody may be a polypeptide having a nucleotide sequence similar to that described herein, such as a nucleotide sequence of the IgG4 Fc domain, an IgG4 Fc domain, or a combination thereof. In some embodiments of any Fc construct, each Fc domain independently has up to 10 (e.g., up to 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10) single amino acids IgG1 Fc domain, IgG2 Fc domain, IgG3 Fc domain with modifications In some embodiments, the antibody is an IgG4 Fc domain, an IgG4 Fc domain, or a combination thereof. Each Fc domain is an IgG1 Fc domain. In some embodiments, each Fc domain is In some embodiments, each Fc domain is an IgG2 Fc domain. In some embodiments, the first, second, and third Fc domains are Each is an IgG1 Fc domain. In some embodiments, each Fc domain is human In some embodiments, each Fc domain is a single Fc domain. For example, 1 or less, 2 or less, 3 or less, 4 or less, 5 or less, 6 or less, 7 or less, 8 and SEQ ID NO:42 having a single amino acid modification (not more than one, not more than nine).

[0074] In some embodiments of any of the Fc constructs described herein, one or more The Fc domain monomers may consist of up to 10 (e.g., up to 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10) of the amino acid modifications. In some embodiments of any of the Fc constructs described in any one of the above, each Fc domain monomer is 10 or fewer (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 or fewer) amino acids In some embodiments of any of the Fc constructs described herein, Each Fc monomer may contain 10 or fewer (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 The sequence of SEQ ID NO:42 includes the amino acid modifications described herein below. In some embodiments of any of the Fc constructs, each Fc monomer comprises five amino acids. The sequence of SEQ ID NO:42 having an acid modification. In some embodiments of the Fc construct, each Fc monomer has 10 amino acid modifications. Any of the Fc constructs described herein comprises the sequence of SEQ ID NO:42. In some embodiments of the construct, each Fc monomer has SEQ ID NO: 8 amino acid modifications. In each case, the modification comprises the amino acid substitutions I253A and R2 92P or both.

[0075] In another aspect, the present disclosure provides a host cell expressing an Fc construct described herein. The host cell comprises a polynucleotide encoding a polypeptide of the present disclosure. wherein the polynucleotide is expressed in a host cell.

[0076] In another aspect, the present disclosure relates to a method for the preparation of a nucleic acid sequence comprising administering to a subject a nucleic acid sequence selected from the group consisting of SEQ ID NOs: 43-79. In another aspect, the disclosure provides a polypeptide comprising an amino acid sequence of SEQ ID NO: an amino acid sequence selected from the group consisting of SEQ ID NO:49 and SEQ ID NO:61-79 The present invention provides a polypeptide comprising:

[0077] In another aspect, the disclosure provides a composition comprising or consisting of SEQ ID NO:78, and In another aspect, a polypeptide comprising or consisting of SEQ ID NO:73 is provided. The present disclosure relates to compositions comprising or consisting of SEQ ID NO:49, and In another aspect, the present disclosure provides a polypeptide comprising or consisting of SEQ ID NO: 0:61. Compositions comprising or consisting of SEQ ID NO:62, and compositions comprising or consisting of SEQ ID NO:61 In another aspect, the present disclosure provides a polypeptide comprising SEQ ID NO:6. Compositions comprising or consisting of SEQ ID NO: 4, and polypeptides comprising or consisting of SEQ ID NO: 63. In another aspect, the present disclosure provides a peptide comprising or consisting of SEQ ID NO:65. and a polypeptide comprising or consisting of SEQ ID NO:63. In another aspect, the present disclosure provides a composition comprising or consisting of SEQ ID NO:67, and In another aspect, a polypeptide comprising or consisting of SEQ ID NO:61 is provided. The present disclosure relates to compositions comprising or consisting of SEQ ID NO:68, and In another aspect, the present disclosure provides a polypeptide comprising or consisting of SEQ ID NO: 0:63. Compositions comprising or consisting of SEQ ID NO:69, and compositions comprising or consisting of SEQ ID NO:63 In another aspect, the present disclosure provides a polypeptide comprising SEQ ID NO:7. 2, and a polypeptide comprising or consisting of SEQ ID NO:70. In another aspect, the present disclosure provides a peptide comprising or consisting of SEQ ID NO:74. and a polypeptide comprising or consisting of SEQ ID NO:73. In another aspect, the disclosure provides a composition comprising or consisting of SEQ ID NO:75, and In another aspect, a polypeptide comprising or consisting of SEQ ID NO:73 is provided. The present disclosure relates to compositions comprising or consisting of SEQ ID NO:77, and In another aspect, the present disclosure provides a polypeptide comprising or consisting of SEQ ID NO: 0: 70. Compositions comprising or consisting of SEQ ID NO:79, and compositions comprising or consisting of SEQ ID NO:73 In another aspect, the present disclosure provides a polypeptide comprising SEQ ID NO:7. Compositions comprising or consisting of SEQ ID NO:6, and polypeptides comprising or consisting of SEQ ID NO:70. Offer petite.

[0078] In some embodiments, the composition comprises the listed amines present in a molar ratio of 1.1:1 to 1:1.1. The polypeptide further comprises a first and a second polypeptide.

[0079] In another aspect, the disclosure provides a method for administering to a patient any one of the compositions described herein. The present invention provides a method of treating a patient, comprising:

[0080] In another aspect, the disclosure provides an Fc construct as described herein (e.g., a three-unit Fc Substantially homogenous (e.g., at least 85%, 9 0%, 95%, 97%, 98%, 99% homogeneous) or a pharmaceutical composition comprising the population of A substantially homogenous population of Fc constructs according to any one of claims 1 to 5 (e.g., a population having three Fc domains). a composition comprising a substantially homogenous population of Fc constructs having one or more pharma- ceutical acceptable salts, and an acceptable carrier or excipient. Such pharmaceutical compositions include compositions comprising Fc The constructs can be made without substantial aggregation or unwanted multimerization.

[0081] In some aspects, the disclosure provides a polypeptide comprising SEQ ID NO:76; and Compositions comprising a polypeptide comprising SEQ ID NO:70 are provided. Thus, the present disclosure relates to a polypeptide comprising SEQ ID NO:78, and In some embodiments, the present disclosure provides a composition comprising a polypeptide comprising O:73. A polypeptide comprising SEQ ID NO:79 and a polypeptide comprising SEQ ID NO:73 Compositions comprising the polypeptide are provided.

[0082] In some aspects, the present disclosure provides a method for administering any one of the compositions described herein to a patient. The present invention provides a method of treating a patient comprising administering

[0083] In some aspects, the disclosure provides a nucleic acid sequence encoding SEQ ID NO:76, and and a nucleic acid sequence encoding SEQ ID NO:70. In this regard, the present disclosure provides a nucleic acid sequence encoding SEQ ID NO:78, and A cell is provided that comprises a nucleic acid sequence encoding NO:73. The nucleic acid sequence encoding SEQ ID NO:79 and the nucleic acid sequence encoding SEQ ID NO:73 are In some aspects, the disclosure provides a cell comprising a nucleic acid sequence encoding the A cell comprising a nucleic acid sequence encoding any one of the Fc constructs described is provided. do.

[0084] In another aspect, the disclosure provides an Fc construct as described herein (e.g., a three-unit Fc Fc constructs having domains) or the substance of the Fc constructs described herein A composition comprising a substantially homogenous population (e.g., an Fc construct having three Fc domains) a composition comprising a substantially homogenous population of In some embodiments, the subject is provided with a method for reducing immune cell activation in a response to a serotonin-dependent inflammatory response. If you have an autoimmune disease.

[0085] In another aspect, the disclosure provides an Fc construct as described herein (e.g., a three-unit Fc Fc constructs having domains) or the substance of the Fc constructs described herein A composition comprising a substantially homogenous population (e.g., an Fc construct having three Fc domains) a composition comprising a substantially homogenous population of or a method for treating an inflammatory disease.

[0086] In another aspect, the disclosure provides an Fc construct as described herein (e.g., a three-unit Fc Fc constructs having domains) or the substance of the Fc constructs described herein A composition comprising a substantially homogenous population (e.g., an Fc construct having three Fc domains) a composition comprising a substantially homogenous population of Methods for enhancing antibody clearance or inhibiting antigen presentation are featured.

[0087] In some embodiments, the Fc constructs described herein (e.g., three Fc domains) are Exemplary diseases that can be treated by administering an Fc construct having an agonist ( are rheumatoid arthritis (RA); systemic lupus erythematosus (SLE); ANCA-associated vasculitis; Antiphospholipid syndrome; Autoimmune hemolytic anemia; Chronic inflammatory demyelinating neuropathy; Transplantation Anti-alloproteinases, anti-self protein in GVHD, anti-substitution protein, IgG therapy, IgG paraproteins Allans; Dermatomyositis; Goodpasture's syndrome; Mediated through antibody-dependent cell-mediated cytotoxicity Type II hypersensitivity syndromes of mediated organ system targets, e.g. Guillain-Barre syndrome, CIDP, skin Myositis, Felty's syndrome, antibody-mediated rejection, autoimmune thyroid disease, ulcerative colitis, Autoimmune liver disease; idiopathic thrombocytopenic purpura; myasthenia gravis, neuromyelitis optica; pemphigus and and other autoimmune blistering diseases; Sjogren's syndrome; antibody-dependent phagocytosis Autoimmune cytopenias and other disorders mediated by the immune system; e.g., synovitis, dermatomyositis, systemic hematopoiesis, Other FcR-dependent inflammatory syndromes include vasculitis, glomerulitis and vasculitis.

[0088] In some embodiments, the Fc constructs described herein comprise an antigen recognition region, e.g. In some embodiments, the Fc construct does not include a variable region or a complementarity determining region (CDR). The construct (or the Fc domain in the Fc construct) may be present in a different polypeptide. In some embodiments, the Fc domain is formed in whole or in part by the association of Fc domain monomers. The Fc constructs described herein include an antigen recognition region, such as a variable region or a CDR. In some embodiments, the Fc construct comprises a polypeptide that promotes association of the two polypeptides. Additional domains (e.g., IgM tailpiece or IgA tailpiece) that In another embodiment, the Fc domain does not include two Fc domains that are linked to form an Fc domain. There are covalent bonds in the Fc construct only between the main monomers. In some embodiments, the Fc construct does not include a covalent bond between the Fc domains. In the present invention, the Fc construct comprises all or substantially all of the Fc domains in the Fc construct. all of which have sufficient structure to simultaneously interact with Fc receptors on the cell surface. In one embodiment, the domain monomers have primary sequences that differ from the wild type or or are different from each other and have a dimerization selection module.

[0089] The Fc domain monomers of the construct have the same primary amino acid sequence. For example, both Fc domain monomers of an Fc domain can be identical. The Fc domain may have a merization selection module, e.g., both Fc domain monomers of the Fc domain may have a merization selection module. Both, C H At least two within a ring of charged residues at the interface between the three domains In some embodiments, the first position can have the same opposite charge mutation. In some embodiments, the polypeptide and the second polypeptide have the same amino acid sequence. In some embodiments, the third and fourth polypeptides have identical amino acid sequences. The sequence of the first Fc domain monomer is different from the sequence of the fifth Fc domain monomer. In some embodiments, the sequence of the third Fc domain monomer is the same as the sequence of the sixth Fc domain monomer. In some embodiments, the sequence of the second Fc domain monomer is different from the sequence of the fourth Fc domain monomer. The sequence is the same as that of the main monomer.

[0090] In any of the Fc constructs described herein, the F of a construct The Fc domain monomer of the c domain is between two Fc domain monomers (i.e., Fc conjugates). between the Fc domain monomer and other monomers of the construct), 0 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 2 20 or fewer amino acids (e.g., 15 or fewer, 10 or fewer amino acids) that are For example, any of the phases of the Fc construct may have different sequences. A complementary dimerization selection module is attached to one of the C domain monomers. H 3. Antibody constant domain The engineered cavity in the other C of the Fc domain monomer H 3. Engineered into Antibody Constant Domains and a manipulated protrusion, the manipulated cavity and the manipulated protrusion being positioned. The projection is inserted into the cavity of the Fc domain monomer to form a pair, as described herein. The Fc domain monomer sequences of the constructs described above may vary. In an embodiment, the Fc construct comprises H Contains amino acid modifications within the 3 domains. In an embodiment, the Fc construct comprises an Fc domain monomer (one) for selective dimerization. or multiple Fc domain monomers) H 3. Exemplary engineered amino acid modifications include those in The cavities and protrusions are shown in Table 1. In another embodiment, the complementary dimerization selection modules The C domain of one of the monomers H 3. Negative sites engineered (substituted) into antibody constant domains Charged amino acids and the other C of the Fc domain monomer H 3. Engineered into Antibody Constant Domains and negatively charged amino acids. and positively charged amino acids are positioned to provide a uniform distribution of Fc domain between complementary domain monomers. Exemplary complementary amino acid changes are shown in Tables 2A-2C. In some embodiments, one or more of the Fc domain monomers have the same sequence. In some embodiments, one or more of the Fc domain monomers have the same modifications. In these, only one, two, three, or four Fc domain monomers have the same modification.

[0091] In some instances, the Fc domain comprises at least one amino acid modification, The amino acid modifications may be directed to (i) binding affinity to one or more Fc receptors, (ii) Fc receptor binding affinity, (iii) the level of sulfation of the Fc domain; (iv) half-life; and (v) promoter function. (vi) Fc domain stability, and / or (vii) susceptibility to degradation. , or more of the amino acid sequence of the Fc domain (e.g., compared to an unmodified Fc domain). In the case of, the Fc domain has 16 or fewer amino acid modifications (e.g., 2 or fewer, 3 or fewer, 4 or fewer , 5 or less, 6 or less, 7 or less, 8 or less, 9 or less, 10 or less, 11 or less, 12 or less, 13 or less , 14 or less, 15 or less, or 16 or less amino acid modifications). The insert is designed to have 16 or fewer amino acid modifications (e.g., 2 or fewer, 3 or fewer, 4 or fewer in the CH3 domain). Below, 5 or less, 6 or less, 7 or less, 8 or less, 9 or less, 10 or less, 11 or less, 12 or less, 13 or less or less than 14, or less than 15, or less than 16 amino acid modifications).

[0092] The present disclosure also includes substantially homogenous populations of any of the Fc constructs described herein. In one embodiment, the pharmaceutical composition is a pharmaceutical composition containing the pharmaceutical composition. contains a pharmaceutical composition in which the sole or main active ingredient is 2. A substantially homogenous population of any one of the Fc constructs described in 1. above. For example, salts, detergents, surfactants, etc. t), one or more selected from fillers, polymers, preservatives and other pharmaceutical excipients; It may contain inactive ingredients.

[0093] In some embodiments, the Fc construct comprises Fc domains present in different polypeptides. In one embodiment, the Fc component is formed at least in part by the association of the main monomers. The constructs are shaped by the association of Fc domain monomers present in different polypeptides. In these embodiments, the Fc construct facilitates the association of the two polypeptides. does not contain additional domains (e.g., IgM tailpiece or IgA tailpiece) that promote In another embodiment, between two Fc domain monomers that are linked to form an Fc domain, In other embodiments, only the Fc component has a covalent bond (e.g., a disulfide bridge). The construct does not contain covalent bonds (e.g., disulfide bridges) between the Fc domains. In yet another embodiment, the Fc construct comprises an Fc domain in the Fc construct. All or substantially all of the Fc receptors on the cell surface can simultaneously interact with In some embodiments of any of these embodiments, The Fc construct may be linked via a linker (e.g., a flexible amino acid spacer). The antibody comprises at least two Fc domains.

[0094] In another aspect, the present disclosure provides a composition that promotes selective dimerization of Fc domain monomers. The present disclosure features methods and methods for detecting Fc domains comprising: The main monomer is C H Within a ring of charged residues at the interface between three antibody constant domains The present disclosure also provides methods for determining whether such an Fc domain is a mutated Fc domain. Including the method of making the C HThe scope of the ring of charged residues at the interface between three antibody constant domains. The two Fc domain monomer sequences have identical mutations at at least two positions within the range. The method further comprises introducing a complementary dimerization selection module that selects for dimerization of the target gene. H 3. Antibody Constant Domains The interface between them consists of a hydrophobic patch surrounded by a ring of charged residues. H 3 When the antibody constant domain is assembled together, these charged residues are paired with oppositely charged residues. The charge of both members of two or more complementary pairs of residues is reversed. Thus, a mutated Fc domain monomer will have a higher affinity for an Fc domain monomer with the same mutated sequence. Although the complementarity is maintained, it is less complementary to the Fc domain monomer without these mutations. In this embodiment, the same dimerization selection module promotes homodimerization. These Fc domains are double mutants, K409D / D399K, K392D / D399K, E357K / K370E, D356K / K439D, K409E / D399 Includes K, K392E / D399K, E357K / K370D or D356K / K439E In another embodiment, the Fc domain comprises an Fc domain monomer having, for example, K409. A quadruple mutant combining any pair of the double mutants D / D399K / E357K / K370E The Fc domain monomers include variants.

[0095] In another embodiment, in addition to the same dimerization selection module, the Fc domain of the Fc domain The main monomers contain non-identical mutations that promote specific association (e.g., engineered cavities and protrusions). As a result, two Fc domains are formed. The monomer comprises two dimer-forming selection modules that maintain complementarity to each other, but It has little complementarity to other Fc domain monomers. This embodiment is similar to the cavity-containing Fc domain. In one embodiment, the heterodimerization is promoted between the protuberance-containing Fc domain monomer and the protuberance-containing Fc domain monomer. , complementary dimers with non-identical mutations in the charge-paired residues of both Fc domain monomers. The formation selection module is a protrusion on one Fc domain monomer and a protrusion on the other Fc domain monomer. Combined with a cavity on the body.

[0096] In any of the Fc constructs described herein, the order of the Fc domain monomers is It is understood that the order can be interchanged. For example, a second Fc In a polypeptide having a first Fc domain monomer connected to a first Fc domain monomer, The carboxy terminus of the Fc domain monomer can be attached to the amino terminus of the linker. , which in turn is linked at its carboxy terminus to the amino terminus of a second Fc domain monomer. Alternatively, the carboxy terminus of the second Fc domain monomer may be linked to an amino acid of the linker. end of the first domain monomer, which in turn can be linked at its carboxy terminus to the and attached to the amino terminus. Both of these configurations are encompassed by the present disclosure.

[0097] Definition: As used herein, the term "Fc domain monomer" refers to an Fc domain monomer that contains at least one hinge domain. a second antibody constant domain and a third antibody constant domain (C H 2nd grade C H 3) or a functional fragment thereof (e.g., (i) another Fc domain) (ii) dimerize with monomers to form Fc domains, and (iii) bind to Fc receptors. The Fc domain monomer refers to, for example, a human, mouse or rat Fc domain. The Fc domain monomers can be of various origins, including IgG, Any immunoglobulin antibody isotype, including IgE, IgM, IgA, or IgD. In addition, the Fc domain monomer can be an IgG subunit. type (e.g., IgG1, IgG2a, IgG2b, IgG3, or IgG4) The Fc domain monomer can be an antigen recognition region, e.g., a variable domain. A portion of an immunoglobulin that can function as a complementarity determining region (CDR). The Fc domain monomers are composed of as many as 10 wild-type Fc domain monomer sequences. and can contain any of the following modifications (e.g., single amino acid modifications) (e.g., 1 to 10 1 to 8, 1 to 6, or 1 to 4 amino acid substitutions, additions, or deletions), the changes being The interaction between the Fc receptor and the Fc domain is altered. Examples of suitable modifications are known in the art. do.

[0098] As used herein, the term "Fc domain" refers to an Fc domain capable of binding an Fc receptor. The term refers to a dimer of two Fc domain monomers. In the wild-type Fc domain, two F Two C domain monomers H 3. Dimerization occurs through interactions between antibody constant domains In addition, there is a hinge domain between the two dimerized Fc domain monomers. Or it forms multiple disulfide bonds.

[0099] In the present disclosure, the term "Fc construct" refers to a construct comprising an Fc domain as described herein. It refers to an associated polypeptide chain that forms an Fc domain (e.g., an Fc constellation having three Fc domains). The Fc constructs described herein may have the same or different sequences. The Fc construct can include three Fc domain monomers. It is possible for the antibody to have two Fc domains, two of which are IgG1 or IgG1-derived Fc domains. the third contains an IgG2 or IgG2-derived Fc domain monomer; In an example, the Fc construct can have three Fc domains, two of which are The first one includes a pair with a protrusion inserted into a cavity, and the third one includes a pair with a protrusion inserted into a cavity. In the present disclosure, the Fc domain is, for example, H , V L , antibody variable region, which is a CDR or HVR The Fc domain does not include, for example, FcγRI, FcγRIIa, FcγRIIb , FcγRIIIa, FcγRIIIb, and FcγRIV. In some embodiments, the Fc constructs described herein form an antibody-specific structure. It does not include the original recognition regions, such as variable regions or complementarity determining regions (CDRs). In the present specification, the Fc constructs include an antigen recognition region, such as a variable region or a CD Contains R.

[0100] As used herein, the term "antibody constant domain" refers to the constant region domain of an antibody (e.g., B, C L Antibody constant domain, C H 1. Antibody constant domain, C H 2 antibody constant domain, or C H The term "antibody constant domain" refers to a polypeptide corresponding to the 3-amino acid sequence of an antibody.

[0101] As used herein, the term "promotes" refers to, for example, the ability of a molecule to bind to another separate Fc domain monomer. Two Fc domain monomers that have higher binding affinity to each other than to the Fc domain This means encouraging and supporting, such as supporting the formation of a As described herein, two Fc domain monomers that combine to form an Fc domain are Each of those C H 3. Compatible amino acid modifications (e.g., manipulations) at the antibody constant domain interface Compatible amino acid modifications The amino acid modifications are similar to those of other Fc domains that do not have these amino acid modifications or that have incompatible amino acid modifications. These Fc domain monomers may preferentially interact with each other rather than with each other. This supports the idea that two interacting C H 3. Amino acids at the antibody constant domain interface Due to the acid modification, the Fc domain monomer may be different from other Fc domain monomers that do not have the amino acid modification. This occurs because the mers have a higher affinity for each other than for each other.

[0102] As used herein, the term "dimerization selection module" refers to a single molecule consisting of two Fc domains. This refers to the sequence of Fc domain monomers that promotes favorable pairing between dimers. The somatic selection module regulates the selective interaction of the two Fc domain monomers with each other. A complementary dimerization selection module is a dimerization selection module that promotes or supports the formation of a dimer. The dimers can have the same sequence or different sequences. A formulation selection module is described herein.

[0103] As used herein, the term "engineered cavity" refers to H3. Intrinsic DNA in Antibody Constant Domains At least one of the amino acid residues has a side chain with a lower molecular weight than the original amino acid residue. By substituting different amino acid residues, C H 3. Formed within the antibody constant domain The term "native amino acid residue" refers to the wild-type C H 3. Antibody constant domain It refers to the naturally occurring amino acid residues encoded by the genetic code of the amino acids.

[0104] As used herein, the term "engineered protrusion" refers to H 3. Intrinsic DNA in Antibody Constant Domains At least one of the amino acid residues has a higher molecular weight side chain than the original amino acid residue. By substituting different amino acid residues, C H 3. Formed within the antibody constant domain The term "native amino acid residue" refers to the wild-type C H 3. Antibody constant domain It refers to the naturally occurring amino acid residues encoded by the genetic code of the amino acids.

[0105] The term "pair with a protuberance inserted into the cavity" refers to a pair in which the first Fc domain monomer has its C H 3 anti The second Fc domain monomer contains an engineered cavity within the C H 3. Contains two Fc domain monomers that contain engineered protuberances within the antibody constant domain. In the pair in which the protuberance is inserted into the cavity, the first Fc domain monomer C H 3. The engineered protrusion in the antibody constant domain is connected to the protrusion and a second F C of the c domain monomer H 3. Engineered cavities in antibody constant domains and H 3. Antibody Constant The dimers are designed to interact without significantly interfering with the normal association of the dimers at the domain-domain interface. will be placed in.

[0106] As used herein, the term "heterodimeric Fc domain" refers to a heterodimeric Fc domain. The term "Fc domain" refers to an Fc domain formed by heterodimerization of two The Fc domain monomers have different binding sites that promote the preferred formation of these two Fc domain monomers. As shown in Figures 1 and 2, There are three Fc domains: one carboxyl-terminal "stem" Fc domain and two amino In an Fc construct having an amino-terminal "branch" Fc domain, Each of the c domains is a heterodimeric Fc domain ("branched heterodimeric Fc domain"). 1 or Fc domain monomers 106 and 114 in FIG. Heterodimeric Fc domain formed by c domain monomers 112 and 116, FIG. Fc domain monomers 206 and 214 or Fc domain monomers 212 and 216 (Heterodimeric Fc domain formed by the addition of

[0107] As used herein, the term "homodimeric Fc domain" refers to a homodimeric Fc domain consisting of two Fc domain monomers. The term "Fc domain" refers to an Fc domain formed by homodimerization of two Fc Domain monomers contain identical oppositely charged mutations (see, for example, the mutations in Tables 2B and 2C). As shown in Figures 1 and 2, there are three Fc domains: Fc constructs having a "stem" Fc domain and two amino-terminal "branch" Fc domains In the present study, the carboxy-terminal "stem" Fc domain is a homodimeric Fc domain ("stem" Fc domain). The Fc domain monomer 1 in FIG. 1 may be a dimeric Fc domain. The homodimeric Fc domain formed by 04 and 110, the Fc domain monomers of FIG. 204 and 210).

[0108] As used herein, the term "heterodimerization selection module" refers to a compatible heterodimerization selection module. Preferred heterodimeric forms of two Fc domain monomers carrying a dimerization selection module To promote synthesis, the C H 3. It can be produced in the antibody constant domain It refers to engineered protuberances, engineered cavities and specific oppositely charged amino acid substitutions that are possible. Fc domain monomers containing heterodimerization selection modules bind to form heterodimers. Examples of heterodimerization selection modules are shown in Tables 1 and 2A. It shows.

[0109] As used herein, the term "homodimerization selection module" refers to a Fc domain monomer. C promotes homodimerization of the IgG1 domain to form a homodimeric Fc domain H The boundary between the three domains Fc domain single-stranded regions at at least two positions within the ring of charged residues on the surface Examples of homodimer formation selection modules are shown in Tables 2A and 2B. It shows.

[0110] As used herein, the term "linking" includes chemical linkages, recombinant means, as well as other methods such as By means of chemical bonds, such as disulfide bonds and amide bonds, two or more components may be bonded together. Combining or adding elements, components or protein domains, e.g., polypeptides For example, two single polypeptides are joined together to form a chemical bond. one adjacent bond via a linkage, chemical bond, peptide linker or other covalent bonding means In some embodiments, the first Fc domain can form a protein structure such as The first Fc domain monomer is linked to a second Fc domain monomer via a peptide linker. and the N-terminus of the peptide linker is linked to the first peptide linker via a chemical bond, e.g., a peptide bond. and the C-terminus of the peptide linker is linked to the C-terminus of the Fc domain monomer of The second Fc domain monomer is linked to the N-terminus of the second Fc domain monomer via a chemical bond that is a tide bond. In an embodiment, the N-terminus of the albumin binding peptide is , Fc domain monomer C via a linker H 3 linked to the C-terminus of an antibody constant domain.

[0111] As used herein, the term "associated" refers to a polypeptide (or a single polypeptide) that is The sequence within the Fc domains may be included in the Fc constructs described herein (e.g., three Fc domains The Fc construct may be arranged to form, for example, a hydrogen bond, a sparse Fc construct having Polypeptides (or within one single polypeptide) that are aqueous or ionic interactions For example, in some embodiments, the interaction between, for example, two Two polypeptides each containing one Fc domain monomer and one Fc domain monomer. Four polypeptides, each of which contains two polypeptides, are assembled to form three F To form an Fc construct having a c domain (e.g., as depicted in Figures 1 and 2), The four polypeptides can associate via their respective Fc domain monomers. The association between the polypeptides does not involve covalent interactions.

[0112] As used herein, the term "linker" refers to a linker that is inserted between two elements, e.g., a protein domain. A linker can be a covalent bond or a spacer. The term "conjugate" refers to a chemical bond, such as an amide bond or a disulfide bond, or a bond such as "Spacer" refers to any type of bond formed by a chemical reaction that is a chemical linkage. The term occurs between two polypeptides or polypeptide domains to differentiate the two polypeptides. Moieties (e.g., For example, polyethylene glycol (PEG) polymers) or amino acid sequences (for example, 3-2 00 amino acids, 3-150 amino acids, 3-100 amino acids, 3-60 amino acids, 3-50 amino acids, 3-40 amino acids, 3-30 amino acids, 3-20 amino acids, 3-10 amino acids, 3-8 amino acids, 3-5 amino acids, 4-30 amino acids, 5- 30 amino acids, 6-30 amino acids, 8-30 amino acids, 10-20 amino acids, 1 0-30 amino acids, 12-30 amino acids, 14-30 amino acids, 20-30 amino acids 15-25 amino acids, 15-30 amino acids, 18-22 amino acids and 20- A spacer is a sequence of 30 amino acids that is part of the primary sequence of a polypeptide. (e.g., polypeptides or polypeptide domains spaced apart via a polypeptide backbone) For example, two hinge regions or two The formation of disulfide bonds between Fc domain monomers is not considered a linker.

[0113] As used herein, the term "glycine spacer" refers to a spacer that separates two Fc domain monomers. A glycine spacer refers to a linker that contains only glycines and is linked in series. At most 4, 8, 12, 14, 16, 18 or 20 glycines (e.g., 4-30, 8-3 0, 12 to 30, 12 to 50, 12 to 100 or 12 to 200 glycine, for example, 12 ~30, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29 or 30 glycine). In some embodiments, the glycine spacer may contain the sequence GGGGG GGGGGGGGGGGGGGG (SEQ ID NO: 27) or consisting essentially of said sequence.

[0114] As used herein, the term "albumin-binding peptide" refers to a peptide that binds to serum albumin. An amino acid sequence of 12 to 16 amino acids that has affinity and function to bind serum albumin Albumin-binding peptides refer to albumin-binding peptides of various origins, for example human, mouse or rat. In some embodiments of the present disclosure, the albumin binding peptide can be Fusion to the C-terminus of Fc domain monomers to increase serum half-life of Fc constructs The albumin-binding peptide is attached to the N-terminus of the Fc domain monomer, either directly or via a linker. Or it can be fused to the C-terminus.

[0115] As used herein, the term "peptide for purification" refers to a peptide for use in the purification, isolation or identification of a polypeptide. The term "peptide for purification" refers to a peptide of any length that can be used for the determination of a polypeptide. and ligating the polypeptide to a nucleotide sequence for purification and / or isolation of the polypeptide, e.g., from a cell lysis mixture. In some embodiments, the purification peptide may aid in the isolation of the peptide. In some embodiments, the purification peptide is a peptide that binds to another moiety with specific affinity. Such moieties that specifically bind to the nucleotide sequence are attached to a solid support such as a substrate, resin or agarose beads. Examples of purification peptides that can be linked to the Fc constructs are described in more detail herein. Please describe in detail.

[0116] As used herein, the term "multimer" refers to at least two associated The term "antibody" refers to a molecule comprising an Fc construct.

[0117] As used herein, the term "antigen recognition region" refers to a region that recognizes and binds to an antigen by an antibody. The antigen recognition region refers to the portion of the antibody's light and heavy chains that recognizes the antigen. The antigen recognition region is the complementarity determining region (CDR, e.g. For example, CDR L1, CDR L2, CDR L3, CDR H1, CDR H2 and CDR RH3) and framework regions (FR), the light and heavy chain variable regions (Fab) Includes.

[0118] As used herein, the phrase "immune cell activation of an immune response" refers to the activation of immune cells by immune complexes or Fc complexes. The construct binds to Fcγ receptors (Fcγ R) (e.g., activating FcγR, e.g., FcγRI, FcγRIIa, FcγRIIc, FcγRIIIa or FcγRIIIb) Refers to an immune response. An immune complex is an antigen-antibody complex formed when an antibody binds to an antigen. Immune complexes often aggregate FcγR and mediate inflammation, infection, and other diseases. Multiple Fc domains that inhibit or activate intracellular processes that play important roles in disease In some embodiments, the Fc constructs of the present disclosure bind to FcγR. , activating FcγR (e.g., FcγRI) that signals on immune cells (e.g., monocytes) , FcγRIIa, FcγRIIc, FcγRIIIa or FcγRIIIb) Kinase phosphorylation (e.g., Syk phosphorylation) in FcγR-expressing cells can be Measurement of certain downstream signaling events, such as mitochondrial activation and calcium influx, can be used to assess immune complexes. Activation of immune cells in the immune response triggered by binding of the Fc construct or Fc-conjugate For example, phosphorylation of kinases (e.g., Syk phosphorylation) in cells can be detected. The level of calcium influx is a function of activation by immune complexes or Fc constructs. levels of kinase phosphorylation (e.g., Syk phosphorylation) or calcitonin, At least 5 times higher, e.g., 5 to 100 times (e.g., 5 to 100 times) than the level of sodium inflow. 00, 10~95, 15~90, 20~85, 25~80, 30~75, 35~70, 4 0 to 65, 45 to 60, or 50 to 55 times, for example, 5 times, 6 times, 7 times, 8 times, 9 times, 10 times , 11x, 12x, 13x, 14x, 15x, 16x, 17x, 18x, 19x, 20x , 21x, 22x, 23x, 24x, 25x, 26x, 27x, 28x, 29x, 30x , 31x, 32x, 33x, 34x, 35x, 36x, 37x, 38x, 39x, 40x , 41x, 42x, 43x, 44x, 45x, 46x, 47x, 48x, 49x, 50x , 51x, 52x, 53x, 54x, 55x, 56x, 57x, 58x, 59x, 60x , 61x, 62x, 63x, 64x, 65x, 66x, 67x, 68x, 69x, 70x , 71x, 72x, 73x, 74x, 75x, 76x, 77x, 78x, 79x, 80x , 81x, 82x, 83x, 84x, 85x, 86x, 87x, 88x, 89x, 90x , 91x, 92x, 93x, 94x, 95x, 96x, 97x, 98x, 99x, or 1 When the antibody titer is 000-fold higher, it induces immune cell activation in an immune response.

[0119] As used herein, the term "phagocytosis" refers to the process by which a cell, often a phagocyte (e.g. A cellular inflammatory process in which a cell (e.g., a monocyte) engulfs another cell, particle, or pathogen (e.g., a microorganism or parasite) and causes a phagocytosis. It refers to a form of endocytosis that results in the formation of phagosomes. Cytosis is a process that is used to detect diseased cells (e.g., cancer cells, infected or dead cells), pathogens and cells. It is the primary mechanism used to remove debris from the body by other cells (e.g., phagocytes (e.g., A cell that is targeted for phagocytosis by a monocyte is referred to as a target cell. For example, The Fc constructs of the present disclosure bind to FcγR (e.g., FcγRI, FcγR by binding to FcγRIIa, FcγRIIc, FcγRIIIa or FcγRIIIb) The immune cells (e.g., monocytes) that are activated by the antibody are then immunized against cancer cells or infected cells in the subject. The endothelial cells can phagocytose target cells that can be infected.

[0120] As used herein, the term "increases" or "increases" phagocytosis of a target cell. "Adding" refers to adding an Fc construct of the present disclosure to an immune cell (e.g., a monocyte). FcγR (e.g., FcγRI, FcγRIIa, FcγRIIc, FcγRIII Phagocytosis induced by binding to F c Increased compared to the level of phagocytosis that occurred without induction by the construct For example, the level of phagocytosis is related to induction by Fc constructs. At least 10% higher than the level of phagocytosis occurring without induction, e.g., 10–20% higher. 100% (e.g. 10-100%, 15-95%, 20-90%, 25-85%, 30 ~80%, 35~75%, 40~70%, 45~65% or 50~60%, for example 10% , 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, 20% ,21%,22%,23%,24%,25%,26%,27%,28%,29%,30% ,31%,32%,33%,34%,35%,36%,37%,38%,39%,40% ,41%,42%,43%,44%,45%,46%,47%,48%,49%,50% ,51%,52%,53%,54%,55%,56%,57%,58%,59%,60% ,61%,62%,63%,64%,65%,66%,67%,68%,69%,70% ,71%,72%,73%,74%,75%,76%,77%,78%,79%,80% ,81%,82%,83%,84%,85%,86%,87%,88%,89%,90% ,91%,92%,93%,94%,95%,96%,97%,98%,99% or 10 0%) is high, phagocytosis of target cells is increased.

[0121] As used herein, the term "treating cancer" refers to the therapeutic treatment of cancer in a subject. Therapeutic treatment may slow the progression of cancer, improve a subject's prognosis, and / or eliminate cancer. do.

[0122] As used herein, the term "treating an infection" refers to the therapeutic treatment of an infection in a subject. Therapeutic treatment refers to a procedure that slows the progression of an infection, improves the prognosis of a subject, and / or prevents infection. Eliminate the disease.

[0123] As used herein, the term "infectious disease" refers to any infectious disease caused by, for example, bacteria, viruses, fungi, parasites, Invasion of cells and tissues of interest by pathogens, including live animals, arthropods, and other microorganisms, parasites, and insects. In some embodiments, a pathogen invades the cells, tissues and / or organs of a subject. In some embodiments, the host may grow, proliferate, and / or produce toxins in the host and / or organ. The subject develops a negative reaction (i.e., an allergic reaction or immune response) to the pathogen. Examples of infections include bacterial infections, viral infections, fungal infections, and parasitic infections. Examples of infectious diseases include, but are not limited to, bacterial and protozoan infections.

[0124] As used herein, the term "bacterial infection" refers to an infection caused by one or more bacteria. Examples of bacteria that cause infections are well known in the art and include Bacteria of the genus Streptococcus (e.g., Strep tococcus pyogenes), Escherichia genus bacteria of the genus Vibrio (e.g., Vibrio cholerae (V cholerae), Enteritis genus bacteria (e.g., Enteritis salmonella) and Salmonella (S almonella bacteria (e.g., Salmonella typhi) )) but are not limited to these.

[0125] As used herein, the term "viral infection" refers to an infection caused by one or more viruses. Examples of viruses that cause infectious diseases are well known in the art. and viruses of the Retroviridae family (e.g., human immunodeficiency virus (HIV)), Viruses of the family Adenoviridae (e.g., Adenovirus), Viruses of the family Herpesviridae (e.g., herpes simplex virus types 1 and 2), viruses of the papillomavirus family (e.g., Human papillomavirus (HPV)), Poxviruses (e.g. smallpox), Picornaviridae viruses (e.g., Hepatitis A virus, poliovirus) , rhinoviruses), hepadnaviruses (e.g., hepatitis B virus), Viruses of the family Labiviridae (e.g., Hepatitis C virus, Yellow fever virus, West Nile virus) viruses of the family Togaviridae (e.g., Rubiviruses), viruses of the family Orthomyxoviridae Viruses (e.g., influenza viruses), Filoviridae viruses (e.g., Ebola Virus, Marburg virus), and Paramyxoviridae viruses (e.g., hemp Examples of viruses that cause infection include, but are not limited to, measles virus, and mumps virus.

[0126] As used herein, the term "fungal infection" refers to an infection caused by one or more fungi. Examples of fungi causing infections are well known in the art and include Aspergillus oryzae. Fungi of the genus Aspergillus (e.g., Aspergillus fumigatus Aspergillus fumigatus, Aspergillus flavus (A. flavus), Aspergillus terreus (A. terreus), Aspergillus niger (A. niger), Aspergillus candidus (A. candidus), Spergillus clavatus (A. clavatus), Aspergillus ochraceus ( A. ochraceus), Candida fungi (e.g., Candida Candida albicans, Candida parapsilosis (C parapsilosis), Candida glabrata (C. glabrata), Can Candida guilliermondii (C. guilliermondii), Candida krusei ( C.krusei), Candida lusitaniae, Candida tropicalis (C. tropicalis), Cryptococcus spp. Cryptococcus fungi (e.g., Cryptococcus neoformans) cus neoformans) and fungi of the genus Fusarium (e.g. Fusarium solani, Fusarium bell F. verticillioides, Fusarium oxysporum (F. oxysporum) but are not limited to these.

[0127] As used herein, the term "parasitic infection" refers to an infection caused by one or more parasites. An example of a parasite is a tapeworm (cestode). ), roundworms (nematodes), flukes (trematodes) and Examples of organisms that may be involved in the development of zoosporidium include, but are not limited to, monogeneans.

[0128] As used herein, the term "protozoan infection" refers to an infection caused by one or more protozoa. Examples of protozoa include the genus Entamoeba. eba protozoa (e.g., Entamoeba histolyti ca)), Plasmodium protozoa (e.g., Plasmodium falciparum Plasmodium falciparum, Plasmodium malariae alariae), Giardia protozoa (e.g., Giardia lamblia) Giardia lamblia and Trypanosoma osoma protozoa (e.g., Trypanosoma brucei) Examples of suitable antibacterial agents include, but are not limited to, Bacillus subtilis.

[0129] As used herein, the term "polynucleotide" refers to an oligonucleotide or a nucleic acid. It refers to any DNA or RNA of genomic or synthetic origin, including fragments or parts thereof. The term "NA" refers to a nucleic acid that is single-stranded or double-stranded and can represent the sense or antisense strand. A single polynucleotide is translated into a single polypeptide.

[0130] As used herein, the term "polypeptide" refers to a molecule in which monomers are linked together through amide bonds. A polypeptide is a single polymer of amino acid residues linked together. , including any amino acid sequence that is either recombinantly or synthetically produced. It is intended.

[0131] As used herein, the term "amino acid position" refers to a region of a protein or protein domain. The amino acid positions of an antibody or Fc construct are indicated by the Kab at numbering system (Kabat et al., Sequences of P roteins of Immunological Interest, Natio nal Institutes of Health, Bethesda, Md., ed. 5, 1991).

[0132] As used herein, the terms "amino acid mutation," "amino acid change," and "amino acid modification" refer to The terms " and " are used interchangeably and refer to a reference Fc domain polypeptide (e.g., wild-type, Refers to a change in the Fc domain polypeptide compared to the unmutated or unaltered Fc sequence. The reference Fc domain polypeptide is a wild-type human IgG1 Fc domain polypeptide. In some embodiments, the amino acid modification includes an amino acid substitution, deletion, and / or insertion. In some embodiments, the amino acid modification is a single amino acid modification. Amino acid modifications include amino acid substitutions, deletions and / or deletions. The description of amino acid modifications includes those for the Fc polypeptide. point mutations (e.g., single nucleotide substitutions) of the nucleotide sequence encoding the exchanges of one or more nucleotides), insertions and deletions (e.g., addition and / or removal of one or more nucleotides), etc. Unless otherwise stated, an insertion is a genetic (i.e., DNA and RNA) modification of a gene. is a sequence of one or more amino acids immediately following the amino acid position at which the insertion is to occur. Unless otherwise specified, amino acid modifications, such as substitutions, insertions, and The deletions and / or deletions are incorporated into both of the Fc domain monomers that make up the Fc domain. In one embodiment, the Fc domain has a first and a second Fc domain monomer. One or more (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 1 0 or more) amino acid modifications in the first Fc domain monomer, e.g., or multiple (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more) In some embodiments, the Fc domain may include, for example, an amino acid modification at amino acid position I2. 53 and / or R292, e.g., the first and second In other embodiments, the Fc domain may include an Fc domain monomer, such as an Fc domain monomer of The main is, for example, a heterogeneous amino acid at amino acid position I253 and / or R292. Fc domain monomers, e.g., first and second Fc domain monomers, having acid modifications. In some embodiments, at least one of the Fc constructs (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more) of the Fc domain In some examples, at least one Fc domain comprises one or more (e.g., , 2, 3, 4, 5, 6, 7, 8, 9, 10 or more) single amino acid modifications. In some instances, the Fc domain contains no more than 16 single amino acid modifications (e.g., no more than 1, no more than 2). , 3 or less, 4 or less, 5 or less, 6 or less, 7 or less, 8 or less, 9 or less, 10 or less, 11 or less, 1 2 or less, 13 or less, 14 or less, 15 or less, or 16 or less amino acid modifications. In some cases, the Fc domain monomer comprises 10 or fewer single amino acid modifications. The Fc domain monomer comprises 12 or fewer single amino acid modifications. The in monomer contains up to 14 single amino acid modifications.

[0133] In some embodiments, at least one of the Fc constructs (e.g., at least one 2) or 3) the Fc domain comprises an amino acid modification. In some embodiments, at least one Fc Domains may be one or more (e.g., 2, 3, 4, 5, 6, 7, 8, 9, 10, or 20 or more) In some embodiments, at least one Fc domain comprises 10 The following single amino acid modifications are included:

[0134] As used herein, the term "percent (%) identity" refers to a sequence similar to that described herein, e.g. Amino acid (or nucleic acid) sequences of candidate sequences that are Fc domain monomer sequences within the Fc construct residues, and align the sequences to achieve maximum percent identity, if necessary. After introducing gaps by Gaps can be introduced in one or both of the reference sequences for comparison purposes. (The non-uniform sequences can be ignored), and the reference sequences such as the wild-type Fc domain monomer sequence are The amino acid (or nucleic acid) residues of the candidate sequence that match those of the reference sequence are The alignment for determining percent identity is In a variety of ways that are within the skill of the art, for example, BLAST, ALIGN, or M Publicly available computer software such as egalign (DNASTAR) software This can be accomplished using software. One of skill in the art can easily perform a sequence analysis over the entire length of the sequences being compared. Alignment methods, including any algorithm necessary to achieve maximal alignment In some embodiments, the appropriate parameters for the measurement of the target object can be determined. The percentage of a given candidate sequence with or against a given reference sequence. amino acid (or nucleic acid) sequence identity (or identity of a given reference sequence) with the has a particular percent amino acid (or nucleic acid) sequence identity to the reference sequence, or The α,α,i,∈{,1,2,3,4,5,6,7,8,9,10,11,12,13,14,15,16,17,18,19,20,21,22,23,24,25,30,31,32,19,26,27,33,34,18,19,28,29,30,31,32,16,26 100×(Fraction A / B) where A is the number of amino acids scored as identical in the alignment of the candidate and reference sequences ( where A is the number of amino acid (or nucleic acid) residues in the reference sequence, and B is the total number of amino acid (or nucleic acid) residues in the reference sequence. In some embodiments, when the length of the candidate sequence is not equal to the length of the reference sequence, The percent amino acid (or nucleic acid) sequence identity of a candidate sequence to a reference sequence relative to the candidate sequence is It should not be equal to the percent amino acid (or nucleic acid) sequence identity of the string.

[0135] In certain embodiments, the amino acid sequence is selected over the entire length of the candidate sequence or over consecutive amino acids (or The candidate sequences are matched by matching the sequence identity (e.g., 50% to 100%) over a selected portion of the nucleic acid residues. For example, 50%~100%, 60%~100%, 70%~100%, 80%~100%, 90 %~100%, 92%~100%, 95%~100%, 97%~100%, 99%~10 For comparison with the candidate sequence, the sequence that shows identity (0% or 99.5% to 100%) is The length of the candidate sequence aligned for comparison purposes may be expressed as the length of the reference sequence. at least 30%, e.g., at least 40%, e.g., at least 50%, of the length of the reference sequence; 60%, 70%, 80%, 90% or 100%. If the corresponding positions in the molecule are occupied by the same amino acid (or nucleic acid) residues, They match in that position.

[0136] In some embodiments, the Fc constructs described herein (e.g., three Fc domains) are The Fc domain monomer in the Fc construct with ribonuclease is a wild-type Fc domain monomer. A nucleic acid sequence that is at least 95% identical (e.g., at least 95% identical) to a ... It may comprise a sequence that is at least 97%, 99% or 99.5% identical to the sequence of In some embodiments, the Fc polypeptide may comprise or consist essentially of the sequence described herein. Fc domains in a construct (e.g., an Fc construct having three Fc domains) The in monomer is any one of SEQ ID NOs: 44, 46, 48, and 50 to 53. A sequence that is at least 95% identical (e.g., at least 97%, 99% or 99.5% identical) to a) may comprise, consist of, or consist essentially of a sequence such as In one embodiment, the Fc domain monomer in the Fc construct has the sequence set forth in SEQ ID NO: : At least 95% identical (e.g., at least 97%, 99% or 99.5% identical) to the sequence The sequence may consist of:

[0137] In some cases, the Fc domain monomers in the Fc constructs described herein are At least 95% identical (e.g., at least 97%, 99% or 99.5% identical) to the sequence In some cases, the Fc in the Fc construct described herein may consist of the sequence. The domain monomer may be up to 10 (e.g., up to 9, 8, 7, 6, 5, 4, 3, 2, or 1 ) a sequence described herein having a single amino acid modification (e.g., a substitution, e.g., a conservative substitution) of It may comprise, consist of, or consist essentially of a sequence which is any one of It could be.

[0138] In some embodiments, two Fc domains alone within the Fc constructs described herein Polypeptides having a mer structure (e.g., polypeptides 102 and 108 in FIG. 1 and polypeptides 103 and 104 in FIG. 2) Peptides 202 and 208) are selected from SEQ ID NOs: 43, 45, 47, and 49. At least 95% identical (e.g., at least 97%, 99% or 99%) to any one of the sequences. 9.5% identical) to the sequence In some embodiments, the two Fc domains in the Fc constructs described herein may be The polypeptide having the in monomer has the sequence of SEQ ID NO:49 and at least 95 % identical (e.g., at least 97%, 99% or 99.5% identical) to the sequence , may consist of, or may consist essentially of, the sequence.

[0139] In some embodiments, the spacer between the two Fc domain monomers may be any of the structures described further herein. The described SEQ ID NOs: 1 to 36 (e.g., SEQ ID NOs: 17, 18, 2 6, and 27) , 77%, 79%, 81%, 83%, 85%, 87%, 89%, 91%, 93%, 95% 97%, 99%, 99.5% or 100% identical to the sequence of the present invention. It may consist essentially of said sequence.

[0140] As used herein, the term "host cell" refers to a cell that is capable of converting proteins into their corresponding nucleic acids. A medium containing the necessary cellular components, e.g., organelles, necessary for expression. Nucleic acids are typically synthesized using conventional techniques known in the art (e.g., transformation, transfection, etc.). into host cells via injection, electroporation, calcium phosphate precipitation, direct microinjection, etc. The host cell may be, for example, a bacterial cell. The cell may be a prokaryotic cell or a eukaryotic cell, for example a mammalian cell (e.g., a CHO cell). As described herein, host cells can be used to subsequently bind and express the desired Fc components. One or more polypeptides encoding desired domains capable of forming a construct. Express the peptide.

[0141] As used herein, the term "pharmaceutical composition" refers not only to an active ingredient, but also to a pharmaceutical composition for administering the active ingredient. The composition may include one or more excipients and diluents that may be suitable for the method. The pharmaceutical composition of the present disclosure refers to a medicinal or pharmaceutical preparation having an Fc construct compatible therewith. The pharmaceutical composition typically is administered intravenously or subcutaneously. In aqueous form for administration.

[0142] As used herein, a "substantially homogenous population" of polypeptides or Fc constructs refers to In a composition (e.g., a cell culture medium or a pharmaceutical composition), At least 50% of the samples were analyzed by non-reducing SDS gel electrophoresis or size exclusion chromatography. The polypeptides have the same number of Fc domains as determined by the Fc domain selection method. Substantially homogenous populations of the antibody or Fc constructs can be prepared prior to purification or by addition of Protein A or other suitable antibody. After protein G purification or any Fab or Fc specific affinity cloning In various embodiments, the polypeptide in the composition can be obtained after chromatography alone. at least 55%, 60%, 65%, 70%, 75% of the peptide or Fc construct, In another embodiment, 80% or 85% of the polypeptides in the composition have the same number of Fc domains. Up to 85%, 90%, 92% or 95% of the peptides or Fc constructs are A substantially homogenous population or composition is at least 85% homogenous (e.g., For example, at least 85%, 90%, or 95% homogeneity.

[0143] As used herein, the term "pharmaceutical acceptable carrier" refers to an excipient or A pharma- ceutically acceptable carrier is a substance that is compatible with the other ingredients of a formulation and is suitable for use in recipes. In this disclosure, pharma- ceutically acceptable carriers are intended to be non-toxic and non-toxic to the subject. The nature of the carrier must provide sufficient pharmaceutical stability to the Fc construct. For example, for oral administration, solid carriers are preferred, and for intravenous administration, Generally, an aqueous carrier (eg, WFI and / or a buffer) is used.

[0144] As used herein, a "therapeutically effective amount" refers to a therapeutically effective amount, e.g., a drug dosage, administered to a subject or patient. for the induction of a desired biological effect in a patient having a condition or disorder described herein. As used herein, a "therapeutically effective amount" refers to an amount that is effective in treating a subject, whether administered in a single dose or in combination. Any dosage or route of administration, either alone or in combination with other therapeutic agents It should also be understood that any such amount can be interpreted as an amount that produces the desired therapeutic effect. [The present invention 1001] a) Below: i) a first Fc domain monomer, ii) a second Fc domain monomer, and iii) linking the first Fc domain monomer to the second Fc domain monomer. Linker A first polypeptide comprising: b) Below: i) a third Fc domain monomer; ii) a fourth Fc domain monomer, and iii) linking said third Fc domain monomer to said fourth Fc domain monomer. Linker and a second polypeptide comprising: c) a third polypeptide comprising a fifth Fc domain monomer; and d) a fourth polypeptide comprising a sixth Fc domain monomer; and An Fc construct comprising: The first Fc domain monomer and the fifth Fc domain monomer are combined to form a first forming an Fc domain, the second Fc domain monomer and the fourth Fc domain monomer are combined to form a second Fc domain, and the third Fc domain monomer and and a sixth Fc domain monomer combined to form a third Fc domain, each of the first and second polypeptides having the sequence TIFF0007611880000106.tif46165 Including, each of the third and fourth polypeptides has the sequence TIFF0007611880000107.tif22164 Including, Fc construct. [The present invention 1002] a) Below: i) a first Fc domain monomer, ii) a second Fc domain monomer, and iii) linking the first Fc domain monomer to the second Fc domain monomer. Linker A first polypeptide comprising: b) Below: i) a third Fc domain monomer; ii) a fourth Fc domain monomer, and iii) linking the third Fc domain monomer to the fourth Fc domain monomer. Linker and a second polypeptide comprising: c) a third polypeptide comprising a fifth Fc domain monomer; and d) a fourth polypeptide comprising a sixth Fc domain monomer; and An Fc construct comprising: The first Fc domain monomer and the fifth Fc domain monomer are combined to form a first forming an Fc domain, the second Fc domain monomer and the fourth Fc domain monomer are combined to form a second Fc domain, and the third Fc domain monomer and and a sixth Fc domain monomer combined to form a third Fc domain, at least one Fc domain monomer comprises an amino acid substitution at position I253; Fc construct. [The present invention 1003] One or both of the first and fifth Fc domain monomers have the amino acid An Fc construct of the present invention 1002, comprising a substitution. [The present invention 1004] one or both of the second and fourth Fc domain monomers has the amino acid The Fc construct of the present invention 1002 or 1003, comprising a substitution. [The present invention 1005] one or both of the third and sixth Fc domain monomers have the amino acid The Fc construct of any one of 1002 to 1004, comprising a substitution. [The present invention 1006] The amino acid substitutions at position I253 are I253A, I253C, I253D, I253E, I253F, and I 253G, I253H, I253I, I253K, I253L, I253M, I253N, I253P, I253Q, Independently selected from the group consisting of I253R, I253S, I253T, I253V, I253W, and I253Y. The Fc construct of the present invention is selected from any one of 1002 to 1005. [The present invention 1007] An Fc construct of the invention 1006, wherein each amino acid substitution at position I253 is I253A. [The present invention 1008] At least one Fc domain monomer comprises an amino acid substitution at position R292. Any of the Fc constructs from 2 to 1007. [The present invention 1009] Any of the above-mentioned antibodies according to any one of claims 1002 to 1008, wherein the first Fc domain comprises an amino acid substitution at position R292. Either Fc construct. [The present invention 1010] 1002 to 1009 according to any one of claims 1002 to 1009, wherein the second Fc domain comprises an amino acid substitution at position R292. Either Fc construct. [The present invention 1011] 10. Any of the above-mentioned embodiments of the present invention, wherein the third Fc domain comprises an amino acid substitution at position R292. Either Fc construct. [The present invention 1012] The amino acid substitutions at position R292 are R292D, R292E, R292L, R292P, R292Q, R 292R, R292T, and R292Y. Either Fc construct. [The present invention 1013] An Fc construct of the invention 1012, wherein each amino acid substitution at position R292 is R292P. [The present invention 1014] The present invention relates to a method for producing an Fc domain comprising the steps of: Any of the Fc constructs, Ming 1002 to 1013. [The present invention 1015] Each Fc domain monomer may contain up to 10, Any of claims 1002 to 1014, comprising 9, 8, 7, 6, 5, 4, 3, 2, or 1 single amino acid modification. These Fc constructs. [The present invention 1016] The Fc monomer of the present invention, Instruct. [The present invention 1017] a) Below: i) a first Fc domain monomer, ii) a second Fc domain monomer, and iii) linking the first Fc domain monomer to the second Fc domain monomer. Linker A first polypeptide comprising: b) Below: i) a third Fc domain monomer; ii) a fourth Fc domain monomer, and iii) linking the third Fc domain monomer to the fourth Fc domain monomer. Linker and a second polypeptide comprising: c) a third polypeptide comprising a fifth Fc domain monomer; and d) a fourth polypeptide comprising a sixth Fc domain monomer; and An Fc construct comprising: The first Fc domain monomer and the fifth Fc domain monomer are combined to form a first forming an Fc domain, the second Fc domain monomer and the fourth Fc domain monomer are combined to form a second Fc domain, and the third Fc domain monomer and and a sixth Fc domain monomer combined to form a third Fc domain, at least one Fc domain monomer comprises an amino acid substitution at position R292; Fc construct. [The present invention 1018] One or both of the first and fifth Fc domain monomers have the amino acid An Fc construct of the present invention 1017 comprising a substitution. [The present invention 1019] one or both of the second and fourth Fc domain monomers has the amino acid The Fc construct of the present invention 1017 or 1018, comprising a substitution. [The present invention 1020] one or more of the third and sixth Fc domain monomers have the amino acid An Fc construct of any one of 1017 to 1019, comprising a substitution. [The present invention 1021] The amino acid substitutions at position R292 are R292D, R292E, R292L, R292P, R292Q, R The present invention relates to a compound according to any one of claims 1017 to 1020, which is independently selected from the group consisting of 292R, R292T, and R292Y. Either Fc construct. [The present invention 1022] An Fc construct of the invention 1021, wherein each amino acid substitution at position R292 is R292P. [The present invention 1023] The present invention relates to a method for producing an Fc domain comprising the steps of: Any of the Fc constructs, Akebono 1017 to 1022. [The present invention 1024] Each Fc domain monomer may contain up to 10, Any of claims 1017 to 1023 of the present invention, which contains 9, 8, 7, 6, 5, 4, 3, 2, or 1 single amino acid modification. These Fc constructs. [The present invention 1025] The Fc monomer of the present invention is an Fc monomer having an amino acid modification in the CH3 domain of the Fc monomer. Instruct. [The present invention 1026] The sequence of each Fc domain is a human IgG1 Fc domain with 10 or fewer single amino acid modifications. Any of the Fc constructs of 1001 to 1025 of the present invention based on the main sequence. [The present invention 1027] The sequence of each Fc domain has SEQ ID NO: 42, and the Fc construct of the present invention 1026. [The present invention 1028] each of the first Fc domain and the third Fc domain comprises the amino acid substitutions I253A and I253B; 1002 of the present invention, wherein the second Fc domain comprises the amino acid substitution R292P. Fc construct. [The present invention 1029] The Fc construct of the present invention, wherein the second Fc domain comprises the amino acid substitution I253A. Lacto. [The present invention 1030] Each of the first Fc domain and the third Fc domain comprises the amino acid substitution I253A. 1002, an Fc construct of the present invention. [The present invention 1031] Each of the first Fc domain, the second Fc domain, and the third Fc domain is An Fc construct of the present invention 1002 comprising the amino acid substitution I253A. [The present invention 1032] The second Fc domain comprises the amino acid substitutions I253A and R292P. c construct. [The present invention 1033] Each of the first Fc domain and the third Fc domain comprises the amino acid substitution I253A. and wherein the second Fc domain comprises the amino acid substitution R292P. Tract. [The present invention 1034] Each of the first Fc domain and the third Fc domain comprises the amino acid substitution I253A. and wherein the second Fc domain comprises the amino acid substitutions I253A and R292P. Fc construct. [The present invention 1035] Each of the first Fc domain and the third Fc domain comprises the amino acid substitution R292P. and wherein the second Fc domain comprises the amino acid substitution I253A. Tract. [The present invention 1036] each of the first Fc domain and the third Fc domain comprises the amino acid substitutions I253A and I253B; The Fc construct of the present invention 1002, comprising R292P. [The present invention 1037] each of the first Fc domain and the third Fc domain comprises the amino acid substitutions I253A and I253B; and the second Fc domain comprises the amino acid substitution I253A. Fc construct. [The present invention 1038] Each of the first Fc domain and the third Fc domain comprises the amino acid substitution R292P. and wherein the second Fc domain comprises the amino acid substitutions I253A and R292P. Fc construct. [The present invention 1039] Each of the first Fc domain, the second Fc domain, and the third Fc domain is An Fc construct of the present invention 1002 comprising amino acid substitutions I253A and R292P. [The present invention 1040] The present invention relates to a method for producing an Fc domain comprising the steps of: Any of the Fc constructs listed in the above table, i.e., 1028 to 1039. [The present invention 1041] The first Fc domain monomer and the fifth Fc domain monomer are and a complementary dimer that promotes dimer formation between the fifth Fc domain monomer and the fifth Fc domain monomer. Any of the Fc constructs of 1002 to 1040 of the present invention, comprising a dimer formation selection module. [The present invention 1042] The second Fc domain monomer and the fourth Fc domain monomer are and a complementary dimerization promoter between the Fc domain monomer and the fourth Fc domain monomer. Any of the Fc constructs of 1002 to 1040 of the present invention, comprising a dimer formation selection module. [The present invention 1043] the third Fc domain monomer and the sixth Fc domain monomer are and a complementary dimerization promoter between the sixth Fc domain monomer and the sixth Fc domain monomer. Any of the Fc constructs of 1002 to 1040 of the present invention, comprising a dimer formation selection module. [The present invention 1044] the first polypeptide and the second polypeptide have an identical amino acid sequence; The third polypeptide and the fourth polypeptide have the same amino acid sequence. Any of the Fc constructs 1002 to 1040 of the present invention. [The present invention 1045] Each Fc domain monomer is compared to the Fc domain monomer sequence of SEQ ID NO:42. and any one of Fc constructs 1002 to 1040 of the present invention, having five or fewer single amino acid modifications. Lacto. [The present invention 1046] Each Fc domain monomer is compared to the Fc domain monomer sequence of SEQ ID NO:42. and any one of the Fc constructs 1002 to 1040 of the present invention, having five or fewer single amino acid substitutions. Lacto. [The present invention 1047] Each Fc domain monomer is compared to the Fc domain monomer sequence of SEQ ID NO:42. and any one of Fc constructs 1002 to 1040 of the present invention, having three or fewer single amino acid modifications. Lacto. [The present invention 1048] Each Fc domain monomer is compared to the Fc domain monomer sequence of SEQ ID NO:42. and any one of Fc constructs 1002 to 1040 of the present invention, having three or fewer single amino acid modifications. Lacto. [The present invention 1049] Each of the second Fc domain monomer and the fourth Fc domain monomer comprises an amino acid substitution Any of F1002 to F1040 of the present invention, including D399K, and either K409D or K409E. c construct. [The present invention 1050] Each of the second Fc domain monomer and the fourth Fc domain monomer comprises an amino acid substitution Any of the Fc constructs of 1002 to 1040 of the present invention, comprising K392D and D399K. [The present invention 1051] Each of the second Fc domain monomer and the fourth Fc domain monomer comprises an amino acid substitution Any of the Fc constructs of 1002 to 1040 of the present invention, comprising E357K and K370E. [The present invention 1052] Each of the second Fc domain monomer and the fourth Fc domain monomer comprises an amino acid substitution Any of the Fc constructs of 1002 to 1040 of the present invention, comprising D356K and K439D. [The present invention 1053] Each of the second Fc domain monomer and the fourth Fc domain monomer comprises an amino acid substitution Any of the Fc constructs of 1002 to 1040 of the present invention, comprising K392E and D399K. [The present invention 1054] Each of the second Fc domain monomer and the fourth Fc domain monomer comprises an amino acid substitution Any of the Fc constructs of the present invention 1002 to 1040, comprising E357K and K370D. [The present invention 1055] Each of the second Fc domain monomer and the fourth Fc domain monomer comprises an amino acid substitution Any of the Fc constructs of 1002 to 1040 of the present invention, comprising D356K and K439E. [The present invention 1056] Each of the first Fc domain monomer and the third Fc domain monomer comprises an amino acid substitution S354C and T366W, wherein the fifth Fc domain monomer and the sixth Fc domain monomer Each of the antibodies comprises the amino acid substitutions Y349C, T366S, L368A, and Y407V. ~1040 Fc constructs. [The present invention 1057] each of the third and fourth polypeptides comprises the amino acid substitutions S354C and T366W; each of the first Fc domain monomer and the third Fc domain monomer comprises the amino acid substitution Y Any one of Fc constructs 1002 to 1040 of the present invention, including 349C, T366S, L368A, and Y407V. Struct. [The present invention 1058] Each of the first Fc domain monomer and the third Fc domain monomer comprises an amino acid substitution E357K or E357R, and the fifth Fc domain monomer and the sixth Fc domain monomer Any one of F1002 to F1040 of the present invention, each of which contains the amino acid substitution K370D or K370E. c construct. [The present invention 1059] The first Fc domain monomer and the third Fc domain monomer have the amino acid substitution K370 D or K370E, and Any one of Fc polypeptides 1002 to 1040 of the present invention, each of which contains the amino acid substitution E357K or E357R. Instruct. [The present invention 1060] Each of the first Fc domain monomer and the third Fc domain monomer comprises an amino acid substitution K409D or K409E, and the fifth Fc domain monomer and the sixth Fc domain monomer Any one of F1002 to F1040 of the present invention, each of which contains the amino acid substitution D399K or D399R. c construct. [The present invention 1061] The first Fc domain monomer and the third Fc domain monomer have the amino acid substitution D399 K or D399R, and Any of the Fc polypeptides of the present invention 1002 to 1060, each of which contains the amino acid substitution K409D or K409E. Instruct. [The present invention 1062] The linker has the sequence TIFF0007611880000108.tif71164 The Fc construct of any one of claims 1002 to 1061, comprising a polypeptide having the formula: [The present invention 1063] each of the first polypeptide and the second polypeptide has the sequence TIFF0007611880000109.tif46165 The Fc construct of the present invention 1002, comprising: [The present invention 1064] each of the first polypeptide and the second polypeptide has the sequence TIFF0007611880000110.tif46165 The Fc construct of the present invention 1002, comprising: [The present invention 1065] each of the first polypeptide and the second polypeptide has the sequence TIFF0007611880000111.tif46165 The Fc construct of the present invention 1002, comprising: [The present invention 1066] each of the first polypeptide and the second polypeptide has the sequence TIFF0007611880000112.tif46165 The Fc construct of the present invention 1002, comprising: [The present invention 1067] each of the first polypeptide and the second polypeptide has the sequence TIFF0007611880000113.tif46165 The Fc construct of the present invention 1002, comprising: [The present invention 1068] each of the first polypeptide and the second polypeptide has the sequence TIFF0007611880000114.tif46165 The Fc construct of the present invention 1002, comprising: [The present invention 1069] each of the first polypeptide and the second polypeptide has the sequence TIFF0007611880000115.tif46165 The Fc construct of the present invention 1002, comprising: [The present invention 1070] each of the first polypeptide and the second polypeptide has the sequence TIFF0007611880000116.tif46165 The Fc construct of the present invention 1002, comprising: [The present invention 1071] each of the first polypeptide and the second polypeptide has the sequence TIFF0007611880000117.tif46165 The Fc construct of the present invention 1002, comprising: [The present invention 1072] each of the first polypeptide and the second polypeptide has the sequence TIFF0007611880000118.tif46165 The Fc construct of the present invention 1002, comprising: [The present invention 1073] each of the first polypeptide and the second polypeptide has the sequence TIFF0007611880000119.tif46164 The Fc construct of the present invention 1002, comprising: [The present invention 1074] each of the first polypeptide and the second polypeptide has the sequence TIFF0007611880000120.tif46165 The Fc construct of the present invention 1002, comprising: [The present invention 1075] each of the first polypeptide and the second polypeptide has the sequence TIFF0007611880000121.tif46165 The Fc construct of the present invention 1002, comprising: [The present invention 1076] each of the first polypeptide and the second polypeptide has the sequence TIFF0007611880000122.tif46165 The Fc construct of the present invention 1002, comprising: [The present invention 1077] each of the first polypeptide and the second polypeptide has the sequence TIFF0007611880000123.tif46164 The Fc construct of the present invention 1002, comprising: [The present invention 1078] each of the first polypeptide and the second polypeptide has the sequence TIFF0007611880000124.tif46165 The Fc construct of the present invention 1002, comprising: [The present invention 1079] each of the third polypeptide and the fourth polypeptide TIFF0007611880000125.tif22165 The Fc construct of the present invention 1002 or 1017, comprising the sequence: [The present invention 1080] each of the third polypeptide and the fourth polypeptide TIFF0007611880000126.tif22165 The Fc construct of the present invention 1002 or 1017, comprising the sequence: [The present invention 1081] each of the third polypeptide and the fourth polypeptide TIFF0007611880000127.tif22165 The Fc construct of the present invention 1002 or 1017, comprising the sequence: [The present invention 1082] each of the third polypeptide and the fourth polypeptide TIFF0007611880000128.tif22164 The Fc construct of the present invention 1002 or 1017, comprising the sequence: [The present invention 1083] each of the first and second polypeptides having the sequence TIFF0007611880000129.tif46165 Including, each of the third and fourth polypeptides has the sequence TIFF0007611880000130.tif22165 The Fc construct of the present invention 1002, comprising: [The present invention 1084] each of the first and second polypeptides having the sequence TIFF0007611880000131.tif46165 Including, each of the third and fourth polypeptides has the sequence TIFF0007611880000132.tif22165 The Fc construct of the present invention 1002, comprising: [The present invention 1085] each of the first and second polypeptides having the sequence TIFF0007611880000133.tif46165 Including, each of the third and fourth polypeptides has the sequence TIFF0007611880000134.tif22165 The Fc construct of the present invention 1002, comprising: [The present invention 1086] each of the first and second polypeptides having the sequence TIFF0007611880000135.tif46165 Including, each of the third and fourth polypeptides has the sequence TIFF0007611880000136.tif22165 The Fc construct of the present invention 1002, comprising: [The present invention 1087] each of the first and second polypeptides having the sequence TIFF0007611880000137.tif46165 Including, each of the third and fourth polypeptides has the sequence TIFF0007611880000138.tif22165 The Fc construct of the present invention 1002, comprising: [The present invention 1088] each of the first and second polypeptides having the sequence TIFF0007611880000139.tif46165 Including, each of the third and fourth polypeptides has the sequence TIFF0007611880000140.tif22165 The Fc construct of the present invention 1002, comprising: [The present invention 1089] each of the first and second polypeptides having the sequence TIFF0007611880000141.tif46165 Including, each of the third and fourth polypeptides has the sequence TIFF0007611880000142.tif22165 The Fc construct of the present invention 1002, comprising: [The present invention 1090] each of the first and second polypeptides having the sequence TIFF0007611880000143.tif46165 Including, each of the third and fourth polypeptides has the sequence TIFF0007611880000144.tif22165 The Fc construct of the present invention 1002, comprising: [The present invention 1091] each of the first and second polypeptides having the sequence TIFF0007611880000145.tif46164 Including, each of the third and fourth polypeptides has the sequence TIFF0007611880000146.tif22164 The Fc construct of the present invention 1002, comprising: [The present invention 1092] each of the first and second polypeptides having the sequence TIFF0007611880000147.tif46165 Including, each of the third and fourth polypeptides has the sequence TIFF0007611880000148.tif22165 The Fc construct of the present invention 1002, comprising: [The present invention 1093] each of the first and second polypeptides having the sequence TIFF0007611880000149.tif46165 Including, each of the third and fourth polypeptides has the sequence TIFF0007611880000150.tif22165 The Fc construct of the present invention 1002, comprising: [The present invention 1094] each of the first and second polypeptides having the sequence TIFF0007611880000151.tif46164 Including, each of the third and fourth polypeptides has the sequence TIFF0007611880000152.tif22164 The Fc construct of the present invention 1002, comprising: [The present invention 1095] each of the first and second polypeptides having the sequence TIFF0007611880000153.tif46165 Including, each of the third and fourth polypeptides has the sequence TIFF0007611880000154.tif22165 The Fc construct of the present invention 1002, comprising: [The present invention 1096] each of the first and second polypeptides having the sequence TIFF0007611880000155.tif46165 Including, each of the third and fourth polypeptides has the sequence TIFF0007611880000156.tif22164 The Fc construct of the present invention 1002, comprising: [The present invention 1097] Construct 18, Construct 5, Construct 6, Construct 7, Construct Construct 9, Construct 10, Construct 11, Construct 13, Construct 14 , Construct 15, Construct 17, or Construct 19 of the present invention 1002. c construct. [The present invention 1098] Construct 5, Construct 6, Construct 7, Construct 9, Construct Construct 10, Construct 11, Construct 13, Construct 14, Construct 15, Construct 16, Construct 17, Construct 18, or Construct 19. , Fc construct of the present invention 1002. [This invention 1099] The first polypeptide, the second polypeptide, the third polypeptide, and / or the fourth polypeptide. Each of the polypeptides of the present invention comprises an amino acid substitution of the N-terminal D to Q. 1017 Fc construct. [The present invention 1100] (i) a first Fc domain monomer comprising a first Fc domain monomer and a second Fc domain monomer; The main course and (ii) a second Fc domain monomer comprising a third Fc domain monomer and a fourth Fc domain monomer. Domain and Including, at least one Fc domain monomer comprises an amino acid substitution at position I253; Fc construct. [The present invention 1101] Each of the amino acid substitutions at position I253 is I253A, I253C, I253D, I253E, I253F , I253G, I253H, I253I, I253K, I253L, I253M, I253N, I253P, I253 Independently from the group consisting of Q, I253R, I253S, I253T, I253V, I253W, and I253Y The Fc construct of the present invention 1100 is selected based on the above. [The present invention 1102] The Fc construct of the invention 1101, wherein the amino acid substitution at position I253 is I253A. [The present invention 1103] Any of F1100 to F1102 of the present invention, comprising at least one amino acid substitution at position R292. c construct. [The present invention 1104] Amino acid substitutions at position R292 include R292D, R292E, R292L, R292P, R292Q, R29 The Fc construct of the present invention 1103 is selected from the group consisting of 2R, R292T, and R292Y. to. [The present invention 1105] The Fc construct of the invention 1104, wherein the amino acid substitution at position R292 is R292P. [The present invention 1106] The first Fc domain monomer and the third Fc domain monomer are connected by a linker. The Fc construct of any one of claims 1100 to 1105, wherein [The present invention 1107] the second Fc domain monomer and the fourth Fc domain monomer are connected by a linker The Fc construct of any one of claims 1100 to 1106, wherein [The present invention 1108] Each linker has the sequence TIFF0007611880000157.tif77165 The Fc construct of the present invention 1106 or 1107 is independently selected from the polypeptides having the following structure: to. [The present invention 1109] The linker TIFF0007611880000158.tif4128 The construct of the present invention 1108. [The present invention 1110] The first Fc domain monomer and the second Fc domain monomer are and a complementary dimerization promoter between the first Fc domain monomer and the second Fc domain monomer. Any of the Fc constructs of the present invention 1100 to 1109, comprising a dimer formation selection module. [The present invention 1111] the third Fc domain monomer and the fourth Fc domain monomer are and a complementary dimerization promoter between the Fc domain monomer and the fourth Fc domain monomer. Any of the Fc constructs of the present invention 1100 to 1110, comprising a dimer formation selection module. [The present invention 1112] (i) a first Fc domain monomer comprising a first F domain monomer and a second Fc domain monomer; In and (ii) a second Fc domain monomer comprising a third Fc domain monomer and a fourth Fc domain monomer. Domain and Including, at least one Fc domain comprises a single amino acid substitution at position R292; Fc construct. [The present invention 1113] Each of the amino acid substitutions at position R292 is R292D, R292E, R292L, R292P, R292Q , R292R, R292T, and R292Y. c construct. [The present invention 1114] The Fc construct of the present invention 1113, wherein the amino acid substitution at position R292 is R292P. [The present invention 1115] The first Fc domain monomer and the third Fc domain monomer are connected by a linker. The Fc construct of any one of claims 1112 to 1114, wherein [The present invention 1116] the second Fc domain monomer and the fourth Fc domain monomer are connected by a linker The Fc construct of any one of claims 1112 to 1115, wherein the Fc construct is linked to the Fc construct of the present invention. [The present invention 1117] Each linker has the sequence TIFF0007611880000159.tif77165 The Fc component of any one of the present inventions 1115 to 1116 is independently selected from the polypeptides having the following structure: Struct. [The present invention 1118] The linker TIFF0007611880000160.tif4128 The Fc construct of the present invention is [The present invention 1119] The first Fc domain monomer and the second Fc domain monomer are and a complementary dimerization promoter between the first Fc domain monomer and the second Fc domain monomer. Any of the Fc constructs of the present invention 1112 to 1118, comprising a dimer formation selection module. [The present invention 1120] the third Fc domain monomer and the fourth Fc domain monomer are and a complementary dimerization promoter between the Fc domain monomer and the fourth Fc domain monomer. Any of the Fc constructs of the present invention 1091 to 1098, comprising a dimer formation selection module. [The present invention 1121] a) Below: i) a first Fc domain monomer, ii) a second Fc domain monomer, and iii) linking the first Fc domain monomer to the second Fc domain monomer. Linker A first polypeptide comprising: b) Below: i) a third Fc domain monomer; ii) a fourth Fc domain monomer, and iii) linking the third Fc domain monomer to the fourth Fc domain monomer. Linker and a second polypeptide comprising: c) a third polypeptide comprising a fifth Fc domain monomer; and d) a fourth polypeptide comprising a sixth Fc domain monomer; and An Fc construct comprising: The first Fc domain monomer and the fifth Fc domain monomer are combined to form a first forming an Fc domain, the second Fc domain monomer and the fourth Fc domain monomer are combined to form a second Fc domain, and the third Fc domain monomer and and a sixth Fc domain monomer combined to form a third Fc domain, each of the first and second polypeptides having the sequence TIFF0007611880000161.tif46165 Including, each of the third and fourth polypeptides has the sequence TIFF0007611880000162.tif22165 Including, Fc construct. [The present invention 1122] a) Below: i) a first Fc domain monomer, ii) a second Fc domain monomer, and iii) linking the first Fc domain monomer to the second Fc domain monomer. Linker A first polypeptide comprising: b) Below: i) a third Fc domain monomer; ii) a fourth Fc domain monomer, and iii) linking the third Fc domain monomer to the fourth Fc domain monomer. Linker and a second polypeptide comprising: c) a third polypeptide comprising a fifth Fc domain monomer; and d) a fourth polypeptide comprising a sixth Fc domain monomer; and An Fc construct comprising: The first Fc domain monomer and the fifth Fc domain monomer are combined to form a first forming an Fc domain, the second Fc domain monomer and the fourth Fc domain monomer are combined to form a second Fc domain, and the third Fc domain monomer and and a sixth Fc domain monomer combined to form a third Fc domain, each of the first and second polypeptides having the sequence TIFF0007611880000163.tif46165 Including, each of the third and fourth polypeptides has the sequence TIFF0007611880000164.tif22165 Including, Fc construct. [The present invention 1123] a) Below: i) a first Fc domain monomer, ii) a second Fc domain monomer, and iii) linking the first Fc domain monomer to the second Fc domain monomer. Linker A first polypeptide comprising: b) Below: i) a third Fc domain monomer; ii) a fourth Fc domain monomer, and iii) linking the third Fc domain monomer to the fourth Fc domain monomer. Linker and a second polypeptide comprising: c) a third polypeptide comprising a fifth Fc domain monomer; and d) a fourth polypeptide comprising a sixth Fc domain monomer; and An Fc construct comprising: The first Fc domain monomer and the fifth Fc domain monomer are combined to form a first forming an Fc domain, the second Fc domain monomer and the fourth Fc domain monomer are combined to form a second Fc domain, and the third Fc domain monomer and and a sixth Fc domain monomer combined to form a third Fc domain, each of the first and second polypeptides having the sequence TIFF0007611880000165.tif46165 Including, each of the third and fourth polypeptides has the sequence TIFF0007611880000166.tif22164 Including, Fc construct. [The present invention 1124] Each Fc domain can be independently selected from IgG1 Fc domain, IgG2 Fc domain, Ig The present invention relates to a method for the production of a IgG1 Fc domain, an IgG3 Fc domain, an IgG4 Fc domain, or a combination thereof. Any of the Fc constructs 02 to 1123. [The present invention 1125] Any one of F1002 to F1123 of the present invention, wherein each Fc domain is an IgG1 Fc domain. c construct. [The present invention 1126] The Fc construct of the present invention, wherein said IgG1 is human IgG1. [The present invention 1127] Each Fc domain comprises SEQ ID NO:42 with up to 10 single amino acid modifications. , Fc construct of the present invention 1126. [The present invention 1128] IgG1 Fc domains, each with up to 10 single amino acid modifications independently Main, IgG2 Fc domain, IgG3 Fc domain, IgG4 Fc domain, or or a combination thereof, any one of the Fc constructs of the present invention nos. 1002 to 1127. [The present invention 1129] one or more of the Fc domain monomers are human with up to 10 single amino acid modifications Any one of the Fc constructs according to claims 1002 to 1127 of the present invention, which is an IgG Fc domain monomer. 。 [The present invention 1130] Each Fc domain monomer has 10 or less single amino acid modifications. Either Fc construct. [The present invention 1131] Each Fc monomer has a sequence of SEQ ID NO:42 with 10 or fewer single amino acid modifications. The Fc construct of any one of claims 1002 to 1127, comprising: [The present invention 1132] Promoting clearance of autoantibodies and enhancing antigen presentation in a subject in need thereof. Suppressing or attenuating immune responses or immune complex-based activation of immune responses The present invention relates to an Fc construct according to any one of claims 1002 to 1131 for use in reducing Kut. [The present invention 1133] Rheumatoid arthritis (RA), systemic lupus erythematosus (SLE), ANCA-associated vasculitis, Phospholipid syndrome, autoimmune hemolytic anemia, chronic inflammatory demyelinating neuropathy, organ transplantation, G VHD, dermatomyositis, Goodpasture's syndrome, mediated via antibody-dependent cell-mediated cytotoxicity Type II hypersensitivity syndromes of organ system targets, e.g. Guillain-Barré syndrome, CIDP, Felix Tee's syndrome, antibody-mediated rejection, autoimmune thyroid disease, ulcerative colitis, autoimmune liver Diseases, idiopathic thrombocytopenic purpura, myasthenia gravis, neuromyelitis optica, pemphigus and other autoimmune diseases. Epidemic bullous disease, Sjögren's syndrome, mediated via antibody-dependent phagocytosis Autoimmune cytopenias and other disorders, heparin-induced thrombocytopenia (HIT), myositis, Polymyositis, antibody-dependent enhancement, stiff-person syndrome, Kawasaki disease, inclusion body myositis, systemic Neurosclerosis, IgA nephropathy, IgG4-related disease, Graves' disease, autoimmune inner ear disease (AIED) ), antiphospholipid syndrome (APS), pemphigus vulgaris, pemphigus foliaceus, pemphigus gestationis, paraneoplastic pemphigus, optic neuritis, Romberg disease, FcR-dependent inflammatory syndrome, synovitis, glomerulitis, and For use in the treatment of inflammatory diseases, autoimmune diseases, or immune diseases selected from the group consisting of vasculitis and vasculitis. The Fc construct of any one of 1002 to 1131 of the present invention. [The present invention 1134] A substantially homogenous population of any one of the Fc constructs of the present invention 1002 to 1131, and one or and a plurality of pharma- ceutical acceptable carriers or excipients. [This invention 1135] The pharmaceutical composition of invention 1134, wherein said homogenous population is at least 85% homogenous. [The present invention 1136] Nucleic acid sequences encoding SEQ ID NO:76 and SEQ ID NO:70 A cell comprising a nucleic acid sequence that [The present invention 1137] Nucleic acid sequences encoding SEQ ID NO:78 and SEQ ID NO:73 A cell comprising a nucleic acid sequence that [The present invention 1138] Nucleic acid sequences encoding SEQ ID NO:79 and SEQ ID NO:73 A cell comprising a nucleic acid sequence that [The present invention 1139] A cell comprising a nucleic acid sequence encoding any one of the Fc constructs of the present invention 1001 to 1131. 。 [The present invention 1140] A method for reducing immune cell activation of an immune response in a subject, comprising the steps of: administering to the subject any of the Fc constructs. [This invention 1141] The method of claim 1140, wherein the subject has an autoimmune disease. [This invention 1142] A method for treating inflammation in a subject, comprising administering to a subject an Fc construct of any one of claims 1001 to 1131 of the present invention. administering a tract to said subject. [This invention 1143] Methods for promoting autoantibody clearance and / or inhibiting antigen presentation in a subject - Patents.com and administering to said subject any one of the Fc constructs of the present inventions 1001 to 1131. Includes, a method. [This invention 1144] A method for preparing any one of the Fc constructs of the present invention 1001 to 1131, comprising: a) providing a host cell comprising a polynucleotide encoding said polypeptide; 、 b) inducing said polypeptide in said host cell under conditions that permit the formation of said Fc construct. expressing a tide; c) recovering the Fc construct; and A method comprising: [This invention 1145] A host cell expressing any one of the Fc constructs according to claims 1001 to 1131 of the present invention, A host cell comprises a polynucleotide encoding the polypeptide, A host cell, wherein the tide is expressed within said host cell. [This invention 1146] A polypeptide comprising an amino acid sequence selected from the group consisting of SEQ ID NO: 43 to 79. Chid. [Invention 1147] A compound selected from the group consisting of SEQ ID NO: 49 and SEQ ID NO: 61 to 79. A polypeptide comprising an amino acid sequence. [This invention 1148] array TIFF0007611880000167.tif46165 A polypeptide comprising or consisting of array TIFF0007611880000168.tif22165 and a polypeptide comprising or consisting of A composition comprising: [This invention 1149] array TIFF0007611880000169.tif46165 A polypeptide comprising or consisting of array TIFF0007611880000170.tif22165 and a polypeptide comprising or consisting of A composition comprising: [The present invention 1150] array TIFF0007611880000171.tif46165 A polypeptide comprising or consisting of array TIFF0007611880000172.tif22165 and a polypeptide comprising or consisting of A composition comprising: [This invention 1151] array TIFF0007611880000173.tif46165 A polypeptide comprising or consisting of array TIFF0007611880000174.tif22165 and a polypeptide comprising or consisting of A composition comprising: [This invention 1152] array TIFF0007611880000175.tif46165 A polypeptide comprising or consisting of array TIFF0007611880000176.tif22165 and a polypeptide comprising or consisting of A composition comprising: [This invention 1153] array TIFF0007611880000177.tif46165 A polypeptide comprising or consisting of array TIFF0007611880000178.tif22165 and a polypeptide comprising or consisting of A composition comprising: [This invention 1154] array TIFF0007611880000179.tif46165 A polypeptide comprising or consisting of array TIFF0007611880000180.tif22165 and a polypeptide comprising or consisting of A composition comprising: [This invention 1155] array TIFF0007611880000181.tif46165 A polypeptide comprising or consisting of array TIFF0007611880000182.tif22165 and a polypeptide comprising or consisting of A composition comprising: [This invention 1156] array TIFF0007611880000183.tif46164 A polypeptide comprising or consisting of array TIFF0007611880000184.tif22164 and a polypeptide comprising or consisting of A composition comprising: [This invention 1157] array TIFF0007611880000185.tif46165 A polypeptide comprising or consisting of array TIFF0007611880000186.tif22165 and a polypeptide comprising or consisting of A composition comprising: [This invention 1158] array TIFF0007611880000187.tif46165 A polypeptide comprising or consisting of array TIFF0007611880000188.tif22165 and a polypeptide comprising or consisting of A composition comprising: [This invention 1159] array TIFF0007611880000189.tif46164 A polypeptide comprising or consisting of array TIFF0007611880000190.tif22164 and a polypeptide comprising or consisting of A composition comprising: [The present invention 1160] array TIFF0007611880000191.tif46165 A polypeptide comprising or consisting of array TIFF0007611880000192.tif22165 and a polypeptide comprising or consisting of A composition comprising: [The present invention 1161] array TIFF0007611880000193.tif46165 A polypeptide comprising or consisting of array TIFF0007611880000194.tif22165 and a polypeptide comprising or consisting of A composition comprising: [The present invention 1162] The first and second polypeptides listed are present in a molar ratio of 1.1:1 to 1:1.1. The composition of any one of claims 1148 to 1161 of the present invention. [The present invention 1163] A method for treating a patient, comprising administering to the patient any one of the compositions of the present invention 1148 to 1162. How to do it. [Brief description of the drawings]

[0145] [Figure 1] FIG. 1 is an illustration of an Fc construct (Fc construct 1, Fc construct 2, or Fc construct 3) containing three Fc domains formed by four polypeptides. The first polypeptide (102) contains one Fc domain monomer (104) that contains an amino acid with a different charge at the CH3-CH3 interface compared to the wild-type Fc domain monomer sequence, linked in tandem with a protuberance-containing Fc domain monomer (106). The second polypeptide (108) contains an Fc domain monomer (110) that contains an amino acid with a different charge at the CH3-CH3 interface compared to the wild-type Fc domain monomer sequence, linked in tandem with another protuberance-containing Fc domain monomer (112). The third and fourth polypeptides (114 and 116, respectively) each contain a cavity-containing Fc domain monomer. [Diagram 2]FIG. 2 is an illustration of an Fc construct (Fc construct 4) containing three Fc domains formed from four polypeptides. The first polypeptide (202) contains one Fc domain monomer (204) that contains a differently charged amino acid at the CH3-CH3 interface compared to the wild-type Fc domain monomer sequence, linked in series with another Fc domain monomer (206) that contains a differently charged amino acid and a protuberance. The second polypeptide (208) contains an Fc domain monomer (210) that contains a differently charged amino acid at the CH3-CH3 interface compared to the wild-type Fc domain monomer sequence, linked in series with another Fc domain monomer (212) that contains a differently charged amino acid and a protuberance. The third and fourth polypeptides (214 and 216, respectively) each contain an Fc domain monomer that contains a differently charged amino acid and a cavity. [Diagram 3] FIG. 3 shows the identification of O-xylosylated Ser in the linker of Fc construct 2 (SGGGSGGGSGGGSGGGSGGG (SEQ ID NO:18)) by LC-MS / MS. [Figure 4] FIG. 4 shows the abundance of O-xylosylation of the linker in an Fc construct having two Fc domains (Fc construct shown in FIG. 13) as determined by LC-MS / MS. [Diagram 5] FIG. 5 shows the formation of monomeric Fc species from Fc constructs 2 and 4 stored at 45° C. as determined by CE-SDS. [Figure 6] FIG. 6 shows the proteolytic products of Fc construct 2 stored at 45° C. for 2 weeks as determined by LC-MS. [Figure 7] FIG. 7 shows the proteolytic products of Fc construct 4 stored at 45° C. for 2 weeks as determined by LC-MS. [Figure 8] FIG. 8 shows inhibition of IL-8 release by THP-1 cells by Fc construct 2 with varying linker length. [Figure 9]FIG. 9 shows inhibition of calcium flux in neutrophils by Fc construct 2 with varying linker length. [Figure 10] FIG. 10 shows the size distribution of Fc construct 2 and Fc construct 4 in crude medium by non-reducing SDS-PAGE. [Figure 11] FIG. 11 shows the expression and assembly of Fc construct 2 ("with electrostatic steering") and another Fc construct having three Fc domains but without the electrostatic steering mutations in the "stem" subunit ("without electrostatic steering"). [Figure 12] FIG. 12 shows that removal of the C-terminal lysine to generate Fc construct 2 did not induce complement-dependent cytotoxicity (CDC) in vitro. [Figure 13] FIG. 13 is an illustration of an Fc construct containing two Fc domains formed by three polypeptides. [Figure 14] FIG. 14 is an illustration of an Fc construct (Fc5X) containing five Fc domains formed by six polypeptides. [Figure 15] FIG. 15 is an illustration of an Fc construct (Fc5Y) containing five Fc domains formed by six polypeptides. [Figure 16] FIG. 16 is an illustration of an Fc construct containing five Fc domains formed by six polypeptides (inverted Fc5Y). [Figure 17] FIG. 17 is an illustration of an Fc construct containing three Fc domains formed by two polypeptides. [Figure 18A]FIG. 18A is an illustration of an Fc construct (Construct 5) containing three Fc domains formed by four polypeptides. The first polypeptide (502) and the second polypeptide (508) each contain one Fc domain monomer that contains a charged amino acid at the CH3-CH3 interface than the wild-type sequence (506 and 512, respectively) linked via a linker to a protuberance-containing Fc domain monomer (504 and 510, respectively). The third and fourth polypeptides (514 and 516, respectively) each contain a cavity-containing Fc domain monomer. The first polypeptide (502) and the second polypeptide (508) each also contain an electrostatic steering mutation, e.g., E357K. Similarly, the third and fourth polypeptides (514 and 516, respectively) each contain an electrostatic steering mutation, e.g., K370D. 506 and 512 each contain the amino acid modification I253A, which is represented as an asterisk. 502 and 508 each have the amino acid sequence of SEQ ID NO: 62. 514 and 516 each have the amino acid sequence of SEQ ID NO: 61. [Figure 18B]FIG. 18B is an illustration of an Fc construct (Construct 6) containing three Fc domains formed by four polypeptides. The first polypeptide (602) and the second polypeptide (608) each contain one Fc domain monomer that contains a charged amino acid at the CH3-CH3 interface than the wild-type sequence (606 and 612, respectively) linked via a linker to a protuberance-containing Fc domain monomer (604 and 610, respectively). The third and fourth polypeptides (614 and 616, respectively) each contain a cavity-containing Fc domain monomer. The first polypeptide (602) and the second polypeptide (608) each also contain an electrostatic steering mutation, e.g., E357K. Similarly, the third and fourth polypeptides (614 and 616, respectively) each contain an electrostatic steering mutation, e.g., K370D. 604, 610, 614, and 616 each contain the amino acid modification I253A, which is represented as an asterisk. 602 and 608 each have the amino acid sequence of SEQ ID NO:64. 614 and 616 each have the amino acid sequence of SEQ ID NO:63. [Figure 18C]FIG. 18C is an illustration of an Fc construct (Construct 7) containing three Fc domains formed by four polypeptides. The first polypeptide (702) and the second polypeptide (708) each contain one Fc domain monomer that contains a charged amino acid at the CH3-CH3 interface than the wild-type sequence (706 and 712, respectively) linked via a linker to a protuberance-containing Fc domain monomer (704 and 710, respectively). The third and fourth polypeptides (714 and 716, respectively) each contain a cavity-containing Fc domain monomer. The first polypeptide (702) and the second polypeptide (708) each also contain an electrostatic steering mutation, e.g., E357K. Similarly, the third and fourth polypeptides (714 and 716, respectively) each contain an electrostatic steering mutation, e.g., K370D. 704, 706, 710, 712, 714, and 716 each contain the amino acid modification I253A, which is represented as an asterisk. 702 and 708 each have the amino acid sequence of SEQ ID NO:65. 714 and 716 each have the amino acid sequence of SEQ ID NO:63. [Figure 18D]FIG. 18D is an illustration of an Fc construct (construct 8) containing three Fc domains formed by four polypeptides. The first polypeptide (802) and the second polypeptide (808) each contain one Fc domain monomer that contains a charged amino acid at the CH3-CH3 interface than the wild-type sequence (806 and 812, respectively) linked via a linker to a protuberance-containing Fc domain monomer (804 and 810, respectively). The third and fourth polypeptides (814 and 816, respectively) each contain a cavity-containing Fc domain monomer. The first polypeptide (802) and the second polypeptide (808) each also contain an electrostatic steering mutation, e.g., E357K. Similarly, the third and fourth polypeptides (814 and 816, respectively) each contain an electrostatic steering mutation, e.g., K370D. 806 and 812 each contain the amino acid modification R292P, which is represented as a diamond. 802 and 808 each have the amino acid sequence of SEQ ID NO: 66. 814 and 816 each have the amino acid sequence of SEQ ID NO: 61. [Figure 18E]FIG. 18E is an illustration of an Fc construct (Construct 9) containing three Fc domains formed by four polypeptides. The first polypeptide (902) and the second polypeptide (908) each contain one Fc domain monomer that contains a charged amino acid at the CH3-CH3 interface than the wild-type sequence (906 and 912, respectively) linked via a linker to a protuberance-containing Fc domain monomer (904 and 910, respectively). The third polypeptide and the fourth polypeptide (914 and 916, respectively) each contain a cavity-containing Fc domain monomer. The first polypeptide (902) and the second polypeptide (908) each also contain an electrostatic steering mutation, e.g., E357K. Similarly, the third and fourth polypeptides (914 and 916, respectively) each contain an electrostatic steering mutation, e.g., K370D. 906 and 912 each contain the amino acid modification I253A, represented as an asterisk, and the amino acid modification R292P, represented as a diamond. 902 and 908 each have the amino acid sequence of SEQ ID NO:67. 914 and 916 each have the amino acid sequence of SEQ ID NO:61. [Figure 18F]FIG. 18F is an illustration of an Fc construct (construct 10) containing three Fc domains formed by four polypeptides. The first polypeptide (1002) and the second polypeptide (1008) each contain one Fc domain monomer that contains a charged amino acid at the CH3-CH3 interface than the wild-type sequence (1006 and 1012, respectively) linked via a linker to a protuberance-containing Fc domain monomer (1004 and 1010, respectively). The third polypeptide and the fourth polypeptide (1014 and 1016, respectively) each contain a cavity-containing Fc domain monomer. The first polypeptide (1002) and the second polypeptide (1008) each also contain an electrostatic steering mutation, e.g., E357K. Similarly, the third and fourth polypeptides (1014 and 1016, respectively) each contain an electrostatic steering mutation, e.g., K370D. 1006 and 1012 each contain the amino acid modification R292P represented as a diamond, and 1004, 1010, 1014, and 1016 each contain the amino acid modification I253A represented as an asterisk. 1002 and 1008 each have the amino acid sequence of SEQ ID NO:68. 1014 and 1016 each have the amino acid sequence of SEQ ID NO:63. [Figure 18G]FIG. 18G is an illustration of an Fc construct (construct 11) containing three Fc domains formed by four polypeptides. The first polypeptide (1102) and the second polypeptide (1108) each contain one Fc domain monomer that contains a charged amino acid at the CH3-CH3 interface than the wild-type sequence (1106 and 1112, respectively) linked via a linker to a protuberance-containing Fc domain monomer (1104 and 1110, respectively). The third polypeptide and the fourth polypeptide (1114 and 1116, respectively) each contain a cavity-containing Fc domain monomer. The first polypeptide (1102) and the second polypeptide (1108) each also contain an electrostatic steering mutation, e.g., E357K. Similarly, the third and fourth polypeptides (1114 and 1116, respectively) each contain an electrostatic steering mutation, e.g., K370D. 1106 and 1112 each contain the amino acid modification R292P represented as a diamond, and 1104, 1110, 1114, and 1116 each contain the amino acid modification I253A represented as an asterisk. 1102 and 1108 each have the amino acid sequence of SEQ ID NO:69. 1114 and 1116 each have the amino acid sequence of SEQ ID NO:63. [Figure 18H]FIG. 18H is an illustration of an Fc construct (construct 12) containing three Fc domains formed by four polypeptides. The first polypeptide (1202) and the second polypeptide (1208) each contain one Fc domain monomer that contains a charged amino acid at the CH3-CH3 interface than the wild-type sequence (1206 and 1212, respectively) linked via a linker to a protuberance-containing Fc domain monomer (1204 and 1210, respectively). The third polypeptide and the fourth polypeptide (1214 and 1216, respectively) each contain a cavity-containing Fc domain monomer. The first polypeptide (1202) and the second polypeptide (1208) each also contain an electrostatic steering mutation, e.g., E357K. Similarly, the third and fourth polypeptides (1214 and 1216, respectively) each contain an electrostatic steering mutation, e.g., K370D. 1204, 1210, 1214 and 1216 each contain the amino acid modification R292P, which is represented as a diamond. 1202 and 1208 each have the amino acid sequence of SEQ ID NO:71. 1214 and 1216 each have the amino acid sequence of SEQ ID NO:70. [Figure 18I]FIG. 18I is an illustration of an Fc construct (construct 13) containing three Fc domains formed by four polypeptides. The first polypeptide (1302) and the second polypeptide (1308) each contain one Fc domain monomer that contains a charged amino acid at the CH3-CH3 interface than the wild-type sequence (1306 and 1312, respectively) linked via a linker to a protuberance-containing Fc domain monomer (1304 and 1310, respectively). The third polypeptide and the fourth polypeptide (1314 and 1316, respectively) each contain a cavity-containing Fc domain monomer. The first polypeptide (1302) and the second polypeptide (1308) each also contain an electrostatic steering mutation, e.g., E357K. Similarly, the third and fourth polypeptides (1314 and 1316, respectively) each contain an electrostatic steering mutation, e.g., K370D. 1304, 1310, 1314 and 1316 each contain the amino acid modification R292P represented as a diamond, and 1306 and 1132 each contain the amino acid modification I253A represented as an asterisk. 1302 and 1308 each have the amino acid sequence of SEQ ID NO:72. 1314 and 1316 each have the amino acid sequence of SEQ ID NO:70. [Figure 18J]FIG. 18J is an illustration of an Fc construct (construct 14) containing three Fc domains formed by four polypeptides. The first polypeptide (1402) and the second polypeptide (1408) each contain one Fc domain monomer that contains a charged amino acid at the CH3-CH3 interface than the wild-type sequence (1406 and 1412, respectively) linked via a linker to a protuberance-containing Fc domain monomer (1404 and 1410, respectively). The third polypeptide and the fourth polypeptide (1414 and 1416, respectively) each contain a cavity-containing Fc domain monomer. The first polypeptide (1402) and the second polypeptide (1408) each also contain an electrostatic steering mutation, e.g., E357K. Similarly, the third and fourth polypeptides (1414 and 1416, respectively) each contain an electrostatic steering mutation, e.g., K370D. 1404, 1410, 1414 and 1416 each contain the amino acid modification R292P, represented as a diamond, and the amino acid modification I253A, represented as an asterisk. 1402 and 1408 each have the amino acid sequence of SEQ ID NO:74. 1414 and 1416 each have the amino acid sequence of SEQ ID NO:73. [Figure 18K]FIG. 18K is an illustration of an Fc construct (construct 15) containing three Fc domains formed by four polypeptides. The first polypeptide (1502) and the second polypeptide (1508) each contain one Fc domain monomer that contains a charged amino acid at the CH3-CH3 interface than the wild-type sequence (1506 and 1512, respectively) linked via a linker to a protuberance-containing Fc domain monomer (1504 and 1510, respectively). The third polypeptide and the fourth polypeptide (1514 and 1516, respectively) each contain a cavity-containing Fc domain monomer. The first polypeptide (1502) and the second polypeptide (1508) each also contain an electrostatic steering mutation, e.g., E357K. Similarly, the third and fourth polypeptides (1514 and 1516, respectively) each contain an electrostatic steering mutation, e.g., K370D. 1504, 1510, 1514 and 1516 each contain the amino acid modification R292P represented as a diamond, and 1506 and 1512 each contain the amino acid modification I253A represented as an asterisk. 1502 and 1508 each have the amino acid sequence of SEQ ID NO:75. 1514 and 1516 each have the amino acid sequence of SEQ ID NO:73. [Figure 18L]FIG. 18L is an illustration of an Fc construct (construct 16) containing three Fc domains formed by four polypeptides. The first polypeptide (1602) and the second polypeptide (1608) each contain one Fc domain monomer that contains a charged amino acid at the CH3-CH3 interface than the wild-type sequence (1606 and 1612, respectively) linked via a linker to a protuberance-containing Fc domain monomer (1604 and 1610, respectively). The third polypeptide and the fourth polypeptide (1614 and 1616, respectively) each contain a cavity-containing Fc domain monomer. The first polypeptide (1602) and the second polypeptide (1608) each also contain an electrostatic steering mutation, e.g., E357K. Similarly, the third and fourth polypeptides (1614 and 1616, respectively) each contain an electrostatic steering mutation, e.g., K370D. 1604, 1606, 1610, 1612, 1614 and 1616 each contain the amino acid modification R292P, which is represented by a diamond. 1602 and 1608 each have the amino acid sequence of SEQ ID NO:76. 1614 and 1616 each have the amino acid sequence of SEQ ID NO:70. [Figure 18M]FIG. 18M is an illustration of an Fc construct (Construct 17) containing three Fc domains formed by four polypeptides. The first polypeptide (1702) and the second polypeptide (1708) each contain one Fc domain monomer that contains a charged amino acid at the CH3-CH3 interface than the wild-type sequence (1706 and 1712, respectively) linked via a linker to a protuberance-containing Fc domain monomer (1704 and 1710, respectively). The third polypeptide and the fourth polypeptide (1714 and 1716, respectively) each contain a cavity-containing Fc domain monomer. The first polypeptide (1702) and the second polypeptide (1708) each also contain an electrostatic steering mutation, e.g., E357K. Similarly, the third and fourth polypeptides (1714 and 1716, respectively) each contain an electrostatic steering mutation, e.g., K370D. 1704, 1706, 1710, 1712, 1714 and 1716 each contain the amino acid modification R292P represented by a diamond, and 1706 and 1712 each contain the amino acid modification I253A represented by an asterisk. 1702 and 1708 each have the amino acid sequence of SEQ ID NO:77. 1714 and 1716 each have the amino acid sequence of SEQ ID NO:70. [Figure 18N]FIG. 18N is an illustration of an Fc construct (construct 18) containing three Fc domains formed by four polypeptides. The first polypeptide (1802) and the second polypeptide (1808) each contain one Fc domain monomer that contains a charged amino acid at the CH3-CH3 interface than the wild-type sequence (1806 and 1812, respectively) linked via a linker to a protuberance-containing Fc domain monomer (1804 and 1810, respectively). The third polypeptide and the fourth polypeptide (1814 and 1816, respectively) each contain a cavity-containing Fc domain monomer. The first polypeptide (1802) and the second polypeptide (1808) each also contain an electrostatic steering mutation, e.g., E357K. Similarly, the third and fourth polypeptides (1814 and 1816, respectively) each contain an electrostatic steering mutation, e.g., K370D. 1804, 1806, 1810, 1812, 1814 and 1816 each contain the amino acid modification R292P represented by a diamond, and 1804, 1810, 1814 and 1816 each contain the amino acid modification I253A represented by an asterisk. 1802 and 1808 each have the amino acid sequence of SEQ ID NO:78. 1814 and 1816 each have the amino acid sequence of SEQ ID NO:73. [Figure 18O]FIG. 18O is an illustration of an Fc construct (Construct 19) containing three Fc domains formed by four polypeptides. The first polypeptide (1902) and the second polypeptide (1908) each contain one Fc domain monomer that contains a charged amino acid at the CH3-CH3 interface than the wild-type sequence (1906 and 1912, respectively) linked via a linker to a protuberance-containing Fc domain monomer (1904 and 1910, respectively). The third polypeptide and the fourth polypeptide (1914 and 1916, respectively) each contain a cavity-containing Fc domain monomer. The first polypeptide (1902) and the second polypeptide (1908) each also contain an electrostatic steering mutation, e.g., E357K. Similarly, the third and fourth polypeptides (1914 and 1916, respectively) each contain an electrostatic steering mutation, e.g., K370D. 1904, 1906, 1910, 1912, 1914 and 1916 each contain the amino acid modification R292P, represented by a diamond, and the amino acid modification I253A, represented by an asterisk. 1902 and 1908 each have the amino acid sequence of SEQ ID NO:79. 1914 and 1916 each have the amino acid sequence of SEQ ID NO:73. [Figure 18P] FIG. 18P is a Western blot showing expression of Construct 4, Construct 5, and Construct 7. [Figure 19]Figure 19A is a graph showing cell binding of IgG1 to Construct 4, Construct 6, Construct 16, and Construct 18 by TR-FRET to FcγRIIb (mean ± standard deviation shown for 3-8 replicates). Figure 19B is a graph showing cell binding of IgG1 to Construct 4, Construct 6, Construct 16, and Construct 18 by TR-FRET to FcγRI (mean ± standard deviation shown for 3-8 replicates). Figure 19C is a graph showing cell binding of IgG1 to Construct 4, Construct 6, Construct 16, and Construct 18 by TR-FRET to FcγRIIa (mean ± standard deviation shown for 3-8 replicates). FIG. 19D is a graph showing cell binding of IgG1 to Construct 4, Construct 6, Construct 16, and Construct 18 by TR-FRET to FcγRIIIa (mean ± standard deviation shown for 3-8 replicates). [Figure 20] 20 is a graph showing binding of Construct 4, Construct 7, Construct 6, Construct 16, Construct 18, and Construct 19 to FcRn by surface plasmon resonance (SPR) at pH 6.0. The normalized maximum binding level is proportional to the number of domains functional for binding to FcRn. [Figure 21] FIG. 21 is a graph comparing the pharmacokinetics of Construct 4, Construct 6, Construct 16, and IVIg in mice. [Figure 22] FIG. 22 is a graph comparing the pharmacokinetics of Construct 4, Construct 5, Construct 6, and Construct 7 to evaluate the effect of the number of domains with reduced FcRn binding, e.g., the I253A mutation, on the pharmacokinetics in mice. [Diagram 23] FIG. 23 is a graph comparing the pharmacokinetics of Construct 20 (Construct 4), Construct 16, Construct 18, and Construct 19 in mice. [Figure 24]FIG. 24 is a graph comparing inhibition of phagocytosis in THP-1 cells induced by IVIg, Construct 4, Construct 6, Construct 16, and Construct 18. [Diagram 25] FIG. 25 is a graph comparing inhibition of IL-8 release in monocytes induced by Pacific Blue-labeled IgG1, Construct 4, Construct 6, Construct 16, and Construct 18. [Figure 26] FIG. 26 is a graph comparing the inhibition of antibody-dependent cell-mediated cytotoxicity (ADCC) induced by Construct 4, Construct 5, Construct 6, Construct 7, Construct 16, Construct 18, and Construct 19. [Figure 27] FIG. 27 is a graph comparing the efficacy of Construct 4, Construct 6, Construct 16, and Construct 18 at various concentrations in the collagen antibody-induced arthritis (CAIA) model as measured by clinical score at day 12. [Figure 28] FIG. 28 is a graph comparing the efficacy of Construct 4 and Construct 18 in the CAIA model administered prophylactically on Day 1, as measured by clinical score. [Figure 29] FIG. 29 is a graph comparing the efficacy of Construct 4 and Construct 18 in the CAIA model administered prophylactically on day −3, as measured by clinical score. [Diagram 30] FIG. 30 is a graph comparing the efficacy of Construct 4 and Construct 18 in the CAIA model administered prophylactically on day −7, as measured by clinical score. [Diagram 31] FIG. 31 is a graph comparing the efficacy of Construct 4 and Construct 18 in the CAIA model administered prophylactically on day −10, as measured by clinical score. [Diagram 32]32 is a graph comparing the efficacy of Construct 4 (AA: black squares, solid line), Construct 18 (Q1: black triangles, dashed line), Construct 19 (Q2: black diamonds, dotted line), or saline (gray circles, dashed line) administered at 100 mg / kg on day 1 in a collagen antibody-induced arthritis (CAIA) model. An equivalent volume of saline (gray circles, dashed and dotted line) was administered on day 1. The mean and standard error of the mean are shown for each time point. [Diagram 33] 33 is a graph comparing the efficacy of Construct 4 (AA: filled squares, solid line), Construct 18 (Q1: filled triangles, dashed line), or Construct 19 (Q2: filled diamonds, dotted line) administered at 100 mg / kg on day -3 in a collagen antibody-induced arthritis (CAIA) model. An equivalent volume of saline (gray circles, dash-dotted line) was administered on day 1. The mean and standard error of the mean are shown for each time point. [Diagram 34] 34 is a graph comparing the efficacy of Construct 4 (AA: black squares, solid line), Construct 18 (Q1: black triangles, dashed line), or Construct 19 (Q2: black diamonds, dotted line) administered at 100 mg / kg on day -7 in a collagen antibody-induced arthritis (CAIA) model. An equivalent volume of saline (gray circles, dash-dotted line) was administered on day 1. The mean and standard error of the mean are shown for each time point. [Diagram 35] 35 is a graph comparing the efficacy of Construct 4 (AA: black squares, solid line), Construct 18 (Q1: black triangles, dashed line), or Construct 19 (Q2: black diamonds, dotted line) administered at 100 mg / kg on day -10 in a collagen antibody-induced arthritis (CAIA) model. An equivalent volume of saline (gray circles, dash-dotted line) was administered on day 1. The mean and standard error of the mean are shown for each time point. [Diagram 36] FIG. 36 is a graph comparing the size-exclusion chromatography profiles of purified constructs X1 (grey) and X2 (black) normalized to the maximum peak. [Figure 37]37 is an image of the results of non-reducing sodium dodecyl sulfate polyacrylamide gel electrophoresis of purified construct X1 (right), purified construct X2 (center), and molecular weight standards (right). Equal masses of the two constructs were loaded. [Figure 38] Figure 38 is a graph comparing the pharmacokinetics of construct X1 (wild-type Fc multimer) (black circles, solid line) and construct X2 (Fc multimer with I253A / R292P mutations) (triangles, dashed line) to assess the impact of reduced FcRn and FcγRIIb binding on pharmacokinetics. Means and standard deviations are shown for each time point. [Figure 39] 39 is a graph showing the pharmacokinetics of Constructs 6 (circles, SEQ ID NOs:64 and 63), 16 (triangles, SEQ ID NOs:76 / 70), and 18 (squares, SEQ ID NOs:78 and 73) administered at 10 mg / kg to cynomolgus monkeys. Construct 4 (inverted triangles, SEQ ID NOs:49 and 48) was administered at 20 mg / kg to cynomolgus monkeys. [Diagram 40] 40 is a graph showing the pharmacokinetics of Constructs 6 (gray circles, SEQ ID NOs: 64 and 63), 16 (diamonds, SEQ ID NOs: 76 and 70), and 18 (inverted triangles, SEQ ID NOs: 78 and 73) administered at 30 mg / kg to cynomolgus monkeys. Construct 4 (SEQ ID NOs: 49 and 48) was administered at 20 mg / kg (squares), 30 mg / kg (circles), and 50 mg / kg (triangles) to cynomolgus monkeys. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0146] Detailed Description of the Invention Therapeutic proteins containing the Fc domain of IgG are used to treat inflammation and immune disorders and inflammation. The present disclosure relates to a method for treating infectious diseases, cancer, and other diseases by using an Fc domain. Fc constructs containing Fc domains (e.g., Fc constructs having 2 to 10 Fc domains) Fc constructs, e.g., Fc constructs having 2, 3, 4, 5, 6, 7, 8, 9 or 10 Fc domains. The present invention features compositions and methods for preparing the Fc constructs described herein. The construct incorporates structural features (e.g., glycine spacers) that significantly improve manufacturing outcomes. Incorporation of the compound into the pharmaceutical composition facilitates the preparation of a homogenous pharmaceutical composition.

[0147] Thus, the present disclosure provides an Fc construct as described herein (e.g., a three Fc domain The present invention is characterized in that the pharmaceutical composition comprises a substantially homogenous population of Fc constructs having a Homogeneity is an important consideration in determining the quality of a pharmaceutical composition, as it affects the pharmacokinetics and performance of the composition in vivo. Traditionally, pharmaceutical manufacturing has relied on the manufacturing method of the product. There are problems with product uniformity that can be caused by several factors. For example, The agent may induce random product cleavage, proteolysis, degradation and / or aggregation, as appropriate. undergo inefficient subunit association and / or inefficient protein folding It can be a cell that has various biosynthetic processes or cellular machinery that is used for pharmaceutical production. Different organisms can also give rise to heterogeneous structures in the product. The initial culture undergoes a rigorous purification process to produce a low heterogeneity composition containing the drug. It is necessary to do so.

[0148] In one aspect, the present disclosure significantly improves the folding efficiency of Fc constructs, and Fc components with structural features that minimize inappropriate subunit association. These Fc constructs are characterized by their high homogeneity. High homogeneity leads to pharmaceutical compositions that are safe, effective, and uniform. The high degree of homogeneity ensures stability and reliability, and prevents contamination by unwanted substances. Possible aggregation or degradation of the drug product (e.g., product degradation and / or product This not only minimizes the formation of aggregates or multimers, but also reduces the risk of contamination caused by unwanted substances. Limit unwanted and harmful side effects.

[0149] As described in detail herein, the present disclosure provides a method for producing a fusion protein comprising the steps of: Not only are substantially homogenous compositions containing the Fc constructs, but also such substantially homogenous compositions The present invention features a method for preparing the composition.

[0150] The Fc constructs described herein contain a glycine spacer between the Fc domains. As is well known in the art, linkers containing both serine and glycine include It provides structural flexibility in proteins and is commonly used to link two polypeptides. Throughout the experiment (see Example 4), it was found that phosphorylation of phospholipids containing both serine and glycine was The linker is O-glycosylated (e.g., O-xylosyl) at multiple serines in the linker. It was observed that the protein undergoes proteolysis at the N-terminus of the serine. We optimized the linker sequence and length to improve the homogeneity of the Fc construct. The present inventors aimed to further improve the efficiency of the ribozyme by replacing all linkers in the construct with , a glycine spacer having only glycines (e.g., at least 12 glycines, e.g., For example, an Fc construct having glycines 12 to 30 (SEQ ID NO: 27) The all-glycine spacer in the Fc construct enhances the This eliminated O-glycosylation in the construct and reduced the rate of proteolysis of the construct. The homogeneity of the Fc construct was further improved by reducing the amount of the antibody (see Example 4). Consequently, the use of an all-glycine spacer in the Fc construct allows c A substantially more homogenous population of constructs could be achieved.

[0151] The homogeneity is a result of the Fc construct composition. For example, the first approach ("A In approach (a)), a linker containing only glycine was incorporated to generate an Fc domain alone. As observed throughout the experiment, the Fc component can be linked to the An all-glycine spacer within the construct (e.g., at least 12 glycines, e.g. , glycines 12 to 30, SEQ ID NO:27) are not O-glycosylated; Are they less susceptible to proteolysis than traditional linkers that contain serine and glycine? (See Example 4).

[0152] In yet another approach ("approach (b)"), the Fc conjugates described herein are Compositions containing a construct (e.g., an Fc construct having three Fc domains) The homogeneity of the lysine residue is improved by removing the C-terminal lysine residue. are highly conserved in immunoglobulins across many species, but are fragmented during protein production. The C-terminal linker of the Fc construct of the present disclosure can be removed completely or partially by a molecular mechanism. Removal of synthins improves the uniformity of the resulting composition, resulting in a more homogenous Fc construct. Preparation is accomplished by the use of the Fc constructs described herein (see Example 8). For example, in some embodiments of an Fc construct having three Fc domains, the C-terminus by removing the codon for the C-terminal lysine, thereby resulting in a polypeptide without a C-terminal lysine residue. Fc constructs and result in a homogenous population of products.

[0153] The Fc constructs described herein (e.g., Fc constructs having three Fc domains) A further approach to improve the homogeneity of compositions containing structures ("Approach ( In "c")", two sets of heterodimerization selection modules were used: ) a heterodimerization selection module with different opposite charge mutations, and (ii) an engineered Heterodimerization selection modules having a cavity and a protrusion. Through experiments, we attempted to form heterodimeric Fc domains within Fc constructs. In the case where both (i) and (ii) are present, uncontrolled formation of the Fc domain monomer is prevented. By reducing aggregation and the resulting unwanted oligomers and multimers, It has been observed that the homogeneity of the resulting pharmaceutical composition is further improved. (i) at least one opposite charge mutation; and (ii) at least one engineered cavity or An Fc domain monomer containing at least one engineered protrusion can be produced. (i) at least one opposite charge mutation; and (ii) at least one engineered protrusion or selectively binds to other Fc domain monomers containing at least one engineered cavity In another embodiment, the charge-reversed mutation K370 D and contains engineered cavities Y349C, T366S, L368A and Y407V Fc domain monomers and the reversed charge mutations E357K and S354C and engineered protuberances and other Fc domain monomers containing T366W and T366W can be generated and selectively bonded. These combine to form the Fc domain.

[0154] As described in detail herein, the Fc constructs of the present disclosure (e.g., three Fc A substantially homogenous composition containing an Fc construct having an Fc domain is An all-glycine spacer is used between the two Fc domain monomers in the tract (A Approach (a) uses a polypeptide lacking a C-terminal lysine in the Fc construct. (approach (b)), and / or two of the heterodimerization selection modules Set ((i) heterodimerization selection module with different opposite charge mutations and (ii) ) Heterodimerization selection module with engineered cavities and protrusions Some Fc domain monomers in the construct promote the formation of heterodimeric Fc domains. This can be achieved by advancing the

[0155] In some embodiments, the Fc constructs described herein (e.g., three Fc domains) are A substantially homogenous composition containing an Fc construct having an IgG1A polypeptide can be prepared by approach (a) )

[0156] In some embodiments, the Fc constructs described herein (e.g., three Fc domains) are A substantially homogenous composition containing an Fc construct having an IgG1A polypeptide can be prepared by approach (b). )

[0157] In some embodiments, the Fc constructs described herein (e.g., three Fc domains) are A substantially homogenous composition containing an Fc construct having an IgG1A polypeptide can be prepared by approach (c). )

[0158] In some embodiments, the Fc constructs described herein (e.g., three Fc domains) are A substantially homogenous composition containing an Fc construct having an IgG1A polypeptide can be prepared by approach (a) This can be achieved by combining (a) and (b).

[0159] In some embodiments, the Fc constructs described herein (e.g., three Fc domains) are A substantially homogenous composition containing an Fc construct having an IgG1A polypeptide can be prepared by approach (a) This can be achieved by combining (a) and (b).

[0160] In some embodiments, the Fc constructs described herein (e.g., three Fc domains) are A substantially homogenous composition containing an Fc construct having an IgG1A polypeptide can be prepared by approach (b). This can be achieved by combining (a) and (b).

[0161] In some embodiments, the Fc constructs described herein (e.g., three Fc domains) are A substantially homogenous composition containing an Fc construct having an IgG1A polypeptide can be prepared by approach (a) This can be achieved by combining (a), (b) and (c).

[0162] In some embodiments, the pharmaceutical compositions containing the Fc constructs described herein are To further improve the quality of the composition, one of the polypeptides in the Fc construct The N-terminal Asp in the or each of the Fc constructs described herein is mutated to Gln. In some embodiments of the composition comprising a substantially homogenous population of Fc constructs, the Fc constructs in the composition The N-terminal Asp in each of the polypeptides within the tract is mutated to Gln.

[0163] In addition, the Fc constructs of the present disclosure (e.g., Fc constructs having three Fc domains) can be used. In the Fc construct, the length of the linker connecting the Fc domain monomers is In some embodiments, at least 4, 8, or 1 2 glycines (e.g., 4-30, 8-30, 12-30 glycines, SEQ ID N The Fc domain in the Fc construct of the present disclosure can be linked using linkers having the following structures: The main monomers can be linked.

[0164] I. Fc domain monomer The Fc domain monomer consists of a hinge domain, C H 2 antibody constant domains, and C H 3 antibody determination The Fc domain monomers may be of various types, e.g., human, mouse, or rat. The Fc domain monomer can be derived from an immunoglobulin antibody isoform. It can be of the type IgG, IgE, IgM, IgA or IgD. Domain monomers can also be used with any immunoglobulin antibody isotype (e.g., IgG1, I The Fc domain monomer may be IgG2a, IgG2b, IgG3 or IgG4. The antibody may also be of a composite type, e.g., a hinge and C from IgG1. H 2 and IgA C H 3 complex or hinge and C from IgG1 H 2 but from IgG3 C H3. Dimers of Fc domain monomers may be present on the surface of leukocytes. FcγRIIIa, which is an Fc receptor that can bind to a certain receptor, e.g., FcγRIIIa In the present disclosure, the Fc domain monomer is C H 3. Antibody constant domain, C H 3-C H 3. Amino acid position at the antibody constant domain interface In another embodiment, the Fc domain may contain a cleavage site to facilitate their association with one another. The monomers may be N- or C-terminal, e.g., albumin-binding peptides or purification peptides. In the present disclosure, an Fc domain monomer can be, for example, a V H , V L Any type of antibody variable region, be it a complementarity determining region (CDR) or a hypervariable region (HVR). It does not contain any region.

[0165] In some embodiments, the Fc constructs described herein (e.g., three Fc domains) are The Fc domain monomer in the Fc construct with ribonuclease is a wild-type Fc domain monomer. 42. 97%, 99% or 99.5% identical), or In some embodiments, the Fc constructs described herein may consist essentially of said sequences. Fc domain monomers in a construct (e.g., an Fc construct having three Fc domains) The organism may contain up to 10 (9, 8, 7, 6, 5, 4, 3, 2, or 1) single amino acid modifications (e.g., A wild-type Fc domain monomer (SEQ ID NO: 1) having a substitution, e.g., a conservative substitution) 42) In some embodiments, the Fc constructs described herein (e.g., three Fc domains) The Fc domain monomer in the Fc construct having the Fc domain has SEQ ID NO: Any one of the sequences 44, 46, 48, and 50 to 53 (see Example 1, Tables 4 and 5) At least 95% identical (e.g., at least 97%, 99%, or 99.5% identical) to the sequence It may comprise, consist of, or consist essentially of a sequence which is In one embodiment, the Fc constructs described herein (e.g., three Fc domains) The Fc domain monomers in the Fc construct (containing up to 10 Fc domains) can be 5, 4, 3, 2 or 1) single amino acid modifications (e.g., substitutions, e.g., conservative substitutions). SEQ ID NOs: 44, 46, 48, and 50-53 (see Example 1, Tables 4 and 5) ) may comprise, consist of, or consist essentially of a sequence In some cases, these amino acid modifications can consist of changes in the length of the glycine spacer. In addition to the above, i.e., up to 10 (9, 8, 7, 6, 5, 4, 3, 2, or The single amino acid modification of all glycine spacers (SEQ ID NO:2 In one embodiment, the length of the Fc construct is in addition to the length of the Fc The domain monomer may have any one of SEQ ID NOs: 48, 52, and 53. , at least 95% identical (e.g., at least 97%, 99%, or 99.5% identical). It may comprise, consist of, or consist essentially of the sequence. In terms of structure, the Fc domain monomers in the Fc construct may be up to 10 (9, 8, 7, 6 , 5, 4, 3, 2 or 1 amino acid modification (e.g., substitution, e.g., conservative substitution). and may comprise any one of SEQ ID NOs: 48, 52, and 53; It may consist of, or consist essentially of, the sequence.

[0166] TIFF0007611880000195.tif198152TIFF0007611880000196.tif82140

[0167] II. Fc Domain As defined herein, an Fc domain is H 3. Interactions between Antibody Constant Domains Thus, it comprises two Fc domain monomers that dimerize. In the present disclosure, an Fc domain is, for example, For example, V H , V L The Fc domain does not include antibody variable regions that are CDRs or HVRs. c receptors, such as Fc-gamma receptors (i.e., Fcγ receptors (FcγR)), Fc- Alpha receptors (i.e., Fcα receptors (FcαR)), Fc-epsilon receptors ( That is, Fcε receptor (FcεR) and / or fetal Fc receptor (FcRn) In some embodiments, the Fc domain of the present disclosure forms a minimal structure that binds to an Fcγ receptor. FcγRI (CD64), FcγRIIA (CD32), FcγRIIB ( CD32), FcγRIIIA (CD16a), FcγRIIIB (CD16b) and and / or binds to FcγRIV and / or fetal Fc receptor (FcRn).

[0168] III. Fc Domain Modifications The unmodified Fc domain monomer may be a naturally occurring human Fc domain monomer or a WT human Fc domain monomer. The Fc domain monomer can be an Fc domain monomer. The Fc domain monomer can be a hinge, a CH2 domain, A naturally occurring human Fc domain monomer containing the main and CH3 domains, or a directed bispecific Fc domain monomer. up to 16 (e.g. up to 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15 or 16 amino acid modifications (e.g., a single amino acid The Fc domain monomer can be an IgA or a variant thereof having an amino acid modification. G1 Fc domain, IgG2 Fc domain, IgG3 Fc domain, IgG4 F The Fc domain monomer may be up to 10 ( 9, 8, 7, 6, 5, 4, 3, 2, or 1) single amino acid modifications (e.g., substitutions, e.g. IgG1 Fc domain, IgG2 Fc domain, IgG 3 Fc domain, IgG4 Fc domain, or a combination thereof. In the case of, the Fc domain monomer may have up to 10 amino acid modifications (e.g., 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, or 16 or fewer amino acids In some cases, the Fc domain is a human IgG Fc domain monomer having A monomer may have 10 or fewer amino acid modifications (e.g., 2, 3, 4, 5, 6, 7, 8, 9, 10 , 11, 12, 13, 14, 15, or 16 or less amino acid modifications) In some cases, the nucleic acid comprises, consists of, or consists essentially of the sequence of DNO:42. In this case, the Fc domain comprises at least one amino acid modification, The modifications may include (i) increasing binding affinity to one or more Fc receptors; (ii) decreasing effector functions; (iii) the level of sulfation of the Fc domain; (iv) half-life; and (v) protease resistance. (vi) Fc domain stability; and / or (vii) susceptibility to degradation. In some cases, the Fc domain may be modified in several ways (e.g., compared to an unmodified Fc domain). , the Fc domain has 16 or fewer amino acid modifications (e.g., no more than 2 in the CH3 domain, no more than 3 in the 4 or less, 5 or less, 6 or less, 7 or less, 8 or less, 9 or less, 10 or less, 11 or less, 12 or less 13 or less, 14 or less, 15 or less, or 16 or less amino acid modifications).

[0169] At least one Fc domain of the Fc construct of the present disclosure may have a nucleotide sequence at position I253 (e.g., For example, I253A, I253C, I253D, I253E, I253F, I253G, I2 53H, I253I, I253K, I253L, I253M, I253N, I253P, I253Q, I253R, I253S, I253T, I253V, I253W, or I2 53Y) and / or position R292 (e.g., R292P, R292D, R292E, R2 The amino acid modifications included R292L, R292Q, R292R, R292T, and R292Y. In some examples, at least one Fc domain has an amino acid sequence at position I, e.g., I253A. In some embodiments, at least one Fc domain comprises an amino acid modification at position 253, e.g., For example, an amino acid modification at position R292, such as R292P. Amino acid modifications at positions 53 (e.g., I253A) and R292 (e.g., R292P) For example, an Fc construct having three Fc domains may contain one at position I253 (e.g., I253A) in two or all three Fc domains may contain amino acid modifications, and additionally or alternatively, one, two, or all three It may include an amino acid modification at position R292 in the Fc domain (eg, R292P). FIG. 1 shows exemplary Fc constructs with I253A and / or R292P amino acid modifications. 2 and Figures 18B to 18O.

[0170] In some embodiments, the half-life-altering Fc domain modification is, for example, an F at position I253. Modification of the Fc domain can reduce binding of the modified Fc domain to FcRn. The modification at position I253 can include an amino acid substitution, where The amino acid may be a natural or unnatural amino acid, a deletion of the amino acid at position I253, or an Fc The substitution is made by the insertion of one or more amino acid residues at position I253 of the Amin domain. Modifications of the acid I253 include multiple modifications (e.g., at other residue positions, e.g., R292) Combinations, e.g., combinations of one or more amino acid substitutions, deletions and / or insertions. In certain embodiments, the Fc construct can be, for example, a triplet. At least one Fc domain may comprise at position I253. For example, the wild type amino acid residue at position I253, e.g., Amino acid (I) may be substituted with a natural or unnatural amino acid, for example, alanine (A). In some examples, each amino acid modification at position I253 is, for example, I253A, I253C, I253D, I253E, I253F, I253G, I253H, I253I, I253 K, I253L, I253M, I253N, I253P, I253Q, I253R, I2 53S, I253T, I253V, I253W, and I253Y .

[0171] In other embodiments, the half-life-altering Fc domain modification is, for example, an Fc domain modification at position R292. Domain modifications can alter the binding of modified Fc domains to FcγRIIb. The modification at position R292 can include an amino acid substitution, where The amino acid may be a natural or unnatural amino acid, a deletion of the amino acid at position R292, or an Fc The substitution is made by the insertion of one or more amino acid residues at position R292 of the Amin domain. The modification of amino acid 292 was consistent with the use of a combination of modifications (e.g., at other residue positions, e.g., I253) Combinations, e.g., combinations of one or more amino acid substitutions, deletions and / or insertions. In certain embodiments, the Fc construct can be, for example, a triplet. At least one Fc domain may comprise an Fc domain of For example, the wild type amino acid residue at position 292 is replaced with an arginine (R ) may be substituted with natural or unnatural amino acids, for example, proline (P). In the examples, each amino acid modification at position R292 is, for example, R292P, R292D, R29 Independently from 2E, R292L, R292Q, R292R, R292T, and R292Y Be selected.

[0172] Exemplary Fc domains with altered binding affinity to Fc receptors include double mutants The Fc monomer contains the S267E / L328F variant. The L328F mutation significantly and specifically enhances IgG1 binding to the FcγRIIb receptor. It has been proven that (Chu et al. Molecular Immunology 45 2008).

[0173] Amino acid modifications to alter the half-life, e.g., at position I253, e.g., I253A, , the Fc domain of the Fc construct, e.g., at least one of construct 4 (Figure 2) In other embodiments, e.g., R An amino acid modification at position R292, such as 292P, is , e.g., at least one of construct 4 (Figure 2) (e.g., 1, 2, 3, 4, or 5). In some embodiments, for example, the amino acid modification can occur at, for example, I253A It can occur both at position I253, e.g., and at position R292, e.g., R292P. For example, an Fc construct, such as construct 4 (Figure 2), may contain, for example, I253A. Any I253 may contain one Fc domain having an amino acid modification, e.g. at least one (e.g., 1, 2) amino acid modification at R292, such as R292P; In another embodiment, the Fc construct may contain one or more Fc domains. Constructs such as construct 4 (Figure 2) have an amine at I253, e.g., I253A. It may contain two Fc domains with amino acid modifications, and may also contain an R domain, such as R292P. 292, at least one (e.g., one, two, or three) Fc domain having an amino acid modification. In yet another embodiment, the Fc construct may contain a mAb, a mAb or a mAb. Construct 4 contains three Fs with an amino modification at I253, e.g., I293A. c domain and may also include an amino acid modification at R292, e.g., R292P. and may contain at least one (e.g., one, two, or three) Fc domains having For example, amino acid positions I253 and / or R292P, Exemplary Fc constructs with modifications at 292 are shown in Figures 18A-18O. Although not shown in 8A-18O, contemplated by the present disclosure are Fc domain-constituting antibodies. In the Fc domain monomer, e.g., It is an Fc construct with a heterogeneous combination of amino acid modifications.

[0174] IV. Dimerization Selection Module In the present disclosure, the dimerization selection module is a module that selects the preferred pairing of two Fc domain monomers. It is the portion of the Fc domain monomer that promotes synthesis to form the Fc domain. The merization selection module is a C domain that is formed by interacting with two Fc domain monomers. H 3. Antibody Constant C of Fc domain monomers containing amino acid substitutions located at the interface between the domains H 3. Antibody Constant The dimerization selection module is the main part of the dimerization selection. As a result of the compatibility of the amino acids selected for substitution, two C H 3. Preferred Antibody Constant Domains The preferred final form of the Fc domain is a dimerization selection module. Amino acids that do not fit in the Fc domain monomer or in the dimerization selection module It is selective for other Fc domains formed from Fc domain monomers that have the substitutions. Amino acid substitutions of this kind can be achieved using techniques such as QuikChange® mutagenesis. This can be accomplished using conventional molecular cloning techniques well known in the art.

[0175] In some embodiments, the dimer formation selection module comprises C H 3. Engineered into Antibody Constant Domains In another embodiment, the dimerization selection model comprises a cavity (described further herein). Joule is C H 3. Engineered protuberances in antibody constant domains (as further described herein) A dimerization selection module that can be adapted to selectively form an Fc domain. Two Fc domain monomers, e.g., one C containing an engineered cavity H 3. Antibody Constant The main and the other C including the operated protrusion H 3. Combine with antibody constant domain to form Fc The protrusion is inserted into the cavity of the domain monomer to form a pair. Cavities are examples of heterodimer formation selection modules, which select for compatible heterodimers. Promotes preferred heterodimer formation of two Fc domain monomers carrying a formation selection module. To advance the Fc domain monomer, H 3. It can be engineered into antibody constant domains. be.

[0176] In another embodiment, a dimer formation selection module containing a positively charged amino acid substitution is The Fc domain monomer has a negative charge and a dimerization selection module containing a negatively charged amino acid substitution. The Fc domain monomers selectively bind to the Fc domain monomers carrying the preferred charge of amino acids. By forming an Fc domain via electrostatic steering (described further herein), In some embodiments, the Fc domain monomer can be composed of the following positively charged amino acids: Substitutions and negatively charged amino acid substitutions may include: K392D, K392E, D39 9K, K409D, K409E, K439D and K439E. In one embodiment, and an Fc domain monomer containing one amino acid substitution, e.g., D356K or E357K, and a negative Fc domain monomers containing charged amino acid substitutions, e.g., K370D or K370E selectively binds to the Fc domain through favorable electrostatic steering of charged amino acids In another embodiment, an Fc domain monomer containing E357K and a K Selectively binds to Fc domain monomers containing 370D and exhibits a preference for charged amino acids. In some embodiments, the opposite charge The amino acid substitutions of the above can be used as heterodimer formation selection modules, Two Fc domain monomers that contain matching oppositely charged amino acid substitutions bind to form a helix. The specific dimerization selection modules are further described below. Further examples are listed in, but not limited to, Tables 1 and 2A.

[0177] In another embodiment, the two Fc domain monomers are H 3. Electric field at the interface between the domains A ring containing identical opposite charge mutations at at least two positions within the ring of charged residues. The homodimerization selection module comprises an Fc domain alone. Oppositely charged amino acids that promote homodimerization of the dimers to form homodimeric Fc domains The substitutions are made by changing the charge of both members of two or more complementary pairs of residues in two Fc domain monomers. By inverting the mutated Fc domain monomer, the mutated Fc domain monomer has the same mutated sequence as the Fc domain monomer. The IgG1-dependent cloning of the Fc domain maintains complementarity to the Fc domain monomer, whereas without these mutations, the IgG1-dependent cloning of the Fc domain maintains complementarity to the Fc domain monomer. In one embodiment, the Fc domain is a double mutant, K409 D / D399K, K392D / D399K, E357K / K370E, D356K / K4 39D, K409E / D399K, K392E / D399K, E357K / K370D or In another embodiment, the Fc domain comprises an Fc monomer comprising D356K / K439E. For example, any pair of double mutants, e.g., K409D / D399K / E357K / K370E The present invention includes an Fc domain monomer that includes a quadruple mutant that is linked to a homodimer formation selection module. Further examples are shown in Tables 2B and 2C.

[0178] In a further embodiment, (i) at least one opposite charge mutation and (ii) at least A single Fc domain containing an engineered cavity or at least one engineered protrusion. The mer is then subjected to (i) at least one opposite charge mutation and (ii) at least one engineered Selectively interacting with other Fc domain monomers containing protrusions or at least one engineered cavity. To form an Fc domain, for example, the reversed charge mutation K37 0D and containing engineered cavities Y349C, T366S, L368A and Y407V. The Fc domain monomer contains the reversed charge mutation E357K and the engineered protuberance S354C. and T366W to selectively bind to other Fc domain monomers containing T366W and T366W. This forms a

[0179] The formation of such Fc domains is H 3. Matching Amino Acid Substitutions in Antibody Constant Domains C H 3-C H At the three interfaces, two dimerization selection modules fit together. For example, engineered cavities are both present when the engineered cavities both contain amino acid substitutions that do not involve engineered cavities. If both contain a spike or both contain an amino acid with the same charge, The formation of heterodimeric Fc domains is not promoted.

[0180] In addition, a method for promoting the formation of an Fc domain from a defined Fc domain monomer is disclosed. Other methods that have been used include the synthesis of monomeric α-terminal leucine zippers, which allow for the formation of heterodimers. The LUZ-Y approach involves C-terminal fusions to each of the Fc domain monomers of LIX. (U.S. Patent Application Publication No. WO2011034605), as well as IgA and IgG C H Heterodimeric Fc domain monomers, each containing three alternating segments The strand-exchange engineering domain (SEED) xchange engineered domain) approach (Davis et al.,Protein Eng Des Sel.23:195-202,2 010), but are not limited to these.

[0181] V. ENGINEERED CAVITIES AND ENGINEERED PROJECTIONS The use of engineered cavities and engineered protrusions (i.e., "knob-into-hole" strategies) T.T. Carter and co-workers (Ridgway et al., Proc. tein 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 favors heterodimer formation, whereas the knob-knob and hole interactions The interaction between the holes is homodimerized due to steric mismatch and loss of favorable interactions. The "knob-into-hole" technology is also described in U.S. Patent No. 5,731,168. It has been disclosed.

[0182] In the present disclosure, engineered cavities and engineered protrusions are incorporated into the Fc constructs described herein. The engineered cavity allows the natural amino acids in a protein to be substituted with lower amino acids. It is the void that is formed when a molecule is substituted with a different amino acid with a side chain of molecular weight. The protuberances are formed when a native amino acid in a protein is replaced by a different amino acid with a higher molecular weight side chain. Specifically, the protrusion is formed when the amino acid to be replaced is is the C of the Fc domain monomer. H 3. Within the antibody constant domain, two Fc domain monomers In some embodiments, one C H 3. Engineering Within Antibody Constant Domains The cavity is the other C H 3. To Accommodate Engineered Protuberances in Antibody Constant Domains Because both C H 3. Antibody constant domains form dimers of two Fc domain monomers A dimerization selection module that promotes or supports dimerization (e.g., a heterodimerization selection module). In another embodiment, one C H 3. Antibody Stationary The engineered cavity in the domain is the other C H 3 Native amino acids in antibody constant domains In yet another embodiment, one C H 3 anti The engineered protuberance in the constant domain of the ribosome is H 3. Native ATPases in Antibody Constant Domains The amino acids are designed to form further interactions with the amino acids.

[0183] The engineered cavity is designed to accommodate amino acids containing longer side chains, such as tyrosine or tryptophan. with amino acids containing shorter side chains, such as alanine, valine, or threonine. In detail, some dimerization selection modules (e.g. For example, the heterodimerization selection module (described further above) can be H 3. Antibody Stationary The domain contains an engineered cavity, such as the Y407V mutation. Similarly, engineered protrusions Substituting amino acids containing shorter side chains with amino acids containing longer side chains In detail, some dimerization selection modules (e.g. For example, a heterodimerization selection module (described further above) can be used in H 3. Antibody Constant In the present disclosure, the engineered cavity contains an engineered protrusion such as the T366W mutation. The engineered protrusions also promote heterodimer formation. H 3-domain disulfide In one embodiment, the engineered cavities Y349C, T366S The Fc domain monomer containing L368A and Y407V has an engineered protuberance S354 Selectively binds to other Fc domain monomers containing C and T366W to form the Fc domain In another embodiment, an Fc domain containing an engineered cavity Y349C can be formed. Selectively bind the main monomer and the Fc domain monomer containing the engineered protuberance S354C. They can be combined to form an Fc domain. Disulfide bond engineering or structural calculations (HA-TF mixture), other engineered cavities and engineered protrusions are shown in Table 1, but are not limited to these.

[0184] (Table 1) TIFF0007611880000197.tif67163

[0185] C H 3. Substituting a different amino acid residue for an original amino acid residue in an antibody constant domain This can be achieved by altering the nucleic acid that encodes the native amino acid residue. The upper limit of the number of original amino acid residues that can be replaced is the number of amino acid residues that can be replaced without sufficient substitution at the interface. Considering that the interaction is maintained, C H 3. Antibody Constant Domain Interface In some cases, the CH3 antibody constant domain is 16 or less (e.g., 2, 4, 6, 8, 10, 12, 14, or 16 or less) single amino acid modifications.

[0186] VI. Electrostatic Steering Electrostatic steering is a method for controlling the formation of higher order protein molecules, peptides, proteins, and Favorable electrostatic interactions between oppositely charged amino acids within protein domains and proteins The electrostatic steering effect can be used to control heterogeneity in the generation of bispecific antibodies. Altering antibody domain interactions to favor dimer formation and reduce homodimer formation A method for doing this is disclosed in U.S. Patent Application Publication No. 2014-0024111.

[0187] In the present disclosure, electrostatic steering is used to regulate the dimerization of Fc domain monomers and the Fc In particular, the formation of Fc domain monomers is controlled using electrostatic steering. Controlling dimerization of the C H 3-C H 3. One or more amino acids forming an interface The acid residues are replaced by positively or negatively charged amino acid residues, so that the introduced Depending on the particular charged amino acid, the interaction may be electrostatically favorable or unfavorable. In some embodiments, positively charged amino acids at the interface, such as lysine, arginine, or histidine, are used. The charged amino acid is replaced with a negatively charged amino acid such as aspartic acid or glutamic acid. In another embodiment, a negatively charged amino acid at the interface is replaced with a positively charged amino acid. Charged amino acids are exchanged with the C H 3. One or both of the antibody constant domains can be introduced into the interacting C H 3. Introducing charged amino acids into antibody constant domains This results in the formation of a dimer formation selection module (described further above). However, it is controlled by electrostatic steering effects as a result of interactions between charged amino acids. In accordance with the present invention, dimers of Fc domain monomers can be selectively formed as controlled by the ribozyme.

[0188] In some embodiments, the Fc domain monomers are coupled to each other as controlled by electrostatic steering effects. A dimerization selective moiety containing an inverted charge that can selectively form dimers of the same molecule is also provided. To form a module, two Fc domain monomers can form heterodimers or homodimers. It can be selectively formed via dimerization.

[0189] Heterodimerization of Fc domain monomers Heterodimerization of Fc domain monomers can be achieved using a variety of methods, including but not limited to those listed in Table 2A. Different but compatible mutations within the two Fc domain monomers, such as identical charged residue pairs, are In some embodiments, this can be enhanced by introducing an Fc domain. The amine monomer contains one of the following positively and negatively charged amino acid substitutions: Available: D356K, D356R, E357K, E357R, K370D, K370E, K392D, K392E, D399K, K409D, K409E, K439D and K43 9E. In one embodiment, the polypeptide includes a positively charged amino acid substitution, e.g., D356K or E357K. and a negatively charged amino acid substitution, e.g., K370D or K37 0E-containing Fc domain monomers selectively bind to the preferred amino acids carrying a charge. In another embodiment, the Fc domain is formed via electrostatic steering. The Fc domain monomer having the K370D moiety selectively binds to the Fc domain monomer having the K370D moiety. These combine to form the Fc domain via favorable electrostatic steering of charged amino acids. do.

[0190] For example, in an Fc construct having three Fc domains, two of the three Fc domains The domains are aligned in a single Fc domain, as facilitated by electrostatic steering effects. The term "heterodimeric Fc domain" refers to a heterodimerization of two or more Fc domains. refers to the Fc domain formed by heterodimerization of two Fc domain monomers. wherein the two Fc domain monomers are a preferred combination of the two Fc domain monomers. Different opposite charge mutations that promote preferred formation (heterodimer formation selection modules) (e.g. As shown in Figures 1 and 2, the Fc domains are one carboxyl-terminal "stem" Fc domain and two amino-terminal "branch" Fc domains and each of the amino-terminal "branch" Fc domains is , a heterodimeric Fc domain (also referred to as a "branched heterodimeric Fc domain"). (e.g., Fc domain monomers 106 and 114 or Fc domain monomer 112 in FIG. 1 ). and 116, Fc domain monomer 2 in FIG. Heterodimers formed by 06 and 214 or Fc domain monomers 212 and 216 The branched heterodimeric Fc domain is the Fc domain containing E357K. It may be formed by a monomer containing K370D and another Fc domain monomer containing K370D.

[0191] (Table 2A) TIFF0007611880000198.tif171128

[0192] Homodimer formation of Fc domain monomers Homodimerization of Fc domain monomers is symmetrical between both Fc domain monomers. By introducing the same electrostatic steering mutation (homodimerization selection module) into In some embodiments, two Fc domain monomers are , C H At least two positions within the ring of charged residues at the interface between the three domains The two Fc domains each contain a homodimerization selection module that contains the same opposite charge mutation. By inverting the charges of both members of two or more complementary pairs of residues in the main monomer. The mutated Fc domain monomers are complementary to Fc domain monomers of the same mutated sequence. The complementarity to the Fc domain monomer is maintained, but is less than that of the Fc domain monomer without these mutations. Electrostatic steering modifications that can be introduced into c domain monomers to promote their homodimerization The differences are shown in Tables 2B and 2C, but are not limited thereto. In one embodiment, the Fc domain The main one is the double opposite charge mutant, e.g., K409D / D399K (Table 2B). In another embodiment, the Fc domain comprises two Fc domain monomers, e.g. The quadruple opposite charge mutant, i.e. K409D / D399K / K370D / E357K (Table 2 C) each of the two Fc domain monomers.

[0193] For example, in an Fc construct having three Fc domains, one of the three Fc domains The domains are aligned in a single Fc domain, as facilitated by electrostatic steering effects. The term "homodimeric Fc domain" refers to a homodimerization of Refers to an Fc domain formed by homodimerization of two Fc domain monomers. wherein the two Fc domain monomers have the same opposite charge mutations (e.g., Tables 2B and 2C). As shown in Figures 1 and 2, the Fc domains are It has one carboxyl-terminal "stem" Fc domain and two amino-terminal "branch" Fc domains. In the Fc construct, the carboxy-terminal "stem" Fc domain is homodimeric. The Fc domain of the IgG1 gene (also referred to as a "stem homodimeric Fc domain") may be a IgG1 gene (e.g., FIG. 2 shows a homodimeric Fc domain formed by Fc domain monomers 104 and 110 of a homodimeric Fc domain formed by two Fc domain monomers 204 and 210 The stem homodimeric Fc domains each contain the double mutant K409D / D399K. It may be formed by two Fc domain monomers.

[0194] (Table 2B) TIFF0007611880000199.tif154128

[0195] (Table 2C) TIFF0007611880000200.tif146166TIFF0007611880000201.tif163166

[0196] C H 3. Substituting a different amino acid residue for an original amino acid residue in an antibody constant domain This can be achieved by altering the nucleic acid that encodes the native amino acid residue. The upper limit of the number of original amino acid residues that can be replaced is the number of amino acid residues that can be replaced without sufficient substitution at the interface. Considering that the interaction is maintained, C H 3. Antibody Constant Domain Interface In some cases, the CH3 antibody constant domain is 16 or less (e.g., 2, 4, 6, 8, 10, 12, 14, or 16 or less) single amino acid modifications.

[0197] VII. Linker In the present disclosure, polypeptide or protein domains and / or associated non-protein moieties A linker is used to describe a bond or connection between two molecules. In some embodiments, A linker is a bond or connection between at least two Fc domain monomers, where and the linker is C of the first Fc domain monomer. H 3. The C-terminus of the antibody constant domain is linked to a second Two Fc domain monomers are alternately linked to the N-terminus of the hinge domain of the Fc domain monomer. In another embodiment, the linker is a Fc domain. It is the bond between the main monomer and other protein domains that are added to it. For example, the linker is the C H 3. Attach the C-terminus of the antibody constant domain to the albumin-binding domain. It is possible to add it to the N-terminus of the peptide.

[0198] The linker may be a simple covalent bond, e.g., a peptide bond, or a polyethylene glycol bond. Synthetic polymers, e.g., PEG polymers, or polymers that are chemically linked, e.g., When the linker is a peptide bond, is a protein domain that is converted to a carboxyl group at the C-terminus of another protein domain through a condensation reaction. Reacting with an amino acid at the N-terminus of a protein domain to form a peptide bond In particular, peptide bonds can be formed by conventional organic chemical reactions well known in the art. These can be produced by synthetic means via synthesis or by natural production from a host cell. For example, two Fc domain monomers are used in a tandem protein. The polynucleotide sequence encoding the NA sequence is inserted into a host cell, for example into a DNA polymerase. The necessary molecular machinery, the enzyme and the ribosome, encodes both proteins. It is possible for the gene to be directly transcribed or translated into a suitable polypeptide.

[0199] In cases where the linker is a synthetic polymer, such as a PEG polymer, the polymer may be Each end is functionalized with a reactive chemical functional group to form a terminal end at the connecting end of the two proteins. It is possible to react with amino acids.

[0200] When the linker (other than the peptide bond shown above) is formed by a chemical reaction, The substituents may be, for example, amines, carboxylic acids, esters, azides or other substituents commonly used in the art. Another functional group, a chemical functional group, is located at the C-terminus of one protein and at the C-terminus of the other protein. It is possible to synthetically add these two functional groups to the N-terminus of the amide-containing amine. Then, the two proteins react to form chemical bonds via synthetic chemistry. Such chemical linking procedures are routine for those skilled in the art. It is.

[0201] Spacer In the present disclosure, the linker between two Fc domain monomers is preferably 3 to 200 amino acids (e.g., 3~200, 3~180, 3~160, 3~140, 3~120, 3~100, 3~ 90, 3~80, 3~70, 3~60, 3~50, 3~45, 3~40, 3~35, 3~ 30, 3~25, 3~20, 3~15, 3~10, 3~9, 3~8, 3~7, 3~6, ​​3 ~5, 3~4, 4~200, 5~200, 6~200, 7~200, 8~200, 9~2 00, 10~200, 15~200, 20~200, 25~200, 30~200, 35 ~200, 40~200, 45~200, 50~200, 60~200, 70~200, 80~200, 90~200, 100~200, 120~200, 140~200, 16 0-200 or 180-200 amino acids) (e.g., 3-150, 3-100, 3-6 0, 3~50, 3~40, 3~30, 3~20, 3~10, 3~8, 3~5, 4~30, 5~30, 6~30, 8~30, 10~20, 10~30, 12~30, 14~30, 2 Amino acids containing 0-30, 15-25, 15-30, 18-22 and 20-30 amino acids In some embodiments, two Fc domain monomers may be used. The linker between the two entities is at least 12 amino acids, e.g., 12 to 200 amino acids (e.g., 12~200, 12~180, 12~160, 12~140, 12~120, 12~ 100, 12~90, 12~80, 12~70, 12~60, 12~50, 12~40, 12~30, 12~20, 12~19, 12~18, 12~17, 12~16, 12~1 5, 12-14 or 12-13 amino acids) (e.g., 14-200, 16-200, 1 8~200, 20~200, 30~200, 40~200, 50~200, 60~200 , 70~200, 80~200, 90~200, 100~200, 120~200, 14 0-200, 160-200, 180-200 or 190-200 amino acids) In some embodiments, the phosphorylation of the two Fc domain monomers is carried out by an amino acid spacer. Car is a peptide that consists of 12 to 30 amino acids (e.g., 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29 or 30 amino acids Preferred peptide spacers are known in the art as amino acid spacers containing are known to be highly flexible and contain flexible amino acid residues such as glycine and serine. In some embodiments, the spacer is, for example, GS, GGS, GG GGS (SEQ ID NO: 1), GGSG (SEQ ID NO: 2) or SGGG (SEQ ID NO:3), which is a multiple motif or a repeat motif. In some embodiments, the spacer can include, for example, TIFF0007611880000202.tif16165, which may contain 2 to 12 amino acids including the GS motif. In certain embodiments, the spacer can be, e.g., It is possible to include 3 to 12 amino acids including the GGS motif, which is TIFF0007611880000203.tif10154. In yet another embodiment, the spacer is, e.g., TIFF0007611880000204.tif10159, which contains 4 to 20 amino acids including the motif GGSG (SEQ ID NO: 2). In other embodiments, the spacer can include, for example, TIFF0007611880000205.tif4130, which may contain the motif GGGGS (SEQ ID NO:1). In some embodiments, the spacer is The file is TIFF0007611880000206.tif4128.

[0202] In some embodiments, the spacer between the two Fc domain monomers consists of only glycine residues. For example, at least four glycine residues (e.g., 4 to 200, 4 to 180, 4~160, 4~140, 4~40, 4~100, 4~90, 4~80, 4~70, 4~ 60, 4~50, 4~40, 4~30, 4~20, 4~19, 4~18, 4~17, 4~ 16, 4~15, 4~14, 4~13, 4~12, 4~11, 4~10, 4~9, 4~8 , 4 to 7, 4 to 6, or 4 to 5 glycine residues) (e.g., 4 to 200, 6 to 200, 8 to 200, 10~200, 12~200, 14~200, 16~200, 18~200, 2 0~200, 30~200, 40~200, 50~200, 60~200, 70~200 , 80~200, 90~200, 100~200, 120~200, 140~200, 1 60 to 200, 180 to 200, or 190 to 200 glycine residues). In embodiments, the spacer is a glycine residue sequence of 4 to 30 glycine residues (e.g., 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22 , 23, 24, 25, 26, 27, 28, 29 or 30 glycine residues). In this embodiment, the spacer containing only glycine residues is glycosylated (e.g., O -linked glycosylation, also called O-glycosylation, or a spacer containing, for example, one or more serine residues. Reduced levels of glycosylation (e.g., reduced levels of O-glycosylation) compared to TIFF0007611880000207.tif4128. sylation) (e.g., xylose, mannose, sialic acid, fucose (Fuc) and / or Reduced levels of O2 by glycans such as galactose (Gal) (e.g., xylose) -glycosylation) (see Example 4).

[0203] In some embodiments, the spacer containing only glycine residues is O-glycosylated ( For example, it may be non-O-xylosylated or may be, for example, one or more A spacer containing a serine residue of TIFF0007611880000208.tif4128, reduced levels of O-glycosylation (e.g., reduced levels of O- silosylation).

[0204] In some embodiments, a spacer containing only glycine residues is not susceptible to proteolysis. or may be a spacer that contains, for example, one or more serine residues. TIFF0007611880000209.tif4128 may have a reduced rate of protein degradation (see Example 4).

[0205] In some embodiments, the spacer can be, e.g., TIFF0007611880000210.tif10158, which may contain the motif GGGG (SEQ ID NO:19). In some embodiments, the spacer can be, e.g. TIFF0007611880000211.tif4132, and may contain the motif GGGGG (SEQ ID NO: 24). In some embodiments, the spacer is The file is TIFF0007611880000212.tif4128.

[0206] In other embodiments, the spacer also contains amino acids other than glycine and serine. For example, It is also possible to include TIFF0007611880000213.tif22161.

[0207] In some embodiments of the present disclosure, a 12 amino acid peptide spacer or a 20 amino acid Using a peptide spacer, two Fc domain monomers, each with the sequence TIFF0007611880000214.tif4148, a 12 amino acid peptide spacer and a 20 amino acid peptide spacer Two Fc domain monomers are connected in series (e.g., polypeptides 102 and 103 in FIG. 1 ). 08, 202 and 208 in FIG. 2). In another embodiment, the sequence An 18 amino acid peptide spacer consisting of TIFF00076118...

Claims

1. (a) a nucleic acid molecule encoding a polypeptide comprising or consisting of the amino acid sequence set forth in SEQ ID NO: 76 and a nucleic acid molecule encoding a polypeptide comprising or consisting of the amino acid sequence set forth in SEQ ID NO: 70; or (b) a nucleic acid molecule encoding a polypeptide comprising or consisting of the amino acid sequence set forth in SEQ ID NO: 78 and a nucleic acid molecule encoding a polypeptide comprising or consisting of the amino acid sequence set forth in SEQ ID NO:

73. Cells containing

2. (a) a polypeptide comprising or consisting of the sequence SEQ ID NO: 76, and a polypeptide comprising or consisting of the sequence SEQ ID NO: 70, or (b) a polypeptide comprising or consisting of the sequence SEQ ID NO: 78, and a polypeptide comprising or consisting of the sequence SEQ ID NO: 73; A composition comprising:

3. (a) a nucleic acid molecule encoding a polypeptide comprising or consisting of the amino acid sequence set forth in SEQ ID NO: 76 and a nucleic acid molecule encoding a polypeptide comprising or consisting of the amino acid sequence set forth in SEQ ID NO: 70; or (b) a nucleic acid molecule encoding a polypeptide comprising or consisting of the amino acid sequence set forth in SEQ ID NO: 78 and a nucleic acid molecule encoding a polypeptide comprising or consisting of the amino acid sequence set forth in SEQ ID NO:

73. A composition comprising:

Images